(Advances in Biochemical Engineering Biotechnology) K.E.L. Eriksson-Biotechnology in The Pulp and Paper Industry-Springer Verlag (19

Advances in Biochemical Engineering
57 Biotechnology
Managing Editor: T. Scheper
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Biotechnolgy
in the Pulp and Paper
Industr~Y
Volume Editor: K.-E. L. Eriksson
With Contributions by
M. Akhtar, D.S. Argyropoulos, P. Bajpai, P. K. Bajpai,
R. A. Blanchette, J. Buchert, J. F. D. Dean,
K.-E. L. Eriksson, R. L. Farrell, M. Guenette, K. Hata,
S. C. Johnsrud, T. K. Kirk, R. C. Kuhad, P. R. LaFayette,
S. B. Menachem, S. A. Merkle, A. Singh, A. Suurn~ikki,
M. Tenkanen, J. S. Tolan, L. Viikari, M. B. Wall
With 41 Figures and 52 Tables
Springer
ISSN 0724-6145
ISBN3-540-61868-6 Springer-Verlag Berlin Heidelberg NewYork
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Managing Editor
Professor Dr. T. Scheper

Institute of Technical Chemistry, University of Hannover
Callinstral3e 3, D - 30167 Hannover, FRG
Volume Editor
Professor Dr. K.-E. L. Eriksson

Center for Biological Resource Recovery, The University of Georgia,
A214 Life Sciences Building, Athens, GA 3 0602-7229/USA
Editorial Board
Prof. Dr. W. Babel Center of Environmental Research

Leipzig-Halle GmbH
Section of EnvironmentalMicrobiology
PeermoserstraBe 15
D - 04318 Leipzig/FRG
Prof. Dr./4~ W. Blanch University of California
Department of Chemical Engineering
Berkely, CA 94720-9989/USA
Prof. Dr. Ch. L. Cooney Massachusetts Institute of Technology
Department of Chemical Engineering
25 Ames Street
Cambridge, MA 02139/USA
Prof. Dr. S.-O. Enfors Department of Biochemistry and Biotechnology

Royal Institute of Technology
Teknikringen 34, S - 100 44 Stockholm/Sweden
Prof. Dr. K.-E. L. Eriksson Center for Biological Resource Recovery

The University of Georgia
A214 Life Sciences Building
Athens, GA 30602-7229/USA
Prof. Dr. A. Fiechter Institute of Biotechnology
EidgenOssische Technische Hochschule
ETH-HOnggerberg,CH-8093 Ztirich/Switzerland
Prof. Dr. A. Iv[ Klibanov Massachusetts Institute of Technology
Department of Chemistry
Cambridge, MA 02139/USA
Prof. Dr. B. Mattiasson Department of Biotechnology

Chemical Center, Lund University
P.O. Box 124, S -221 00 Lund/Sweden
Prof. Dr. S. B. Primrose 21 Amersham Road
High Wycombe, Bucks HP13 6QS/UK
VI Editorial Board
Prof. Dr. H. d. Rehm Westf'alische Wilhelms Universit/it

Institute of Microbiology
Corrensstr. 3, D - 48149 Miinster/FRG
Prof. Dr. P. L. Rogers Department of Biotechnology

Faculty of Applied Science
The University of New South Wales
Sydney2052/Australia
Prof. Dr. H. Sahm Institute of Biotechnology

ForschungszentrumJiilich GmbH
D - 52428 Jtilich/FRG
Prof. Dr. K. Schi~gerl Institute of Technical Chemistry

University of Hannover
Callinstr. 3, D - 30167 Hannover/FRG
Prof. Dr. G. T. Tsao Director, Lab. of Renewable Resources Eng.

A. A. Potter Eng. Center, Purdue University
West Lafayette, IN 47907/USA
Prof. Dr. K. Venkat Phyton Inc., 125 Langmuir Lab.

95 Brown Road, Ithaca, NY 14850-1257/USA
Prof. Dr. John Villadsen Department of Biotechnology

Technical University of Denmark
Bygning223, DK-2800 Lyngby/Denmark
Prof. Dr. U. yon Stockar Swiss Federal Institute of Technology Lausanne

Institut de G6nie Chimique
CH- 1015 Lausanne/Switzerland
Prof. Dr. C. Wandrey Institute of Biotechnology

Forschungszentrum Jtilich GmbH
P.O.Box 1913, D - 52428 Jtilich/FRG
Preface
One of natures most important biological processes is the conversion of wood

and other lignocellulosis to carbon dioxide, water and humic substances.
Biotechnology, by definition, is the technical utilization of biological reactions.
Since wood and other lignocellulosics constitute the raw material for the forest
industries there should be ample opportunity for biotechnology in manipulating
these resources. There are, however, difficulties for implementation o f such
technologies in this industry. While biotechnology in areas like medicine and
pharmacology concerns production o f expensive products on a small scale,
biotechnical utilization and conversions of lignocellulosics means production
of inexpensive products on a large scale. Biotechnological utilization of
lignocellulosic materials is therefore a very difficult task, and the commercial
utilization of this technology has, therefore, only recently gained momentum.
One reason for this was the lack of basic knowledge about enzyme mechanisms
involved in the degradation and conversion of wood, other lignocellulosics and
their individual components. However, the worldwide efforts devoted over the
past few decades to research for a better understanding of these mechanisms
now provide a solid base for successful development of biotechnology for the
pulp and paper industry. For those deeply involved in these investigations, it is
obvious that the investments have been a dazzling success. This issue of
Advances in Biochemical Engineering/Biotechnology presents, in great de-
tail, recent findings about microorganisms and their enzymes involved in the
degradation of wood and wood components, cellulose, the hemicelluloses and
lignins. The issue covers not only biotechnology in the pulp and paper industry,
but also in forestry.
The chapter on Forest Tree Biotechnology demonstrates how trees, unlike
such inanimate resources as metallic ores, have the potential to be modified
genetically, essentially transforming lead into gold. The new methodologies
being used to address problems in forest biotechnology are described with
respect to their potential impact on forest tree improvement. A whole chapter
is devoted to ,,Lignin", it's role in wood, it's biosynthesis and structure as well
as methods for it's analysis. The chapter on the use of white-rot fungi for a
specific delignification of wood chips, i.e .... Biomechanical Pulping" describes
how this technique is now being run in pilot plant scale and how it approaches
commercialization. Pitch problems in pulping processes are addressed with
respect to how they can be minimized by the use of enzymes or fungi. The
technique for how to do this is described in great depth.
VIII Preface
No less than three chapters are devoted to pulp bleaching. One is concerned
with purification of effluents containing organochlorine compounds using
biotechnology. Two other chapters are devoted to the use of hemicellulases for
bleaching, one of which describes the basic research in this area, while the other
presents results from the use of enzymes in the pulp mills.
The last chapter focuses on biotechnology to solve slime problems caused
by microorganisms growing in the water systems of pulp and paper mills. It is
inevitabte that there will be an increasing need for closing of these systems,
thus there will likely be increasing slime problems in these systems in the
future.
These papers convey the current and developing applications ofbiotechnology
in the pulp and paper industry. Wherever possible, the authors have attempted
to peer into their crystall balls to see what lies ahead. However, the age of
biotechnology is only just dawning, but I have no doubt that surprises that lie yet
ahead for us will only serve to further enthuse this field of research.
September, 1996 Karl-Erik L. Eriksson

Table of Contents
Forest Tree Biotechnology

J. F. D. Dean, P. R. LaFayette, K-E. L. Eriksson, S. A. Merkle. 1
Microorganisms and Enzymes Involved

in the Degradation of Plant Fiber Cell Walls
R. C. Kuhad, A. Singh, K.-E. L. Eriksson . . . . . . . . . . . . . . . . . . 45
Lignin
D.S. Argyropoulos, S.B. Menachem . . . . . . . . . . . . . . . . . . . . . . 127
Fungal Delignification and Biomechanical Pulping of Wood

M. Akhtar, R. A. Blanchette, T. K. Kirk . . . . . . . . . . . . . . . . . 159
Solving Pitch Problems in Pulp and Paper Processes

by the Use of Enzymes or Fungi
R. L. Farrell, K. Hata, M. B. Wall . . . . . . . . . . . . . . . . . . . . . . . . 197
Reduction of Organochlorine Compounds in Bleach

Plant Effluents
P. Bajpai, P. K. Bajpai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Hemicellulases in the Bleaching of Chemical Pulps

A. Suurn~ikki, M. Tenkanen, J. Buchert, L. Viikari . . . . . . . . . . . 261
Using Enzymes in Pulp Bleaching: Mill Applications

J. S. Tolan, M. Guenette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Biotechnology for Solving Slime Problems in the Pulp

and Paper Industry
S. C. Johnsrud . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Author Index Volumes 51 - 57 . . . . . . . . . . . . . . . . . . . . . . . . . 329
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

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Forest Tree Biotechnology
Jeffrey F.D. Dean 1, Peter R. LaFayette 2, Karl-Erik L. Eriksson z,

and Scott A. Merkle 1
Daniel B. Warnell School of Forest Resources and
Department of Biochemistry and Molecular Biology, University of Georgia,
Athens, GA-30602, USA
List of S y m b o l s a n d A b b r e v i a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1 The C h a l l e n g e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............... 4
2 Key P r o b l e m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1 W o o d and P a p e r P r o d u c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 Breeding a n d Life-Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 M a n - M a d e a n d E n v i r o n m e n t a l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3 Propagation ............................................. 7
3.1 Cell a n d Tissue C u l t u r e of F o r e s t Trees . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2 In V i t r o P r o p a g a t i o n T e c h n i q u e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.3 A x i l l a r y S h o o t M u l t i p l i c a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.40rganogenesis ......................................... 10
3.5 S o m a t i c E m b r y o g e n e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.6 P r o t o p l a s t C u l t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.7 In Vitro Screening a n d S o m a c l o n a l V a r i a t i o n . . . . . . . . . . . . . . . . . . . . . . . 14
3.8 C r y o p r e s e r v a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.9 Artificial Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4 Genetic Engineering ........................................ 15
4.1 Tissue C u l t u r e C o n s i d e r a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.2 Biological G e n e Transfer ( A g r o b a c t e r i a - M e d i a t e d T r a n s f o r m a t i o n ) . . . . . . . . . . 17
4.3 Physical G e n e Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.3.1 T r a n s f o r m a t i o n by Microprojecti,~e B o m b a r d m e n t . . . . . . . . . . . . . . . . 20
4.3.2 T r a n s f o r m a t i o n of P r o t o p l a s t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.3.3 A l t e r n a t i v e T r a n s f o r m a t i o n T e c h n i q u e s . . . . . . . . . . . . . . . . . . . . . . . 21
4.4 Selection Systems, P r o m o t e r s , a n d R e g u l a t o r y E l e m e n t s . . . . . . . . . . . . . . . . 22
4.5 C u r r e n t a n d F u t u r e T a r g e t s for G e n e t i c E n g i n e e r i n g in F o r e s t Trees . . . . . . . . . 23
4.5.1 L i g n i n C o n t e n t a n d C o m p o s i t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.5.2 Sterility a n d E a r l y F l o w e r i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.5.3 H e r b i c i d e Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.5.4 Insect a n d P a t h o g e n Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.5.5 Tree F o r m a n d F i b e r M o r p h o l o g y ......................... 26
4.5.6 N o v e l T r a i t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.6 R e g u l a t o r y C o n s i d e r a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5 M o l e c u l a r Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5.1 D N A M a r k e r T e c h n i q u e s .................................. 28
5.1.1 R e s t r i c t i o n F r a g m e n t L e n g t h P o l y m o r p h i s m ( R F L P ) .............. 28
5.1.2 R a n d o m Amplified P o l y m o r p h i c D N A ( R A P D ) . . . . . . . . . . . . . . . . . . 28
5.1.3 B u l k e d Segregant Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5.1.4 M i c r o s a t e l l i t e R e p e a t P o l y m o r p h i s m s . . . . . . . . . . . . . . . . . . . . . . . . 29
5.1.5 Amplified F r a g m e n t L e n g t h P o l y m o r p h i s m s ( A F L P ) . . . . . . . . . . . . . . . 30
5.1.6 Microsatellite H y b r i d i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5.2 A p p l i c a t i o n s of D N A M a r k e r s in F o r e s t R e s e a r c h . . . . . . . . . . . . . . . . . . . . 31
5.2.1 G e n e t i c L i n k a g e M a p s ................................ 31
5.2.2 M a p p i n g Projects ................................... 32
5.2.3 M a r k e r - A s s i s t e d Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Advances in Biochemical Engineering/

Biotechnology, Vol. 57
9 Springer-Verlag Berlin Heidelberg 1997
2 Jeffrey F.D. Dean et al.
5.3 Other Applications of D N A Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

5.3.1 Quantification of Genetic Diversity . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.3.2 Genotype Verification and Delineation . . . . . . . . . . . . . . . . . . . . . . . 34
6 Electronic and Computational Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
The forest products industry has traditionally viewed trees as merely a raw, and more or less
immutable, natural resource. However, unlike such inanimate resources as metallic ores, trees have
the potential to be modified genetically, essentially transmuting lead into gold. Increasingly, m o d e r n
alchemists are applying the tools of biotechnology in efforts to reduce the biological constraints on
forest productivity. Several new methodologies being used to address problems in forest biology are
described with respect to their potential impact on forest tree improvement. In addition to
addressing problems inherent to the current use of trees for production of pulp and paper or solid
wood products, genetic manipulation of trees brings with it the potential to create new industries
based on the novel characteristics of transgenic trees, e.g. trees containing transgenes to detoxify
specific pollutants could be used in the remediation of sites contaminated with hazardous wastes.
Efforts to modify trees through biotechnology are in their infancy, and this review seeks to outline
the underpinnings of what will undoubtedly be an area of increased emphasis in the next millennium.
Forest TreeBiotechnology
List of Symbols and Abbreviations
Symbol Description
2,4-D 2,4-dichlorophenoxyacetic acid
AFLP amplified fragment length polymorphism
BAP benzylaminopurine
Bt Bacillus thuringiensis endotoxin
CAD cinnamyl alcohol dehydrogenase
CaMV cauliflower mosaic virus
CAT chloramphenicol acetyltransferase
EPSP 5-enolpyruvylshikimate-3-phosphate
F5H ferulate-5-hydroxylase
GFP green fluorescent protein
GUS [3-glucuronidase
IAA indoleacetic acid
NAA naphthylacetic acid
OMT o-methyltransferase
PCR polymerase chain reaction
PEG polyethylene glycol
QTL quantitative trait locus
RAPD random amplified polymorphic DNA
RFLP restriction fragment length polymorphism
STFI Swedish Pulp and Paper Research Institute
vir a class of genes controlling the virulence of Agrobacterium
tumefaciens
1 The Challenge
Since the advent of agriculture, one of the principal challenges for mankind has
been to increase the ability of plants to capture incoming solar energy for the
production of food, fuel, and fibers. As the human population continues to
burgeon, it becomes clear that the demand for increased production efficiency in
crops and trees will not diminish. While the most significant advances of the
"green revolution" have resulted in a doubling and tripling of yields from
agronomic crops, the potential for increasing forest productivity, for instance
through reduced rotation times, could be much greater given the minuscule
effort to date in applying the techniques of modern biotechnology to potentials
in forest tree biology. The increasing use of plant polysaccharides for the
production of fuel-grade ethanol is one example suggesting that the future
products derived from forest trees may not remain just paper and timber. Thus,
new products derived from wood also have the potential to drive demand for
greater forest productivity. However, the greatest pressure for increased tree
productivity is likely to result from preservation efforts that remove forests on
public lands from the role of harvestable inventory. Regardless of corporate
sentiment, current trends in this area are unlikely to be reversed, and, as
a consequence, plantation forestry is likely to become a major production
paradigm for the next century. Tree productivity will need to be increased
significantly if we are to have any chance at meeting projected fiber demands
using the diminished acreage allotted to production forestry.
Forestry has never faced challenges as great as those before it now. In some
parts of the world misguided land use policies have led to extensive deforesta-
tion, while, elsewhere, productivity has stagnated due to the detrimental effects
of environmental pollutants such as acid rain. Population growth in every
corner of the globe increases pressure to produce more fodder and fuel wood. In
the past, answers to these complex issues usually centered on social, political, or
economic redress, and such is likely to remain the case for the near future.
However, plant biotechnology has advanced to a point where we can now
envisage biological solutions to some of the more intractable forestry problems.
This chapter is intended to convey the optimism that currently pervades this
field by highlighting a few of the most exciting areas in which biotechnology is
being applied to problems of forest biology.
2 Key Problems
2.1 Wood and Paper Products
The term "wood quality" is often used to describe the overall suitability of
a particular wood source for a specific end use. Thus, wood considered to be of
Forest Tree Biotechnology 5
excellent quality for one type of product may be useless for another application
and vice versa. For example, spruce fibers generally tend to have thinner walls
than those from pine, and, as a result, spruce pulps form more compact and
denser sheets of paper. Similarly, softwood fibers, which are always longer than
hardwood fibers, increase the tear strength index of paper, but the tight packing
of short hardwood fibers yields a smoother, more uniform writing surface. Solid
wood products are dependent on a completely different set of parameters to
define wood quality. Zobel and Van Buijtenen [1] have reviewed the ways in
which variations in wood properties influence the quality of wood and paper
products, while Karenlampi [-2] has discussed the role of fiber properties in
product quality.
Wood quality is a function of the morphology and chemistry of all the
various cell types and the ratio of their occurrence in a particular species of tree.
Thus, wood quality is constrained by the genetics, age, environmental condi-
tions, and cultivation practices imposed on the individual tree. G r o w t h condi-
tions can significantly alter such cellular parameters as fiber volume and
morphology as well as cell wall architecture and chemistry. Cell walls are
actually a composite material, and their ultrastructure thus derives from the
ways in which the various molecular components are arranged and oriented to
form the fiber wall. In general, embedded cellulose microfibrils give the walls
tensile strength, while the surrounding matrix of hemicelluloses and lignin bind
and rigidities the composite. Lignin also serves to hold the individual fiber cells
together. The carbohydrate components, i.e. cellulose and hemicelluloses, are
hydrophilic materials, while lignin is hydrophobic, and the relative proportions
of these two classes of material in a given fiber wall define many of the bulk
properties of the fiber.
Structural features of importance for paper production include fiber length,
diameter, wall thickness and ultrastructure. Softwood fibers used in pulp pro-
duction can have a mean length anywhere from 2.5 mm to more than 10 mm;
however, the majority of softwoods have an average fiber length of 3-5 mm.
Most softwood pulp fibers have a diameter of less than 0.1 ram. Hardwood
fibers are, on the average, about 1/3 the length and about 1/2 the width of
softwood fibers. It seems likely that the fibers used for paper manufacturing in
the future will increasingly come from plantations of fast-growing trees where
juvenile fibers will predominate [3]. It is relatively easy to predict the effect this
will have on products made with hardwood fibers since the morphology and
chemistry of juvenile fibers of hardwoods are more similar to mature fibers than
is the case for softwood fibers [4, 5]. However, juvenile softwood fibers are
significantly shorter and weaker than mature fibers, and, as a consequence, they
do not have the superior strength characteristics needed for making strong
paper using current technology. Thus, the long fibers currently obtained from
mature temperate softwoods may well become a limiting factor for future paper
production.
There is currently a fundamental lack of knowledge about the relationship
between fiber quality and pulp product properties. One reason for this is that the
6 JeffreyF.D. Dean et al.
parameters which are used to characterize the fibers at different steps in the
processing chain are non-uniform. Foresters describe wood quality with respect
to tree growth and density, pulp makers focus on measurements of viscosity and
kappa number, while papermakers test freeness and handsheet properties. All of
these players are looking at the bulk properties of fiber mixtures, but no one has
studied purified, uniform fiber preparations in sufficient detail to explain how
individual fiber types might influence the bulk properties of the mixture. An
understanding of individual fiber properties should make it possible to model
how different combinations of fiber mixtures might improve paper processing.
To collect such information on fiber mixtures, the Swedish Pulp and Paper
Research Institute (STFI) has developed a measurement system called STFI
Fiber Master. This equipment can rapidly determine fiber form, length, width,
and flexibility for more than 1000 particles per second in a diluted fiber
suspension. When combined with information on the chemistry and binding
parameters for the fibers, this new analytical tool provides a powerful technique
for optimizing fiber mixtures to suit the production of a particular product.
2.2 Breeding and Life-Cycle
Although they have been exploited by man for millennia, forest trees constitute
one of the last major plant groups that has not been subjected to significant
genetic manipulation with an eye toward improvement. The reason for the lack
of progress with forest trees compared to agronomic and horticultural species
can be attributed to the fact that these plants are characterized by a number of
unique biological features which have made their breeding a slow and difficult
process. The most obvious of these is the relatively large size of tress of
reproductive age, which makes controlled breeding in the greenhouse difficult.
However, two facets of their long life histories currently present major barriers
to rapid improvement of trees via traditional breeding: (1) the long lag period
between seed germination and flower production, and (2) the lengthy interval
between seedling and mature phenotype. Average time to flower production
varies greatly among species, from as short as 5 years for some species of Pinus
to as long as 25 years for some species of Quercus [6] Consequently, the tree
breeder may have to wait more than two decades between breeding cycles.
Furthermore, the major traits for which improvement is desired, such as volume,
wood specific gravity, and form or growth habit, usually cannot be assessed until
the tree assumes its mature phenotype, perhaps 20-30 years from the seedling
stage. Attempts to predict mature tree performance from that of seedlings has so
far met with limited success [e.g. 7, 8]. To make matters worse, many of these
desirable traits are not inherited in a simple fashion, but instead result from
complex interactions of genes at multiple loci. As a consequence, improvement
of quantitative traits requires breeding programs designed to make incremental
advances in a population mean for a desired trait using such tools as mass
selection. While this approach has led to measurable improvement in some
commercial tree species such as Pinus taeda [9], these gains have required
massive cooperative efforts over multiple decades. For the majority of trees,
traditional breeding approaches are simply not a realistic means for achieving
genetic improvement.
2.3 Man-Made and Environmental
In addition to the natural constraints that limit forest tree productivity, man has
added new problems as a consequence of population growth and industrial-
ization. One of these problems is pollution of the soil, air and water. While some
forest trees seem able to tolerate high levels of air pollution, surviving well, for
example, in urban environments, others are severely damaged by high levels of
pollutants such as ozone. Unless we are satisfied with being limited to a few
species of trees for growth in polluted areas, trees that currently cannot survive
airborne toxins will have to be engineered to tolerate certain levels of these
pollutants. Population growth in some countries has led to increasing competi-
tion for land. For example, in the south eastern United States, old agricultural
fields were once plentiful enough to be employed for pine plantation forestry.
Today, with the growth of cities and suburbs, these lands are no longer available
for agriculture, let alone forestry. Thus, forests will probably have to be planted
on lands that are less than optimal with regard to fertility and water availability.
In these cases, we will need trees that are specifically adapted for growth on sites
where trees available today would be unable to survive. An extreme case might
be the need to produce populations of trees adapted to growing on sites which
are unsuitable for any other use, such as those contaminated by heavy metal
residues.
3 Propagation
3.1 Cell and Tissue Culture o f Forest Trees
The goal most often cited for in vitro culture of forest trees is propagation. As
with conventional vegetative propagation (macropropagation) techniques such
as rooted cuttings, the primary objective of in vitro propagation (micropropaga-
tion) is to capture the total genetic superiority of the parent material, including
both additive and nonadditive genetic components. In addition, clonal propaga-
tion systems allow the application of a very high selection differential, since
whole new populations of plants can be cloned from just a few elite individuals.
Theoretically, since somatic tissues are used as the starting material, each tree
regenerated from these tissues should be an exact clonal copy of the ortet.
The ideal result should therefore be a highly uniform clonal population that
replicates the phenotype of the source tree. However, unlike macropropagation

systems, some in vitro propagation methods have the potential to introduce
significant new genetic or epigenetic changes into propagules regenerated from
them, so that the parent material is not truly replicated (see section on soma-
clonal variation). Furthermore, the nature of much in vitro propagation techno-
logy for forest species excludes clonal propagation of proven genotypes, since
many of these systems are limited to employing explants from juvenile geneti-
cally unproven material.
A second principal goal of micropropagation that parallels conventional
vegetative propagation is simply to mass propagate plants, bypassing sexual
reproduction, which for a particular species may be too expensive, inefficient or
impractical. In vitro propagation can also be used to overcome problems
associated with macropropagation, including limitations on available parent
material or space. In addition, just as propagation through seeds may be limited
by biological factors, certain species or even individual genotypes may be
recalcitrant to conventional vegetative propagation techniques such as rooting
of stem cuttings. In these cases, in vitro techniques may succeed where macro-
propagation fails.
In addition to the advantages shared with conventional vegetative propaga-
tion, in vitro culture has a number of associated potential and real applications
that are unique. These include protoplast culture and fusion for generation of
somatic hybrids, in vitro screening, generation of useful somaclonal variants,
generation of artificial seeds, and long-term storage of germplasm using cryo-
preservation. Finally, in vitro culture currently provides the only route for
generation of genetically engineered genotypes of forest trees. Gene transfer
technologies including Aorobacterium-Ti plasmid mediated gene transfer, elec-
troporation, microinjection, and microprojectile bombardment all depend on
the ability to culture cells in vitro in order to select the transformed cells, usually
by employing drug-resistance marker genes. Once transformed cells are ob-
tained, these can be cultured to regenerate transformed plantlets (see section 4.2
on gene transfer).
3.2 In Vitro P r o p a g a t i o n T e c h n i q u e s
Micropropagation systems fall into three broad categories: axillary shoot (or
bud) multiplication, organogenesis, and somatic embryogenesis. Axillary shoot
methods rely on multiplication of preformed structures, while organogenesis
and somatic embryogenesis rely on de novo generation of either plant organs or
embryos, respectively (i.e. morphogenesis). All these regeneration systems have
great potential to be applied for mass propagation of forest tree species. As the
examples below show, the success of a given method appears to be highly
species-specific, with shoot multiplication or organogenic systems working
well for some species, while embryogenic systems are clearly superior for
others.
Forest trees were among the first plants cultured in vitro. Gautheret [10]
reported formation of adventitious buds in cultured cambial explants of Ulmus
campestris, demonstrating that an exogenous source of sugar was required for
bud production, while high levels of indoleacetic acid (IAA) in the medium
inhibited bud formation. Jacquiot [11] extended the research with this species,
using trees up to 180 years old as tissue sources. Mathes [12] reported in vitro
production of both roots and shoots in callus cultures of Populus tremuloides,
although apparently no plantlets were produced. It was not until four years later
that Wolter [13] produced entire P. tremuloides plantlets in vitro by inducing
shoot formation with benzylaminopurine (BAP) and subsequently rooting the
shoots in vitro. Winton [14], working with triploid P. tremuloides callus, also
obtained complete plantlets, demonstrating for the first time in vitro propaga-
tion of a forest tree with a superior genotype. Among coniferous species,
adventitious shoots were first produced in callus cultures of Sequoia semper-
virens [15]. However, it was not until 25 years later that complete plantlets of
a conifer, Pinus palustris, were regenerated in vitro [16].
Since the pioneering work with Populus and P. palustris, hundreds of tree
species have been propagated in vitro, although most of this work has been done
on an experimental rather than an operational scale. It would be unrealistic to
attempt to review here all of the in vitro propagation research with forest trees
reported to date. The most recent complete review of forest tree micropropaga-
tion is that of Thorpe et al. [17]. In addition, a number of books have been
published in the past 10 years containing chapters describing in vitro propaga-
tion of many of the important woody angiosperm and gymnosperm genera. We
would refer those desiring information on manipulation of individual woody
plant genera in vitro to books edited by Bajaj [18-20] or Bonga and Durzan
[21]. Here, we will briefly review the basics of each of the three systems along
with their advantages and disadvantages, citing examples of some of the more
advanced systems developed for commercially important forest species.
3.3 A x i l l a r y S h o o t M u l t i p l i c a t i o n
Production of plantlets from axillary shoots is similar to vegetative propagation

via rooted cuttings in that shoots are excised from source tissue and rooted
individually to multiply the original genotype. The differences are that, with
axillary shoot systems, the procedures are performed in vitro under aseptic
conditions, and subsequent manipulations in vitro can be used to greatly
multiply the number of propagules obtained. These manipulations may include
treatment with a cytokinin such as BAP or zeatin, or culture conditions which
induce axillary buds in the cultured tissues to elongate into shoots. Such shoots
can be excised and either rooted in vitro or placed back into culture for further
enhancement of axillary branching. Thus, unlike rooted cutting macropropaga-
tion, treatments can be applied in vitro which promote multiplication of new
shoot material ad infinitum.
One of the principal advantages of axillary shoot methods for forest tree
multiplication is that all propagules are derived from preformed buds, thereby
enhancing the likelihood that propagules will be true to type. Because shoots
arise from meristems present in the explant, there is little chance of introducing
somaclonal variation into the propagules. The greatest advantage of axillary
shoot multiplication systems, however, is the fact that this method has to date
proven to be the most effective technique for multiplying mature selected trees
[17]. Some examples of mature hardwood forest species propagated using this
method are Robinia pseudoacacia [22], Liquidambar styraciflua [23], Eucalyptus
citriodora [24] and Acer saccharinum [25, 26].
While axillary shoot multiplication systems have been the primary in vitro
propagation method applied to hardwoods, the successful application of this
technique to conifers has been less frequently reported. In pines, this appears to
be due to difficulty in stimulating elongation of the axillary buds found at the
bases of needle fascicles [27]. An exception to this occurs with Pinus radiata, for
which workers in New Zealand have developed an axillary shoot multiplication
system which has enabled them to plant thousands of clonal trees in the field
since the mid-1980s [28].
Given that production of plants from axillary shoots has some similarities to
propagation from rooted cuttings, it might be expected that these techniques
would share some common disadvantages. One such disadvantage is a relatively
low frequency of propagule production. Although repeated cycles of in vitro
culture can ultimately produce thousands of propagules, there is often a substan-
tial lag phase before operational numbers of plants can be produced regularly.
Axillary shoot multiplication methods are also relatively labor intensive, requiring
significant amounts of handling, for both cycling of cultures and production of
plantlets. Plantlet production can require multiple steps, including induction,
shoot elongation, shoot excision, rooting, and acclimatization of plantlets, each of
which may require significant inputs of labor. Therefore, although axillary shoot
multiplication remains the major route for the regeneration of woody angio-
sperms in vitro, its labor-intensive nature has limited its application mainly to
woody ornamentals which have relatively high single-tree values compared to
forest species. McCown and McCown [29], who described axillary shoot multi-
plication systems for Quercus, Amelanchier, Ulmus, Betula, and Populus, argued
that tree microculture must become more automated to reduce labor costs before
this technique can be economically applied to forest species. However, because of
multiple handling steps, axillary shoot multiplication methods may not be as
amenable to scale-up or automation as other methods for forest tree propaga-
tion, although advances in robotics may change this potential [30].
3.40rganogenesis
Organogenesis is the de novo production of plant organs (buds, shoots, and

roots) from organized tissues or callus. As with axillary shoot multiplication
systems, organogenic cultures are usually stimulated to produce buds by addi-

tion of a cytokinin to the medium. In many cases, initial explants are induced to
form callus and subsequently buds. Then, as with axillary shoot methods, the
buds are elongated into shoots, excised, and rooted individually.
The primary advantage of organogenic regeneration methods over axillary
shoot methods is a potential for higher frequency plantlet production in a shor-
ter period of time. Callus can be grown in large quantities with less demand for
labor and space, and adventitious shoot production can be used to achieve high
multiplication rates. However, like axillary shoot methods, organogenesis-based
methods may require the labor-intensive steps of shoot elongation, excision,
rooting, and plantlet acclimatization to produce field-plantable stock. In addi-
tion, unlike axillary shoot methods, organogenic methods, especially those
requiring an intermediate callus, may be associated with the production of
significant amounts of somaclonal variation in the regenerated plantlets. Thus,
the resulting plantlets from a given clone may display unacceptable phenotypic
variation. Lester and Berbee [31], for example, observed a wide range of
variation in plantlets derived from callus cultures of Populus.
Organogenic regeneration systems have been developed for a number of
coniferous genera, including Pinus [e.g. 16], Picea [e.g. 32], Pseudorsuga [33]
and Abies [e.g. 34]. In most cases, adventitious buds were induced to form
directly from tissues such as seedling cotyledons. Among conifers, only Pinus
eldarica has produced long-term callus cultures that could be induced to form
adventitious buds from which plants were successfully regenerated [35]. In a few
cases, plantlets produced from adventitious conifer buds have been tested in the
field. In the cases of Pinus taeda and Pseudotsuga menziesii, plantlets derived
from adventitious buds survived well, but lagged behind seedlings in height
growth [36, 37]. However, over the past 10 years the emphasis of conifer
micropropagation research has shifted to somatic embryogenesis, since it is
believed by many working in the area that this technique offers the best
potential for scale-up to commercially viable levels.
As of 1987, McCown and McCown [29] were unable to cite a single case of
commercial application of a micropropagation system based on adventitious
bud generation for a North American hardwood. Although to our knowledge
this situation remains unchanged, a number of promising adventitious bud
systems have been reported for hardwoods in the past few years. Among these,
high-frequency adventitious bud regeneration systems have been reported for
Liquidambar styraciflua [-38~41], several Populus species and hybrids [e.g.
42-45], Ulmus spp. [46], and Robinia pseudoacacia E47-50].
3.5 Somatic Embryogenesis
Somatic embryogenesis is the de novo production of structures resembling

zygotic embryos, either from organized tissues or from callus. Structures
classified as somatic embryos must be bipolar (possessing both root and shoot
poles) and have no vascular connection to the source tissue. Somatic embryos
may be derived either through direct or indirect embryogenesis. In direct
embryogenesis, embryos are formed essentially by multiplication of a zygotic
embryo explant, i.e. by embryo cloning [51]. Indirect embryogenic systems
involve a dedifferentiation of non-embryonic tissue to form a callus from which
somatic embryos arise [51]. As with most other plant species, somatic em-
bryogenesis in forest trees is usually induced by exposure to an auxin such as
NAA or 2,4-D, although there are a few cases of embryogenesis being induced
by cytokinins [e.g. 52, 53] or no exogenous growth regulators at all [e.g. 54].
Since continuous exposure to auxin usually results in repetitive cycles of embryo
production, growth regulators are usually removed from the medium following
induction to allow the somatic embryos to complete development. In some
cases, only a few weeks or even days of exposure to auxin is required to induce
repetitive embryogenesis [e.g. 55, 56].
The first report of somatic embryogenesis in a hardwood forest species was
for Santalum album [57]. Since that time, somatic embryogenesis has been
reported in hundreds of angiosperm trees (see reviews by Tulecke [-58] and
Warm [59]). However, it was not until 1985 that somatic embryogenesis was
reported in a coniferous species, Picea abies [60]. In the last decade, somatic
embryogenesis has been reported for most commercially important conifers,
including Pinus [e.g. 61-63], Abies [52], Larix [e.g. 64] and Pseudotsuga [65].
For a full summary of somatic embryogenesis in coniferous trees, the reader is
referred to reviews by Attree and Fowke [66] and Tautorus et al. [67]. The most
advanced systems with regard to high-frequency plantlet production have been
developed for Picea. Canadian researchers working with Picea 91auca have used
abscisic acid, osmotica and desiccation treatments to obtain high levels of
vigorous plant production from somatic embryos [68]. Workers at BC
Research planted the first large-scale field tests of somatic embryo-derived
conifers from cultures of interior spruce (Picea 91auca engelmannii complex)
[69].
Somatic embryogenesis has been cited by many authors as the in vitro
regeneration system of choice for economical production of clonal populations
of forest trees [e.g. 70]. Certainly, this type of system has a number of powerful
advantages over axillary shoot multiplication methods and organogenesis.
A major advantage is the potential for very high frequency regeneration.
Depending on the species, virtually unlimited numbers of embryos can be
generated from a single explant. In addition, embryogenic cultures of many
species can be grown in liquid, allowing production and handling of thousands
of embryos at one time. Thus, in comparison to axillary shoots and organogen-
esis, somatic embryogenesis offers the potential for high-volume large-scale
propagation which can translate into significant labor savings. Greater econo-
mies of scale may be possible if bioreactor and continuous culture technologies
can be applied to embryogenic systems [e.g. 71,72]. The feature of somatic
embryogenesis which may ultimately have the largest impact for mass propaga-
tion of forest trees is the fact that the product is an embryo. The morphological
and physiological similarity of somatic embryos to zygotic embryos means that
they are complete propagules in themselves, with embryonic roots, shoots and
leaves, and, most importantly, the "program" to make a complete plant. As
a consequence, no separate shoot elongation or rooting steps are required for
plantlet production. This characteristic further lowers labor inputs and gives
somatic embryos the potential for direct delivery to the greenhouse or field (see
section 3.9 on artificial seeds), thereby eliminating the need for labor-intensive
transplanting.
One drawback of current forest tree embryogenic systems is the low fre-
quency of plantlet production or "conversion" of somatic embryos to plantlets.
Although numerous systems have been reported, and some of these produce
embryos at high frequencies, a major bottleneck has been induction of embryo
maturation and subsequent production of field-plantable stock. Currently, only
a handful of tree species such as Liriodendron tulipifera and some Picea species
can be propagated via somatic embryogenesis in sufficient numbers to enable
establishment of useful field tests [69, 733. Another major disadvantage of
embryogenic propagation methods for forest species is that the bulk of the
systems reported to date rely on immature tissues (i.e. from seeds or seedlings) as
explanting material. Thus, the material being propagated is of unproven genetic
value. Most reports of somatic embryogenesis in tree species are in reality
reports of "embryo cloning," in which the zygotic embryo is induced to replicate
itself indefinitely. However, in the past few years, a few reports have appeared in
which embryogenic cultures were initiated from mature tissues. For instance,
embryogenesis cultures have been initiated from the male flower parts of
Quercus [743 and Aesculus [75], while Michler and Bauer [76] used leaf tissues
of a Populus hybrid of known genetic value to obtain somatic embryos.
3.6 Protoplast Culture
Another potential application of in vitro culture which may eventually succeed

for forest tree improvement is generation of somatic hybrids via protoplast
fusion. For example, protoplasts of Citrus species from different genera which
were unable to hybridize sexually were fused, and somatic hybrid plantlets were
regenerated [77]. Although regeneration of somatic hybrids has not been
reported to date for a forest tree, protoplasts have been isolated from a number
of forest species and induced to regenerate cell walls and eventually divide to
produce protoplast-derived plantlets. Angiosperms regenerated from proto-
plasts include Populus spp. [78], Ulmus spp. [79] and Liriodendron tulipifera
[80]. Among gymnosperms, embryogenic cultures have provided a reliable
source of totipotent protoplasts, and many conifers have been regenerated from
protoplasts derived from embryogenic cultures. These include Pinus taeda [62],
Pinus caribaea [81], Picea glauca [82], Abies alba [83], and Larix species
[84, 85],
3.7 In Vitro Screening and Somaclonal Variation
As mentioned earlier, organogenic and embryogenic regeneration systems may

be characterized by significant levels of somaclonal variation. This phenomenon
may be regarded as a nuisance if the goal is production of truly clonal
propagules. However, somaclonal variation may also be applied as a tool for
generating useful variants. Although not frequently reported in forest trees,
screening of genotypes for such traits as disease resistance can be accomplished
in vitro, and this can be followed by production of propagules from the selected
material. This technique has been applied to the selection of some coniferous
trees [86]. It has also been used to generate hybrid Populus variants that were
resistant to the herbicide glyphosate [87] as well as eastern cottonwood
(Populus deltoides) variants that displayed increased or decreased resistance to
leaf rust caused by Metampsora medusae [88].
3.8 Cryopreservation
Another technique that is likely to come into widespread use in operational

propagation of forest trees is long-term cryopreservafion of culture tissues. The
ability to store germplasm for long periods will become critical if in vitro
propagation methods are to be used commercially. This is because most of the
forest tree embryogenic systems reported to date depend on materials of un-
known genetic value. Consequently, trees derived from these cultures will need
to be tested for field performance prior to their release into production pro-
grams. Even if researchers are eventually able to clone mature tissues from elite
genotypes of particular forest species, there will still be a need for field evalu-
ation of trees derived from these cultures. In addition, under continuous culture,
the useful life of most embryogenic cultures does not exceed a few years.
Therefore, embryogenic cultures must be held in a suspended state while trees
derived from them are tested. Once field testing is complete, cultures of those
clones showing the best field performance can be scaled up for production of
propagules. Fortunately, embryogenic cultures of both gymnosperms and angio-
sperms seem to be relatively amenable to cryopreservation. This technique has
already been applied to the long-term storage of embryogenic cultures of Betula
[89], Picea glauca [90], Pinus caribaea [91] and Abies nordmanniana [92].
3.9 Artificial Seeds
One of the advantages of somatic embryogenesis over the other in vitro propa-
gation systems noted above is the potential of somatic embryos to be directly
delivered to the greenhouse or field as synthetic seeds. Efforts have concentrated
on preparing somatic embryos to emulate seeds with regard to such character-
istics as desiccation tolerance, resistance to mechanical damage and ability to
sustain ex vitro post-germinative growth. These features would facilitate mech-

anical handling and automated planting of somatic embryos. A number of
encapsulation techniques have been employed, including the use of such materials
as hydrated gels and water-soluble resins [93]. While development of artificial
seed technology has mainly focused on species of agronomic importance, such as
alfalfa, the same technology has been tested on somatic embryos of a few forest
trees. Artificial seeds have been generated by alginate gel encapsulation of somatic
embryos of Pinus taeda [61, 62], Picea species [94], Santalum album [95] and
Robinia pseudoacacia [96]. Although plantlets have been regenerated from some
of these artificial seeds, none of these systems has been developed to the point
where the somatic embryos could be employed as if they were true seeds.
4 Genetic Engineering
As illustrated by the wide variety of specialized cultivars available within

agronomic and horticultural crop species, traditional plant breeding based on
controlled sexual crosses has the potential to tailor the traits of virtually any
plant to be more suitable for its end use. However, this process generally
requires selection and back-crossing through nine or more generations just to
introduce a single trait into a plant line and, thus, the life-cycle characteristics of
long-lived and slow-maturing forest trees make such breeding efforts difficult at
best. Additionally, such plant improvement schemes are limited to modification
of traits already available within the species or within closely-related hybridizing
species. Fortunately, recent advances in biochemistry and molecular biology
have provided techniques for the direct transfer of foreign genes to plant tissues,
thereby circumventing some of the most troublesome bottlenecks inherent to
traditional plant improvement strategies.
Only slightly more than a decade ago, researchers in several laboratories
accomplished the first controlled introductions of foreign genes into plants
[97-100]. This process, commonly referred to as transformation, has been used
to introduce into plants a variety of foreign genes whose expression confers
novel traits to the transformants [101, 102]. A wide variety of genetic constructs
designed to modulate the expression of existing plant genes in such a way as to
enhance the properties of the desired plant products have also been tested, and
the product of one such transformation, the Flavr-SavrTM tomato, recently
became the first genetically engineered plant product approved for human
consumption [103]. Most plant transformation work has so far focused on
agricultural crops [104], but efforts to produce genetically engineered trees are
on the increase and several reviews of the work to date are available [105-111].
The treatise by Haines [112] is a particularly useful guide to the opportunities
presented by biotechnology with respect to tree improvement.
As discussed in greater detail in the following sections, the minimum require-
ments for successful gene transfer into forest trees are: (1) a tissue culture system
Table 1. Tree species that have been successfully transformed and regenerated.
Species Method Reference

Angiosperms
Allocasuarina verticillata Agrobacterium [114]
Azadirachta indica Agrobacterium [115]
Carica papaya Microprojection [116]
Agrobacterium [117]
Carya illinoensis Agrobacterium [118]
Citrus jambhiri Protoplast/Chemical [1 I9]
Eucalyptus sp. Electroporation [120]
Juglans regia Agrobacterium [121]
Liquidambar styraciflua Agrobacterium [122, 123]
Microprojection [ 124]
Liriodendron tulipifera Microprojection [125]
Malus Pumila Agrobacterium [126]
Populus sp. Agrobacterium [127]
Microprojection [128]
Prunus domestica Agrobacterium [129]
Prunus persica Agrobacterium [130]
Robinia pseudoacacia Agrobacterium [49]
Cymnosperms
Larix decidua Agrobacterium [131]
Picea glauca Microprojection [ 132]
allowing for the regeneration of intact plants (see the preceding section); (2)
a delivery system for stable introduction of genes into the cultured cells; and (3)
a selection system for recovering those cells that have received the introduced
genes. It then remains for the researcher to identify the appropriate foreign gene
to be introduced or endogenous gene requiring altered expression, while of final
concern are the promoter and regulatory elements that will serve to express the
introduced gene construct in the correct tissues at the proper developmental
stage. Table 1 lists a variety of horticultural and forest trees that have been
successfully transformed and regenerated, although in most cases the inserted
genes served only as markers of transformation and not as sources of new traits.
It should be noted that, owing to the ease with which they may be transformed
and regenerated, members of the genus Populus have been the predominant
model system to date for demonstrating new transformation techniques for trees
[e.g. l 13]. Thus, numerous reports of transformation and regeneration of species
and hybrids in this genus appear in the literature, but no attempt has been made
to present an exhaustive list of that work here.
4.1 Tissue Culture Considerations
All of the considerations previously discussed for the regeneration of intact

plantlets from in vitro tissue culture are important when considering a system for
gene transfer work. However, the two most important considerations are that
the culturing system should be one in which the new plantlet can be derived
from a single cell (i.e. organogenic, embryogenic or protoplast culture), and that
the frequency with which plantlets can be generated from the system should be
as high as possible. Regeneration from single cells ensures that all cells in the
resulting plantlet will carry the introduced transgene at the same position in the
genome, thereby standardizing its expression and inheritance in subsequent
progeny. A high frequency of regeneration is required, particularly when using
physical gene transfer techniques (see below), because as few as 1:1000 cells
receiving the engineered gene will insert it into their genetic material in such
a way that it will be stably retained and expressed in subsequent generations of
cells. Unfortunately, tissue culture systems that are highly regenerable are not
always highly transformable [133, 134], and even when transgenes are stably
inserted into the recipient genome, a variety of positional and cell state effects
may lead to a loss of expression in regenerated plantlets [135]. Although tissue
culture systems meeting all these requirements are still relatively rare for forest
tree species, the increasing number of successes noted in the foregoing section
suggest that the availability of competent systems will not be a limiting factor for
long.
4.2 Biological Gene Transfer (Agrobacteria-Mediated

Transformation)
The techniques by which exogenous DNA is transferred into plant cells may be
classified as either physical or biological, depending upon whether the DNA is
"naked" or passed along by an intermediary organism. Transformation systems
based on plant viruses were at one time thought to hold great potential for
genetic engineering and some effort has continued in this area [136, 137], but
results for the most part have been disappointing [101]. In contrast, the best
characterized and most accessible plant transformation systems available today
are biological and use domesticated varieties of the plant pathogens, Agrobac-
terium tumefaciens or A. rhizogenes, to mediate transfer of DNA to the host
tissues. Manuals describing comprehensive protocols for performing such work
are available [138, 139], and the book edited by Croy [140] provides an
invaluable resource enumerating a vast number of vectors, marker genes, regula-
tory elements and other tools for gene transfer into plants.
In their pathogenic forms, A. tumefaciensand A. rhizogenes cause crown-gall
and hairy-root diseases, respectively [141]. The diseases appear primarily in
woody and herbaceous dicots, and actually represent unusual parasitic interac-
tions that are initiated when the bacteria colonize wounded tissues. The bacteria
locate these wounds through a chemotactic response to phenolic compounds
(e.g. acetosyringone or sinapic acid) released into the soil from the wound [142].
Once inside the wound, the bacteria use a specialized process resembling bacterial
conjugation to transfer into the plant cells DNA copied from an extra-chromo-
somal element referred to as the Ti-plasmid [143]. A pair of 25-base-pair direct
repeats on the Ti-plasmid, the so-called T-DNA borders, facilitate transfer and
integration of any genes lying between the borders into the host plant genome
where they can subsequently be replicated and expressed. Only that DNA lying
between the T-DNA borders is copied and transferred as single stranded DNA
to the host plant cells. Until recently, this process was considered the only
example of inter-kingdom DNA transfer. However, a related system appears to
be capable of transferring DNA between bacteria and yeast [144].
In the wild-type disease-state interaction, some of the genes carried between
the T-DNA borders encode enzymes that alter phytohormone (auxin or
cytokinin) levels in the transformed tissues, and thereby cause tissue deforma-
tion (i.e. galls or hairy roots). Another set of genes transferred from the wild-type
Ti-plasmid catalyze the production of opines, a class of amino acid derivatives
unique to Agrobacterium infections, which are utilized by the bacteria as
a source of both carbon and nitrogen [145]. Several other (vir) genes Iie in
regions of the Ti-plasmid outside the T-DNA borders, and are expressed only in
the bacterium. The vir gene products control such aspects of the interaction as
conjugative transfer of T-DNA into the plant cell, integration of the DNA into
the host genome, and the range of plant species recognized as hosts for the
bacterium [146-148]. Several detailed reviews of the molecular basis for
Agrobacterium-mediated transformation are available [143, 148, 149].
The realization that any DNA flanked by T-DNA sequences could be
transferred from the Ti-plasmid and integrated into the genome of infected plant
tissues was the key to development of transformation systems based on
Agrobacteria. Of course, crown galls and hairy roots are not desirable features
for trees of the future, so the Ti-plasmids used for routine plant transformation
have been domesticated by removal of the "oncogenes" (phytohormone and
opine biosynthesis genes) which cause these tissue deformations. The oncogenes
have been replaced in the T-DNA by a variety of selectable marker genes, such
as those whose products confer resistance to antibiotics or herbicides (see
below). To facilitate recombinant gene manipulations, the T-DNA and vir gene
regions of the Ti-plasmid have, in many cases, been placed on separate plasmids
to yield what are commonly referred to as binary vector systems [99, 150]. The
shuttle plasmid of a typical binary system contains the T-DNA, and is often
simply referred to as the binary vector. These shuttle plasmids generally contain
a selectable marker gene, a multiple cloning site for insertion of the genetic
construct, and a variety of gene expression modulators (promoters, enhancing
elements, and terminators), all of which are flanked by the T-DNA sequences
[149]. Elsewhere on the shuttle plasmid are replication origins competent for
maintaining the plasmid in either Agrobacterium or E. coli - - the latter primarily
to facilitate access to the well-developed molecular biology systems available for
this organism. A wide variety of shuttle vectors are available, and improved or
specialized versions are continually being announced [151-156]. The vir genes
required for T-DNA transfer and integration with the binary system are
maintained in Agrobacterium on so-called helper plasmids, and a variety of these

plasmids have been developed in an effort to improve the efficiency of T-DNA
transfer and integration and to expand the range of host plants which may be
transformed using the Agrobacterium system [157, 158].
This last point, in particular, is significant with respect to efforts at trans-
forming commercial gymnosperm tree species. The range of host plants suitable
for Agrobacterium transformation was long thought to be limited to the dicoty-
ledonous angiosperms, since tumors are seldom formed when monocots or
gymnosperms are treated with these bacteria [159 161]. However, tumor
formation is a result of a complex series of events that occur after DNA transfer
and integration, and, consequently, transformation may occur in the absence of
tumor formation [157]. Since recognition of the separate nature of these events,
a significant number of monocots have been verifiably transformed using
Agrobacterium [162]. In some cases where DNA was not transferred to any cells
in treated monocots, specific strains of Agrobacterium were shown to have
difficulty in attaching to the cells in the wound [163], while in other cases the
intended hosts were found to secrete compounds that blocked expression of the
vir genes [164]. An increasing array of bacterial strains and plasmids have
provided ways to circumvent some of these problems [165, 166]. As a conse-
quence, various combinations of broad host range Agrobacterium strains and vir
genes are now routinely tested against different genetic lines of recalcitrant host
species during the development and optimization of appropriate transformation
systems [167]. The observation that vir gene expression can be induced by tissue
explants from a variety of algae, bryophytes, pteridophytes, and gymnosperms,
as well as the monocotyledonous and dicotyledonous angiosperms, suggests
that the host range for Agrobacterium transformation may yet be extended far
beyond its current use [168]. However, it seems unlikely that Agrobacterium-
mediated transformation could ever become universally applicable to all species
of plants.
As can be seen from Table 1, a variety of woody dicots, including species
important for the wood and wood-product industries, have been regenerated
from tissues transformed using Agrobacterium. Sadly, amongst the conifers,
Agrobacterium-mediated transformation followed b y regeneration has so far
only been accomplished with larch [-131,169]. Although Agrobacterium has
been shown to be capable of introducing DNA into the cells of other conifer
species [ 170-173], regeneration of plants from such transformed tissues remains
problematic. It seems that those conifer cells predisposed to regenerate are
difficult to transform with Agrobacterium, while easily transformed tissues are
recalcitrant to regeneration [174]. It appears that a variety of factors, including
the genetic background and metabolic state of the host cells, play a significant
role in governing the susceptibility of these plants to Agrobacterium-mediated
transformation [135, 175]. As a consequence, it is difficult to determine at this
time whether the lack of progress in this area could best be addressed through
focused research into improved Agrobacterium systems or more reliable regen-
eration protocols for the commercial gymnosperms.
4.3 Physical Gene Transfer
4.3.1 Transformationby Microprojectile Bombardment
Increasingly, microprojectile bombardment is becoming the transformation

technique of choice for plants that are recalcitrant to Agrobacterium-mediated
systems [176-179]. In this method, DNA containing the transgene is coated
onto small (1-5 ~tm) particles of gold or tungsten which are subsequently shot
into the target tissues with sufficient force to pierce the plant cell walls. The
particles lodge within the cells without killing them, and the DNA is released for
incorporation into the recipient genome. This technique is not restricted to
particular plant species or even to plants in general, as it has also been used to
transform a wide variety of animal cells [180-182], yeast [183, 184], fungi
[185, 186] and even bacteria [187]. Microprojectile bombardment can even be used
to transform the genomes of chloroplasts [188, 189] and mitochondria [183].
In the original instrument, tungsten microcarriers were accelerated by a
gunpowder charge (hence the common reference to "gene guns") [190], but the
commercial instrument developed from that early work uses a controlled burst
of pressurized helium for improved consistency [191]. Subsequent variations on
this general theme have resulted in the development of instruments optmized for
increased control over the bombardment conditions [192, 193] or decreased
cost of the instrumentation [194, 195]. The equipment designs described in the
latter papers put this technology well within reach of any moderately well-
funded laboratory contemplating plant transformation experiments.
As is the case for binary vectors used in Agrobacterium-mediated trans-
formation, vectors containing various selectable markers, multiple cloning sites,
and expression modulating sequences (promoters/terminators) have been
designed specifically for microprojectile transformation work [196]. The princi-
pal difference between these vectors and those used in Agrobacterium-mediated
systems is the elimination of DNA required by Agrobacterium, such as the
T-DNA borders and the Agrobacterium-competent origin of replication. The
smaller vectors that result from omission of these sequences are more tractable
to many of the molecular biological manipulations used to prepare transgene
constructs, i.e. plasmid preparation, restriction digests, ligations, and bacterial
transformation, but a further reason for minimizing extraneous DNA in these
vectors lies in the nature of the integration event. With Agrobacterium-mediated
transformation, only the DNA lying between the T-DNA borders is transferred
into the recipient cell, and the number of copies of T-DNA integrated into the
host genome is usually very low [149]. However, with microprojectile trans-
formation, many copies of the entire plasmid containing the transgene are
introduced into every cell, and it is often the case that many copies of this DNA
are integrated in the recipient genome [149]. Given the current regulatory
concerns with regard to characterization of the integrated DNA prior to field
release of transgenic plants, it is surprising that more effort has not gone into
developing minimal DNA vectors specialized for microprojectile transformation
similar to those developed by During [155] for Agrobacterium-mediated trans-

formation. On the other hand, efforts are underway to develop innovative
systems for site-specific transgene insertion [197, 198], and, in this respect,
elements from both the Saccharomyces FLP/FRT system [199, 200] and the
bacteriophage P1 Cre-loxP system [201,202] have been successfully tested in
plants. The specificity inherent in these systems would appear to be ideal for the
development of transgenic plants with precise insert structure and location.
Further flexibility in future transgenic plant development may result from using
derivatives of the Ac/Ds transposable element system from maize to remove
specific sequences, such as selectable marker genes, after transformation has
been accomplished [203].
4.3.2 Transformation of Protoplasts
Direct uptake of foreign DNA by plant cells can potentially avoid the difficulty
presented by limited susceptibility of some woody plants to infection with
Agrobacteria, as well as the high equipment costs usually associated with
microprojectile transformation. Protoplasts can be induced to efficiently take up
naked DNA under the influence of polyethylene glycol (PEG) [204-206] or
a polarizing electric field (electroporation) [207-209]. Several forest tree species
of commercial importance having limited transformability by Agrobacterium,
including various spruces and eucalypts [109, 174], have demonstrated at least
transient expression of foreign genes introduced by one or more of these
techniques. Systems for regenerating spruce, pine, and eucalyptus trees from
protoplasts have also been reported [210]. However, the high labor demand
required to isolate and culture protoplasts, as well as the need for a reliable
regeneration system, remain significant drawbacks to widespread use of this
transformation technique. In addition, the increased risk of isolating somaclonal
variants from transformed protoplasts limits the usefulness of this system for
developing commercial products [174]. On the other hand, the number of
woody species for which protoplast regeneration systems are available is con-
stantly increasing [109, 210], and in some situations protoplast transformation
may provide unique opportunities, for example, as an alternative to micro-
projectile bombardment in organelle transformation [211,212].
4.3.3 Alternative Transformation Techniques
The transformation techniques described above are by far the most widely used
and accepted. However, each has its drawbacks. Consequently, a variety of
alternatives have been proposed, but most have met with marginal success at
best [101]. Sawahel and Cove [213] have provided a fairly concise catalog of
most of these alternative techniques, but two interesting techniques for trans-
forming intact cells, sonication [214] and silicon carbide fiber microinjection
[215], were developed too recently to be covered by these authors. The latter
technique seems particularly simple and inexpensive, and has been used success-
fully in several different laboratories [216-219]. The essence of the technique is
that DNA is coated onto minute (0.6 ~tm diameter, 10-80 lain length), rigid fibers
of silicon carbide, and these are subsequently vortexed with suspension-cultured
cells of the recipient plant. Like tiny syringe needles, the fibers penetrate the cell
walls without killing the cells and deliver the DNA for subsequent integration.
This technique would appear to have the potential to gain widespread accept-
ance once its less precise aspects, e.g. how best to immobilize the DNA on the
fibers, are more rigorously optimized. In terms of cost efficiency and ease of use,
silicon carbide fiber transformation appears to hold great potential for enabling
any laboratory equipped for plant tissue culture to successfully embark on plant
transformation projects.
4.4 Selection Systems, Promoters, and Regulatory Elements
To identify those cells that have received the transgene, transformation vectors
carry either screenable or selectable markers [104, 220]. Selectable markers
encode enzymes that enable the transformed cells to grow under conditions that
kill non-transformed cells; thus, the most commonly used selectable markers
encode enzymes that detoxify antibiotics or herbicides added to the culture
medium. Neomycin phosphotransferase (kanamycin-resistance) [221], hy-
gromycin phosphotransferase [222], gentamicin acetyltransferase [223, 224],
and the Tn5 gene encoding bleomycin resistance [225] are the most widely
used antibiotic resistance markers, while phosphoinothricin acetyltransferase
(biaphalos-resistance) [226], modified 5-enolpyruvylshikimate-3-phosphate
(EPSP) synthase (glyphosate-resistance) [227], modified acetolactate synthase
(chlorsulfuron-resistance) [228], and bromoxynil nitrilase (bromoxinyl-resist-
ance) [229] are commonly used herbicide resistance markers.
It is not unusual to find that cell lines recovered from selective media have
lost their regenerative capacity, and this is a principal reason for using screen-
able markers. The most widely used screenable markers have been chloram-
phenicol acetyltransferase (CAT) [230] and/~-glucuronidase (GUS) [231], but
these have the drawback of requiring destructive sampling of the putatively
transformed tissues in order to assay the enzyme activity. Alternatively, a gene
from the jellyfish, Aequoria victoria,which encodes a protein (GFP) that fluores-
cences green under UV irradiation, is showing great promise as a non-destruc-
tive screenable marker for transgenic organisms [-232]. Modified versions of the
gene have already been developed to express proteins with altered emission
spectra so that the expression from multiple gene constructs may be monitored
simultaneously [233]. GFP has been used as a reporter gene in several plant
systems, including suspension-cultured Citrus cells [234, 235], after the gene was
modified to eliminate problematic sequences, such as a cryptic splice site [236].
However, its use in woody species may be limited, since transformed cells of
L. tulipifera or L. stryraciflua which expressed the G F P polypeptide did not

fluoresce (Kim and Merkle, unpublished observations), possibly because these
particular cells could not catalyze the post-translational event necessary to form
the fluorophore.
The promoter and terminator sequences most widely used for expression of
transgenes in plants have been derived from one of three sources: the 35S RNA
of cauliflower mosaic virus (CaMV), the nopaline synthase (hop, nos) gene from
one class of A. tumefaciens Ti-plasmids, or the octopine synthase (ocs, oct) gene
from a second class of A. tumefaciens Ti-plasmids. These promoters all lead to
expression of chimeric genes at high levels in a wide variety of plant tissues, and
various combinations have been developed to improve constitutive expression
of gene constructs [220]. To efficiently modify characteristics of specific plant
tissues, e.g. wood, increased efforts need to be made to identify promoters that
can drive gene expression to high levels in the specific tissues of interest (106).
Relatively few such promoters have been identified in plants, and woody plants,
in particular, have received only cursory examination with respect to the
promoters that control gene expression. To some extent this may be due to
a perception that research in this direction should be supported by those
commercial enterprises that stand to gain the most from application of
tissue-specific promoters.
4.5 Current and Future Targets f o r Genetic Engineering

in Forest Trees
4.5.1 Lignin Content and Composition
Lignin makes up 20~30% of the total dry weight of wood and constitutes the
principal barrier to production of pulp and paper. Estimates suggest that
altering the composition of lignin in gymnosperms so that it resembles the more
easily extracted lignin in angiosperms could provide the US industry alone with
an annual saving in excess of $6 billion [237]. The potential savings that would
accrue from reductions in total lignin content of the order of 10-15% have been
suggested to be of a similar magnitude.
Because the lignin biosynthetic pathway is relatively well understood, it has
provided an opportune target for early experiments in genetic engineering of
forest trees, and detailed reviews of efforts to date are available [107, 238].
Reduced cinnamyl alcohol dehydrogenase (CAD) activity in transgenic plants
leads to incorporation of hydroxycinnamoyl aldehydes into lignin, and the
resultant red-brown polymer is much easier to remove from fibers using chem-
ical pulping techniques [239,240]. Transgenic plants having reduced
hydroxycinnamoyl O-methyltransferase (OMT) activity have shown a variety of
effects in terms of both lignin content and composition [241-244]. However, it is
hoped that co-expression of an angiosperm OMT along with a ferulate-5-
hydroxylase (F5H), such as was recently cloned from Arabidopsis [245, 246], will
lead to the production of sinapyl alcohol in transgenic gymnosperms. A success-

ful outcome would result in the conversion of the softwood lignin from the less
easily degraded guaiacyl-rich polymer to a more easily degradable guaiacyl-
syringyl copolymer, such as that which distinguishes hardwood species. Ex-
tracellular glucosidases with high specificity for monolignol glucosides have
been purified from conifer cambium, and these would appear to provide excel-
lent targets for efforts to alter lignin deposition [247, 248].
The oxidative polymerization step in lignin biosynthesis presents another
interesting possibility for manipulating lignin via genetic engineering [-107].
Attempts to down-regulate lignification by reducing peroxidase expression in
transgenic plants have not yet yielded positive results [249], and efforts to use
antisense laccase genes in a similar manner have so far been inconclusive
E250, 251].
4.5.2 Sterility and Early Flowering
The release of genetically engineered trees into environments in which they can
interbreed with wild populations is unlikely to be approved by regulatory
agencies unless mechanisms to control sexual reproduction are in place [-252].
Thus, if the value of genetically engineered trees is to be realized, it is essential
that sterile tree lines be created. In addition to solving problems associated
with release, sterile tree lines will have the added benefit that proprietary
genetic materials will be better protected from acquisition by competitors. It is
also possible that by blocking formation of reproductive structures, energy
resources will be redirected into vegetative growth, thereby increasing
growth yields. The production of cellular toxins under the control of promoters
specific for gene expression in pollen-producing tissues has been shown to result
in male sterility [253, 254], and such a system should function equally well in
trees.
Tree breeding could be greatly accelerated if trees could be induced to flower
while they were still seedlings, i.e. within 1 or 2 years following germination. Not
only would this dramatically shorten the breeding cycle, but it would also make
breeding in the controlled environment of the greenhouse possible. Finally,
induction of early flowering in forest trees would enable rapid characterization
of inheritance patterns of transgenes in the progeny of transgenic trees. Recent
research with a group of flower-meristem-identity genes from the herbaceous
model plant, Arabidopsis, has culminated in the production of transgenic
Arabidopsis, in which precocious flower development was induced by over-
expression of the inserted transgene [255]. Hybrid aspen transformed with the
same Arabidopsis LEAFY gene (LFY) under the control of the cauliflower
mosaic virus 35S promoter produced flowers after only five months in the
greenhouse [-256]. This work has obvious potential to completely revolutionize
the entire field of tree breeding.
4.5.3 Herbicide Resistance
Herbicides are widely used in site preparation to minimize competition from

weedy species in newly replanted forests. However, these chemicals often cannot
be applied directly to the trees themselves, or even to the area immediately
around them, without causing significant damage or even death. Glyphosate is
a broad-spectrum low-toxicity herbicide that blocks the aromatic amino acid
biosynthetic pathway at EPSP synthase, and genes encoding mutant forms of
this enzyme have been shown to confer resistance to the herbicide when
expressed in transgenic plants [227]. Resistance to glyphosate has been intro-
duced into commercial hardwood [127, 257] and softwood [169] tree species,
and resistance to other herbicides has been conferred to various other transgenic
plants and trees [258-260]. As shading and competition for nutrients are known
to be significant factors limiting tree growth rates in young plantations, develop-
ment of herbicide resistance would appear to be a logical target for tree
improvement. However, widespread application of these compounds can have
a profoundly detrimental effect on the environment, and concern for this was
highlighted in a recent survey that showed a widespread understanding among
forestry professionals that aerial spraying of herbicides in forests would not
easily be accepted by the general public [261]. On the other hand, as noted by
Jouanin et al. [110], herbicide-resistant trees would probably be most helpful in
a nursery setting where herbicide application can be more rigorously controlled.
4.5.4 Insect and Pathogen Resistance
Endotoxin (Bt) produced by the Bacillus thuringiensis soil bacterium has enjoy-
ed widespread forest application as a non-toxic biopesticide that is highly
specific for phytophageous lepidopteran, coleopteran, and dipteran insects
[262]. The biology and mode of action of this biopesticide has been reviewed
[263-265], and various studies have examined factors influencing its use in
forest settings as well as its persistence in forest ecosystems [266, 267]. Trans-
genic tobacco and tomato plants expressing this protein were shown to be
protected from feeding by lepidopteran larvae [268, 269], and similar protection
from gypsy and forest tent moth caterpillars was demonstrated in transgenic
poplar [270-272]. Transgenic larch trees expressing the Bt endotoxin have been
recovered by Shin et al. [169], but there are no reports, as yet, regarding the
resistance of these softwoods to insect attack. Although it is anticipated that
such genetically engineered trees will eventually be deployed in commercial
plantings, insects have been shown to be capable of developing resistance to
these toxins [273]. There is thus a need to develop appropriate deployment
strategies [274].
Protease inhibitor proteins are produced in many plants as a response to
wounding or insect feeding, and the role of these proteins is to interfere with
digestion in the insect gut [275]. A trypsin inhibitor protein from cowpea was
used to protect transgenic tobacco from Heliothis virescens larvae [276], and
a cysteine protease inhibitor from rice was recently shown to protect poplar
from a species of boring beetle [277].
Schuerman and Dandekar [111] reviewed efforts to date to engineer plants
for viral resistance, but work with woody plants has so far been limited to
horticultural species [278, 279]. A variety of strategies using transgenes to
control fungal diseases have been discussed and tested [280, 281]. However,
none of these techniques have yet been tested in trees.
4.5.5 Tree Form and Fiber Morphology
Wood formation requires carbon partitioning to favor the plant stem, and
Timmis and Trotter [237] noted the potential for increasing carbon deposited in
the tree bole by altering the growth habit of the tree. They used several lines of
evidence to suggest that tree growth habit might be controlled by a limited
number of genes, and speculated on the possible significance of work performed
by Klee and co-workers [282] in which transgenic petunias having altered auxin
metabolism showed significant increases in xylem and phloem formation. Re-
cent work has shown that alteration of auxin metabolism in transgenic poplar
leads to a variety of changes in tree form and wood characteristics [283].
4.5.6 Novel Traits
There has recently been significant interest in using plants to clean up sites
contaminated with heavy metals and toxic organic wastes [284 288]. Stomp et
al. [289] noted a variety of tree growth habit characteristics that would make
these plants very good candidates for use in phytoremediation.
4.6 Regulatory Considerations
Like all other technological advances, the production of transgenic plants brings
with it the potential for unforeseen consequences [290-292]. To minimize the
possibility that such consequences could lead to irreversible problems when
these organisms are introduced into the environment, all transgenic plants are
subjected to a process of risk assessment prior to field release. The regulations
governing such risk assessments vary from country to country, but Raffa [293]
presents a set of guiding principles that are valid in most cases. In addition to the
explicit governmental regulations on the subject, several recent reviews discuss-
ing the possible impacts of transgenic plant release should be considered by
researchers attempting to create transgenic plants for use in the commercial
sector [294-299]. Although most of these guidelines and rules were developed to
address situations encountered in the cultivation of agricultural crops, it can
readily be argued that these considerations are even more critical in the case of
genetically engineered trees, since these organisms have a longer life-span and
hence a much longer period in which to pass their transgenes to wild popula-
tions.
5 Molecular Breeding
Classical plant breeding strategies are generally untenable for most forest tree
species. Long generation times (5 to 20 years), coupled with the fact that many
traits important to forest product industries can only be fully assessed after the
tree has reached maturity, preclude rapid analyses of test crosses and have, thus,
effectively limited breeding programs to the most economically important tree
species. Even in these cases, family pedigrees of more than three or four
generations are rare.
To avoid the delay of having to score mature phenotypes, breeding experi-
ments have increasingly relied on biochemical markers whose expression has
been correlated with particular mature phenotypes. For example, isozymic
variation has been widely used in agronomic as well as forest breeding programs
[e.g. 112, 300]. For isozyme analyses, proteins contained in tissue extracts are
resolved in a gel matrix on the basis of their physical properties (size, shape, or
electrical charge). Subsequent staining based on the catalytic activity of the
enzyme results in specific visualization of the isozymes, even in the presence of
numerous other unrelated enzymes. An advantage of this method is that it yields
co-dominant markers, since both alleles of a heterozygous locus can be detected.
Unfortunately, there are relatively few biochemical markers whose expression
has been matched with desirable phenotypes, and the expression of these
markers is in many cases highly dependent upon environmental factors. It is also
important to note that only a small amount of the genetic variation in a popula-
tion is displayed phenotypically. Furthermore, since only about one-third of
amino acid substitutions effect changes in the protein that can be detected by
electrophoretic techniques, no more than one third of the total genetic variation
is discernible through the use of isozyme analysis. With respect to the potential
variation contained in the entire genome, isozymic markers are even more
limited, since only about 0.5% of the typical eukaryotic genome consists of
coding sequences [301].
Using DNA-based markers, researchers can potentially access all of the
variation contained within a given genome, thereby increasing their chances of
finding a marker that segregates with the specific phenotype of interest. Thus,
the main advantage of molecular markers is that they are based on the polymor-
phisms occurring naturally in the DNA of a given species, and, as forest trees are
among the most genetically variable organisms known [302], molecular
markers useful for tree breeding programs should not be difficult to identify. In
addition to constituting a larger pool of potential markers, DNA-markers have

the advantage that they do not change in response to environmental factors or
the developmental stage of a particular plant tissue. For a general review on the
use of molecular genetic markers in plants see Rafalski and Tingey [303], and
for comprehensive discussions of the ways in which molecular markers may be
applied to forest tree improvement programs consult Neale et al. [304] and
Haines [112].
5.1 DNA Marker Techniques
5.1.l Restriction Fragment Length Polymorphism (RFLP)
RFLP (restriction fragment length polymorphism) analysis was first described

by Botstein et al. [305], and has been reviewed elsewhere [306-308]. Briefly,
DNA is extracted and cut into discrete fragments using restriction endonuc-
leases. The DNA fragments are then separated by gel electrophoresis and
transferred to a membrane filter so that they can be detected using specific RNA
or DNA probes in a process known as Southern blotting. Polymorphisms are
then detected by the presence or absence of labeled probe in bands on the blot
[309].
Advantages of this method are that RFLPs are multi-alleleic and co-domi-
nant (i.e. heterozygotes can be distinguished from either homozygote), and there
are a virtually unlimited number of potential probes. Drawbacks include the
relatively large amount of DNA required (2-15 pg per gel lane, depending on the
genome size of the species, although it should be noted that a single blot can be
reprobed many times), as well as the length of time and high cost required for
each analysis. More problematic is the potential for artifacts which arise from
incomplete digestion of the DNA by restriction endonucleases. In order to
maximize sample throughput, procedures for preparing DNA are often stream-
lined, and, as a result, the DNA can be impure. Impurities in turn lead to partial
digestion which becomes manifest as extra bands on the Southern blots.
5.1.2 Random Amplified Polymorphic DNA (RAPD)
RAPDs (random amplified polymorphic DNA) are dominant molecular

markers pioneered by Williams et al. [310] and Welsh and McClelland [311].
The method is based on PCR (polymerase chain reaction) and uses random
oligonucleotide primers (usually 10-mers) to amplify genomic DNA. The use of
short primers and low annealing temperatures leads to the generation of PCR
products from multiple sites in the genome, and polymorphisms become appar-
ent when sequence differences affecting primer binding sites give rise to different
banding patterns when the reaction products are analyzed by gel electro-
phoresis.
By way of advantages, RAPD analyses do not require prior sequence

information, and none of the plant-derived DNA requires passage through
bacteria. In addition, the technique requires very little DNA (10-25 ng), radio-
isotopes are unnecessary, and once the DNA has been prepared, the experi-
mental procedure is relatively simple and amenable to automation [312]. The
principal disadvantage with RAPDs is that only one strand of the DNA is
amplified, and, as a consequence, dominant alleles are visualized whether the
individual is heterozygous or homozygous at the particular locus. Haploid
tissues, such as occur in conifer megagametophytes, can be used to circumvent
this limitation [313]. A further disadvantage is that PCR-based technologies on
the scale required for plant breeding are relatively costly because of the expense
of the polymerase.
5.1.3 Bulked Segregant Analysis
Bulk segregant analysis, described by Michelmore et al. [314], is a useful

technique for finding new markers, typically RAPDs, that are linked to a par-
ticular locus or to an area that is sparsely populated with markers. The method
uses two groups of individuals that arise from a single cross and are
homozygous for alternate alleles governing a particular trait. If members of
a segregating population are pooled on the basis of their phenotype, then the
groups can be expected to be heterozygous at all unlinked loci but markedly
skewed in the direction of one or other parental allele for a linked locus.
Advantages of the procedure are that arbitrary primers can be used as probes,
and the ability to pool individuals speeds analysis. One particular disadvantage
of this technique for the purposes of forest tree breeding is linkage equilibrium,
which is highly outbred species may mask the segregation of loci. In the case of
conifers, this particular problem can again be minimized by analyzing haploid
megagametophyte tissues from a sufficient number of individuals within a family
[3133.
5.1.4 Microsatellite Repeat Polymorphisms
Microsatellite repeats are highly polymorphic regions of DNA that occur

frequently in eukaryotic genomes and are most commonly represented by the
dinucleotide repeats [AC]n, [AG]n, and [AT]n [315-317]. PCR primers based
on DNA sequences from regions flanking microsatellite sequences may be used
to amplify the genomic DNA spanning the repeat region. The resultant products
may then be resolved by gel electrophoresis to reveal allelic variations in the
lengths of repeats. Advantages of this technique are that microsatellite repeats
occur with moderate abundance, are highly polymorphic, and provide co-
dominant markers. In addition, because the technique is based on PCR,
it requires very little input DNA, and no radioisotopes are required.
Disadvantages include a requirement for cloning and sequencing to characterize

multiple microsatellite loci, not all of which will prove to be useful for analysis,
as well as a need to use high resolution gels in order to differentiate polymor-
phisms as slight as two or three nucleotides.
5.1.5 Amplified Fragment Length Polymorphisms (AFLP)
A DNA fingerprinting technique referred to as amplified fragment length poly-

morphism (AFLP) uses genomic DNA digested with restriction endonucleases
as a PCR template [318]. The technique is similar to RFLP analysis, but relies
on the power of PCR, rather than hybridization, to reveal subtle differences in
the DNA from different sources. The binding between short PCR primers and
DNA restriction fragments is very specific, and may not even occur between
sequences that differ by as little as one or two nucleotides. Advantages of the
method include no need for prior sequence data and the ability to selectively
amplify sequences from a large number of restriction fragments. In addition,
AFLP mapping can be used to detect corresponding genomic clones in large
fragment libraries, e.g. cosmid or yeast artificial chromosomes. On the other
hand, the technique is limited in practice to the resolving power of the gel
electrophoresis system. Thus, reactions that generate more than 50 to 100
fragments are generally too complex to analyze. Also, the method functions best
when used in conjunction with established breeding programs and documented
multigenerational lines. The procedure also shares the high cost inherent in
RAPD and RFLP analyses as well as the lengthy time requirements of RFLP
mapping.
5.1.6 Microsatellite Hybridization
Another recently developed method takes advantage of the fact that, in a typical
RAPD analysis, abundant PCR products may be visualized by staining, but
additional products in quantities below the staining threshold of ethidium
bromide are also resolved in the gel. These minor products can be visualized by
hybridization with microsatellite probes (e.g. [GT]8, [CT]8, [CC]12, [GA]12)
to produce multiple independent and polymorphic fingerprints [319,320].
Advantages of this combined method include no need for prior sequence
information, low input of template DNA, and when using non-isotopic detec-
tion methods, the possibility of using probes repeatedly. The method is espe-
cially useful for displaying polymorphisms in species where little variation is
revealed by RAPD analyses alone. However, the method also suffers from the
relatively high cost associated with RAPD analyses, as well as the time required
for RFLP analyses.
5.2 Applications o f DNA Markers in Forest Research
The long generation times of forest trees places a premium on the development
of methods that will allow for early genetic selection of the desired phenotype.
Indirect selection based on DNA markers can be practiced at a very early age
once linkages to important traits are identified. The advantages are obvious in
that cost would be much lower than with progeny testing, selection intensities
would be much higher, and selection would be potentially more efficient because
of the higher heritabilities of the markers. One recent development that has
great potential for forest tree breeders came from studies of the genes that
regulate floral development in the model herbaceous plant, Arabidopsis thaliana.
By introducing the LEAFY gene from Arabidopsis into aspen it was possible to
induce flowering in transgenic aspen during their first year of growth as opposed
to waiting the typical 10-20 years [-256]. Undoubtedly, this technology will find
widespread application in forest tree improvement programs, particularly when
coupled with marker-assisted selection.
5.2.1 GeneticLinkage Maps
Genetic linkage maps are constructed by analyzing RFLP and/or RAPD

markers and monitoring the segregation of a marker among progeny of a test
cross. The high degree of polymorphisms allows for a virtually unlimited
number of markers to be mapped using a single segregating population.
A database of markers is made from the same set of F2 or backcross plants.
When a map containing 100-200 well-dispersed markers has been constructed,
virtually any new marker can be linked to one that has been previously mapped.
The accumulated markers for a segregating population form a database that
becomes a valuable resource for placement of new markers, and the database
can be expanded by distributing the mapping data to other research groups. The
practical strategies for building genetic linkage maps in plants was recently
reviewed [321].
A major obstacle to the assembly of genomic maps for marker-aided tree
breeding is that most tree species are highly outcrossed, resulting in significant
linkage equilibrium between marker loci and the genetic locus of interest [322].
As a consequence, relationships established from the analysis of one cross do not
necessarily hold for a second cross and, therefore, conventional mapping of
forest trees in natural populations is extremely difficult. It has been argued that
these hurdles severely limit the utility of marker-assisted selection in forest tree
breeding. However, an opposing view, based on recent technological and theor-
etical advances, has been offered by O'MaUey et al. [323]. These authors note
that by screening a sufficient number of potential RAPD markers and choosing
the most reliable, i.e. markers that are consistently easy to score, one is able to
quickly map the genome of individual trees. With maps of individual trees,
breeders can minimize the difficulties brought about by linkage equilibrium and
circumvent the need for the extended pedigrees commonly used for agronomic
crop improvement.
Of special interest to forest tree geneticists is the advantage provided by
using conifer megagametophyte tissue as a DNA source. The haploid nature of
this DNA allows for recombination and segregation events to be followed
among open-pollinated seeds from a single tree, and circumvents the dominant
allele problem inherent in using RAPDs with diploid samples. One is able to
search for markers that are consistently found together in the same seeds,
indicating that they are on the same chromosome [313]. This unique advantage
of gymnosperms serves to partially offset the difficulties inherent in the large
genome sizes typical of many softwood tree species.
The lack of a suitable haploid stage in angiosperms led to the development of
a "pseudo-test cross" method, which, in conjunction with RAPD technology,
allows construction of single-tree genetic linkage maps [324]. The method ~elies
on the observation that a cross between two heterozygous individuals results in
many single-dose RAPD markers segregating 1 : 1 in their F1 progeny. Although
this mapping strategy requires a controlled genetic cross to be made, the
additional effort enables one to survey twice the heterozygosity, i.e. that from
each parent. The method may also be applied to conifers to quickly generate
single-tree linkage maps [-324].
5.2.2 Mapping Projects
DNA-based molecular marker maps are being made for a number of hardwood
and softwood species. RAPDs of megagametophyte tissue have been used to
build a map covering 90% of the maritime pine (Pinus pinaster) [325] and 85%
of the longleaf pine (Pine palustris Mill.) genomes [326]. Maps are being
constructed for Norway spruce (Picea abies Karst.) [327], slash pine (Pinus
elliotti Engelm. var. elliotti) [328], and white spruce (Picea 91auca (Moech) Voss)
[313]. RFLPs were used to construct a map for loblolly pine (Pinus taeda L.) in
a three-generation pedigree strategy [329]. More recently it was shown that
RFLP probes used in building a loblolly pine map could be used in related
species for the purpose of comparative genome mapping [330]. For eucalyptus,
an outbred third-generation pedigree was used to build a map using RAPDs,
RFLPs, and isozyme markers [331], and the pseudo-test cross strategy was used
for Eucalyptus 9randis and E. urophylla [332]. A genomic map was made for
peach [333] using bulk segregant analysis and RAPDs.
5.2.3 Marker-Assisted Selection
Molecular markers provide a powerful tool for monitoring traits difficult to

select for using traditional techniques, as well as for the simultaneous selection
of multiple traits. To use this technique, linkages between marker loci and
phenotypically (economically) important traits must be identified. Then, instead
of waiting for the tree to reach maturity before it displays the trait of interest, the
breeder can select the offspring of a cross that carry the specific linked DNA
marker(s). With forest trees, marker-aided selection is best applied to single
traits that are relatively well characterized, e.g. wood specific gravity [334].
Neale and co-workers have identified quantitative trait loci (QTLs) influencing
wood specific gravity in an outbred pedigree of loblolly pine [335]. RAPDs have
been used to identify QTLs which influence early height growth in pines [336],
while RAPDs and the pseudo-test cross strategy were used to identify QTLs
affecting vegetative propagation in Eucalyptus [332]. Bradshaw and Stettler
[337] mapped QTLs for stem growth and form as well as spring leaf flush in
poplar. The molecular genetics of rust resistance in poplars was studied by Villar
et al. [338] who used RAPDs and bulk segregant analysis to quickly identify
suitable markers which were subsequently validated in a 2 x 2 factorial mating
design.
Markers may also be used to locate valuable genes to facilitate their cloning
by map-based or positional cloning. The technique is based on the premise that
any gene consistently inherited with a marker must lie near it on the same
chromosome. Once isolated and characterized, the genes could be used to
transform recipient forest trees or used as markers for the early selection of
desirable phenotypes in wild-type (non-transformed) trees. The approach shows
the greatest immediate potential when applied to traits governed by single genes.
Devey et al. [339] have combined the power of RAPDs and bulked segregant
analysis of haploid megagametophyte tissues to make significant progress with
this approach by identifying ten RAPD markers that map close to a sugar pine
(Pinus lambertiana Dougl.) gene for resistance to white pine blister rust, one of
the most damaging pathogens of Southern pines. Fusiform rust is a devastating
disease of loblolly pine for which resistance was for many years assumed to have
a polygenic basis [9]. However, RAPDs and bulk segregant analysis were
recently used to identify a region of the host genome that behaves as a single
dominant gene and is responsible for resistance to this disease [340]. Narrow-
crown growth habit is a desirable phenotype for high-density forest plantations,
and RAPDs, in conjunction with bulked segregant analysis, have been used to
identify a locus linked to pendula, a single gene controlling the narrow-crown
phenotype in Norway spruce (Picea abies L.) [341]. Three markers linked to
resistance to black leaf-spot disease in Chinese (Ulmus parvifolia) and Siberian
(U. pumila) elms [342], and two markers linked to scab resistance in apple
(Malusfloribunda) [343] have been identified using a similar strategy. Consider-
ing the difficulties (transformation, vegetative propagation, government regula-
tions) currently besetting genetic engineering of forest trees, it is conceivable that
marker-assisted selection may have greater near-term impact on forest tree
improvement [323].
5.3 Other Applications o f DNA Markers
5.3.1 Quantification of Genetic Diversity
RAPD markers have been used to investigate the distribution of variability in

natural populations of Eucalyptus 91obulus and provide the foundation for
effective breeding and gene conservation strategies [344]. RAPDs have revealed
an extensive amount of genetic diversity in trembling aspens (Populus tremulo-
ides) in a study that also highlighted the benefits of careful PCR primer selection
[345]. The authors identified a single RAPD primer that was able to separate
83% of the trees sampled in any given population. Microsatellite markers have
also been used to determine the level of genetic variation in tropical rain forest
species where they proved to be powerful tools for analyzing population struc-
ture with respect to gene flow and paternity [346].
5.3.2 Genotype Verification and Delineation
RAPD markers have proven useful in identifying the parentage of progeny. In

European white birch (Betula pendula Roth), a commercial set of random 10-mer
primers was used to confirm lineages [347]. The authors pointed out that
although robotics are becoming more commonplace in repetitive PCR routines,
there is still considerable labor involved in the preparation of amplification-
quality DNA templates. To minimize this problem, they developed a pooled-
progeny strategy similar to that used in bulk segregant analysis [314], in which
leaf samples of equivalent fresh weight from different individuals were pooled
and used as the source of DNA for subsequent RAPD analyses. RAPDs have
also been used to analyze somatic embryos and plants regenerated from em-
bryogenic lines of Norway spruce (Picea abies (L.) Karst) to determine the level
of somaclonal variation [348]. It was shown that material from the same cell
line shared identical banding patterns, whereas regenerants from different cell
lines were clearly distinguishable. RAPD patterns were reported to be somati-
cally stable, thereby enabling researchers to trace clone identity from the
laboratory to the field [349]. However, because of the dominant nature of
RAPD markers, as well as the difficulty in reproducing banding patterns both in
different laboratories and between pedigrees, the authors concluded that RAPD
markers were not likely to replace isozyme analyses for this purpose. RAPD
mapping of peach (Prunus persica) was reported by Pooler and Scorza [350],
who cautioned that spontaneous somatic rearrangements (bud sports) could
lead to unexpected inheritance of RAPD markers. RAPDs were used to identify
mislabeled clonal material (ramets) in a sitka spruce (Picea stichensis) breeding
program [351], and were further used to identify and assess the extent of natural
introgression between populations of interior spruce, white spruce (Picea 9lauca
(Moench) Voss) and Engelmann spruce (Picea engelmannii Parry) [352].
6 Electronic and Computational Resources
Although not a biotechnology, sensu stricto, the Internet hosts a wide variety of
information resources that are of increasing use to researchers in all areas of
forestry. As with all other aspects of the Internet, forestry-related resources are
being added at an exponential rate, and providing a current and comprehensive
list of useful sites is becoming impossible. However, Table 2 contains a highly
subjective list of relatively stable sites that should act as excellent starting points
for those wishing to explore and sample the wide variety of forestry resources
online.
Table 2. A Selection of Internet Resources Related to Forest Biotechnology (5/1/96)

Server Page Title Internet Address
General Starting Points
Yahoo Forestry List http://www.yahoo.com/science/agriculture/
forestry/
Galaxy Forestry List http://galaxy.einet.net/galaxy/Engineering-and-
Technology/Agriculture/Forestry.html
TAPPIWeb List of Forestry
Servers http://www.tappi.org/resource.htm
Natural Resources: Institutions and Organiza- http://sfbox.vt.edu: 10021/Y/yfleung/forestry.
tions html
More Forestry Stuff ht tp://forestry.bangor.ac.uk/forst uff.htm
The Pulp and Paper Jumplist http://www.nlbbs.com/~ dc001/paper/j umplist.
html
Other Related Forestry Servers http://www.chem.csiro.au/extpulp.htm
The WWW Virtual Library - Forestry http://www.icfrnet.nnp.ac.za/mirrors/Forestry.
html
Governmental Organizations
CSIRO Forestry and Forest Products http://www.chem.csiro.au/fordiv2.htm
Dendrome-Tree Genome Mapping Project http://s27w007.pswfs.gov/
Finnish Forest Research Institute (METLA) http://www.metla.fi/
Japanese Forest Research Institute ht tp://ss.ffpri.affrc.go.jp/
National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/
Natural Resources Canada http://www.emr.ca/
US Forest Service ht tp://www.fs.fed.us/Homepage,html
Educational Organizations
Auburn University http://www.forestry.auburn.edu/
Institute for Paper Science and Technology http://www.ipst.edu/
Louisiana State University ht tp://wwwlfpl.forestry.Isu.edu/
NYForest Online http://149. 119.1.26/default.html
Oxford University http://ifs.plants.ox.ac.uk/
Poplar Molecular Network gopher://poplar 1,cfr.washington.edu:70/1
Texas A&M University http://165.91.48.43/
University of Alberta http://www.rr.ualber ta.ca/
University of Illinois http://gopher.ag.uiuc.edu:80/NRES/forest ry-re-
search.html
University of Maine http://www.ume.maine.edu/~ nfa/for mgt/
wse.htm
University of Minnesota http://mercury.forestry.umn.edu/FP/ForP.html
University of Washington http://weber.u.washington.edu: 80/--,cfrwww/
Table 2. (Continued)
Server Page Title Internet Address

Independent Organizations
American Forest & Paper Association http://www.infobahn.com.tw/adv/afpa/afpa.htm
Technical Association of the Pulp and Paper
Industry http://www.tappi.org/
United Nations - FAO http://www.fao.org/WAICENT/Forestry.htm
Corporations
ForestNet http://www.forestnet.com/
ForestPro http://www.forestpro.com/
Lej6 International http://www.transport.com/~ leje/homepg.html
Individuals
Jeff Lindsay http://www.athenet.net/~ jlindsay/Paper.shtm 1
:~top
Knut's Pages http://www.carleton.ca/~kmenard/forest.html
Steve Shook's Directory http://weber.u.washington.edu/~ esw/fpm.htm
Miscellaneous
BiotechnologyLaw Web Server http://biotechlaw.ari.net/
BiotechnologyPermits Home Page http://www.aphis.usda.gov/BBEP/BP/
Usenet Newsgroups
Agroforestry bionet.agroforestry
Pulp and Paper misc.industry.pulp-and-paper
Listservs
Wood Net wood-net@ esusda.gov
Wood Science wood-science @unixg.ubc.ca
7 Conclusions
The true innovator is often characterized by an ability to see the far-reaching

trends and changes in technology and economy as well as their connections to
global, social, and industrial patterns. The long rotation times required for forest
tree production demand that the forester should have a particularly good crystal
ball in order to anticipate future trends and needs for the trees being planted.
When rotation times are short, e.g. Brazilian eucalyptus cut after seven years, it
is relatively easy to predict that a newly planted tree will probably be used for
fibers. Even with rotation times of the order of 25 to 30 years, as is the case for
southern pine in the United States, one may be able to make a reasonably
correct prediction regarding the future use of the tree. However, when rotation
times run to 100 years or more, as is the case in Scandinavia, you need a crystal
ball of increasingly high quality to make any reasonable predictions.
Regardless of what will be the particular end use of trees in the next century,
there is no denying that there will be an urgent need for them to grow faster.
Demand for fibers and solid wood will continue to grow even as harvestable
forest acreage decreases due to population increases and set-asides that reserve
forested areas for recreational purposes. In addition, advances in wood chem-
istry and biotechnology may also make trees the ideal feedstock for "bio-
refineries" that could produce alternatives to most of the current petrochemical
products of oil-refineries. It is unlikely that we will forever be stuck with the
dogma that forestry is only for the production of paper and timber. Thus, we
anticipate that trees will eventually be cultivated like other plants for faster
growth, greater yield, and a more diversified and efficient use.
New silvicultural practices will certainly be needed to speed the growth of
trees, and as a consequence, biotechnological techniques are likely to become
commonplace in forestry. In vitro propagation will enable us to produce large
numbers of genetically improved trees without having to establish large breed-
ing orchards. Molecular markers will help in identifying superior genotypes as
well as in monitoring the products of improved breeding schemes. Genetic
engineering will provide the means to rapidly bring new genetic materials to
bear on problems such as acid rain and attack by exotic insects. By combining
traditional practices with the techniques of biotechnology, tomorrow's foresters
will likely produce a revolution in the way forestry is practiced, as well as in the
products which can be derived from trees.
There is no doubt that there is a way ahead if only we have a head for the
way.
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Microorganisms and Enzymes Involved in the
Degradation of Plant Fiber Cell Walls
Ramesh Chander Kuhad 1, Ajay Singh 2, and Karl-Erik L. Eriksson 3

Department of Microbiology, University of Delhi South Campus,
BenitoJuarez Road, New Delhi-ll0021, India
2 Department of Biology, University of Waterloo, Ontario, Canada N2L 3G1
3 Center for Biological Resource Recovery, Department of Biochemistry and
Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA
1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
1.1 Composition of W o o d and Other Plant Fibers . . . . . . . . . . . . . . . . . . . . . . 47
1.2 Structure and Composition of Wood and Other Plant Cell Walls . . . . . . . . . . . 48
1.2.1 The Cellulose C o m p o n e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
1.2.2 The Hemicellulose Components . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
1.2.3 The Lignin C o m p o n e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
1.2.4 Cell Wall Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
1.2.5 Other Cell Wall Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2 Microorganisms and Their Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.1 Microorganisms Involved in the Degradation of Lignocellulosic Materials . . . . . . 56
2.1.1 Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.1.2 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.2 Enzymes Involved in the Degradation of Plant Fiber Cell Wall C o m p o n e n t s . . . . 61
3 Degradation of Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.1 Microorganisms Producing Cellulose-Degrading Enzymes . . . . . . . . . . . . . . . 63
3.2 Cellulolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.2.1 Regulation of Cellulase Production . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.2.2 Molecular Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2.3 Cellulases are Organized in D o m a i n s . . . . . . . . . . . . . . . . . . . . . . . . 73
3.2.4 Catalytic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.3 Assay of Cellulolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.4 Possibilities for Biotechnology Based on Cellulolytic Enzymes . . . . . . . . . . . . . 80
4 Degradation of Hemicelluloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.1 Microorganisms Producing Hemicellulose-Degrading Enzymes . . . . . . . . . . . . 84
4.2 Hemicellulolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.2.1 Xylan-Degrading Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
94.2.2 M a n n a n - D e g r a d i n g Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.3 Assay of Hemicellulose-Degrading Enzymes . . . . . . . . . . . . . . . . . . . . . . . . 93
4.4 Possibilities for Biotechnology Based on Hemicellulolytic Enzymes . . . . . . . . . . 94
5 Degrad ation of Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5.1 Microorganisms Involved in Lignin D e g r a d a t i o n . . . . . . . . . . . . . . . . . . . . . 97
5.2 Ligninolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.2.1 Lignin Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.2.2 Manganese Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.2.3 Laccase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.2.4 HzOz-Producing Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.2.5 Oxidoreductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.3 Assay of Ligninolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5,4 Possibilities for Biotechnology Based on Ligninolytic Enzymes . . . . . . . . . . . . 108
6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Advancesin BiochemicalEngineering/
Biotechnology,Vol. 57
ManagingEditor: T. Scheper
9 Springer-VerlagBerlinHeidelberg1997
46 R.C. Kuhad et al.
One of natures most important biological processes is the degradation oflignocellulosic materials to
carbon dioxide, water and humic substances. This implies possibilities to use biotechnology in the
pulp and paper industry and consequently, the use of microorganisms and their enzymes to replace
or supplement chemical methods is gaining interest. This chapter describes the structure of wood
and the main wood components, cellulose, hemicelluloses and lignins. The enzyme and enzyme
mechanisms used by fungi and bacteria to modify and degrade these components are described in
detail. Techniques for how to assay for these enzyme activities are also described. The possibilities
for biotechnology in the pulp and paper industry and other fiber utilizing industries based on these
enzymes are discussed.
Microorganismsand EnzymesInvolvedin the Degradationof Plant Fiber Cell Walls 47
1 Background
Because of growing consumer demand for environmentally safe processes and

products faced with market and legislative pressures, the pulp and paper
industry is modifying its pulping, bleaching and effluent treatment technologies
to reduce the environmental impact. However, new approaches to pulp and
paper manufacture are rare because of technical and economical constraints [1].
Biotechnology has received increasing attention during the past two decades
because of its commercial potential in many fields. The virtue of biotechnology
lies in its potential for more specific reactions, to provide less environmentally
deleterious processes, to save energy, and to be used where non-biological
processes are impractical. The raw material in the forest product industries is
wood and its components. Thus, possibilities for employing biotechnology in
these industries must be numerous since one of nature's most important biolo-
gical processes is the degradation of lignocellulosic materials to carbon dioxide,
water, and humic substances. Consequently, the use of microorganisms and
their enzymes to replace or supplement older chemical methods in the pulp and
paper industry is gaining interest.
The accessibility of enzymes to wood and fibers is limited due to factors such
as adsorption to surface areas, low fiber porosity, and low median pore size of
fibers [1]. In addition, the molecular organization of the different components of
the plant fiber cell wall, i.e. cellulose, hemicellulose, and lignin, which are jointly
termed the lignocellulose complex, also limits the accessibility of microorgan-
isms and their enzymes to wood and its fiber components. Thus, the proper and
useful application of microorganisms and their enzymes in improving pulp and
paper production processes requires a more complete understanding of the
molecular architecture of the substrates, mechanisms of enzyme action, and
methods for the study of the specific degradation of each component of the fiber
cell wall.
This article reviews the composition of wood and other plant fibers, micro-
organisms, and their enzymes involved in the degradation of wood and its
components cellulose, hemicellulose, and lignin, techniques for the assay of
enzymes modifying and degrading these components, and the possibilities for
biotechnology based on these enzymes.
1.1 Composition of Wood and Other Plant Fibers
Lignocellulosics in the form of wood and agricultural residues are virtually

inexhaustible, since their production is based on the photosynthetic processes.
They account for more than 60% of total biomass produced. The net photosyn-
thetic production of the dry biomass by plants on earth has been estimated to be
155 billion tons per year [2]. Regardless of source, lignocellulosic materials
contain cellulose, hemicellulose and lignin as major components. Table 1 shows
Table 1. Composition of some wood and agricultural lignocellulosic residues
Residue % Dry weight
Hexosans Pentosans Lignin Ash
Hardwoods
Aspen 50 28 15 0.3
Beech 47 20 23 0.2
Birch 41 26 25 1.0
Cottonwood 46 19 24 0.6
Oak 48 18 28 0.4
Poplar 45 19 20 0.1
Red maple 39 33 23 1.0
Softwoods
Douglas fir 57 8 24 0.4
Eastern hemlock 43 10 32 0.4
Jack pine 41 10 27 0.1
White pine 44 11 28 0.1
Red spruce 43 12 27 0.2
White spruce 44 I0 27 0.3
Agricultural Residues
Bagasse 33 30 29 4
Barley straw 40 20 15 11
Corn cob 42 39 14 2
Cotton stalks 42 12 15 6
Groundnut shells 38 36 16 5
Oat straw 41 16 11 12
Rice straw 32 24 13 18
Rye straw 37 30 19 4
Wheat straw 30 24 18 10
the composition of several hardwoods, softwoods and agricultural residues. The

compositions of hardwoods and softwoods are significantly different. The lignin
content of softwood is generally higher than that of hardwoods, whereas the
hemicellulose content of hardwoods is higher than that of softwoods. With a few
exceptions, straw species are more uniform in composition than wood species.
Generally straws have lower cellulose content than wood, but, in spite of this,
the holocellulose (cellulose + hemicellulose) fraction is approximately equal to
that of wood.
1.2 Structure and Composition of Wood and Other Plant Cell Walls
W o o d is m a d e u p o f cells o r fibers. A m a t u r e tree s t e m h a s a n o u t e r m o s t l a y e r o f

b a r k ( p h l o e m ) s e p a r a t e d f r o m t h e w o o d (xylem) b y a t h i n layer, t h e c a m b i u m .
T h e c a m b i u m is t h e l a y e r w h e r e t r a c h e i d s ( w o o d fibers) a r e p r o d u c e d [3].
T h e fibers a r e p r o d u c e d in a n a q u e o u s e n v i r o n m e n t a n d exist in a l i v i n g t r e e in
a m a x i m a l l y s w o l l e n state. T h e g r o w i n g tree f o r m s l a r g e d i a m e t e r t h i n - w a l l e d
fibers in the early part of the growing season, small diameter thick-walled fibers
in the late growing season. Thin-walled early wood fibers are much more flexible
than the thick-walled late wood fibers.
Wood is a porous material consisting of a matrix of fiber walls and air space.
The air space exists mostly in the form of fiber cavities (lumens) and to a much
lesser extent as voids within fiber walls. The wood fiber wall has three major
constituents: cellulose, hemicelluloses, and lignin. Juvenile wood, heart-
wood/sapwood proportion, wood density, and fiber length are the wood quality
attributes important to the pulp and paper industry. Among these, fiber length is
of particular importance in pulp and paper quality.
Wood is classified in two major groups: softwoods, i.e., gymnosperms (pine,
spruce, larch, etc.) and hardwoods, i.e., angiosperms (birch, aspen, beech, maple,
oak, etc.). They differ considerably in cell type.
Softwoods have mainly two types of cells, the long (2-5 mm) tracheids, which
give strength to the wood and are responsible for vertical water transport, and
the smaller ray cells, which transport water in the horizontal direction. In
addition, there are resin channels in both horizontal and vertical directions [3].
Softwood tracheids are connected by bordered pits [-4].
Hardwoods possess more diverse types of cells. In general, the annual
growth ring of hardwoods is composed of an array of vessels, fibers, and ray
parenchyma cells in various arrangements. These cells provide for water con-
duction, strength support, and transport and storage of nutrients. The thick-
walled fiber tracheids and libriform fibers (fiber length 0.64-2.30mm) are
located around the vessels and are connected to other cells via bordered or
simple pits [4]. Vessels are thin and short (0.03-0.13 ram), but when located on
top of each other they form tubes up to several meters in length. These vessels
are more effective for water transport than the softwood tracheids [3].
Each tracheid cell is initially surrounded by a primary cell wall low in lignin
content. As growth progresses, a secondary cell wall, into which lignin is
successively incorporated, develops centripetally to the primary wall. The sec-
ondary wall constitutes the largest proportion of the total cell wall, and most of
cell wall lignin (60-80%) is located here [-5-9]. Lignification is always preceded
by deposition of cell wall polysaccharides, and polymerization of monolignols
occurs within the carbohydrate gel. However, the type of carbohydrate changes
during the course of cell wall development, i.e., the formation of cell wall layers
[10]. A model illustrating the arrangement of lignin, hemicellulose, and cellulose
within the cell wall has been proposed by Kerr and Goring [11]. Terashima and
Fukushima [10] have schematically pictured the deposition of cell wall compo-
nents and their irreversible assembly to form a lignified cell wall in tree xylem
(Fig. 1).
Every supporting cell (dead) has a lumen, which can be more or less empty or
filled with water. In all softwoods and many hardwoods, the inner or warty layer
is a heterogenous mixture of components of unknown composition. The second-
ary cell wall outside the warty layer is made up of three layers, $1, $2, and $3,
with $3 closest to the lumen, Sz the middle layer, and $1 the outermost layer.
Cambium Deposition of celt walt components , ,~Mature

9ML,CC-" ( Laps of time ) cell wall
w p --..
Formation of cell wall layers -S1--
w 52-"
~S3~
, Pectic substances
------ Arabinogalactan
Deposition of carbohydrates 1Xylan '
- - - MannQn
Ceilulo'~e
Llgnin i[Ageing
Deposition of [lgnin and , p-Coumary| alcohol
type of monolignol 1 Conlferyl alcohol .!!
~ G $1napyt alcohol 'I
/Gymnosperm { ;C H ~ ' ~ I ~ S ~ I~176 1
Monomer / Tracheid 7.,ML ~ ~ _ . _ ~ ~
composition | SW
. and . ~ Angiosperm H ~ ~ r :
condensation I Vessel { CCML H ~ , condensation I
Fig. 1. Schematic representation of the process of deposition of cell wall components and the middle
lamella in gymnosperms and angiosperms. M L middle lamella, CC cell corner, P primary wall,
C M L compound middle lamella, $1 outer, $2 middle, and $3 inner layer of secondary wall, H, G, and
S, p-hydoxy-, guaiacyl-, and syringyl-propane units [10]
These layers consist of cellulose microfibrils embedded in an amorphous mix-

ture of different hemicelluloses and lignin. The microfibrils form helices around
the cell with different angles and directions for the different layers. The Sz layer
is the thickest (1-5 gm) and the $3 layer the thinnest (0.1 gm). The width of
$1 layers varies from 0.2-0.3 gm [3]
It is important to note that cellulose, hemicelluloses, and lignin are present in
each layer of the cell wall. The concentration of cellulose is highest in sub-layer
$2 and decreases toward the middle lamella, where it is present only in small
amounts. The $3 layer is rich in hemicelluloses, while lignin is the dominant
compound in middle lamellae [12]. While lignin concentration is high in the
middle lamella, this layer is extremely thin, and most of the lignin is in the
secondary wall.
Straw is a much more heterogenous material than wood. However, cell wall
structures of straws have been less studied than wood fiber walls. Straw fibers,
principally derived from cells and internodes, are fairly long and slender with
sharply pointed ends. In addition to fibers, straw contains short nonfibrous
tissues consisting of epidermal cells, platelets, serrated cells, and spirals which
are derived from the pitch, nodes, chaffs, and rachises. While 96% of softwood
cells may be considered as fibers, only about 35-39% of the cells in straw are
fibers [12].
1.2.1 The Cellulose Component
Cellulose is considered to be a linear homopolymer of hydroglucose units linked

with [3-1,4-glucosidic units, although the true repeating stereochemical unit of
cellulose is cellobiose ([3-1,4-D-glucosyl-D-glucose) [13]. Glucose and cello-
dextrins are the products when cellulose is hydrolyzed. Some natural materials,
e.g., cotton, are almost pure cellulose. Cotton is mostly a-cellulose, the form of
cellulose insoluble in 17.5% NaOH. Wood and other plant celluloses generally
contain [3-cellulose as well, a material soluble in the above NaOH solution. The
and [3 forms of cellulose also differ in their intermolecular hydrogen bonding
patterns. The cellulose molecule is a polymer with a degree of polymerization
(DP) of up to about 15 000. However, DPs as high as 25 000 have been reported
for the algae Valonia.
The cellulose molecule is thread-like, existing as bundles of molecules form-
ing fibrils stabilized laterally by hydrogen bonds between hydroxyl groups of
adjacent chains. The arrangement of cellulose molecules in the fibrillar bundles
is so regular that cellulose has a crystalline X-ray diffraction pattern. The
individual cellulose chains are packed in groups of about 30 to form elementary
fibrils (microfibrils), approximately 100 of which are packed to form fibrils.
These fibrils are further packed to form the cellulose fiber [14]. The microfibril
has been considered to be stronger than steel of corresponding size and thus
significantly contributes to the strength of wood [15].
Cellulose is known to exist in four structures (cellulose I-IV) as revealed by
X-ray diffraction patterns [16, 17]. Cellulose I is the native form of cellulose in
the secondary cell wall. Cellulose I can be converted into types II and III by
chemical or physical treatment. The transition from cellulose I to cellulose II is
irreversible. Cellulose II may, however, be the thermodynamically most stable
form. Cellulose IV, the dominant structure in the primary cell wall, is believed to
have a form similar to but more disordered than that of cellulose I. Cellulose
I can be transformed to cellulose IV via cellulose III [-18-20]. In cellulose I the
chains are arranged partially in flat sheets. These sheets are thought to be kept
together by van der Waals forces, hydrophobic interactions, and interchain
hydrogen bonds.
The degree of crystallinity of cellulose depends very much on origin and
type of pretreatment. It may vary from 0% (amorphous and acid-swollen cellu-
lose) to approximately 100% (cellulose from Valonia macrophysa) [21]. The
degree of crystallinity of cotton cellulose is about 70%, while the figures
vary from 30 to 70% for most other commercial celluloses [22]. However, the
crystalline forms of cellulose are still not completely understood. Crystalline
cellulose is highly resistant to microbial attack and enzymatic hydrolysis,
whereas amorphous cellulose is degraded at a much faster rate [23]. As
its crystallinity increases, cellulose becomes increasingly resistant to further
hydrolysis.
1.2.2 The HemicelIulose Components
Hemicelluloses are easily hydrolyzed [24] short chains of branched hetero-

polysaccharides composed of both hexoses and pentoses. D-Xylose and L-ara-
binose are the major constituents of the pentosans (xylans), while D-glucose,
D-galactose, and D-mannose are the constituents of the hexosans (mannans).
The major hemicellulose components in softwood are mannan-based, and those
in hardwood xylan-based. The close association of the hemicelluloses with
cellulose and lignin in the fiber cell walls contributes both to rigidity and
flexibility. Hemicelluloses are composed both of neutral sugars, all present as
their respective anhydrides, i.e. xylan, araban, glucan, galactan, and mannan
(substituted with acetyl groups) and of uronic acids. Hemicelluloses constitute
on average about 26% of hardwood, 22% of softwood, and 30% of various
agricultural residues [25-28]. They usually have DPs of 100 to 200. Apart from
galactose-based hemicelluloses, which are characterized by 13-1,3-1inkages, most
of the hemicelluloses are built up by ~-l,4-1inkages between their backbone
sugars. Besides wood, hemicelluloses are also found in grasses, cereals, and some
very primitive plants [24]. The type and amount of hemicellulose varies widely,
depending on plant materials, type of tissues, growth stage, growth conditions,
storage, and method of extraction [29-31].
The mannan hemicelluloses, galactoglucomannans and glucomannans in
softwoods and hardwoods, are both branched heteropolysaccharides. At least
two galactoglucomannans, often termed "glucomannans" with different sugar
ratios are predominant in softwoods. The less soluble polysaccharide and the
major (water-soluble) polysaccharide, consist of galactose: glucose: mannose in
the ratio of 0.1 : 1 : 3 and 1 : 1 : 3, respectively. Their backbones are built up of
1,4-1inked [3-D-glucopyranose and 13-D-mannopyranose units, largely distrib-
uted at random. The mannose and glucose units in the backbone are partially
substituted in C-2 and C-3 positions by acetyl groups, approximately 1 per 3-4
hexose units [23, 32]. Depending on the wood species, the glucose: mannose
ratio has been reported to vary from 1 : 1 to 1 : 2. Galactose is not present in
hardwood mannans [23, 32].
The major class of hemicelluloses is xylans, which are found in large quan-
tities in annum plants and deciduous trees, and in smaller quantities in conifers.
Xylans in grasses and cereals are generally characterized by the presence of
L-arabinose linked as a single unit substituent to a D-xylose backbone. Substan-
tial differences in substituents are found in wood xylans. These xylans are
characterized by the presence of 4-O-methyl-D-glucuronic acid, L-arabinose, or
acetyl groups as substituents on the D-xylose backbone. In general, the content
of 4-O-methyl-D-glucuronic acid is higher in softwood than in hardwood.
Unbranched linear xylan homopolysaccharides are rare [33]. Xylans comprise
15-30% of annual plants, 20-25% of hardwoods, and 7 12% of softwoods.
Xylans appear to be a major interface between lignin and other carbohydrate
components in many isolated phenolic-carbohydrate complexes [-34-38], where
they are probably covalently linked to phenolic residues via the arabinosyl [39]
Microorganisms and EnzymesInvolvedin the Degradation of Plant Fiber Cell Walls 53
and/or glucuronosyl residues [-40]. Xylans tend to adsorb onto cellulose and to
aggregate with other hemicellulosic components, probably as a result of hydro-
gen-bonding interactions [41, 42]. Xylan may play a major role in cell wall
cohesion since its selective removal from delignified wood fiber results in
a substantial increases in fiber porosity [43]. There have been observations
which suggest that cellulose is protected from enzymatic attack by xylan and
mannan [44, 45].
Naturally occurring hemicelluloses differ from isolated hemicelluloses. Be-
sides impurities of other cell wall materials, isolated hemicelluloses are altered
during oxidative delignification, which may lead to reduction in the chain length
of the polysaccharides. The structure of xylan isolated from wood is dependent
upon the type of polymer originally present in the wood and also on the pH of
the cooking liquor used in pulping. In Kraft cooking, with its high pH, xylans or
arabinoxylans are found in good yield dependent upon whether the wood
contains 4-O-methylglucuronoxylan or arabino-4-O-methylglucuronoxylan.
The Kraft process begins with extreme alkaline conditions which causes losses
of hemicelluloses. Two-thirds of the glucomannans are dissolved very quickly
and degraded by alkaline peeling reactions [46]. The peeling reaction in xylans
is much slower than those occuring with cellulose or glucommannans because of
the unique sequence of sugar units at the reducing end of xylans [46].
1.2.3 The Lignin Component
Lignin is not a definite uniform compound, but is a collective form for substan-
ces that have similar chemical properties but very different molecular weights.
The molecular weight of lignins may be 100 kDa or greater [47]. A considerable
part of the photosynthetic activity in plants is devoted to the conversion of
atmospheric carbon dioxide to lignin. Lignin is found in the highest concentra-
tion in the middle lamella, but is most abundant in the secondary walls of
vascular plants [48]. It performs important functions in the life of plants as
a permanent bonding agent between cells, making a wood composite material,
and is a UV light stabilizer, antioxidant, and water-proofing agent. It also
protects plants, wood in particular, from attack by microorganisms. The water
permeation-reducing property of lignin plays an important role in the internal
transport of water, nutrients, and metabolites in the plant.
Lignins are highly branched polymeric molecules consisting of phenyl-
propane-based monomeric units linked together by different types of bonds,
including alkyl-aryl, alkyl-alkyl, and aryl-aryl ether bonds. The relative propor-
tions of three cinnamyl alcohol precursors incorporated into lignin, i.e.,
p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, vary not only with
the plant species but also with the plant tissues and location of the lignins within
the plant cell wall. Ecological factors such as age of the wood, climate, plant
sustenance, and amount of sunlight also affect the chemical structure of lignins.
A major problem in studying the chemistry of lignins has been the difficulty in
isolating intact lignins from plant materials. The hydrolyzable linkages in lignins
are suggested to be of two types: I]-aryl ether and 0~-aryl ether [49, 50]. The
predominant [3-aryl ether type bond is more resistant to cleavage. Under mild
hydrolytic conditions, the cleavage of the ether bond is exclusively restricted to
the a-aryl ether type [51, 52].
In terms of physical properties, lignins are amorphous polymers that have no
crystallinity. The amorphous nature of lignin has been studied using various
techniques such as broad-line nuclear magnetic resonance, differential scanning
calorimetry, viscoelasticity, and X-ray diffractometry [53]. The mode of polym-
erization during lignin biosynthesis makes it optically inactive. Lignins are
insoluble in water and difficult for microorganisms to penetrate and degrade.
They are generally acid stable but can be solubilized under alkaline conditions.
Lignins are closely associated to cellulose and hemicelluloses in plant cell walls,
and it has been shown that some hemicelluloses are linked by covalent bonds to
lignin [54]. Recently, Joseleau et al. [33] have discussed covalent bonding
between lignin and xylan. The best documented are ester linkages between
glucuronoxylans and lignin via benzyl ester bonds with the carboxyl group of
4-O-methylglucuronic acid [40, 55, 56]. This bond is likely to be established
between quinonemethide intermediates and D-glucuronic acid during the pol-
ymerization processes [57]. The second most reported covalent bonds between
xylans and lignins are ether linkages involving the L-arabinose side chains [58]
or xylose units [59, 60]. Lignin-xylan complexes have been isolated from
hardwoods, while from softwoods both lignin-mannan and lignin-xylan com-
plexes were obtained [54]. Several studies have suggested substituents of the
backbone of the hemicelluloses, such as arabinose, galactose and 4-O-methyl-
glucuronic acid, are the connecting links to lignin [61-63]. Possible links
between hemicelluloses and lignins are demonstrated in Fig. 2. Lignin isolates
from woody materials contain, beside carbohydrates, significant amounts of
protein [64].
Fig. 2. Schematic representation of connections

betweensoftwoodhemicellulosesand lignin [54]
1.2.4 Cell Wall Proteins
The plant cell wall contains, besides cellulose, hemicelluloses and lignins, struc-
tural proteins called extensins. Three kinds of cell wall proteins, which differ in
amino acid composition, have been found in plants [65]. These are hydroxypro-
line-rich glycoproteins, glycine-rich proteins and proline-rich proteins [65, 66],
all considered to be important structural components of plant cell walls. Re-
cently Bao and co-workers [67] found an extensin-like protein in mature wood
of loblolly pine (Pinus taeda L). These authors suggested that such structural
proteins play important roles in the differentiation of xylem, and could thereby
affect the properties of wood. However, the existence of lignin-protein com-
plexes suggests a role for proteases in the degradation of woody and other plant
materials, particularly in delignification processes such as pulp bleaching. Use of
such enzymes might be beneficial for the improvement of pulp and paper
manufacturing processes.
1.2.5 Other Cell Wall Components
Plant cell walls also contain extraneous materials, including extractives

and non-extractives. Depending upon species, wood contains from 0.4 to
8.3% extractives on a dry weight basis, whereas agricultural residues may
contain even greater amounts [68, 69]. The extractives can be broadly divided
into three groups, i.e., terpenes, resins, and phenols. The terpenes can be
regarded as isoprene polymers, with terpene alcohols and ketones as build-
ing blocks. The resins include a wide variety of non-volatile compounds, in-
eluding fatty acids, alcohols, resin acids, phytosterols, and some less known
neutral compounds. The most important phenols are tannins, heartwood
phenols, and related substances. In addition, low molecular weight carbo-
hydrates, alkaloids, gums, and various other cytoplasmic constituents are
present [70-73].
The non-extractives make up 0.2q18% of the dry weight and include
inorganic components such as silica, carbonates, oxalates, and non-cell wall
substances such as starch, pectin, and protein [69]. In agricultural residues, the
non-extractives make up about 10% of the dry weight. Silica, deposited as
crystals, is especially abundant in straw. In spite of the small quantity of
extraneous materials their role is significant in that they provide an obstacle to
the hydrolysis of cellulosic materials. Pectic substances (protopectin), though in
small proportions, occur notably in the middle lamellae of the parenchymatous
tissues, where they impart a structural function in binding and supporting
cells.
2 Microorganisms and their Enzymes
2.1 Microorganisms Involved in the Degradation o f Lignocellulosic

Materials
Wood and other lignocellulosic materials are degraded by a variety of fungi and
bacteria. The structural architecture and chemical composition of wood play
a significant role in its resistance to degradation by microorganisms.
2.1.1 Fungi
Most of the fungi able to produce the necessary enzymes for the degradation of
lignocellulosic materials belong to the Ascomycetes, Deuteromycetes, or
Basidiomycetes groups. Fungi living on dead wood that preferentially degrade
one or more of the wood components cause three types of wood rot, i.e., soft rot,
brown rot and white rot [23, 74].
Important fungi causing soft-rot include Chaetomium cellulolyticum, Asper-
gillus niger, Trichoderma viride (reesei) , Fusarium oxysporum, Thielavia terres-
tris, Penicillium jenthillenum, Dactylomyces crustaceous and different species of
Paecilomyces, Papulaspora, Monodictys, Allescheria, Hypoxylon, Xylaria, and
Graphium [23, 75, 76]. They all efficiently attack wood carbohydrates but
modify lignins only to a limited extent. A unique feature of soft-rot attack is the
production of chains of biconical and cylindrical cavities within the secondary
wall. This unusual microscopic pattern of decay in the $2 layer is characteristic
of the soft-rot type of deterioration. Two patterns of soft-rot attack have been
identified. One form of attack comprises cavities formed within the secondary
cell wall, and the second form causes an erosion of the entire secondary wall
originating from hyphae in cell lumina and progressing towards the middle
lamella [77, 78]. The rate and extent of decay by soft-rot fungi depend on the
type of wood they attack. In general, hardwoods are degraded to a greater
extent than softwoods [79]. The ability of fungi to cause soft-rot attack of
aromatic moieties of lignins has also been reported [79, 80]. However, they
mainly cause demethylation of lignin and degrade the side chains and aromatic
rings to a lesser extent.
Fungi causing the brown-rot type of decay include Poria placenta, Tyromy-
ces balsemeus, Gloeophyllum trabeum, Lentinus lepidius, Lenzites trabeam, Con-
iophora puteana, Laetiporus sulphureus and Fomitopsis pinicola [23, 76, 81, 82].
These also exhibit preference for cellulose and hemicelluloses, lignins being
degraded only to a limited extent. These fungi can cause rapid and extensive
degradation of cellulose early in the decay process [23, 83]. The hyphae of
brown-rot fungi are normally localized in the wood cell lumen and penetrate
adjacent cells either through existing openings or by producing boreholes in
wood cell walls. During the decay process, brown-rot fungi remove cell wall
substances first from the S 2 layer of the secondary wall, while the $3 layer,
adjacent to the lumen, remains virtually unchanged. The $1 layer may also be
attacked, but the primary wall and the middle lamella are very resistant because
of their high lignin content [84]. Although hyphae are in direct contact with the
S 3 layer, the ultrastructural changes are most obvious deep within the secondary
wall. For a long time, involvement of brown-rot fungi in lignin degradation
eluded clear demonstration. However, there is much evidence that brown-rot
fungi can cause substantial degradation of lignins in wood [23, 85, 86]. The
efficient demethylation of lignins or of simple lignin-related model compounds
by G. trabeum has been reported. This fungus was also found to degrade
[O14CH3]-labelled dimeric lignin model compounds in the presence of spruce
sapwood relatively soon after inoculation compared to the white-rot fungus
Phanerochaete chrysosporium [81].
In spite of the fact that brown-rot fungi primarily degrade wood carbohy-
drates, most of them are unable to degrade and utilize pure cellulose, parti-
cularly in submerged liquid cultures [87-89]. However, a few species, several of
which are important degrades of forest products, do degrade pure cellulose [90]
when in contact with inoculated pinewood blocks [87]. Lignified jute fibers were
also found to be easily degraded by brown-rot fungi, whereas delignified ones
were not [87]. Moreover, Highley [91] reported the degradation of cotton
cellulose in contact with wood. It seems that the degradation of cellulose by
brown-rot fungi is activated by the presence of lignin or lignin-like compounds
[91]. It may be dependent upon prior or concomitant removal of the hemicel-
lulose components [92, 93]. However, conclusive evidence does not yet exist
that lignin activates the cellulolytic system of brown-rot fungi.
Recently, the presence of spruce sapwood has been reported to greatly
stimulate the demethoxylation of labelled lignin by brown-rot fungi [81]. This
finding suggests that wood has some stimulatory effect on these fungi to utilize
cellulose or lignin. A great deal of research seems to be necessary to establish the
exact relationships.
Brown-rot fungi differ substantially from white-rot fungi with respect to the
cellulolytic enzymes produced and the pattern of cellulose degradation. The
involvement of oxidative systems in the depolymerization of cellulose during the
early stages of decay by brown rot has been demonstrated [88]. Considering
that most of the pore sizes in sound wood are too small to allow cellulolytic
enzymes to penetrate the wood, this might offer an advantage. A non-enzymatic
hypothesis involving the generation of oxygen-derived radical species has been
suggested to be involved in the initial attack on the cellulose polymer [94, 95].
However, this hypothesis has not been satisfactorily explored.
White-rot fungi are the only wood-rotting fungi which, to any extent, can
attack all the components of plant cell walls. The most studied fungi of this
group are Phanerochaete chrysosporium, Trametes versicolor, Dichomitus
squalens, Phlebia radiata, Heterobasidium annosum, Phellinus pini, C yathus ster-
coreus, Pleurotus ostreatus, Ceriporiopsis subvermispora, Polyporus anceps and
Ustulina vulgaris [23, 96, 97]. Most of the fungi belong to Basidiomycetes except
for Ustulina vulgaris, which is classified as an Ascomycete. The normal pattern

of wood decay by these fungi involves simultaneous attack on both polysacchar-
ides and lignin [23]. The ability of the white-rot group to efficiently remove
lignin from wood makes them suitable for industrial applications where lignin
or other phenolic compounds need to be modified or removed [23, 98]. Some
white-rot fungi such as P. chrysosporium and T. versicolor have an unselective
mode of wood degradation, i.e., they degrade cellulose, hemicellulose, and lignin
simultaneously, while others such as Phlebia tremellosa, C. subvermispora and
P. pini degrade the lignin component more selectively [99]. Some white-rot
fungi have been reported to attack both selectively and non-selectively in
different regions of the same piece of wood [100]. Several strains of P. chrysos-
porium and C. subvermispora degrade the different components of plant fiber cell
walls with a different mode of attack and to a different extent [98]. The factors
responsible for the different modes of degradation, i.e., a rather specific delignifi-
cation or a simultaneous decay of lignin and carbohydrates, remain unexplained
[23]. Dill and Kraepelin [101] suggested that environmental factors such as
nitrogen concentrations in wood may be responsible in governing white-rot
decay type. Also, white-rot fungi have a different production pattern with
respect to the extracellular lignin-degrading enzymes - a trait likely to affect the
degradation pattern [102].
White-rot fungi grow in all the different types of cells of hardwoods and
softwoods. Fungal hyphae enter the cell lumen, first colonize the ray paren-
chyma cells, and then penetrate from cell to cell either via pits or by the
development of boreholes directly through the cell walls [23, 84, 103]. Soon
after the easily metabolizable nutrients are depleted, cell wall degradation
starts. A separation between cells within or adjacent to the compound middle
lamella has been reported both at early [103] and late [104] stages of wood
decay.
White-rot fungi that simultaneously attack cell wall components erode
localized regions of cell wall layers, and these further extend through the
secondary cell wall and middle lamella. The extensive erosion of cell walls and
numerous holes in adjacent walls as later stages of decay have been observed in
white-rot fungi that selectively degrade lignin. Lignin degradation by fungal
hyphae in cell lumen progresses from the lumen edge of the secondary wall
towards the middle lamella. Several reports suggest that white-rot fungi degrade
lignin from the secondary wall before the middle lamellae between cells is
degraded [105, 106]. The separation of cell occurs as a result of degradation of
the middle lamella.
It is well established that white-rot fungi often cause a progressive thinning
of the cell walls, starting from the lumen and continuing towards the middle
lamella [103, 107]. P. chrysosporium, when growing on aspen wood chips
supplemented with nutrients, either produced boreholes or hyphae that entered
and advanced through cell walls using natural wood pits [107, 108]. P. chryso-
sporium was frequently found to progress along the axis of vessels or fibers, with
secreted enzymes etching the $3 and $2 layers [108, 109]. Likely, fungal decay
involves enzymatic softening and swelling of wood cell wall fibers as well as
thinning and fragmentation of the wood cell walls.
White-rot fungi produce an extracellular slime sheath around the hyphal
cells which establishes close contact between hyphae and wood cell walls
[110-112]. With the progress of the decay, the fibrillar slime materials become
intrinsically associated with the swollen and delignified wood cell wall and form
a connection between the fungal hyphae and wood substrates [113]. Several
suggestions have been put forward for the roles of the fungal slime sheath. For
example, studies have revealed the localization of lignin peroxidase and manga-
nese peroxidase to be associated with the slime during decay of wood [113].
A more detailed investigation concerning the role of fungal slime would possibly
contribute to the knowledge of mechanisms of wood attack by white-rot fungi.
Recently, Akin et al. [72] studied the chemical and structural modifications
of Bermuda grass (Cynodon dactylon) cell walls caused by the two white-rot
fungi C. stercoreus and C. subvermispora. Both fungi were observed to extensive-
ly colonize the cut end of the stem section, disrupt the parenchyam cell walls,
and partially degrade sclerenchyma cells. UV absorption microspec-
trophotometry indicated that ester-linked phenolic acids were totally removed
from the parenchyma cell walls, and these cells were readily and completely
degraded by both fungi. However, aromatic constituents were only partly
removed from the recalcitrant sclerenchyma cell walls. For more extensive
details about the various patterns of wood decay by white-rot fungi, readers are
referred to Eriksson et al. [-23].
In addition to aerobic fungi, anaerobic fungi, inhabitants of the alimentary
tract of herbivorous animals, play key roles in the degradation of plant cell wall
material. Anaerobic fungi degrade the major structural polysaccharides, but
cannot utilize the lignin moieties. Some anaerobic (rumen) fungi have the ability
to solubilize small amounts of phenolic compounds [114-118]. These fungi
colonize materials such as soybean hulls and grasses [119, 120]. Moreover,
highly recalcitrant plant materials like mestome sheath of leaf blades [121] and
palm press fibers and wood [-122] have been reported to be colonized by the
anaerobic fungi.
Among anaerobic fungi, the most studied are Neocallimastix frontalis,
N. Patriciarum, Piromyces (Piromonas) communis, and Caecomyces (Sphaeromonas)
communis [118, 123]. Electron microscopic studies have revealed that rumen
fungi preferentially attach to specific regions of plant particles, i.e., the cut ends
or stomata [124]. The fungal rhizoids readily invade the plant cell wall lumen
and the middle lamellae between individual plant cell walls [14, 118]. Ho and
co-workers [125] showed that some anaerobic fungi produced appressorial-like
structures "penetration pet" which penetrated undamaged cell walls of guinea
grass and rice straw. The fungal hyphae grow extensively and ramify throughout
the interior of plant cell walls, which causes physical disruption of the cell wall
[121]. Furthermore, Joblin [126] has also suggested the physical disruption of
the plant fibers by Caecomyces spp. The ability of anaerobic fungi to disrupt
plant tissues could well be attributed to their ability to produce an array of
extracellular enzymes, i.e., cellulases, hemicellulases, proteases, amylases, phen-

olic acid esterases, and pectinases [120]. For more details about rumen fungi,
the reader is referred to the excellent reviews by Weimer [,14], Wubah et al.
[118] and Trinci et al. [120].
2.1.2 Bacteria
Bacteria generally degrade wood slowly. Degradation takes place on wood

surfaces with a high moisture content. Because of lack of penetrating ability,
bacteria usually invade wood cells simultaneously with fungi. They appear to
attack both softwoods and hardwoods by first colonizing the parenchyma cells.
After utilizing the cell contents, they may also attack the parenchyma cell walls
[73]. They move into adjacent cells and trachieds with fast disruption of pits
[-127]. Although bacteria can directly attack fibers, vessels, and trachieds, few
species or strains can degrade all the cell wall components [-23]. However, some
bacteria have been found to degrade lignified wood cells, which was confirmed
by ultrastructural investigations [128]. Cell wall erosion [129, 130], tunnel
formation [131], and removal of lignin [132] by bacteria have also been
reported. Different patterns of cell wall decay, cavitation, and tunneling by
bacteria have been found both in natural and laboratory environments
[131,133, 134].
In contrast, studies in some laboratories have shown that bacteria are unable
to degrade lignified plant cell walls [135, 136], but they were able to do so after
chemical pretreatment of the cells. Efficient bacterial degradation of wood
already treated with cellulase-less mutants of P. chrysosporium and Phlebia
9igantea has been observed [137].
Rumen bacteria are major degraders of plant fiber cell walls by production
of enzymes active against structural components of these cell walls [138]. Some
of the most extensively studied rumen bacteria include Fibrobacter succinogenes,
Ruminococcus albus, and R. flavifaciens. These bacteria have a complete set of
polysaccharide-degrading enzymes and also the ability to adhere to fibers
[138, 139]. These species adhere strongly to partially degraded cell walls, but
erode the components only if they are adjacent and in direct contact with the
bacteria. Often, the plant cell walls are totally degraded, but at other times
digestion seems to be interrupted before the hydrolysis is completed.
Some actinomycetes, the filamentous eubactria, also actively degrade
lignocellulosic plant materials. Different species of Streptomyces have been
reported to colonize vessels, fibers, and parenchyma cells [140]. S. flavovirens
rapidly colonizes the phloem and degrades parenchyma cells as well as thick-
walled, highly lignified sclereids [129]. In advanced stages of degradation,
parenchyma cells were found to be completely destroyed, and sclereids showed
evidence of eroded cell walls. Various studies have established that several
actinomycetes attack grass lignocellulosics, leading to the partial solubilization
of the substrate rather than to its mineralization [141]. Several species of
Streptomyces have the ability to degrade and remove lignin from softwood,
hardwood, and graminaceous substrates [142, 143]. Streptomyces viridosporus
oxidatively depolymerizes lignin as it degrades cellulose and hemicellulose
components of plant residues [144]. Lignin degradation by actinomycetes is
reviewed by Zimmerman [128], and the varying abilities of different species of
Streptomyces to delignify lignocellulosics is discussed in detail.
2.2 Enzymes Involved in the Degradation of Plant Fiber Cell Wall

Components
Because of the complex nature of wood and other plant cell walls, a number of
different enzymes are required to degrade the wall components. The generally
accepted picture of enzymatic degradation of cellulose is that it proceeds by the
synergistic action of at least three major types of hydrolytic enzymes. In
addition, oxidative and phosphorolytic enzymes also participate in cellulose
degradation in some organisms. The hydrolytic enzymes (Fig. 3) involved in
degradation of native cellulose to glucose are: (1) endoglucanases (endo-l,4-[3-
D-glucan-4-glucanohydrolase, EC 3.2.1.4), which randomly attack the cellulose
chains and split 1,4-[~-glycosidic linkages, (2) exoglucanases, generally cel-
lobiohydrolases (exo-l,4-[3-D-glucan-4-cellobiohydrolase, EC 3.2.1.91), which
release either cellobiose or, in some cases, glucose from the non-reducing end of
cellulose, and (3) 1,4-[3-glucosidases (EC 3.2.1.21), which hydrolyze cellobiose
and other water-soluble cellodextrins to glucose [23]. Two types of oxidative
enzymes, cellobiose oxidase (CBO), now called cellobiose dehydrogenase (CDH)
(EC 1.1.9.18), and cellobiose: quinone oxidoreductase (CBQ) (EC 1.1.5.1), which
oxidize the reducing end group in cellobiose or higher cellodextrins in the
presence of a suitable electron acceptor, have been identified in many wood-
degrading fungi and may have a key role in cellulose degradation [23]. In
addition, lactonase (EC 3.1.1.17) has been found to operate synergistically with
cellulases in the degradation of cellulose [145]. Some aerobic and anaerobic
bacteria lack [~-glucosidases and produce cellobiose phosphorylase (EC 2.4.1.20),
which cleaves cellobiose but not cellotriose or higher cellodextrins [146, 147].
Because of the complex structure of hemiceUuloses, several different enzymes
are needed for their enzymatic degradation. The two main enzymes responsible
for the depolymerization of hemicellulose backbone are endo-l,4-[3-D-xylanase
(EC 3.2.1.8) and endo-l,4-13-D-mannanase (EC 3.2.1.78). While small oligosac-
charides are hydrolyzed by 1,4-]3-D-xylosidase, 1,4-[3-D-mannosidase, and 1,4-[3-
m-glucosidase, the side groups are split off by ~t-L-arabinosidase, a-D-
glucuronidase and cx-D-galactosidase. Esterified side groups are released by
acetylxylan esterase [23, 148-150].
Since the process of biodegradation of lignin by white-rot fungi is oxidative
in nature, the role of phenoloxidases in lignin degradation has been extensively
studied [23]. At least three phenoloxidases (Fig. 3) have been identified
as important in the ligninolytic enzyme system, i.e., laccase [151,152],
~__CH3iH MeOXi= HCOH+ H202

~ I Glucose~ Glu II Ox Araoino-2-hexulose
. . I CO
+2
'~ I .r }, I H20
. . . . ', ][ s.'- u2 H202 ~ ", II 9
~T--~ 0 r o ~ ~
~. I HO i ~ I
glyoxa, % ' "~ glyoxylic /' : ,/
-,~>o ~ -+'~o acid
~OH
,~," , 9
-~ UnP k~_ k.2J J VA , Low Mol. Wt.
[
. . . . . Laccase 02 ~H202
' mediator"" # Products
.c~ .-'+" LiP .-) 02. /
,.
o
HO O''" Jg~-@ + VA
k @ + Mn2+ rPhenoxy radicals]
- Lignin 9 / Cation radicals |
. ~'@ +mediator r - L Quinones J~,-.,~
" T "~ 02 H202+@l
.o H202.(~1
=E O2xQred y Medoxy Lignin O2+(~1
.~ Phenols
H20 Q0X J~"Medrsd WW'/"*'Ligninox @ hems ~'
o @ ~ (~ + domain )
E ~3+ H202 + Fe2+

/
Cellobiono-1,5-1actone
~ "OH H2o~I spont......
"'., Cellulose ( ~ (~) 9 Cellobiose ; or @ '4"-"
- I| Cellobionic acid
....... .| | | ....
,,-" @ :
,, Oligosaccharides ,, Glucose ( r ~ , " Gluconolactone
, ~i 02 H202 I~H20
.. | | ',
" ' Gluconic
s
,j ~. ~ H 2 ~ + (O) acid
c Hypha - "
l R chrysosporium ~ Hypha "-,L
Fig. 3. Extra- and intracellular enzyme mechanisms involved in degradation oflignin and cellulose
by the white-rot fungus Phanerochaete chrysosporium. Enzymes involved: (1) glyoxal oxidase, (2)
lignin peroxidase, (3) manganese peroxidase, (4) laccase, (5) cellobiose dehydrogenase, (6) cellobiose:
quinone oxidoreductase, (7) Protease, (8) Lactonase, (9) endo-l,4-~-glucanase, (10) exo-l,4-~-
glucanase, (11) 1,4-[~-glucosidase,(12) glucose-l-oxidase, (13) catalase. Meox methanol oxidase, Glu
II Ox glucose-2-oxidase, VA veratryl alcohol. Vanillic acid metabolism: VH vanillate hydroxylase,
Ph.Ox phenol oxidase, Q-red NAD(P)H: quinone oxidoreductase, Diox dioxygenase, M-red
maleylacetate reductase. Metabolic products from lignin degradation: A vanillin, B vanillic acid,
C methoxyhydroquinone (MHQ), D hypothetical ortho-quinone (II), E hydroxyquinol, F maleyl
acetate, G Jbketoadipate
Microorganisms and Enzymes Involved in the Degradation of Plant Fiber Cell Walls 63
lignin peroxidase (LIP), and manganese-dependent peroxidase (MnP)

[153-155]. Both peroxidases belong to the class of horseradish peroxidases (EC
1.11.1.7) and oxidize their substrate by two consecutive one-electron oxidation
steps with intermediate cation radical formation. LiP, because of its high redox
potential, can attack non-phenolic methoxyl-substituted lignin subunits, where-
as MnP acts exclusively as a phenol oxidase on phenolic substrates using Mn 2
and Mn 3 as an intermediate redox couple [156]. Laccase (EC 1.10.3.2), a true
phenoloxidase, has a broad substrate specificity. It oxidizes phenols and phen-
olic lignin substructures with formation of radicals. The enzyme can cause both
polymerization and depolymerization by production of phenoxy radicals [157].
However, laccase is generally considered to have a redox potential too low to
allow for oxidation of non-phenolic lignin structures.
Other enzymes of importance in lignin degradation are the H2Oz-producing
ones, (Fig. 3). Such enzymes are glucose-l-oxidase and glucose-2-oxidase
[158, 159]. Other oxidases producing HzO2 have been detected in P. chryso-
sporium, namely methanol oxidase and glyoxal oxidase [160, 161]. The oxida-
tive action of methanol oxidase yields formaldehyde. Glyoxal oxidase is produc-
ed extracellularly and uses several aldehydes and hydroxycarbonyl compounds
found as secondary metabolites in the fungal culture filtrate [161]. In addition,
the enzymes reducing phenoxy radicals may also be involved in lignin degrada-
tion processes [1]. Such enzymes are CDH, CBQ, and NAD(P) H: quinone
oxidoreductase [1,162].
The novel extracellular enzymes capable of releasing aromatics (feruloyl and
p-coumaroyl groups) from isolated grass cell walls and from their methyl esters
are feruloyl and p-coumaroyl esterases [163, 164]. An esterase which unlocks
p-coumaroyl groups from lignocellulose has been reported from Streptomyces
viridosporus T7A [165], while feruloyl esterase has been detected in cultures of
Schizophyllum commune [-166], F. succinogenes [167, 168], and Butyrivibrio
fibrisolvens [167]. Both feruloyl and p-coumaroyl esterases have been reported
to be produced by anaerobic fungi [163, 164]. The effective attack and penetra-
tion of lignified tissues, although without mineralization of lignin per se, by the
anaerobic fungi strongly suggest that phenolic acid esterases play an important
role in the biodegradation of realcitrant cell walls in grasses [169]. Use of these
enzymes, in combination with specific hemicellulolytic enzymes, may offer
a strategy to remove aromatics and specific sugars from lignocellulosics [170].
3 Degradation of Cellulose
3.1 Microorganisms Producing Cellulose-Degrading Enzymes
A large number of microorganisms have the ability to produce cellulose-degrad-

ing enzymes (Table 2). However, relatively few are capable of producing all the
Table 2. Important microorganisms with respect to cellulolytic potential
Soft-rot fungi Aerobic bacteria

Aspergillus niger Bacillus circulans
Chaetomium cellulolyticum Bacillus subtilis
F usariurn ox ysporum Cellulomonas fimi
Neurospora crassa Cellvibrio gilvus
Penicillium pinophilum Microbispora bispora
Trichoderma reesei Pseudomonas fluorescens
Brown-rot fungi Anaerobic bacteria
Coniophora puteana Acetovibrio cellulolyticus
Lanzites trabeum Clostridium cellulovorans
Poria placenta Clostridium thermocellum
Tyromyces palustris
White-rot fungi Rumen (anaerobic) bacteria
Phanerochaete chrysosporium Butyrivibrio fibrisolvens
Sporotrichum thermophile Fibrobacter succinogenes
Coriolus versicolor Ruminococcus albus
Rumen (anaerobic) fungi Actinomycetes
Caecomyces (Sphaeromonas) communis Streptomyces lividans
Neocallimastix frontalis rhermoactinomyces curvata
Neocallimastix patriciarum Thermomonospora fusca
Piromyces communis
necessary enzymes for degradation of crystalline cellulose [171]. While bacteria

degrade cellulose by cell-bound enzymes, fungi secrete most of these enzymes
into the surrounding growth medium [172, 173]. All organisms able to degrade
crystalline cellulose secrete more or less complex systems of cellulolytic enzymes
with different specificities and modes of action, which act in synergism to
hydrolyze or oxidize cellulose. Fungi are the most studied organisms with
respect to degradation of cellulose and production of cellulolytic enzymes.
Fungal cellulases are sometimes produced in very high concentrations in sub-
merged cultures and do not seem to form physical complexes with each other as
do bacterial cellulases. However, fungal cellulases often act in a strongly synerg-
istic manner [174-180].
Fungi causing soft-rot mainly degrade the polysaccharides. The ability to
produce all the necessary enzymes for degradation of cellulose varies among the
different organisms in this group. The best known of those producing a complete
set of cellulases is T. viride (reesei) [181, 182]. Trichoderma strains secrete large
amounts of different cellulolytic enzymes which, by their combined action,
degrade crystalline cellulose. T. reesei produces at least three endoglucanases,
two exoglucanases, and one or two 13-glucosidases [183, 184]. Other well known
fungi causing soft-rot are listed in Table 2.
Fungi causing brown-rot degrade cellulose rapidly, but the enzyme system
seems to operate differently from those of T. viride (reesei) and the white-rot
fungus P. chrysosporium. P. placenta, L. trabeum, and Tyromyces palustris are
brown-rot fungi among the most studied for their cellulolytic activities [23].
Brown-rot fungi have been suggested to employ a different mechanism of

cellulose degradation than that operating in soft-rot and white-rot fungi.
Brown-rot fungi depolymerize cellulose rapidly during the early decay of wood,
whereas white-rot fungi do so more slowly and utilize the degradation products
simultaneously [23,185]. Brown-rot fungi produce endoglucanases and
]3-glucosidases but no detectable levels of exoglucanases [23, 91]. In contrast to
these reports, C. puteana (a wide spread brown-rot fungus causing severe
damage to buildings) has recently been reported to secrete two exoglucanases
(cellobiohydrolases) when growing on pure cellulose [186, 187]. The presence of
an exoglucanase in a brown-rot fungus is certainly intriguing. These workers
have further reported that C. puteana gives a positive Bavendamm reaction
[188], a feature used to distinguish between brown-rot and white-rot fungi.
According to this definition, C. puteana is not a brown-rot fungus.
The induction and repression mechanisms of cellulases differ remarkably in
brown-rot and white-rot fungi. While the brown-rot fungi produce ample
amounts ofcellulases on glucose as carbon source [189], this is not the case with
white-rot fungi, at least not with P. chrysosporium, whose cellulase production is
induced by cellobiose and repressed even by low concentrations of glucose
[190].
Since brown-rot fungi apparently lack exoglucanases, there is no synergistic
cooperation between endo- and exoglucanases to degrade crystalline cellulose
[23, 91, 191]. No other enzyme systems are known to substitute for these effects
[23]. The mechanisms of cellulose degradation by brown-rot fungi remain
a mystery and need to be clarified. However, one hypothesis includes the
involvement of the Fenton reagent mechanism, i.e. H 2 0 2 and Fe 2 +, as critical
for predisposing cellulose to attack by cellulolytic enzymes during early degra-
dation [74, 192]. Contrary to earlier reports [191,192] on the accumulation of
H202 in culture media, Veness and Evans [193] could not detect extracellular
H202 in cultures of several brown-rot fungi. However, Fenton's reagent was
observed to give rise to acid production from cellulose. The same acids were
formed by a crude enzyme mixture from T. reesei. Recently brown-rot fungi
have been observed to secrete low molecular mass glycoproteins which chelate
ferrous ions [194]. It was proposed that these iron-containing glycoproteins are
small enough to penetrate the $2 and Ss layers of wood cell walls to catalyze the
oxidation of electron donors by molecular oxygen to produce oxygen species
such as 02" and H202. According to the hypothesis, all reactants required for
cell wall degradation by a Fenton-like reaction would be present simultaneously
in the Sz layer.
More recently, Backa et al. [195] have reported the generation of hydroxyl
radicals by brown-rot fungi grown in liquid media, agar media, or wood. Their
observations have indicated that hydroxyl radicals (HO.) contribute signifi-
cantly to the initial degradation of wood. The hydroxyl radical is one of the few
agents that can disrupt the crystalline structure of cellulose. In biological
systems, most hydroxyl radicals are produced by Fenton reactions [196]. Kirk
et al. [197] have reported that Fenton's reaction causes a cellulose degradation
similar to that resulting from brown-rot decay. Other non-enzymatic agents

such as siderophores [198] and oxalic acid [199] have been reported to be
involved during degradation of wood by brown-rot fungi. The mechanism by
which these agents work along with the enzymes in situ to degrade cellulose is
not known.
The brown-rot fungi form extensive hyphal sheaths, which appear to provide
a functional interface between the fungal hyphae and the wood [200-202].
Degradation of wood by brown-rot fungi has been reported to occur at a dis-
tance from the fungal hyphae and to be characterized by early removal of the
hemicellulose components. The following functions have been assigned to the
extracellular sheath: (1) modification of the extracellular ionic environment and
pH-level, (2) recognition of and adhesion to the substrate, (3) concentration,
storage, and transport of decay agents, (4) protection against dehydration and
other environmental impacts, (5) conditioning of the wood substrate to degrada-
tion, (6) storage of nutrients, (7) regulation of the decay process, (8) increase of
the surface area for aerobic respiration, and transportation and presentation of
wood-degrading enzymes during the degradation process [201,203-205]. Green
et al. [-205] have revealed the localization of enzymes on fibrillar elements of the
sheath structure, on the hyphal surface, and within the soluble sheath matrix.
The group of fungi causing white-rot is rather heterogenous, but these fungi
have in common the ability to degrade lignin as well as other lignocellulosic
components [206]. The most studied white-rot fungus was first isolated from
wood chip piles and was originally given the name Chrysosporium lignorum
[207]. The name was later changed to Sporotrichum pulverulentum for its
imperfect stage [208] and then to Phanerochaete chrysosporium for its perfect
stage [209]. The fungus grows optimally at 38-39 ~ and hence is characterized
as thermotolerant. At least five endoglucanases, three exoglucanases, and two
[3-glucosidases have been characterized in cultures of P. chrysosporium
[210-213].
The anaerobic rumen-inhabiting fungi secrete cellulases, often described as
a complex of enzymes which, by acting together, solubilize cellulose efficiently
[214]. An extracellular enzyme system from Neocallimastixfrontalis was found
to have a several-fold higher specific activity in solubilizing cotton fibers per unit
of endo-I~-glucanase activity than the endoglucanase of T. reesei mutant, Rut
C-30, known to produce a very active cellulolytic system [215]. The major end
product of the N.frontalis-cellulases, acting on Avicel cellulose, was glucose and
not cellobiose, which also indicates an efficient 13-glucosidase activity [216].
Wilson and Wood [217] showed that a minor component (4% of the total
extracellular protein) of the cellulolytic enzymes of N. frontalis is present in
a cellulosome-like structure which is secreted and adsorbed on to cellulose. In
this respect, N.frontalis resembles the bacterium Clostridium thermocellum. The
component which degrades crystalline cellulose contains several enzymes
arranged in a complex called the cellulosome, although the cellulosome of
N.frontalis (700 kDa) is smaller than that of C. thermocellum (2 106 kDa) and
also differs in other ways [120]. Recently, Wood and Wilson [218] reported on
Piromyces communis, another anaerobic rumen fungus capable of producing

a highly active cellulase system. This enzyme system was capable of solubilizing
highly crystalline cotton fibers at a rate greater than that of any other cell-free
cellulase system reported in the literature.
Cellulose is also degraded by both aerobic and anaerobic bacteria. However,
the cellulolytic enzyme systems of bacteria are not directly comparable to those
of fungi. Bacteria often produce cellulases in small amounts ( < 0.1 g/L), and
degradation of cellulose seems to take place by a cluster of multienzyme
complexes which are difficult to disrupt without loss of total activity as well as of
the individual components [219]. The most studied bacteria with respect to
their cellulase systems are species of Clostridium, Cellulomonas, Bacillus and
Pseudomonas. The cellulase complex of C. thermocellum cellulosome, consists of
several endoglucanases, an exoglucanase, a cellobiose phosphorylase that
breaks down cellobiose to glucose and glucose-l-phosphate, a cellodextrin
phosphorylase which phosphorolyzes 13-1,4-oligoglucans, and two 13-gluco-
sidases [220]. The cellulosome produced by C. thermocellum and other Clos-
tridia is a cellulose-binding and cellulase-containing complex, which is respon-
sible for most of the cellulase and hemicellulase activities observed in culture
broth of such bacteria [219]. During exponential growth on cellulose, the
cellulosome is anchored to the bacterial cell surface, and anchors the cells to
the insoluble substrate. When the cellulose is solubilized, the bound enzyme
clusters are released into the culture medium [221]. Other bacteria which may
produce cellulosome-like entities similar to those produced by C. thermocellum
include Acetovibrio cellulolyticus, Bacterioides cellulosolvens, Butyrivibrio
fibrisolvens, Cellulomonas sp., Clostridium cellobioparum, C. papyrosolven,
Fibriobacter succinogenes, Ruminococcus albus, R. flavefaciens, and Thermomono
spora curvata [222]. The extracellular cellulase systems of Pseudomonas fluor-
escens and Cellvibrio gilvus are also similar to those mentioned above, and
degradation products of enzymatic action are mainly cellobiose and cellotriose
[223].
Filamentous prokaryotes such as Actinomycetes are also important mem-
bers of the microbial community responsible for the degradation of cellulose.
Mesophilic species of Streptomyces and thermophilic species of Thermomono-
spora and Thermoactinomyces are the most studied of these organisms
[224, 225]. Their extracellular cellulases degrade cellulose by mechanisms sim-
ilar to those of fungi, i.e., non-associated enzymes excreted into the culture
medium.
3.2 Cellulolytic E n z y m e s
3.2.1 Regulation of Cellulase Production
The production of cellulases appears to be controlled by induction and repres-

sion mechanisms. In most microorganisms, cellulase biosynthesis is induced by
68 R.C. Kuhadet al.
cellulose degradation products [21]. The endoglucanases have been the most
studied in this respect. P. chrysosporium [190] and G. trabeum [226] have been
shown to form endoglucanases in media containing only carboxymethylcel-
lulose (CMC), while this was not the case with T. reesei. Cellobiose is an inducer
of endoglucanases in both P. Chrysosporium [190] and G. trabeum [226], but
does not induce any endoglucanase activity in T. reesei [190]. In contrast,
sophorose is the most potent cellulase inducer in T. reesei [227, 228]. However,
cellobiose has been shown to induce cellulases in T. reesei, but only when
cellobiose hydrolysis is artificially decreased by addition of nojirimycin [229].
Other cellulose degradation products such as cellobiono-l,5-1actone 1-230] or
oxidized cellulose [231] have also been demonstrated to enhance the formation
of cellulases in T. reesei.
Glucose is the end product of cellulose hydrolysis and causes catabolite
repression of endoglucanases in P. chrysosporium [190] and T. reesei [232]. In
contrast, the brown-rot fungi P. placenta and G. trabeum produce endog-
lucanases with glucose or mannose as the sole carbon source, even if the
expression of these enzymes was four to five times as high with cellulose or
cellobiose as carbon sources [88,226]. The addition of glucose to induced
cultures of G. trabeum to a concentration of 40 mM or higher does not repress
the production of endoglucanases [226].
It appears from various studies that the regulation of cellulase synthesis in
anaerobic fungi also involves both induction by cellulose and catabolite repres-
sion by glucose [233-237]. However, considering the low concentration of free
glucose in the rumen, it is unlikely that glucose regulates cellulase synthesis in
vivo [14].
Production of cellulases in fungi is also regulated by phenomena other than
induction and catabolite repression by cellulose degradation products. Various
phenols have thus been demonstrated to repress the production of cellulases and
xylanases in S. commune and C. 91obosum. In addition, phenols repressed endo-
glucanase in phenoloxidase-less mutants of P. chrysosporium, but caused no
significant repression in phenoloxidase-less revertants and in the wild type [147].
Moreover, cellulose activity in culture filtrates of fungi is dependent not only
on regulation of cellulase biosynthesis but also on the presence of specific
inhibitors in the culture. Gluconolactone is a powerful inhibitor of [3-glucosidase
in P. chrysosporium and T. reesei [-211,238]. Inhibition of ~-glucosidase activity
by gluconolactone or nojirimycin prevented induction by cellulose, but not by
sophorose [238]. However, sophorose fails to induce all components of the
cellulolytic system, indicating that other degradation products of cellulose might
contribute to regulation of this system [239]. The true inducer of cellulase
expression, in T. reesei as well as enzyme(s) responsible for its formation,
remains unexplained.
However, not only inhibition but also activation of cellulases seems to occur
in culture solutions of fungi. Thus, two acidic proteases produced under cellu-
lolytic conditions have been demonstrated to enhance endoglucanase activity
(Fig. 3) in P. chrysosporium up to tenfold [23].
Induction of cellulase biosynthesis by cellulose degradation products is

likely to occur in actinomycetes and bacteria as well. Addition of cellulases
exogenously to a cellulose culture medium of Thermomonosporafusca prompted
the induction of cellulases [240]. Cellobiose and sophorose have both been
shown to act as inducers of cellulase synthesis in Cellulomonas mutants insensi-
tive to catabolite repression [241]. Recently, Weimer [14] has discussed the
regulation of the cellulase systems in rumen bacteria. The complete cellulase
complexes in these bacteria do not seem to be highly regulated by the carbo-
hydrate concentration. However, the regulation of individual proteins must not
be ignored. The cellulase synthesis does not appear to be induced by cellulose
and cellobiose, and glucose neither represses enzyme synthesis nor inhibits
enzyme activity, except at concentrations well above those encountered in vivo
[242, 243].
3.2.2 Molecular Properties
Most of the fungal and some of the bacterial cellulases are glycoproteins with
sugars attached to asparagine (N-linked) or serine and threonine (O-linked)
residues. The carbohydrate content of cellulases varies from 1 to about 10%,
and the principal sugar is mannose. However, other sugars such as glucose,
galactose, xylose, N-acetyl glucosamine, and galactosamine have also been
detected [244]. Nitrogen-linked glycosylation appears to impart a specific
conformation and stabilize the structure of the cellulases, thereby protecting
them from proteolytic attack during secretion while O-linked glycosylation
seems to be required for secretion of an active cellulase [-245].
Endoglucanases hydrolyze ]3-1,4-glucosidic bonds in a random fashion over
a cellulose chain. As a result, there is a rapid decrease in chain length and a slow
increase in reducing end groups. Three different endoglucanases, i.e EG I, EG II,
and EGIII are produced by T. reesei [239, 246]. The major EG component, EG
I, represents approximately 5-10% of the totally secreted proteins in T. reesei
cultures [247]. In spite of clear differences in their mechanism of actions, the
N-terminal sequences indicate considerable homology between EG I and CBH
I [-248]. Multiple forms of EG III have been purified exhibiting Mr values of 48,
48 and 37 kDa with pIs corresponding to 5.4, 5.7, and 4.8 [-184]. A number of
other endoglucanases have also been isolated from T. reesei cultures which
could not be identified as either EG I or E G I I I [249].
Cellobiohydrolases (exoglucanase, EC 3.2.1.91) degrade cellulose by splitting
off cellobiose from the non-reducing end of the chain. These enzymes have very
limited action on substituted cellulose such as carboxymethyl cellulose (CMC)
and hydroxyethyl cellulose (HEC). Heterogeneities of the cellobiohydrolase
components from T. reesei have been studied in detail, and two immunolo-
gically distinct cellobiohydrolases, CBH I and CBH II, have been detected in the
extracellular medium of T. reesei using polyclonal antibodies [-250]. Both are
glycoproteins differing in the amount of carbohydrates attached to the protein.
They lack any apparent homology in their amino acid sequences. The differ-
ences in the active centers of these enzymes are reflected by differences in their
mode of action. While CBH I preferentially binds to crystalline regions, CBH II
binds to both crystalline and amorphous regions [251]. CBH I comprises
the major part (ca. 60%) of the cellulolytic enzymes synthesized by T. ressei
[252].
~-Glucosidases, secreted by cellulolytic organisms, represent a small portion
of the total extracellular proteins. They catalyze the hydrolysis of water-soluble
cellodextrins as well as alkyl- and aryl-13-D-glucosides. Intracellular and plasma
membrane-bound 13-glucosidases have also been identified. However, the exact
genetic and biochemical relationships between these different [~-glucosidases are
not yet clearly understood. It has been suggested that extracellular enzymes may
result from the release of intracellular or membrane-bound enzymes to the
outside medium upon autolysis [253]. Species of Aspergillus and Phanerochaete
seem to produce 13-glucosidases of higher molecular weights than does T. reesei
[23]. From Sporotrichum thermophile, two distinctly different [3-glucosidases
were isolated [254]. One with a Mr of 440 kDa had only aryl-[3-glucosidases
activity, while the other, with a Mr of 40 kDa, had cellobiase activity and only
low activity toward aryl-[3-glucosides. Purified [3-glucosidase from C. 9ilvus
attacks cellobiose slowly as compared to its activity towards higher oligo-
saccharides [255].
Cellobiose dehydrogenase (CDH), previously termed cellobiose oxidase
(CBO) [256-258], is produced by several cellulolytic fungi (soft-rots, white-rots,
and one brown-rot, i.e., C. puteana). Moreover, recently, CDH production from
a cellulolytic bacterium Cyotophaga sp. LX-7 has also been reported [259].
CDH carries both FAD and heme as prosthetic groups. Various investigations
have revealed that a proteolytic cleavage product of CDH, previously known as
cellobiose: quinone oxidoreductase (CBQ) [260], represents the flavin-contain-
ing domain of CDH [261-263]. Both CDH and CBQ, in the presence of an
appropriate electron acceptor, oxidize the reducing end groups of cellobiose,
higher cellodextrins, and even cellulose to the respective onic acids via the
corresponding lactone [257, 258]. Both CDH and CBQ are known to utilize
quinones and their analogs, such as 2,6-dichlorophenol-indophenol (DCPIP), as
electron acceptors. Cytochrome c acts as an electron acceptor for CDH but not
for CBQ [264, 265, 257]. This ability of CDH is used to differentiate between
CDH and CBQ. The total activities of both CDH and CBQ can be determined
using either ferricyanide DCPIP or other reducible quinones as electron
acceptors.
CDH is a monomeric protein in which the heme group has been shown to be
of the cytochrome b type [266]. The enzyme reduces cytochrome c about 200
times as fast as it reduces oxygen [264], whereas Fe(III) compounds such as
Fe(III) acetate and Fe(CN)~ are reduced 35-50 times as fast as molecular
oxygen [267]. CDH could be split into the FAD and the heme domains by
papain [261] and by proteases from P. chrysosporium [263], although, in the
latter case, only when the CDH was bound to cellulose. Only the FAD domain
was reduced by the addition of cellobiose [261]. This indicates that the FAD
domain is directly responsible for the oxidation of cellobiose and that the
electrons must then be transferred from the reduced FAD to an appropriate
acceptor, i.e the heme domain, quinone, Fe(III), or even molecular oxygen.
Small-angle X-ray scattering studies have suggested that CDH as well as its
fragments appear to be of prolate shape, and that the cross-section of the FAD
(4.3-5.1 nm) is considerably larger than that of the heine fragment (3.3 nm)
[268]. The observations further suggested a co-linear arrangement of the two
domains of CDH. Thus, according to their model, CDH appears very much to
be a linear particle. However, the possibility that CDH may have a bent shape
cannot be ruled out.
Based on the research conducted with these oxidative enzymes, some pos-
sible physiological roles of these in cellulose and lignin degradation have been
suggested [257]: (1) oxidation of cellulose and introduction of carboxyl groups,
resulting in a distortion of the crystalline structure of cellulose, (2) conversion of
cellobiose, which inhibits hydrolytic enzymes, into cellobionic acid, (3) oxidation
of reducing end groups in cellulose to prevent reformation of glycosidic bonds
broken by cellulases, (4) direct energy uptake by coupling with electron transfer
chains on the fungal cell wall, (5) generation of active species (superoxide anion
radicals) to disrupt the crystalline structure of cellulose, thus facilitating hy-
drolysis of cellulose, and (6) reduction of phenoxy radicals to prevent repolymer-
ization of lignin degradation products.
Interestingly, CDH has also been demonstrated to act as a Fenton's reagent
to generate hydroxyl ('OH) radicals (Fig. 3) in the presence of Fe(III) and H202
[267, 269]. The hydroxyl radicals are strong enough oxidants to attack not only
cellulose but also other plant cell wall components in a non-specific manner
[162]. However, a physiological operation of such a mechanism requires some
special extracellular compartmentation arrangements since the production of
these radicals could also be harmful to the fungus [270].
Bacterial cellulases seem to be quite a complex mixture. In Cellulomonasfimi
cultures, up to 10 components with CMCase (endoglucanase) activity have been
identified [271]. The glycosylated components bind strongly to Avicel and are
stabilized by this substrate, whereas enzymes free in solution are unstable and
are altered by proteolysis and deglycosylation [272]. The endoglucanase of B.
subtilis exhibits almost twice the specific activity of the T. reesei endoglucanase
[273]. Recently, a bifunctional cellulase was isolated from Bacillus sp. D 04
[274]. The enzyme was a single polypeptide (Mr 35 kDa) with both endo- and
exoglucanase activities, each activity existing at a separate site. One cell-bound
and two extracellular (A and B) endoglucanase components of P.fluorescens var.
cellulosa have been identified [275]. All three of these enzymes hydrolyzed
a variety of substrates, and the mode of action toward CMC substrates appears
to be similar to that shown by fungal endoglucanases. The major endoglucanase
( > 80% of total endoglucanase activity) of T. curvata exhibits its highest
activity on CMC with a high degree of polymerization [276]. Glutamic and
aspartic acids constitute 24% of the total amino acid composition.
Bacterial cellulase systems differ from fungal cellulase systems by forming

aggregates of multienzymes (cellulosomes) rather than existing as individual
extracellular enzymes [222]. Cellulosomes are known to be produced by several
anaerobic cellulolytic bacteria, Clostridia in particular. The cellulase system of
C. thermocellum is very active against crystalline cellulose, with a specific activity
higher than that observed for the T. reesei cellulase system [277]. The cellulo-
somes have molecular masses ranging from 2 x 106 to 6.5 x 1 0 6 Da, diameters of
about 18 nm, and contain 14 to 26 different polypeptides ranging in size from 37
to 210 kDa [219, 221,278]. Cellulosomes form larger complexes, polycellulo-
somes, with masses from 50 x 1 0 6 to 80 1 0 6 Da [279]. Protuberances covering
the surface of the bacterial cells are packed with polycellulosomes, each of which
seems to contain several hundred cellulosomes [280].
The cellulosome efficiently hydrolyzes both amorphous and crystalline cellu-
lose, whereas individual peptides alone or as a mixture do not [281]. Many of
the polypeptides in the cellulosome complex are catalytically active and can be
characterized as endoglucanases, xylanases, and cellodextrinases. The occur-
rence of cellobiohydrolase in cellulosomes has been suggested [282, 283],
but its presence still needs reconfirmation [281]. In addition, 13-glucosidase,
[3-xylosidase, J3-galactosidase, and [3-mannosidase activities have also been
observed in cellulosomes [284].
Several polypeptides in cellulosomes have been sequenced, including the
largest (210-250 kDa), a cellulosome-integrating protein previously termed S1
or SL and now designated CelL or CipA [285]. CipA may act as a cellulose-
binding factor and/or a scaffolding protein anchoring the various catalytic
subunits of the cellulosome, which, in addition, binds to the cellulosic substrate
and also anchors the complex to the bacterial cell surface [281,286]. Another
important subunit from the cellulosome has been identified as CelS (82 kDa),
termed the catalytic unit [286]. CipA and CelS are thus two of the components
of the cellulosome of C. thermocellum [287]. An "anchor-enzyme model"
proposed to explain the cooperative action between these two subunits [-288]
was confirmed by DNA sequencing of the genes encoding these two proteins
[289]. Based on these observations, the model was recently expanded into
a more sophisticated one [290]. It is now evident that CipA provides the anchor
for the other catalytic components and that CelS provides the cellobiohydralase
or exoglucanase activity necessary for crystalline cellulose degradation. For
details about the mechanism of cellulose degradation according to the an-
chor-enzyme model, readers are referred to Wu [-290] and Wang et al.
[286]. Also, an excellent review on the cellulosome of the anaerobic ther-
mophilic C. thermocellum has recently been published by Felix and Ljungdahl
[281].
The cellulosomes in other Clostridia such as C. cellulovorans, C. cellu-
lolyticum and C. jsui appear to have properties very similar to those of the
C. thermocellum cellulosome [291]. The cellulosome of C. cellulovorans consists
of three major subunits called CbpA, P100, and P70 and of at least six different
enzyme subunits [292].
Some aerobic and anaerobic bacteria employ phosphorylases for cellulose

degradation [-147]. Cellobiose and higher cellodextrins are metabolized by
C. 9ilvus, and the cell yield per glucose equivalent increases with the degree
of polymerization (DP) of the cellodextrins. This suggests that cleavage
of glucosidic bonds is brought about by phosphorylation and thus conservation
of energy of these bonds [23]. The physiological role of cellobiose phos-
phorylase (EC 2.4.1.20) is to convert cellobiose into glucose-l-phosphate, which
is utilized more efficiently than glucose by these microorganisms. The presence
of cellobiose phosphorylase was first demonstrated in C. thermocellum 1-293].
Two types of enzymes have been identified, namely, those specific for cellobiose
and those utilizing higher cellodextrins for the formation of a-glucosyl phos-
phate [146, 294]. The major products of enzymatic action are glucose-l-phos-
phate and glucose. The purified cellobiose phosphorylase from C. 9ilvus was
found to consist of four subunits and to have specificity for cellobiose, requiring
Pi and Mg 2+ for phosphorylation [146].
3.2.3 CelluIases are Organized in Domains
Proteolytic cleavage of cellulases into separate catalytic and cellulose-binding

regions demonstrated the presence of these two domains within these enzymes
1-295]. On the basis of existing amino acid sequence identities in their putative
catalytic domains, fungal and bacterial cellulases have been classified into
families of homologous proteins. This classification is confirmed by hydropho-
bic cluster analysis [296, 297]. For details of classification based on catalytic
domains, their occurrence and properties, readers are referred to the excellent
reviews available on this aspect [298, 299]. In brief, family A includes cellulases
from a diverse range of organisms including Gram-negative and Gram-positive
bacteria, aerobic and anaerobic bacteria, and the fungus T. reesei (Table 3).
Besides typical patterns of hydrophobic amino acids, some of the conserved
regions contain residues that are identical in most of the sequences. In family B,
three clearly related endoglucanases from C. fimi, Streptomyces sp. KSM-9 and
Microbispora bispora, and CBH II from T. reesei sharing significant similarity
are included (Table 3). Family C comprises three cellulases of fungal origin,
exhibiting at least 45% similarity (to each other). Among three bacterial cellu-
lases in family D, Erwinia ehrysenthemi EGY and C. uda EG are more closely
related. Families E, F, and G are also of bacterial origin, with most of the
enzymes from F and G having xylanase activity. Out of nine families, A, B, F,
and H contain both fungal and bacterial enzymes. Family C contains only
fungal enzymes, and families D, G and I contain only bacterial enzymes. Family
E contains both bacterial and plant enzymes (Table 3).
Cellulose-binding domains (CBDs) form distinct functional units for most
cellulolytic enzymes. Although these CBDs are not essential for catalytic activ-
ity, they do modulate the specific activities of the enzymes on soluble and
insoluble cellulosic substrates. Table 4 summarizes the different CBDs identified
Table 3. Cellulase and xylanase families of homologous catalytic domains

Family organism Enzyme Terminus Reference
A Bacillus sp. 1139 EG N [637]
Bacillus sp. N-4 EG A N [638]
Bacillus sp. N-4 EG B N [638]
Bacillus sp. N-4 EG C N [639]
Bacillus polymyxa EG [640]
Bacteroides ruminicola EG N [641]
Butyrivibriofibrisolvens END 1 N [642]
Caldocellum saccharolyticum EG B C [643]
Clostridium acetobutylicum EG 1 N [644]
Clostridium cellulolyticum EG A N [627]
Clostridium thermocellum Cel B N [645]
Clostridium thermocellum Cel C [646]
Clostridium thermocellum Cel E N [647]
Clostridium thermoceUum Cel H C [648]
Erwinia chrysenthemi EG Z N [628]
Fibrobacter succinogenes EG 3 C [649]
Ruminococcus albus SY 3 CEL A [650]
Ruminococcus albus SY 3 CEL B [650]
Trichoderma reesei EG III C [635]
Xanthomonas campestris EngXCA N [651]
B Cellulomonas fimi Cen A C [623]
Microbispora bispora Cel A N [629]
Streptomyces sp. KSM-9 Cas A [652]
Trichoderma reesei CBH II C [633]
C Humicola grisea CBH I [653]
Neurospora crassa CBH I N [636]
Phanerochaete chrysosporium CBH I N [631]
Trichoderma reesei CBH I N [632]
Trichoderma reesei EGI N [634]
Trichoderma viride CBH N [654]
D Bacillus circulans Bgc [655]
Cellulomonas uda EG [656]
Clostridium thermocellum Cel A N [657]
Erwinia chrysenthemi EG Y [658]
E Butyrivibriofibrisolvens CED I [622]
Cellulomonasfimi Cen B N [624]
Cellulomonas fimi Cen C Internal [625]
Clostridium thermocellum Cel D N [659]
Clostridium stercorium Cel Z N [660]
Dictyostelium discoideum SGSP27(~6 N [661]
Persea americana EG [662]
Persea americana Cel 1 [663]
Persea americana Cel 2 [663]
Pseudomonas fluorescens Cel A N [630]
Pseudomonasfluorescens Cel B C [303]
Pseudomonas fluorescens Cel E N [304]
F Bacillus sp. C-125 Xyn A [664]
Butyrivibrio fibrisolvens Xyn A N [665]
Caldocellum saccharolyticum Cel B N [643]
Caldocellum saccharolyticum Xyn A [666]
Caldocellum saccharolyticum ORF 4 [666]
Cellulomonas fimi Cex N [626]
Table 3. (Continued)
Family organism Enzyme Terminus Reference

Clostridium therrnocellum Xyn Z C [667]
Cryptococcus albidus Xyn [668]
Pseudomonasfluorescens Xyn A C [669]
Pseudomonasfluorescens Xyn B C [670]
Thermoascus aurantiacus Xyn [671]
G Bacillus circulans Xyn [672]
Bacillus pumilus Xyn A [673]
Bacillus subtilis Xyn [674]
Clostridium acetobutylicum Xyn B [675]
H AspergiUus aculeatus EG [676]
Erwinia carotovora Cel S [310]
I Ruminococcus albus Cel A [311]
Table 4. Cellulose binding domains in bacterial and fungal cellulases
Organism Enzyme Terminus Reference

Bacteria
Butyrivibrio fibrosolvens EG C [622]
Cellulomonas fimi Cen A N [623]
Cellulomonasfimi Cen B C [624]
Cellulomonas fimi Cen C N [625]
Cellulomonas fimi Cex C [626]
Clostridium cellulolyticum EG A C [627]
Erwinia chrysenthemi EG Z C [628]
Microbispora bispora Cel A C [629]
Pseudomonasfluorescens vat. cellulosa Cel A C [630]
Pseudomonasfluorescens var. cellulosa Cel B N [303]
Pseudomonasfluorescens var. cellulosa Cel E C [304]
Fungi
Neurospora crassa CBH I C [636]
Phanerochaete chrysosporium CBH I C [631]
Trichoderma reesei CBH I C [632]
Trichoderma reesei CBH II N [633]
Trichoderma reesei EG I C [634]
Trichoderma reesei EG II N [635]
in b a c t e r i a l a n d fungal cellulases. Sequence a n a l y s e s of four cellulases f r o m T.

reesei reveal the presence of a s h o r t c o n s e r v e d r e g i o n (AB region) at either
N - t e r m i n a l ( C B H I a n d E G III) o r C - t e r m i n a l ( C B H I a n d E G I) of these
enzymes [-300]. T h e r e m o v a l of the A B region f r o m C B H s h a s been s h o w n to
decrease significantly their affinity to m i c r o c r y s t a l l i n e cellulose. C B D of E G
I has significantly h i g h e r c e l l u l o s e - b i n d i n g affinity t h a n t h a t of C B H I [301].
M o s t of the differences are a s c r i b e d to a r e p l a c e m e n t of a t y r o s i n e b y a
t r y p t o p h a n in tile c e l l u l o s e - b i n d i n g d o m a i n .
76 R.C. Kuhadet aL
The most important features of bacterial CBD sequences are: low contents of
charged amino acids, high contents of hydroxy amino acids, and conserved
tryptophan, asparagine, and glycine residues. Din et al. [302] have demon-
strated that the isolated CBD of CenA (endoglucanase) from C. fimi, while
having no detectable hydrolytic activity, disrupts the structure of cellulose fibers
and releases small cellulolytic particles. The CBD of an endoglucanase (CelB)
from P. fluorescens var. cellulosa binds the enzyme to cellulose as does CBD of
CenA from C. fimi [303]. Recently, a novel endoglucanase (CelE), containing
N-terminal catalytic and C-terminal cellulose-binding domains, was character-
ized by the same group of scientists [304]. A truncated form of the enzyme,
which lacked the CBD, displayed the same activity of a full-length CelE against
soluble cellulose and acid-swollen cellulose, but substantially lower activity than
the full-length enzyme against Avicel, a microcrystalline type of cellulose.
The cellulosome of C. thermocelIum binds strongly to cellulose, and this
binding may be mediated by a non-catalytic component of the cellulosome
[-278]. A CBD has been reported to be present in a cellulosome-integrating
protein A (CipA) [305]. The cellulose-binding protein (CbpA) has been shown to
mediate the interaction between crystalline cellulose substrates and the cellulase
enzyme complex of Clostridium cellulovorans [-306]. Mutation analysis revealed
that the entire 163-amino acid region of CBD was required for the maximal
binding to crystalline cellulose [307].
The cellulose binding of CDH from P. chrysosporium was discovered inde-
pendently in two different laboratories [,261,308]. The binding was found to be
comparable with that of T. reesei CBH I and to be associated with the FAD
domain and probably independent of the catalytic site. A comparison of the
binding isotherms of CDH and CBH I has shown that the dissociation constant
of CDH is much lower than that of CBH I. The capacity (the amount of enzyme
bound per unit mass of cellulose) is also lower, which indicates that CDH binds
more sparsely than does CBH I [-309]. Recently, cDNA encoding CDH from
P. chrysosporium has been cloned and characterized [310, 311]. The low se-
quence similarity with the conserved cellulose binding sequences of cellulases
suggested that CDH might possess a specific sequence for cellulose binding
which is different from that of cellulases [311].
3.2.4 Catalytic Properties
It is now a well-established fact that degradation of crystalline cellulose is

carried out by a multicomponent enzyme system wherein the individual compo-
nents interact in a synergistic way to convert the insoluble substrate glucose
[23, 180]. The synergistic action of cellulases depends on the ratio of individual
enzymes, the degree of substrate saturation, and the type of substrate [-312]. The
action of CBH I and CBH II and the EG I and EG III, purified from T. reesei,
has been evaluated against different substrates [313]. Synergism between the
EGs and CBH II follows the normal pattern for endo/exo type synergism,
whereas the synergism between the EGs and CBH I depends on the structural
and ultrastructural features of the substrate. The activity of individual enzymes
from T. reesei is greatest toward amorphous cellulose, whereas binary combina-
tions CBH I/EG III and CBH I/CBH II exhibit a greater degree of synergism
toward crystalline cellulose [314-3163. The synergistic action of two immunolo-
gically distinct cellobiohydrolases (I and II) from Penicillium pinophilum was at
its maximum on Avicel when the two enzymes were mixed in the ratio 1 : 1 [317].
It was suggested that these may be two stereospecific enzymes concerned with
the hydrolysis of two different configurations of non-reducing end groups, one
being exposed on the cellulose surface and the other being buried as shown by
Henrissat et al. [318]. While only CBH II of P. pinophilum acts synergistically
with cellobiohydrolases of T. koningii and Fusarium solani, only CBH I shows
synergistic action with the endoglucanases of T. koningii or F. solani [-317].
About 45 years ago, Reese et al. [319] postulated that microbial conversion
of native cellulose to soluble sugars involved two enzymes that acted consecut-
ively. The model proposed that an unidentified enzyme called C1 acted first,
making the substrate more accessible to a hydrolytic enzyme called Cx. How-
ever, the precise action of C1 was not clear, and a strictly nonhydrolytic C~ has
never been identified. Intramolecular synergism was recently shown in endog-
lucanase CenA from C. fimi, which is composed of a catalytic and a non-
hydrolytic CBD that can function independently [320]. The individual domains
interact synergistically in the disruption and hydrolysis of cellulose fibers. It was
suggested that the catalytic domain corresponds to the hydrolytic Cx system,
and the CBD corresponds to the nonhydrolytic C~ system postulated by Reese
and his colleagues.
The cleavage of [3-1,4-glycosidic linkages catalyzed by cellulases is com-
monly assumed to proceed with a lysozyme-type mechanism through protona-
tion of the glycosidic oxygen by an acidic amino acid residue and stabilization of
the resulting carbonium ion by another amino acid residue [321]. The active
sites of many enzymes have been found to involve aspartic and glutamic acid
residues. There are pronounced differences in the mode of hydrolysis even
between CBH I and CBH II and EG I and EG IlL CBH I attacks at sites other
than the non-reducing end in the higher homologs (cellodextrins), and hy-
drolyzes both cellobiosides and lactosides. On the other hand, CBH II shows
strict substrate specificity, three to four contiguous ~-l,4-1inked glycosyl resi-
dues being required [322]. It has also been deduced that CBH I acts with overall
retention of configuration, whereas CBH II acts with reversion of configuration
[-323]. Endoglucanases act via retention of configuration. EG 1 belongs to the
group of non-specific endoglucanases because it also hydrolyzes xylan [324]. Its
hydrolytic reaction proceeds with transfer reactions, which indicates that it
probably possesses several subsets, each capable of binding a glucose molecule,
and the glucosyl intermediates formed during the hydrolytic reactions are then
transferred to acceptor molecules [323].
The structure of some members of cellulase families is now known. Small-
angle X-ray diffraction studies suggest that both CBH I and CBH II from
T. reesei are essentially tadpole-shaped [325, 326]. The core is estimated to be

60 to 7OA long with a diameter of 40 A, and to be attached to a tail estimated to
be 150 A long. Bergfors et al. [327] successfully crystallized the catalytic core of
the T. reesei CBH II after removing 82 amino acids from the N-terminus by
papain cleavage. It consists of a TIM-like a/[3-barrel with the active site residues
(two aspartates) located in the center of a tunnel [328]. Later, the catalytic cores
of CBH I and EG I from T. reesei were also crystallized using the hanging drop
vapor diffusion method [329].
Joliff et al. [330] purified and crystallized enduglucanase D from C. ther-
mocellum. Crystals were trigonal and diffracted X-rays to a resolution of 2.8 A.
CelD of C. thermocellum consists of a 12-0t-helical barrel with the active site
residues situated in a long groove across the surface of the enzyme [331]. It
contains an extended NHz-terminal segment sticking out from the enzyme core
to interact with a symmetry-related molecule through an intermediate salt
bridge (Lys 38-Asp 201) [332].
EG I from Hurnicola isolens was crystallized in different forms using various
precipitants by vapor diffusion methods [333]. Two forms obtained from
ammonium sulfate precipitation grow as tetragonal bipyramids, while third and
fourth forms obtained from PEG 8000 grow as monoclinic plates and long
hexagonal rods, respectively. EG V from H. isolens consists of a six-stranded
[~-barrel domain with interconnecting loops with a 40 A groove along the
surface of the enzyme containing the catalytic residues Asp 10 and Asp 121
[334].
A comparison of the structures of the catalytic cores of CBH II from T. reesei
[328] and the catalytic domain (E2cd) of Thermomonosporafusca revealed large
differences in their active site accessibility and supported the hypothesis that the
main differences between endo- and exoglucanases is the degree to which their
active sites are accessible to substrates [335].
3.3 A s s a y o f Cellulolytic E n z y m e s
A wide variety of substrates have been used for the assay of cellulose-degrading
enzymes (Table 5). However, due to the lack of specific substrates and lack of
standardization of activity determination, it has been difficult to compare
cellulase production in one fungal or bacterial strain with another.
The most commonly used assay of complete cellulase activity is based on the
hydrolysis of filter paper [336]. The activity is defined as the amount of reducing
sugars released in one hour when using 50 mg of Whatman No. 1 filter paper as
substrate under standardized conditions. Released reducing sugars are quanti-
fied by the dinitrosalicylic acid (DNS) method [337], and the results are
expressed as glucose equivalents. However, this method registers only the
number of reducing end groups and is very much dependent upon the
[3-glucosidase levels in the enzyme mixture [338]. The use of standardized dyed
insoluble cellulosic substrates (dyed Avicel or filter paper) has also been
Table 5. Assay of cellulolytic enzymes
Enzyme Substrate Measurement
Total cellulase Filter paper/Avicel Reducing sugars

Dyed cellulose Release of dye
Cellulose agar Agar clarification
14C-cellulose 14C_products
Endoglucanase Carboxymethyl cellulose Reducing sugars viscosity
Hydroxyethyl cellulose
Exoglucanase Amorphous cellulose Reducing sugars
p-Nitrophenyl-13-cellobioside p-Nitrophenol
p-Nitrophenyl-13-1actoside
fI-Glucosidase Cellobiose Glucose
p-Nit rophenyl- ~-D-glucoside p-Nitrophenol
4-Methylumbelliferyl-[3-glucoside 4-Methylumbelliferone
(Cellobiose: quinone Reducible quinones such as Decrease in absorbance
oxidoreductase) 2,6-Dichlorophenol-indophenol,
3-Methyl-5-t-butyl-benzoquinone
in the presence of cellobiose or
lactose
Cellobiose phosphorylase Cellobiose Glucose- 1-P
recommended for the comparison of cellulase preparations [339]. An alternative

is to use a X4C-labelled cellulose, where the amount of 14C set free in solution
can be easily related to the total amount of 14C in the solid substrate [340].
The release of reducing sugars from water-soluble cellulose derivatives such
as CMC and HEC has most commonly been used for the determination of
endoglucanase activity, since these derivatives are not hydrolyzed by exog-
lucanases. Viscometric methods, in which the viscosity changes of a CMC
solution can be calculated in absolute enzyme units, have also been developed
[341,342]. However, this method requires thorough knowledge of the proper-
ties of the cellulose derivative to allow for accurate calculations. A zymogram
technique using viscous solutions of CMC for the detection of endoglucanases in
polyacrylamide gels after isoelectric focusing was also developed [343]. When
CMC solutions are applied on blotting papers to be put on top of the gels for
a short period of time, detectable reducing sugars are produced where the
endoglucanases are located.
Measurement of exoglucanase activity in a mixture of cellulolytic enzymes is
more difficult. Deshpande et al. [344] used the heterobiosides, p-nitrophenyl-[3-
D-cellobioside (pNPC) or p-nitrophenyl-13-D-lactoside (pNPL) as selective sub-
strates for the measurement of exoglucanase (cellobiohydrolase) activity. Exog-
lucanases, splitting off cellobiose units from the non-reducing end of cellulose
chains, specifically act on the agluconic bond (between p-nitrophenyl and the
disaccharide moiety) and not on the holosidic bond (between the two glucose
units in cellobiose). Another procedure used for assay of exoglucanase activity
takes advantage of the enzyme cellobiose: quinone oxidoreductase (CBQ), which

in the presence of cellobiose or higher cellodextrins reduces quinones [345].
Exoglucanase activity in mixtures with endoglucanases can be determined
using amorphous cellulose [346]. The number of endoglucanase units in the
enzyme mixture is determined preferably by the viscometric method by Almin
and Eriksson [341], and the amount of reducing sugars (A) produced by the
endoglucanase is computed from a standard curve prepared by the use of known
amounts of purified endoglucanase towards the same amorphous cellulose. The
amount of reducing sugars (B) obtained with the enzyme mixture is then
measured. The reducing sugars content (C) released due to exoglucanase activity
is estimated as C = B - A . Any problem caused by the presence of [3-
glucosidase activity in the enzyme mixture can be overcome by addition of
6-gluconolactone, which effectively inhibits the [3-glucosidase activity. Crystal-
line cellulose cannot be used in this assay, since the synergistic effect obtained
from the endo- and exoglucanases will conceal the results.
The most commonly used assay methods for [3-glucosidases include
measurement of the release of glucose from cellobiose or of p-nitrophenol from
p-nitrophenyl-[3-D-glucophyranoside (pNPG) 1-347]. The most specific method
used to study the production of glucose from cellobiose is the glucose
oxidase/peroxidase method [348]. [3-Glucosidase activity can also be assayed by
determining the release of 4-methylumbelliferone from the substrate 4-methyl-
umbelliferyl-[3-glucoside [349].
The most commonly used substrates for the assay of CBQ are reducible
quinones such as DCPIP or 3-methoxy-5-tert-butylbenzoquinone (DTBB)
[260, 350-352]. The substrate reduction is monitored by the decrease in absorb-
ance at a specific wavelength, 600 nm and 420 nm for DCPIP and DTBB
respectively [351,263]. CDH can also reduce both of these substrates. However,
only CDH can reduce cytochrome c.
Cellobiose phosphorylase activity is commonly assayed spectrophotometri-
cally by measuring the formation of glucose-l-phosphate from cellobiose [146].
3.4 Possibilities for Biotechnology Based on Cellulolytic Enzymes

Cellulases have been commercially available for the last 15-20 years. They have
attracted a lot of commercial interest because of their many industrial applica-
tions in areas such as bioconversion of cellulosic materials, food, textiles,
pharmaceuticals, detergents, wastewater treatment, and fruit and vegetable
processing (Table 6) [1, 2, 353].
Interest in the application of enzymes in the pulp and paper industry has
increased dramatically in recent years. The main application now is to remove
inks from recycled papers. More than 40% of the total paper used in the United
States is now being recycled. In Europe and Japan this figure is about 55%.
Production of market deinked pulp in the US has, over the past six years,
increased from 210000 tons to about 1 million tons by the end of 1995 and is
Table 6. Applications of cellulolytic enzymes
Field Applications
Bicconversion Conversion of cellulosic materials to sugars for production of
ethanol, other solvents, organic acids, and single cell protein
Textiles Biostoning and biopolishing of jeans and other fibers
Detergents Cellulasesas a componentin detergents
Food Improvementofyieldsin starchand proteinextraction;macerationand colour
extraction of fruits and vegetables
Medical Additivein digestiveenzymes
Livestock Feed additiveand silagequalityimprovement
Pulp and paper Enzymaticdeinking,increaseof freenessof fiber suspensions
Environment Waste water treatment/purification
expected to reach 1.8 million tons by the end of 1997. It is estimated that
approximately 90% of pre-consumer paper waste and 42% of post-consumer
office waste paper (OWP) is likely to be recovered and reused to produce
printing/writing paper [354]. The conventional chemical methods for deinking
are less well suited for deinking of laser and xerox printed paper for cost-effective
production of high quality pulp [355]. Cellulases alone, or in combination
with a few other enzymes such as hemicellulases, have been found useful for
deinking different types of paper waste. Several reports and patents describing
enzymatic deinking have been issued [356-359]. Endoglucanases and endo-
xylanases have been demonstrated to effectively deink selected old newsprint
waste [360] and to improve optical and strength properties of paper from
enzymatically deinked pulp. Successful enzymatic deinking of mixed waste-
papers, i.e., old newspapers/old magazines (ONP/OMG), using specially
formulated mixtures of enzymes followed by flotation to separate the enzy-
matically released ink from the fibers, has been demonstrated [355]. The
enzymatic deinking process also improves freeness (water drainage of the
recycled fibers) compared to chemically deinked fibers. Enzymatic deinking
represents the first promising biotechnological application of cellulases in the
pulp and paper industry. In a short period of time, the use of cellulases has thus
made an impressive leap forward. The cellulase-based deinking process offers an
environmentally benign way to improve pulp and paper production from
recycled waste papers.
Cellulases have been successfully used in the textile industry for bio-stoning
of jeans and bio-polishing of cotton and other cellulosic fabrics. Traditional
stone-washing of jeans involves three steps. After removal of the starch coating
of amylases (desizing), the jeans are abraded with pumice stone (1-2 kg/pair
of jeans) in large washing machines (abrasion). After this treatment the jeans
are washed to remove excess dye. However, the high loading of stones
in the washing machines not only decreases the capacity of jean loading
but also causes a lot of mechanical wear to both machines and jeans. Cellu-
lases have been shown to effectively facilitate the removal of indigo dye
82 R.C. Kuhadet al.
from the fiber surfaces [361 363]. Enzymatic stone-washing allows for up to
50% higher jean load and provides the desired soft finish. Typically, 100 kg of
stones are replaced by 1 kg of enzyme [363]. Two different types of cellulases,
neutral and acidic, are generally used, but neutral cellulases are the enzymes of
choice because of the decrease in indigo black staining and a wider working
pH range.
During the last few years, cellulases have also been used for quality im-
provement of cellulosic fabrics. This process is called "Bio-polishing."
Enzymatic bio-polishing is a novel biological finishing process for textiles
made from cellulosic fibers such as cotton, rayon, ramie, and tencel. The
controlled treatment with acidic cellulases improves softness and water ab-
sorbency of the fibres, strongly reduces the tendency for pill formation, and
provides a clearer surface structure with less fuzz [361,363]. Also, bio-poli-
shing permanently enhances fabric look, feel and color without any chemical
coating of the fibres.
Enzymes like proteases and lipases are now commonly used in the detergent
industries, but the use of cellulases in this industry is a more recent approach.
Soil in the interfiber space of cotton is the most difficult contaminant to remove
with common detergents [364]. An alkaline cellulase from a Bacillus sp. has
been shown to interact selectively with cellulose in the interior of fibers and
remove soil in the interfiber spaces in the presence of usual detergent ingredients
[364]. The cleaning power of cellulase-containing detergents shows remarkable
improvement.
Cellulases are also used to improve silage of cattle feed, i.e., to produce high
quality silage from grasses containing only small amounts of water-soluble
carbohydrates. Addition of cellulases and hemicellulases helps to release low
molecular mass sugars to promote fermentation by lactic acid-producing bac-
teria. Cellulases, used as feed additives alone or with proteases, have been shown
to significantly improve body weight gain in piglets and to improve the quality
of pork meat [365].
Enzymatic saccharification of lignocellulosic materials produces monosac-
charides, which subsequently can be fermented to a variety of products such
as ethanol, other alcohols, organic acids, single cell protein, and lipids.
Among various pretreatment methods studied, steam explosion pretreatment
at temperatures of 170-250~ was reported to be the most efficient method
for preparing lignocellulosic substrates for enzymatic hydrolysis [366]. Steam-
exploded sugar cane bagasse was saccharified by cellulases produced by
T. reesei C-30 [367]. Cellulase preparations, 20 FPU g of substrate, gave 70%
saccharification, and could, if supplemented with 13-glucosidase, result in 90%
saccharification. A similar effect by addition of [~-glucosidase was observed
for saccharification of autohydrolyzed Eucalyptus regnans sawdust [368].
A certain amount of 13-glucosidase is thus critical for achieving efficient
saccharification [366].
The cost of enzymes is an important factor for the economy of enzymatic
saccharification of lignocellulosic materials. Therefore, the extent to which
enzymes can be recycled has a significant impact on the production costs of

sugar and alcohol [369]. Various recycling methods for cellulases have been
discussed, some of which have certain drawbacks. However, the one proposed
by Vallander and Eriksson [366], i.e., mixing already saccharified lignocellulosic
materials to which enzymes are adsorbed with new substrate, seems to be an
efficient way to recycle the enzymes.
Bioconversion of glucose of ethanol is a well-documented technology. How-
ever, utilization of pentose sugars for ethanol production, (pentoses account for
approximately 25% by weight of lignocellulosic materials [23]) is in its infancy.
A suitable pentose fermentation technology needs to be developed using pentose
(xylose) metabolizing microorganisms [366, 370, 371]. The bioconversion of
pentose and hexose sugars to alcohol poses a challenging task for fermentation
scientists.
Fungal cellulases have been shown to improve the yield of sugars from
unmodified malts in the brewing process [372]. In production of alcohol from
cassava, addition of Trichoderma cellulases increases ethanol yields by conver-
sion of cellulose to glucose [373]. The isolation of starch from tubers and cereals
requires a release of the integrated particles as cleanly as possible. Cereal
starches are released and improve yield when amylase-free cellulases and
xylanases are applied to the starch slurry prior to centrifugation and washing.
Cellulases used to improve and speed up color extraction by pectinases from the
skin of fruits also have an important application as a part of an enzyme complex
in the maceration of fruits and vegetables.
Lignocellulosic wastes from pulp and paper industries, as well as wastes from
dairy and agricultural industries, are potential substrates for the production of
microbial protein. Two processes have been in commercial operation for such
production from wood sugars. The Candida process, using the yeast Candida
utilis, started in the beginning of this century, while the Pekilo process, using
the fungus Paecilomyces variotii, is more recent [374, 375]. Two other processes
for the direct conversion of solid lignocellulosic wastes to protein by fungi
have been developed to pilot plant scale. One process using the white rot
fungus S. pulverulentum (P. chrysosporium) was developed at the Swedish
Pulp and Paper Research Institute (STFI) [376]. The other process, using
the mold C. cellulolyticum, was developed at the University of Waterloo,
Canada [377]. However, these processes were found not to be economically
feasible, since the protein produced could not compete on a cost basis with
soybean protein unless the substrate used in the fermentation had a negative
value [378].
The STFI approach to protein production based on lignocellulosic substra-
tes was later changed to a waste water treatment/purification process in which
the dissolved substances in the white-water system from mechanical pulp pro-
duction were used as substrates. The process was tested on a large pilot scale in
a newsprint paper mill, and was found to have a good potential as a technique to
be used for closed white-water systems of mechanical pulp and paper mills
[378].
4. D e g r a d a t i o n o f H e m i c e l l u l o s e s
4.1 Microorganisms Producing Hemicellulose-Degrading Enzymes

Hemicellulases are widespread, since they are produced by fungi, bacteria from
terrestrial and marine environments, rumen microorganisms (protozoa, fungi,
and bacteria), yeasts, and marine algae. Fungi, yeasts, and some bacteria secrete
hemicellulases extracellularly, although cell wall-bound and intracellular
hemicellulolytic enzymes have also been reported [149, 379]. Table 7 lists some
important microorganisms producing hemicellulose-degrading enzymes. They
are produced both constitutively and inductively. Hemicellulolytic activity is
generally associated with an enzyme complex consisting of a variety of enzymes
Table 7. Important microbial sources of hemicellulolytic enzymes
Enzyme Bacteria Fungi
[3-1,4-Xylanases Bacillus pumilus Aspergillus niger

Bacillus subtilis Fusarium oxysporum
Clostridium acetobutylicum Aspergillus wentii
Cellulomonas ada Trichoderma koningii
Streptomyces xylophagus Neurospora crassa
[3-1,4-Xylosidases Clostridium thermocellum Aspergillus niger
Bacillus pumilus Corticium rolfsii
Acetovibrio cellulolyticus Penicillium wortmanni
Trichoderma reesei
et-Arabinosidase Streptomyces pupurascens Aspergillus niger
Bacillus subtilis Cortisium rolfsii
Ruminococcus albus Trochoderma reesei
a-Glucuronidase Trichoderma reesei
Agaricus bisporus
Pleurotus ostreatus
Esterases Fibrobacter succinogenes Aspergillus niger
Bacteroides cellulosolvens Aspergillus phoenicus
Clostridium thermocellum Trichoderma reesei
[3-1,4-Mannanases Aerobacter aerogenes Aspergillus niger
Bacillus subtilis Thielavia terrestris
Caldocellum saccharolyticum Trichoderma reesei
Streptomyces lividans Paecilomyces variotii
[3-1,4-Mannosidase Bacillus subtilis Aspergillus niger
Aspergillus awamori
Thielavia terrestris
Polyporus sulphureus
et-Galactosidase Bacillus subtilis Aspergillus niger
Cellulomonas sp. Sclerotium rolfsii
Aspergillus tamarii
Mortierella vinacea
Microorganismsand EnzymesInvolvedin the Degradationof Plant Fiber CellWalls 85
splitting off substituents from the backbone of the polysaccharides as well as

depolymerizing this backbone [-23, 148, 380a, 380b]. Most filamentous fungi
secrete these complexes of hydrolytic enzymes in response to inductive stimuli
[23, 381,382]. Xylanases, mannanases, cellulases and other related enzymes are
usually secreted together into the culture media, and selective induction of
individual enzymes is not a common mechanism in fungi [,190, 383-386].
Hemicellulolytic enzymes of fungal origin are well characterized and have
been studied in detail [23, 387]. Production and characterization of xylanases
from several Trichoderma, Aspergillus and Fusarium strains have been reported
[149, 388]. Brown-rot fungi such as Tyromyces palustris, Poria placenta and
Coniophora cerebella are also potential producers of these enzymes [389, 390].
Of the fungal hemicellulases, those from A. niger have been best characterized
[-23, 150]. The three-dimensional structures of several low molecular mass
xylanases have recently been reported ~391-393]. The Trichoderma xylanase is
ellipsoidal with dimensions 32 A x 42 A.
Only a few yeast species such as some species of Aureobasidium, Crypto-
coccus, and Trichosporon are known to produce xylan-degrading enzymes and
are therefore identified as xylanase producers [-23].
Hemicellulases of bacterial origin are less studied, because eukaryotic
microbes, such as the filamentous fungi, are better producers of these enzymes.
Most of the bacterial studies are confined to species of Bacillus, Streptomyces,
Cellulomonas, Thermomonospora, and Chainia sp. Among actinomycetes, the
xylan-degrading enzyme systems of some Streptomyces have been studied in
some detail, whereas little information is available on the nature and function of
hemicellulolytic enzymes produced by other actinomycetes [-23, 394].
The anaerobic bacteria have been reported to be producers of hemicellulose-
degrading enzymes. The major bacteria are of the genera Clostridium, Ther-
moanaerobacter, Acetovibrio, and Bacteroides [23, 395-397]. Khan and co-
workers [397] have studied the production of endoxylanases, xylosidases, and
esterases from C. thermocellum, A. cellulolyticus, A. cellulosolvens, and
B. cellulosolvens. More recently, two acetyl-xylan esterases, two 13-xylosidases,
an a-glucuronidase, and a cell-associated endoxylanase have been purified and
characterized from the Thermoanaerobacterium sp. strain JW/SL-YS485
[-398-400]. Six distinct ~-xylosidase genes of C. stercorarium 9F-9 have been
cloned. One of them (XylA) has been reported to encode a bifunctional protein
(xylosidase A) with [3-D-xylosidase and a-L-arabinofuranosidase activities [-401].
However, the gene products do not seem to have two catalytic domains, as both
activities resided in one catalytic site.
4.2 Hemicellulolytic Enzymes
Xylose and mannose form the backbone of the hemicellulose polymers in wood.
Because of complex structure of the hemicelluloses, several different types of
enzymes are required for their enzymatic degradation and modification. Among
these, the two major hemicellulose-degrading enzymes are endo-l,4-[3-D-

xylanase and endo-l,4-[3-D-mannanase. The water-soluble oligosaccharides are
then further hydrolyzed by 1,4-13-D-xylosidase. Moreover, enzymes splitting off
side groups and notably, a-c-arabinosidase, a-D-glucuronidase, 0t-D-galac-
tosidase, acetylxylan esterase, and acetylgalactoglucomannan esterases have
been found to be of importance for a complete degradation of these hetero-
polymers [23, 402, 403].
4.2.1 Xylan-Degrading Enzymes
A complete degradation of branched xylans requires the concerted action

of several different enzymes (Fig. 4), i.e. endoxylanase, [3-xylosidase,
e-glucuronidase, e-arabinosidase and acetylxylan esterase. Endoxylanase, the
H H H H H
. u. " I . o. .Io. . O,e

! !
COOH o 0
H OH H OH
Araf
OI
Ac lO
- - 4XyN1--4XylOl--4Xyl/31--4Xyl.81--4Xyl/~ 1--4XylOl--4XylOl--4XyL81--4Xyl.81 --4XylO 1--
2 2 2
Of I~
MeGIcA MeGlcA
Xyl~1--4Xyl/31--
endo-l,4-O-xylanase (EC 3.2.1.8)
O-xylosidase (EC 3.2.1.37)
9E ~ =-glucuronidase (EC 3.2.1. )

'~ =-L-arabinofuranosidase (EC 3.2.1.55)
acetylesterase (EC 3.1.1.6) or acetyl xylan esterase ?
Fig. 4. A hypothetical structure of plant xylan and the linkages attacked by microbial xylan-
degrading enzymes.The fragmentcomprising5 D-xylose(Xyl)monomersis shown in the upper part
of the figure.Ac acetylgroup, ArafL-arabinofuranose, MeGlcA 4-O-methyl-D-glucuronicacid [148]
most widely studied and best characterized xylanolytic enzyme, attacks the
xylan backbone to produce both substituted and nonsubstituted shorter
oligomers, xylobiose and xylose. [3-Xylosidase is employed to convert oligomers
to xylose and acts in concert with endoxylanases, a-glucuronidase, a-ara-
binosidase and acetyl-xylan esterase to achieve total hydrolysis of xylans to
monosaccharides [148].
Endoxylanases (1,4-[3-D-xylan xylanohydrolase, EC 3.2.1.8) hydrolyze the
1,4-[3-D-xylopyranosyl linkages of xylans such as L-arabino-D-xylan, L-arabino-
D-glucurono-D-xylans and D-glucurono-D-xylans. The existence of an exoen-
zyme (1,4-[3-D-xylan xylohydrolase, EC 3.2.1.37), although much less studied,
has also been reported [379]. It appears that the xylosidic linkages in lignocel-
lulosics are not all equivalent and equally accessible to xylanolytic enzymes. The
accessibility of some linkages also changes during the course of hydrolysis due
to removal of substituents and shortening of the backbone chain [404].
The existence of two types of fungal endoxylanases has been demonstrated,
i.e., debranching or arabinose-releasing xylanases and non-debranching or
xylotriose-cleaving xylanases [405]. Both types of xylanases are capable of
attacking glucuronoxylans and unsubstituted 1,4-[3-D-xylans [149]. A. niger van
Tiegham produces three endoxylanases, Xyl I, Xyl II and Xyl III [406]. Hy-
drolysis of rice straw arabinoxylan by Xyl I resulted in the accumulation of
arabinoxylotriose and arabinoxylobiose. These were degraded by Xyl II. How-
ever, Xyl III, which had both arabinose-releasing and xylotriose-cleaving activ-
ities, did not enhance the hydrolysis of arabinoxylan in addition to what was
already accomplished by Xyl I and Xyl II.
The non-debranching xylanases degrade heteroxylans randomly. Five
xylanases of different specificities were isolated from a commercial enzyme
preparation (Rhozyme HP-150 from A. niger) that did not debranch arabinog-
lucuronoxylans E407]. One of these (20.8 kDa, pI 6.7) degraded heteroxylans
and xylose oligosaccharides to mainly xylobiose and xylose. The four other
xylanases (1~28 kDa) degraded heteroxylan to xylose oligosaccharides (sub-
stituted heteroxylan) but without release of xylose. Vrsanska et al. [408] studied
the quantitative binding and hydrolysis of xylose oligosaccharides by an A. niger
xylanase. The substrate binding site of the enzyme had seven D-xylosyl binding
subsites. Bond cleavage frequencies of oligosaccharides by this enzyme were
dependent on substrate concentration. At low substrate concentration, hydroly-
sis of xylo-oligosaccharides occurred via a (unimolecular) mechanism that
yielded xylose from xylotriose, xylobiose from xylotetrose, and xylobiose and
xylotriose from xylopentose as major end products. At higher substrate concen-
trations, there was a deviation from a unimolecular mechanism to one ap-
proaching a termolecular shifted hydrolysis with subsequent change in product
distribution. Thus, hydrolysis of xylotriose yielded xylobiose, while xylotetrose
and xylopentose were prone to transglycosylation reactions resulting in xylo-
oligosaccharides of higher DP, which in turn were subsequently degraded to
mainly xylotriose and xylobiose. Both xylobiose and xylose could be utilized as
glycosyl acceptors in transglycosylation reactions.
Sporotrichum dimorphosporum degraded redwood arabinoglucuronoxylan

mainly to xylose, xylobiose, arabinoxylobiose, arabinoxylotriose, glucarabino-
xylotriose, and glucarabinoxylotetrose [409]. Although L-arabinose and D-glu-
curonic acid were identified at the branch points of the reducing ends of the
D-xylosyl chains of the oligosaccharides, they were not released on hydrolysis.
The wood-rotting fungus, Trametes hirsutus, was found to produce a xylanase
(23 kDa), which degraded willow 4-O-methylglucuronoxylan mainly to xylotet-
rose, xylopentose and 4-O-methyl derivatives [410].
Species of Trichoderma have long been known to produce xylanases. These
enzymes are generally produced along with cellulases during growth on ligno-
cellulosic materials. Trichoderma xylanases have been found to be active on
xylans from different sources, usually producing xylooligomers, xylobiose and
xylose. Xylose is not the major product and it is typically produced after an
accumulation of xylo-oligomers [411]. Partially deacetylated xylo-oligomers
were found to be more accessible than acetylated xylo-oligomers to hydrolysis
by xylanases. Most of the Trichoderma endoxylanases appear to lack activity for
removing the arbinosyl substituents from arabinoxylan. However, the xylanase
from T. koningii was found to release arabinose. Non-specific xylanases from
Trichoderma spp. have also been reported. They not only hydrolyze xylan but
may also attack cellulose, CMC, p-nitrophenyl-~-glucoside, cello-oligomers,
and laminarin [411].
Multiple xylanases (electrophoretically distinct endoxylanases) of microbial
origin have been found [404]. The production of a system of enzymes, each
enzyme having a specialized function, is probably one strategy that a microor-
ganism uses to achieve efficient substrate hydrolysis. More comprehensive
studies are required before the basis of xylanase multiplicity can be properly
understood. The regulation, cross-substrate specificity, and post-translational
modifications of these enzymes must be considered [411]. In fungi some of the
multi-enzymes may also be allozymes, products of different alleles of the same
gene. On the other hand, each of the multiple xylanases may be a distinct gene
product produced by a microorganism to enhance xylan utilization.
Cooperative interactions among multiple endoxylanases (electrophoretically
distinct) from N. crassa, S. exofoliatus, T. byssochlamydoides, and T. harzianum
have been demonstrated [412]. They can increase the extent of hydrolysis of
xylan. Thus, cooperative interactions involving all the three endoxylanases from
T. harzianum are required to achieve maximal hydrolysis of aspen xylan [413].
The degree of cooperativity apparently increased with increased complexity of
the substrate, from the form of the deacetylated polymer to that of the acetylated
polymer and, most significantly, to the form of aspen holocellulose. Hydrolysis
of corn cob arabinoxylan releases arabinoxylotriose containing an interposed
L-arabinose residue attached to a terminal D-xylose unit [-414].
Yeasts belonging to the genera Aureobasidium, Cryptococcus, and Trichos-
poron have been recognized to produce xylanases [23, 415]. T. cutaneum pro-
duces a single xylanase (Mr 45 kDa), which degrades several xylans but not
xylobiose or cellulose [416]. Degradation of oat husk arabinoxylan by
T. cutaneum xylanase yielded xylose, xylobiose, and xylotriose as major end

products. Endoxylanase from C. albidus exhibits a low affinity for p-nit-
rophenyl-[3-D-xyloside [380a]. Xylobiose was attacked at a very slow rate by
this xylanase and cleavage was detected only when radio-labelled [1-3H]xyl2
was used [417].
One of the most studied bacteria, an alkalophilic Bacillus sp. C-59-2, pro-
duces a xylanase which degrades rice straw arabinoxylan to xylobiose and
xylotriose as major end products with smaller amounts of longer xylo-oligosac-
charides [418]. A xylanase from an acidophilic Bacillus sp. 11-1S degraded
xylans of larchwood and rice straw in an endo manner, liberating xylobiose,
xylotriose, and xylose as the major end products of hydrolysis. Streptomyces
endoxylanases preferentially attack the xylan chain where the degree of branch-
ing by arabinose is low [419]. These xylanases yield mainly xylotriose in the
early stages of hydrolysis. As the hydrolysis proceeds, xylo-oligosaccharides
with a degree of polymerization of 3 5 are further degraded into xylobiose and
some xylose [420423]. A purified endoxylanase from Streptomyces sp. hy-
drolyzed corn hull and cob arabinoxylans and several other xylans to xylotriose,
D-xylose and L-arabinose [414]. The arabinoxylans are not completely hy-
drolyzed by Strepromyces endoxylanase and a mixture of substituted oligosac-
charides is obtained as products [424]. A Streptomyces isolate has been ob-
served to exhibit a very low arabinosidase activity [-423]. For details about
hemicellulases from actinomycetes see ref. [394].
Cellulose-binding domains have been conclusively demonstrated in
xylanases of P. fluorescens var. cellulosa [425, 426]. The amino acid sequences
have been determined by cloning and expression of the genes encoding two
endoxylanases (XylA and XylB), one an arabinofuranosidase (XylC) and the
other an acetyl esterase (XylD). The N-terminal sequence of XylA is remarkably
similar to the C-terminal sequence of endoglucanase A [427] and to the
N-terminal sequence of endoglucanase B from P. fluorescens var. cellulosa
[428]. Within these regions of all three enzymes is a homologous sequence of
about 100 amino acid residues followed by two serine-rich regions flanking
a second smaller homologous region. XylA binds strongly to crystalline cellu-
lose, but not to insoluble xylan. It can also hydrolyze soluble xylan while bound
to cellulose [429]. The non-specific xylanases of Myrothecium verrucaria and
Penicillium capsulatum also bind to cellulose and are active while still bound
[430]. However, they are easily eluted with buffer, and it is not clear as yet
whether they bind non-specifically or via cellulose-binding domains. Recently,
an extracellular endoxylanase from F. oxysporum has been reported to contain
a CBD, a peptide (--,2 kDa) consisting of 18 amino acids. The amino acid
sequence showed no homology with any known CBD [431], and therefore
could depict a new class of fungal CBDs.
13-D-Xylosidase (1,4-[~-D-xylan xylohydrolase, EC 3.2.1.37) hydrolyzes xylo-
oligosaccharides to xylose and are essential for the complete hydrolysis of
xylans. This enzyme is produced in appreciable amounts by A. niger, and
xylosidases have also been characterized from T. reesei, Sclerotium rolfsii,
Chaetomium trilaterale and B. pumilus. Two [3-xylosidase genes from B. pumilus

have been isolated and their products compared [432]. The purified
[3-xylosidases I and II were both dimers consisting of 65-70kDa subunits.
Xylosidase I converted oligosaccharides to xylose, while xylosidase II had little
activity on xylobiose. The xylosidase from B. pumilus does not transfer xylose
residues to nucleophiles other than water and operates with inversion of product
configuration, suggesting a single displacement mechanism without formation
of an intermediate enzyme-xylosyl complex [433].
Fungal xylosidases exhibit several different properties compared to B. pum-
ilus enzymes. The molecular masses of fungal enzyme proteins are generally
higher, and they do exhibit transferase activity. Strong transferase activities have
been observed in [3-xylosidases from A. niger [434] and Penicillium wortmanni
[435]. Most of the reported enzymes show highest activity for xylobiose and no
activity for xylan. The activity toward xylo-oligosaccharides decreases rapidly
with increasing chain lengths. T. viride xylosidase hydrolyzed xylo-oligosac-
charides in the rate order X2 > X3 > X4 > X5 [436].
The production of e-D-glucuronidase (EC 3.2.1.??) by the fungi Agaricus
bisporus, Pleurotus ostreatus [437], and T. reesei [438] has been demonstrated.
The importance of this enzyme for the hydrolysis of acidic xylo-oligomers was
pointed out by Puls et al. [438, 439]. The enzyme was found to act in synergism
with endoxylanases and to liberate 4-O-methylglucuronic acid from 4-0-
methylglucuronic acid-substituted xylo-oligomers. Among the different organ-
isms tested, only T. reesei seemed to produce all the side-group cleaving activity
[440]. F. oxysporum and S. olivochromogenes produced acetyl-xylan esterase,
while other side-group cleaving activities were low in the culture filtrates
of A. awamori and B. subtilis. The molecular masses of the purified a-
glucuronidases from T. reesei [440] and A. bisporus [439] were 70 and 45 kDa,
respectively.
a-L-Arabinofuranosidase (EC 3.2.1.55) hydrolyzes nonreducing ~-L-ara-
binofuranosyl groups of a-L-furanosides, arabinans, arabinoxylans, and
arabinogalactans. Although several a-arabinosidases have been purified and
partially characterized, relatively little has been reported about their role in the
hydrolysis of xylan. The enzyme has been characterized from A. niger, Corticium
rolfsii, Streptomyces purpurascens, B. subtilis, Thermoascus aurantiacus and
Ruminocoecus albus [441~443]. All a-arabinosidases hydrolyze beet arabinan to
arabinose. Arabinose is also liberated by purified xylanase from A. niger [444]
and T. koningii [445]. However, these xylanases did not show any activity
toward p-nitrophenyl-a-L-arabinofuranoside, arabinotriose, or arabinotetraose.
Some a-arabinosidases have been reported to produce arabinose from ara-
binoxylan [441]. Although arabinose-substituted xylo-oligosaccharides,
produced by endoxylanases, were the preferred substrates for T. reesei a-ara-
binosidase, the purified enzyme also released arabinose from long arabinoxylan
chains [446].
The existence of acetyl-esterases in unpurified enzyme preparations was
demonstrated by Frohwein et al. [447]. These enzymes were shown to hydrolyze
acetylated carbohydrates, i.e. acetyl-mannose, acetyl-glucose, acetyl-maltose

and acetyl-cellobiose. Williams and Withers [448] isolated more than 100
bacterial cultures from bovine rumen and observed acetyl-esterase activity in
nineteen of these isolates. The existence of acetyl xylan esterase (EC 3.1.1.6) in
fungal cultures was first reported by Biely et al. [449]. The esterases of T. reesei,
A. niger, S. commune and A. pullulans efficiently de-acetylated steamed xylan.
Partially purified acetyl-esterases and endoxylanases from T. reesei and S.
commune exerted a synergistic action in the hydrolysis of acetylated xylan, and
xylo-oligomers, xylose, and acetic acid were produced. The acetyl-esterase fi-om
T. reesei had a Mr of 45 kDa, and the activity could be separated by
chromatofocussing into two isoenzymes with pI 6.8 and 6.0, respectively [-450].
The enzymes exhibited activity toward naphthyl acetate, triacetin, and glucose-
and xylose-acetate.
Feruloyl and p-coumaroyl esterases have also been detected in the culture
supernatants of fungi and bacteria, including S. commune, A. niger, A. phoenicus,
A. awamori and F. succinogens [451, 452]. These enzymes are responsible for the
selective hydrolysis of ester bonds between L-arabinosyl residues and ferulic
(4-hydroxy-3-methoxycinnamic) or p-coumaric (4-hydroxycinnamic) acids. Re-
cently, McCrae et al. [452] isolated and characterized these enzymes from A.
awamori. Wheat straw xylan was de-esterified by the esterases without prior
degradation of the polysaccharide. However, esterified short-chain xylo-
oligosaccharides, which were generated by a purified fungal xylanase, were
better substrates.
4.2.2 Mannan-Degrading Enzymes
1,4-[3-D-Mannanase (1,4-[3-D-mannan mannanohydrolase, EC 3.2.1.78) is

capable of hydrolyzing the 1,4-13-D-mannopyranosyl linkages of D-mannans and
D-galacto-D-mannans. Highly purified enzyme preparations from B. subtilis and
A. niger are also capable of hydrolyzing the D-gluco-D-mannans of konjac and
arum root, providing D-glucose, D-mannose, and a series of manno- and
glucomanno-oligosaccharides. Both endo and exo types of D-mannanases have
been characterized and are produced by various microorganisms including
intestinal and rumen bacteria and fungi. Microbial endomannanases have been
reported to be both inductive and constitutive, usually being secreted extracel-
lularly [453]. However, in some bacteria such as Sporocytophaga myxococco-
ides, Aerobacter mannanolyticus, and Xanthomonas campastris, intracellular or
membrane-bound endomannanases have been reported [23].
Endomannanases hydrolyze ~-D-mannans to D-mannose and a series of
mannose oligosaccharides of DP 2-6. Their action on larch glucomannan
(mannose:glucose ratio 3:1) also yield D-glucose in addition to mannose
[454]. Galactoglucomannans from spruce and Canadian hemlock were de-
graded by A. niger endomannanase to mannobiose, mannose, glucose, and
mannose-oligosaccharides [-455]. Several mannose oligosaccharides containing

glucose and galactose have also been identified in enzymatic hydrolysates of
Canadian hemlock. Araujo and Ward [456] isolated and characterized four
~-D-mannanases and one ~-mannosidase components from Thielavia terrestris.
The enzymes were glycoproteins with carbohydrate contents ranging from 6 to
37%. The enzyme components hydrolyzed coffee bean mannan in a synergistic
manner. Probably due to a binding requirement near or at the active site,
most fungal endomannanases studied to date need four or more mannosyl
residues for efficient hydrolysis [457]. Some mannanases are able to hydrolyse
mannotriose but at a slow rate [458]. A multiplicity of mannanases have
been observed in several fungal species including Tyromyces palustris [459],
Polyporus versicolor F460], and T. terrestris [-456].
1,4-~-Mannosidases (~-D-1,4-mannoside mannohydrolase, EC 3.2.1.25)
hydrolyze 1,4-1inked-[~-D-mannosyl groups from the non-reducing end of
their substrates, manno-oligosaccharides and mannose-containing glyco-
pepetides [-461]. Its absence from mannanase preparations results in the
accumulation of oligosaccharides during mannan hydrolysis. A purified man-
nosidase from Polyporus sulphurius liberates ~-D-mannopyranosyl units from
various natural substrates [-462]. A [3-mannosidase from A. niger exhibited
high activity towards p-nitrophenyl-[3-D-mannoside, but did not hydrolyze the
corresponding 0t-D-mannoside or other tested p-nitrophenyl glycosides [463].
The enzyme activity against 1,4-[3-mannotriose was higher than the activity
against mannobiose. However, the rate of hydrolysis of 1,4-[3-1inked D-manno-
oligosaccharides by ~-mannosidase from an edible mushroom, Tremella
fuciformis, was found to be markedly reduced with increasing degrees of
polymerization [464]. Thus [3-mannotriose was hydrolyzed at a rate which was
30%, and ~-mannotetrose at a rate which was 13% of the rate of hydrolysis of
mannobiose.
~-Galactosidase (a-galactoside galactohydrolase, EC 3.2.1.22) occurs widely
in microbes, plants, and animals. The enzyme catalyzes the hydrolysis of
melibiose, methyl-, ethyl-, phenyl-, and o-nitrophenyl-0t-D-galactosides. Addi-
tion of any one of the above 0t-galactosides to an exponentially growing culture
of A. aerogenes causes as much as a 100-fold increase in the rate of ~-galac-
tosidase production [465]. The enzyme can remove the 0t-D-galactosyl units
found as branched substituents on softwood O-acetylgalactoglucomannans.
The purified enzyme of A. niger was found to be highly specific for the anomeric
configuration of the glycosidic linkages [-466]. Purification and crystallization
of an ~-galactosidase from the fungus Mortierella vinacea have been re-
ported [-467]. This hydrolyzes the usual ~-D-galactopyranosides but does
not liberate D-galactose from galactoglucomannan of guar gum. Similarly,
two 0t-galactosidases from Aspergillus tamarii catalyzed the hydrolysis of
o-nitrophenyl-0t-D-galactoside, melibiose, raffinose, and stachyose but did not
attack the galactoglucomannans [468]. However, a T. reesei 0t-galactosidase
has been shown to release galactose from polymeric galactomannan
[469].
4.3 Assay of Hemicellulose-Degrading Enzymes

The activity of endo-xylanase is generally assayed by measuring the increase of
reducing sugars released from xylan substrates (Table 8). However, there are
many factors causing variations in the determination of xylanase and xylosidase
activities. It has been demonstrated that the reported activities in international
units (IU) varied by a factor of 3 to 107, depending on the dilution of the enzyme
prior to assay [470]. Xylanase activity is also affected by the type of substrate,
varying from 1.5 to 104 IU/ml among the different xylan preparations from
larchwood. The units of enzyme activity obtained also vary with the availability
of easily degradable xylo-oligosaccharides in the substrate. The measurement of
xylanase activity is clearly dependent upon the differences in the mode of action
of different xylanases from different sources and also upon the considerable
heterogeneities in xylans from different sources. It is therefore necessary to use
standardized methods for preparation of xylan substrates and standardization
of enzyme dilution to minimize variations. Other methods such as viscometry
[471] and nephelometry [472] have also been used to assay xylanase activity.
The substrates p-nitrophenyl-[3-D-xylopyranoside and methyl-13-D-xylano-
pyranoside are commonly used for the assay of 13-xylosidase activity, p-Nitro-
phenol and xylose, respectively, released by enzymatic action on these
Table 8. Assay of hemicellulose-degradingenzymes
D-Xylanase Xylan Reducing sugars

Viscometry
[3-Xylosidases p-Nitrophenyl-[3-D-xylopyranoside p-Nitrophenol
Methyl-13-D-xylanopyranoside Xylose
Xylobiose
ct-Arabinosidase p-Nitrophenyl-~-L-arabinofuranoside p-Nitrophenol
Phenyl-ct-L-arabinofuranoside Reducing sugars
~-Glucuronidase 2-O-(4-O-methyl-ct-D-glucupyrano-xyluronic Uronic acid
acid)-xylobiose
Esterases p-Nitrophenyl acetate p-Nitrophenol
Birch hemicellulose Acetic acid
Qt-Napthyl acetate ct-Napthol
Methyl esters of ferulic and Ferulic acid
p-Coumaric acid p-Coumaric acid
D-Mannanases Locust bean galactomannan Reducing sugars
RBB-dyed carob galactomannan
~-Mannosidase p-Nitrophenyl-[3-D-mannopyranoside p-Nitrophenol
1,4-13-D-mannooligosaccharides Mannose
~-Galactosidase p-Nitrophenyl-~-D-galactopyranoside p-Nitrophenol
Melibiose Reducing sugars
Raffinose
substrates, are then measured. ~-Glucuronidase activity is generally determined

by using 2-O-(4-O-methyl-ct-D-glucupyranoxyluronic acid)-xylobiose as a stan-
dard substrate which is obtained by alkali-extraction of (4-O-methyl-D-
glucurono)-D-xylan from birchwood [439]. In assays of a-arabinosidase activ-
ity, p-nitrophenyl-0t-c-arabinofuranoside and phenyl-~-L-arabinoside have been
used as substrates. However, it is difficult to evaluate how specific these assay
techniques might be, since 13-xylosidase of A. niger [473] and Penicillium wor-
tmanni [474] were also reported to show activity toward p-nitrophenyl-0t-L-
arabinofuranoside.
For estimation of acetyl-esterase activity, the rate of release of p-nitrophenol
from p-nitrophenyl acetate can be used. Biely et al. [449] used a solution of
a non-dialyzable fraction of birch hemicellulose for acetyl-xylan esterase activity
measurement. One enzyme unit was defined as the amount of enzyme liberating
one gmol acetic acid per min from 10 mg of the substrate dissolved under
standardized conditions. Recently, McCrae et al. [452] described the assay
methods for feruloyl- and p-coumaroyl-esterase activities, where methyl esters of
ferulic and p-coumaric acid, respectively, were used as substrates, and the
amounts of released ferulic and p-coumaric acid were determined by HPLC.
Locust bean galactomannan and Remazol brilliant blue-dyed carob galac-
toglucomannan have been routinely used as substrate for the assay of endoman-
nanase activity [475]. The measurement of the rate of cleavage of glycosidic
bonds by the rate of release of p-nitrophenol is the most common assay of
13-mannosidase activity. Measurement of the release of D-mannose reducing
sugar equivalents from 1,4-13-D-manno-oligosaccharide substrates is another
assay technique for this enzyme [23]. Several different substrates have been used
for the assay of a-galactosidase activity. Melibiose and raffinose, presumed to be
natural substrates, and p-nitrophenyl-0t-D-galactopyranoside are commonly
used for the assay of this activity [476].
4. 4 Possibilities f o r Biotechnology Based on Hemicellulolytic

Enzymes
Important criteria for industrial implementation include the existence of inex-

pensive and highly active enzyme preparations which can be obtained in bulk
quantities. The judicious use (proper enzyme/enzymes and enzyme/substrate
ratio) of proper mixes of xylan- and mannan-degrading enzymes could result in
cleaner reactions, higher yields, and lower consumption of enzyme and energy
[404]. Table 9 shows some industrial applications of hemicellulose-degrading
enzymes.
For bioconversion of polysaccharides in lignocellulosic materials to fermen-
tation products, maximal utilization of the polymeric sugars is desirable. Com-
plete cellulolytic and hemicellulolytic enzyme systems are required to achieve
maximum hydrolysis of complex substrates to yield monomeric sugars. In order
Microorganisms and EnzymesInvolved in the Degradation of Plant Fiber Cell Walls 95
Table 9. Applications of hemicellulosedegrading enzymes

Field Applications
Bioconversion Enzymatic hydrolysisof polymericsugars to monomericsugars which can be
fermented to ethanol, xylitol, other chemicals,and single cell protein
Pulp and paper Enzymatic prebleaching,debarking, beating, pulp fiber refining,and produc-
tion of dissolving pulps
Food Extracting coffeeand plant oils, improving starch recovery,processingcereal
flours, producingfood thickeners,providing differenttextures to bakery prod-
ucts, clarifyingjuices and wines, and production of xylo-oligosaccharidesfrom
xylans
Feed Feed supplementationto improve nutritional properties of agricultural silage
Textile Retting of flax
to obtain high yields of sugar, the lignocellulosic materials need to be pretreated

prior to enzymatic hydrolysis. Steam explosion is one such pretreatment that
has proven to be efficient and to require less energy than most other pretreat-
ments. Sugars obtained in saccharification processes can be further utilized for
the production of xylitol, organic acids, ethanol, and other solvents via fermen-
tation [477]. Pentoses can also be used as substrates for microorganisms
(C. utilis) for the production of protein-rich biomass (single cell protein), and
various processes have been developed for this purpose [23].
Xylanolytic enzymes are now being used in the pulp and paper industry for
the improvement of existing processes. These enzymes are used in the treatment
of cellulosic pulps to remove residual xylans in the production of dissolving
pulps. Hemicellulases have also been studied for enzymatic debarking and pulp
refining to reduce energy demands in mechanical pulping processes [478]. In an
enzymatic beating process, the enzymes are added to bleached pulp fibers to
increase external fibrillation and thus improve paper-making properties [479].
Xylanase treatment produces pulp fibers with properties similar to those of
slightly beaten pulps. Crude enzyme preparations containing both hemicel-
lulases and cellulases have been used to improve fibrillation and drainage
properties of recycled fibers. Mill trials of this concept have been carried out
successfully using a commercial T. reesei enzyme called Liftase A40 [-480].
Xylanases probably enhance the process of fibrillation as well as facilitate
enzymatic deinking of recycled fibers in combination with cellulases [481].
Dissolving pulps are used to produce cellulose derivatives such as acetates,
cellophanes, and rayons. Hemicellulose contaminants lead to color and haze in
the product and to poor cellulose derivatization. The use of xylanases to obtain
a cellulose free of xylan was first proposed by Paice and Jurasek [482]. Xylanase
treatment may reduce the chemical loading required in caustic extraction of
xylan from kraft pulps. However, the feasibility of using xylanases for the
production of hemicellulose-free cellulose will be dependent on a high selectivity
of the enzymes used. The absence of cellulolytic activities is a particular require-
ment.
Unbleached kraft pulps have a low brightness due to chromophoric groups

in the lignin, the latter being removed in successive bleaching stages. Bleaching is
necessary to attain the target brightness with retention of pulp strength proper-
ties [483]. The most reactive bleaching chemicals are elemental chlorine (C),
ozone (Z), and peroxy acids, which react with the aromatic structures of lignin.
Chlorine dioxide (D) and oxygen (O) react with lignin structures containing free
hydroxyl groups. Sodium hypochlorite (H) and hydrogen peroxide (P) react
with certain functional groups such as double bonds. Effective delignification
can thus be achieved by the action of different types of bleaching chemicals
active on different sites in lignin. Addition of an enzymatic step, i.e., xylanase, to
any conventional chemical bleaching sequence results in a higher final bright-
ness value of the pulp. The incorporation of xylanase prebleaching also permits
the use of lower chlorine charges and lower charges of unselective bleaching
chemicals such as peroxide and ozone. The dosages of xylanase reported to be
used in enzyme-aided bleaching have varied between 30 nkat/g and about
8300 nkat/g. However, an economically realistic dosage seems to be about
100 nkat/g [483].
Yang et al. [484] developed a non-chlorine bleaching process (The EnZone
Process) consisting of a xylanase treatment (X) in combination with O, Z, and
P bleaching stages for bleaching hardwood (eucalyptus) kraft pulp. Pulp deligni-
fled with an OXZP sequence readily yielded a pulp brightness of 85-90% (ISO)
compared with a brightness of 78.0-84.7% for OZP bleached pulp. When
evaluated against a reference pulp (ODEDED-bleached to 90.2% brightness),
Enzone-bleached hardwood pulp (OXZP) had a higher brightness stability,
similar tensile index, and slightly lower tear index. More recently, bleaching of
softwood pine kraft pulp by the Enzone Process using a bleaching sequence of
OXEpZP was reported [485].
Not only xylanases but also endomannanases and side-group cleaving
enzymes have been shown to aid lignin extraction. Treatment of kraft pulps by
endomannanase from T. reesei [486], B. subtilis, and A. niger [487] have been
shown to improve the bleachability in subsequent bleaching stages. In xylanase-
aided delignification side-group cleaving enzymes such as a-arabinosidase and
a-glucuronidase provide some beneficial effects, as was shown by a lower kappa
number and a higher final brightness for pine kraft pulp [488].
More than 100 mill trials of enzyme-aided bleaching processes have been
carried out, about half of them in Europe. The amount of chlorine needed after
xylanase prebleaching has been shown to be reduced by 20-30% [483]. The
enzymatic treatment is fully compatible with existing industrial equipment, and
generally no expensive investments in the mills are necessary [489]. The price of
enzymatic treatment is estimated to be 2-5 USD per ton of pulp, which is
expected to decrease with adaptation of more efficient enzyme-producing strains
and technologies [483].
Xylanases have also been used for clarifying juices and wines, for extracting
coffee and plant oils, for improving nutritional properties of silage, for macerat-
ing plant cell walls, for producing food thickeners, and for providing different
Microorganisms and EnzymesInvolved in the Degradation of Plant Fiber Cell Walls 97
textures to bakery products [490]. Pure xylanase is not required in many of

these applications. Xylo-oligomers produced by enzymatic hydrolysis of xylan
may be valuable for their rheological properties [491].
Xylanases have also been proposed for use in the processing of cereals.
Addition of xylanase causes loss in wheat flour dough water absorption and
consistency, as well as changes in loaf texture [4923. Xylanases have also been
used to enhance the recovery of starch from wheat flours [493]. The major
industrial use of ~-galactosidase is in the hydrolysis of raffinose in the sugar beet
industry [494]. Limited endo-hydrolysis or removal of side-groups may be used
for the modification of polymers, e.g., galactoglucomannans used as thickeners
in the food industry.
5 Degradation of Lignin
5.1 Microorganisms Involved in Lignin Degradation
Lignin is degraded to different extents by different microorganisms, of which

wood-rotting fungi are the most effective, white-rot fungi in particular [23, 52].
These predominantly degrade wood from deciduous trees (angiosperms), but
coniferous wood trees (gymnosperms) are also degraded [23, 495]. Most white-
rot fungi degrade wood by a simultaneous attack on the lignin, cellulose, and
hemicelluloses, but a few are rather specific lignin degraders [23, 98, 99]. The
white-rot fungi produce an array of extracellular oxidative enzymes (Table i0),
the best characterized of which are lignin peroxidase (LIP), manganese per-
oxidase (MnP), and laccase [52, 496 5003. The regulation of the production of
individual enzymes and lignin degradation is a complex phenomenon. Results
obtained in laboratory conditions cannot easily be extrapolated to the decay
process in the cell walls of woody plants. However, Lip and MnP have also been
isolated from rotting wood [501]. The effects of nutritional and cultivation
conditions on lignin degradation and production of ligninolytic enzymes have
been studied by various groups throughout the world and discussed in detail in
Eriksson et al. [233. The role of regulatory factors such as Mn on lignin
degradation and enzyme production is not fully understood. During degrada-
tion studies, Mn has been found to be deposited in white-rotted wood [23, 100],
and has been reported to play a regulatory role in the degradation of lignin and
the expression of laccase, MnP, and LiP [502, 5033.
Soft-rot fungi efficiently degrade wood polysaccharides but degrade lignin
slowly and incompletely [47]. They have been observed more commonly on
hardwoods than on softwoods. Some species of soft-rot fungi studied for their
degradation of lignin and lignin model compounds are shown in Table 11.
Degradation of lignin in beechwood by Chaetomium globosum and lignin loss
along with wood weight loss was correlated with demethoxylation of lignin
[504]. Chaetomium globosum has been reported to release 20-30% 1 4 C 0 2 from

differently labelled corn stalk lignins in seven weeks [80]. Daldinia concentrica
was found to cause some lignin degradation, syringyl-propane units being
degraded before guaiacyl-propane units [505]. Many soft-rot fungi produce
enzymes which demethoxylate vanillic acid in the presence of NADH and
oxygen to form protocatechuic acid and formaldehyde [506]. Although soft-rot
fungi have been shown to demethoxylate lignin in wood, demethoxylation of
isolated lignins has not been reported. Soft-rot fungi have also been reported to
possess low levels of protocatechuate 3,4-dioxygenase [507]. Low levels of
peroxidase activity in C. cellulolyticum and C. piluliferum have also been
reported [508].
Brown-rot fungi (Table 11) prefer coniferous wood. According to a survey of
substrate/fungal relationships, 19% of North American Polyporus species are
brown-rot fungi, among which 60 out of 71 are found primarily on coniferous
wood, i.e., brown-rot fungi are mainly softwood degraders [97]. These fungi
cause extensive degradation of the polysaccharides and only a limited degrada-
tion of lignin [-100]. In advanced stages of degradation, the residue is a brown
mass, which mostly consists of lignin. The limited change in lignin caused by
brown-rot fungi involve (1) demethoxylation, and (2) aromatic hydroxylations
and limited side-chain oxidations. These fungi do not cleave the aromatic rings
in lignin efficiently.
Brown-rot fungi have the capability of cleaving and metabolizing methoxy
groups of vanillic and ferulic acids [508]. The fungi remove the methoxy groups
as methanol, whereas soft-rot fungi appear to release formaldehyde as the initial
product. Brown-rot like white-rot fungi have been observed to decarboxylate
vanillate to methoxy-hydroquinone, and the enzyme vanillate hydroxylase was
detected in brown- and white-rot fungi but not in soft-rot fungi [507]. Most of
the brown-rot fungi [509] were found to produce ethylene from 2-keto-4-
methiolbutyric acid, which can be used as a characteristic for ligninolytic
activity [510]. At least four nonenzymatic agents, e.g., oxalic acid [199], sid-
erophores [198], Fenton's reagent (leading to hydroxyl radical production)
[511], and glycopeptides [512] have been reported to be implicated in the lignin
degradation process by brown-rot fungi. However, the mechanisms by which
these agents function with enzymes for in situ degradation of lignin is not
understood [513].
White-rot fungi, based on the production pattern of their extracellular
enzymes, have been classified in four groups by Hattaka [499] and six groups by
Tuor et al. [97]. Both types of classification have some overlaps and exceptions
with respect to different strains which vary in their ability to produce one or the
other enzyme. An attempt to group the white-rot fungi to avoid the confusion of
their belonging to one or the other group has been made by Eggert et al. [514].
Table 10 shows the white-rot fungi categorized on the basis of their enzyme-
producing ability. For details, readers are referred to refs. [97, 499].
Much less attention has been given to lignin-degrading bacteria compared to
fungi [515]. Most of the studies of bacterial degradation of lignin were based on
Table 10. Phenol-oxidase production by various lignin-degrading white-rot

fungi
LIP-, MnP-, and laccase-producing LiP- and laccase-producing

Coriolus versieolor Oudemansiella radicata
Cyathus buUeri Pleurotus florida
Phlebia radiata Polyporus pletensis
Pycnoporus sanguineus Polyporus brumalis
Pleurotus ostreatus Phlebia tremellosus
Pleurotus sajo-caju Phlebia ochraceofulva
LiP- and MnP-producing LiP-producing
Coriolus pruinosum Bjerkandera adusta
Phanerochaete chrysosporium Daedaleopsis confragosa
Polyporus varius
Laccase-producing
Pycnoporus c.innabarinus
MnP- and laccase-producing Novel oxidase*-producing
Ceriporiopsis subvermispora Bjerkandera adusta
Dichomitus squalens Pleurotus eryngii
Lentinus edodes Pleurotus ostreatus
Panus tigrinus Trametes versicolor
Rigidoporus lignosus Pluerotus sajor-caju
* Aryl alcohol oxidase (AAO), Veratryl alcohol oxidase (VAO)
species of Pseudomonas and filamentous Actinomycetes [516]. Bacteria gener-

ally cause a low percentage of mineralization of lignin in 14C-labelled lignin in
lignocellulosic materials or in DHPs as observed in pure cultures [517]. Actino-
mycetes have been reported to mineralize lignin successfully, although not as
fast or comprehensively as white-rot fungi [128].
A number of Gram-negative bacterial species (Table 11) belonging to the
genera Pseudomonas, Xanthomonas, Acinetobacter, Achromobacter, Agrobac-
terium, Aerobacter, and Erwinia have been reported to degrade lignin [-128].
However, much of the present knowledge has been obtained from studies of the
species Xanthomonas and Pseudomonas. Xanthomanas sp. was reported to
convert 30% of 14C-labelled DHP to 14CO2 [518]. However, lignin polymers
were degraded only up to a molecular weight of 1000 Da, suggesting the
involvement of intracellular enzymes for lignin degradation [517]. P. fluorescens
produces benzaldehyde lyase which cleaves the acyloin linkage of 1,2-diaryl-
ethane compounds anisoin and benzoin [519]. Pseudomonas spp. have also been
reported to degrade biphenyls via oxidation to 2,3-dihydroxyphenyl and 2-
hydroxy-6-oxo-phenylhexa-2,4-dienoate to benzoic acid [520]. P. paucimobilis
SYK6 produces a protocatechuate-4,5-dioxygenase which catalyzes ring fission
of protocatechuate and 3-methylgallic acid, an intermediate of the metabolism
of lignin model compounds with biphenyl structure [521].
Actinomycetes (Table 11) degrade grass lignins by release of lignin-rich,
water-soluble fragments called acid precipitable polymeric lignin (APPL) [522].
Table 11. Lignin-degrading brown- and soft-rot fungi and bacteria
Brown-rot fungi Actinomycetes

Fomitopsis pinicola Arthrobacter sp.
Gleophyllum trabeum Microbispora sp.
Poria placenta Nocardia sp.
Lentinus lepideus Streptomyces badius
Pholiota adiposa Streptomyces cyaneus
Spongiporus sinuosus Streptomyces setonii
T yromyces palustris Streptomyces viridosporus
Thermomonospora mesophila
Soft-rot fungi Other bacteria
Chaetomium 91obosum Acinetobacter sp.
Daldinia concentrica Xanthomonas sp.
Lecythophora hoffmannii Pseudomonas sp.
Petrillidium boydii Achromobacter sp.
Pialophora mutabilis Aerobacter sp.
Erwinia sp.
APPL is not metabolized further by Actinomycetes, and APPL production

seems to be a unique property of these microorganisms, observed mainly in
Streptomyces and Thermomonospora [516, 522, 523]. Purified lignin peroxidase
(ALip-P3) from S. viridosporus was found to catalyze C-C bond cleavage in the
side chains of phenolic and nonphenotic lignin models in the presence of H z O 2
1-524]. The enzyme also oxidizes polymeric lignin, as shown by the rapid H202
consumption upon addition of purified enzyme to lignin. In addition, S. viridos-
porus also produces aromatic acid esterases [525] and aromatic aldehyde
oxidases [526]. Ball et al. [527] screened 20 Actinomycetes for ligninolytic
activity and found S. badius, T. mesophile, and S. cyaneus to be the most active
lignin degraders. S. badius secretes four extracellular peroxidases similar to those
of S. viridosporus [528]. With the discovery of extracellular LiP from Streptomy-
ces spp., it seems likely that these Actinomyces attack lignin by a similar method
to that of white-rot fungi [529]. S. viridosporus has also been reported to
produce one or more extracellular HzOz-generating oxidases [526], but no
evidence of their role in supplying H 2 0 2 to LiP for lignin degradation was
demonstrated.
5.2 Ligninolytic Enzymes
In the complex ligninolytic enzyme system, peroxidases, laccases, and H 2 0 z-

producing oxidases are the most studied (Fig. 3). Peroxidases (LiP and MnP)
and laccase are defined as phenol oxidases [23]. The reactions catalyzed by
these enzymes are very similar (Fig. 3). They oxidize phenolic compounds,
thereby creating phenoxy radicals, while non-phenolic compounds are oxidized
to the corresponding cation radicals [530-533]. While all lignin-related phen-
olic compounds are oxidized by phenol oxidases, the different enzymes have
altogether different substrate ranges for the non-phenolic ones [533].
LiP and MnP from P. chrysosporium have been crystallized and their tertiary
structure examined, while no such report has yet appeared about laccase. The
crystallographic structure of LiP from P. chrysosporium has been studied at
different resolutions, i.e., at 2.0, 2.5, and 2.6 A [534 536]. The model comprises
all 343 amino acids, one heme molecule, and three sugar residues. The crystal
structure revealed that the enzyme consists mostly of helical folds with separate
domains on either side of the catalytic heme. The enzyme also contains four
disulfide bridges formed by eight cysteine residues [535, 536] and two structural
calcium ions, which appear to be important for maintaining the integrity of the
active site [536].
MnP presents a good sequence homology with LiP. However, except for
preliminary information [537], no detailed reports on the structure of MnP
have yet been published. Presently, an attempt to crystallize laccase is in
progress in this laboratory at the University of Georgia, Athens (UGA).
5.2.1 Lignin Peroxidase
Lignin peroxidase (LIP) (ligninase, EC 1.11.1.14), was first discovered in lig-

ninolytic cultures of P. chrysosporium [153, 154], and seems to constitute a
major component of the ligninolytic system. LiP catalyzes a large variety of
reactions, e.g., cleavage of 13-O-4 ether bonds and C~-C~ bonds in dimeric lignin
model compounds - the basis for the depolymerization reactions catalyzed by
LiP. The enzyme also catalyzes decarboxylation of phenylacetic acids, oxidation
of aromatic C~-alcohols to C~-oxo compounds, hydroxylation, quinone forma-
tion, and aromatic ring opening [538]. LiP oxidizes its substrate by two
consecutive one-electron oxidation steps, with intermediate cation-radical
formation. Due to its high redox potential, LiP can also oxidize non-phenolic
methoxy-substituted lignin subunits. Several studies have indicated the import-
ance of LiP in the degradation of lignin and xenobiotics. The enzyme can
depolymerize dilute solutions of lignin [539], oxidize and degrade a variety of
dimers and oligomers structurally related to lignin in vitro [532], and catalyze
the production of activated oxygen species [540]. Studies of LiP oxidation of
DHPs [541] confirm the involvement of LiP in the initial degradation of lignin
[542].
LiP has a similar catalytic cycle to that of horseradish peroxidase. The five
redox states of LiP have been characterized, and its catalytic cycle has been
investigated by various groups of scientists [543 545]. In the catalytic cycle of
LiP, the native Fe 3 + enzyme is first oxidized by H 2 0 / to compound I, and then
a one-electron reduction of compound I with veratryl alcohol takes place [546]
or H 2 0 2 oxidation results in compound II. One further electron reduction of
compound II with veratryl alcohol brings the enzyme back to its native form,
and in this way the catalytic cycle is maintained [543,547]. However, in
competition with a reducing substrate, compound II can react with H 2 0 2 and
result in the formation of the catalytically less active compound III [548].
Compound III is stable but becomes inactivated in the presence of H 2 0 2 [544].

It has further been shown that compound III can transform back to the native
enzyme either spontaneously or in the presence of H202 and veratryl alcohol
[545], and the cycle can get restarted.
The generation of phenoxy radicals during oxidation of phenolic substrates,
in demethoxylation, and in ether cleavage reactions [549] explains the re-
polymerization of lignin in reactions with LiP [550]. Oxidation of phenolic
compounds causes inhibition of LiP [551]. This inhibition can be overcome by
lowering the H202 level relative to phenol and enzyme levels. Moreover, LiP
has a preference for phenolic compounds as compared to veratryl alcohol. Thus,
in order to achieve maximum enzyme (LIP) turnover and oxidation of non-
phenolic compounds, the presence of phenolic compounds in the vicinity of LiP
needs to be low [552].
Harvey et al. [552] have shown that veratryl alcohol is oxidized to a cation
radical capable of participating in intermolecular charge transfer reactions.
Based on this property of the cation radicals, it was proposed that the cation
radicals of veratryl alcohol, the product of LiP catalysis, may mediate in the
oxidation of lignin. In addition, these radicals may assist in the reaction of LiP
compound II with the reductant and thereby maintain the active peroxidase
cycle. The reduction of LiP II to native enzyme results in the release of two
substrate cation-radicals (Fig. 5a). The mechanism by which substrate cation-
radicals promote reduction of compound II is not clear. The cation-radicals may
form a pathway of charge transfer away from the vicinity of the porphyrin so
that the chance of radical generation in the vicinity of the active site of LiP is
HzOz
H202
native enzyme ~'. - Con'.pound [
notive er~yme Compouncl !
FemP Pel~lO)P"
FelIIP FeZglO2P"
e'b
\
e"
Compound11
Compound
FelglOl P ~ V A +"
~
FeIXIO) p ~ X P" -,--- Fe~IO]P__X ~
,.,,.. ~ O z
,- Compound'mr
Compound ~r
F~mtOa'lP FenttOl-}P
b
Fig. 5. Schematicillustrationof the catalyticcycleof LiP showingthe oxidationof (a) VA veratryl

alcohol,(substratemodifiedcompoundII) and (b) X phenoliccompounds(enzymefeedbackcontrol)
1-552]
minimized. This is unlikely to happen with phenoxy radicals which are in-
capable of participating in charge transfer reactions. Their formation would also
result in LiP inhibition (Fig. 5b). Results from different laboratories have shown
that veratryl alcohol can act both as a stabilizer of LiP and as a charge transfer
mediator. An excellent review concerning the physiological importance of aryl
alcohols in ligninolytic fungi has recently been published 1-533].
5.2.2 Manganese Peroxidase
Manganese peroxidase (MnP), another peroxidase found in ligninolytic cultures

of P. chrysosporium, was discovered by Kuwahara et al. 1-155]. The enzyme acts
exclusively as a phenoloxidase on phenolic substrates using Mn 2 +/Mn 3 as an
intermediate redox couple. MnP has also been shown to produce H202 in the
oxidation of glutathione, NADPH, and dihydroxy-malic acid 1-553]. However,
H202 production is not the major function of MnP. Instead, this enzyme is
involved in the oxidation of phenols and phenolic lignin structures. It oxidizes
Mn(II) to Mn(III) in the presence of a proper chelating agent, and Mn(III) must
form a complex with the chelator before it oxidizes phenolic substrates 1,554].
Organic acids are good chelators, and white-rot fungi are producers of oxalic
acid, malonic acid, pyruvic acid, and malic acid 1-533]. Mn(III)/oxalate and
Mn(III)/malonate form very stable chelators which probably also function in
vivo. Malonate facilitates Mn(III) dissociation from the enzyme and has a rela-
tively low Mn(II) binding constant 1,554]. MnP has a catalytic cycle very similar
to that of LiP. However, in this case, compound II is readily reduced by Mn 2
to the native enzyme to complete the catalytic cycle (Fig. 6) 1-156, 555].
A Mn(III) complex can oxidize phenolic lignin substructures by acting as
a mediator between the enzyme and the polymer, and leads to the formation of
AH2
Fe 3" )~tH-
.~j31'q ~ MnlTIT
AH l
~AH" Fig. 6. The catalytic cycle of

manganese peroxidase (MnP)
and its five oxidation states.
Compound TIT Compound I ROOH H202, AH2 phenolic sub-
F e 3 + O ~ FeZ+ O2 Fe'I~T= O [P]t strate [555]
phenoxy radicals as intermediates [530]. Subsequently C~-C~ cleavage or

alkyl-phenyl cleavage would yield depolymerized fragments, including quinones
and hydroxyquinones. The purified MnP has been reported to depolymerize
D H P [556] and also to degrade high molecular mass chlorolignins [557]. MnP
appears earlier than LiP in cultures of P. chrysosporium and can oxidize
phenolic lignin structures, while LiP can also oxidize non-phenolic lignin
substructures [531]. Thus, MnP and LiP jointly provide a good system for the
degradation of lignin-rich woody and non-woody plant materials under proper
reaction conditions.
Several white-rot fungi have been found to produce peroxidases which are
different from LiP and MnP, and which are generally termed manganese-
independent peroxidase, MIP [558-560]. However, information about the func-
tion of MIP in lignin degradation is scanty [533, 561].
5.2.3 Laccase
Laccase (benzenediol: 02 oxidoreductase, EC 1.10.3.2) appears to play an

important role in the degradation of lignin and is produced by most white-rot
fungi. Both constitutive and inductive forms of laccases are known
[-102, 562-564]. All laccases are glycoproteins [565], and they generally contain
four copper ions [566]. These are distributed among three different binding
sites, and each copper ion appears to play an important role in the catalytic
mechanism. Laccase is a true phenoloxidase with broad specificity towards
aromatic compounds containing hydroxyl and amine groups [500]. The enzyme
oxidizes phenols and phenolic substructures by one-electron abstraction with
formation of radicals that can either repolymerize or lead to depolymerization
[567].
Laccase catalyzes demethoxylation reactions of terminal phenolic units. It
can also degrade [3-dimers and [3-0-4 dimers via Ca oxidation alkyl-aryl
cleavage, and C~-C~ cleavage [500]. Laccase has also been shown to catalyze the
cleavage of aromatic rings in a similar way to LiP [-568]. It was observed that
laccase from C. versicolor transformed 4,6-di-tert-butylguaiacol into a ring-
opening product, the muconolactone derivative 2,4-di-(tert-butyl),4-(methoxy-
carbonylmethyl)-2-buten-4-olide. This work clearly demonstrated that 1SO in-
corporated into the muconolactone was from 1802 but not from H2180. It is
obvious from the results that laccase, like LiP, can cleave aromatic rings. Studies
of side-chain cleavage and ring-opening of lignin model compounds indicated
that both laccase and LiP, which catalyze one-electron oxidation of either
phenolic or non-phenolic compounds, are involved in the initial degradation of
lignin substructure model compounds [542].
Until 1990, laccase had been considered to be able to degrade only phenolic
lignin model compounds [567]. However, Bourbonnais and Paice [569] then
reported that oxidation of non-phenolic lignin substructures by laccase from
T. versicolor took place in the presence of a suitable redox mediator, i.e., the dye
2,2'-azinobis(3-ethylthiazoline-6-sulfonate) (ABTS). More recently, laccase

along with ABTS has been shown to also delignify kraft pulp [570]. The
presence of the mediator prevented re-polymerization of kraft lignin by
T. versicolor laccase [571]. Other phenolic reagents have also been proposed
and found to work as mediators [572]. Currently, brightening and delignifica-
tion of kraft pulp has been shown with another mediator, 1-hydroxybenzot-
riazole (HBT) [573]. Moreover, the production of a fungal metabolite which
acts as a physiological laccase/redox mediator has recently been shown in our
laboratory (UGA) to be produced by the white-rot fungus Pycnoporus cinna-
barinus [514].
Laccase and other phenoloxidases act in cooperation with other enzymes.
Intracellular NAD(P)H-dependent quinone-reducing enzymes have been re-
ported from P. chrysosporium [574 576]. These enzymes may constitute part of
a metabolic system which facilitates fungal utilization of small fragments of
lignin to degrade them intracellularly [575]. The reaction of laccase with high
molecular mass fractions of lignosulfonates in the presence of glucose oxidase
gives rise to more depolymerization than polymerization. Glucose oxidase
causes a decrease in the generation of quinone intermediates by laccase [577].
Rapid reduction of phenoxy radicals and quinone compounds by the coopera-
tion oflaccase with oxido-reductase type enzymes could be one possible route to
shift the polymerization/depolymerization equilibrium towards depolymeriz-
ation [23]. Laccase has been reported to act synergistically with MnP to bring
about efficient degradation of radiolabelled 1-Ievea lignins in vitro [578]. Re-
cently, laccase from T. versicolor has been demonstrated to generate Mn(III)
chelates from Mn(II) [579], which have been shown to be implicated in the
degradation of various natural [578, 580] and synthetic lignins [156, 556]. More
recently, a FAD-dependent aryl alcohol oxidase (veratryl alcohol oxidase, VAO)
has been observed to reduce synthetic DCPIP and guaiacyl quinonoids and
phenoxy radicals, generated by laccase, with concomitant oxidation of veratryl
alcohol to veratryl aldehyde. The cooperative action of laccase and VAO could
prevent the polymerization of phenolic compounds and depolymerize (reduc-
tion of Mr) soluble lignosulfonates considerably [581]. Continued investigations
are necessary to elucidate the role of laccase and other cooperative enzymes in
lignin degradation.
5.2.4 HeO2-Producing Enzymes
White-rot fungi also produce several H202-generating enzymes. Intracellular

enzymes are glucose-l-oxidase, glucose-2-oxidase, methanol oxidase, [23] and
fatty acyl-CoA oxidase [582]. Extracellular H202-generating enzymes are
glyoxal oxidase secreted by P. chrysosporium [161] and aryl-alcohol oxidase
found in cultures of T. versicolor, P. sajor-caju, P. ostreatus, and P. adusta
[533].
Both glucose oxidases have been purified and shown to exhibit the following
reactions:
glucose-1-oxidase
[5-D-glucose + 02 , ~-D-gluconolactone + H202
glucose-2-oxidase
I~-D-glucose + 02 , D-arabino-2-hexulose + H202
Methanol oxidase oxidizes methanol to formaldehyde and H202 [160]. In

addition to methanol, ethanol is also oxidized, probably to acetaldehyde.
Glyoxal oxidase (GLOX) is thought to be very important for H202 generation
since it oxidizes several aldehydes and ~-hydroxy carbonyl compounds which
occur as secondary metabolites [583]. Recently, a role of GLOX activity in the
regulation of ligninolysis by peroxidase has been suggested. The inactive GLOX
can be reactivated when coupled to LiP with veratryl alcohol as the substrate for
LiP [584]. Aryl-alcohol oxidase oxidizes aromatic alcohols to aldehydes and
reduces 02 to H202 [533]. Moreover, MnP has also been found to generate
HzOz by catalyzing the oxidation of NADP(H), glutathione, dithiothreitol, and
dihydroxymaleic acid [585, 586].
5.2.50xidoreductases
Other enzymes of importance in lignin degradation are NAD(P)H: quinone

oxidoreductase, aryl alcohol dehydrogenase, and probably also CDH(CBQ).
P. chrysosporium produces at least two different intracellular NAD(P)H:
quinone oxidoreductases [-23,574, 576, 587]. These enzymes reduce
methoxyquinone using either NADH or NADPH as electron donors. Laccase
can interact with this enzyme to increase decarboxylation of lignin model
compounds. In addition, glucose-l-oxidase from C. versicolor could function as
glucose: quinone oxidoreductase and use quinone as co-substrate in place of
0 2 at low O2 tension [588].
CDH or CBQ (the proteolytic breakdown product of CDH[263, 309]) have
probably not beyond doubt been shown to play an important role in lignin
Table 12. Electronacceptorsfor CDH (CBO)and CBQ

ABTScation-radical 3-Methoxy-5-tert-butyl-benzoquinone
4-Aminopyridinecation-radical 2-Methoxy-benzoquinone
Benzylviologen Methyleneblue
Cerulignol Mn3+ malonate
Cytochromec Osmium(II1)
2,6-Dichlorophenol-indophenol TMB radical
Ferricyanide Triiodine ion
Ferriacetate Tetramethoxy-azo-p-methylenequinone
Ferricitrate Vanillicacid phenoxyradical
Guaiacylphenoxyradical Veratryt alcohol cation-radical
Oxygen(slow)
degradation [257], but, when first discovered, C B Q was suggested to be a link

between cellulose and lignin degradation. Both C D H and C B Q have been
discussed in depth in Section 3. Both enzymes reduce a number of electron
acceptors including quinones, phenoxy- and cation-radicals, complexed ferric
ions, c o m p o u n d II of LiP, MnP, and molecular oxygen (Table 12). Quinone
reduction is more rapid than oxygen reduction, oxygen being slowly reduced to
superoxide and/or H 2 0 2 [589].
The following are suggested to provoke the participation of C D H in lignin
degradation: (1) reduction of quinones for detoxification [260], (2) reduction of
phenoxy radicals to prevent their polymerization [264], (3) reduction of compound
II of LiP and M n P [590] (4) production of - O H in order to degrade lignin and
other wood components [162], and (5) support of M n P action/function
by means of solubilization of Mn(IV) O2(s) and production of chelating agents
for manganese ions [591].
5.3 Assay of Ligninolytic Enzymes

Assays for various ligninolytic enzymes are presented in Table 13. Lignin
peroxidase (LIP) activity is routinely assayed in a reaction mixture containing
veratryl alcohol and H 2 0 2 in a suitable buffer. The reaction is initiated by the
addition of H202, and the oxidation of veratryl alcohol to veratraldehyde is
monitored at 310 nm [592].
Table 13. Assay of major ligninolytic enzymes

Lignin peroxidase Veratryl alcohol Monitored at 310 nm
H202
Manganese peroxidase Vanillylacetone Monitored at 336 nm
Phenol red Monitored at 610 nm
MBTH and D M A B Monitored at 590 nm
H202
Laccase Syringaldehyde Monitored at 525 nm
ABTS Monitored at 436 nm
MBTH and D M A B Monitored at 590 nm
Cellobiose: quinone DCPIP Monitored at 600 nm
oxidoreductase MTBBQ (decrease in absorbance)
Cellobiose
Cellobiose oxidase Cytochrome c Monitored at 550 nm
(CBO/CDH) Cellobiose
ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulphonate;MBTH, 3-methyl-2-ben-
zothiazoline hydrazone; DMAB, 3-dimethylaminobenzoicacid; DCPIP, 2,6-dich-
lorophenol-indophenol; MTBBQ, 3-methyl-5-t-butyl-benzoquinone
There are several methods for the assay of manganese peroxidase activity.
However, most of them have certain disadvantages, including lack of definition
of units of activity, low sensitivity, measurement in the UV range, and no
determination of extinction coefficients [593]. The most common method is
monitoring the disappearance of the substrate (vanillylacetone) at 336 nm [553].
The reaction mixture contains vanillylacetone, H2Oz, and MnSO4 in a suitable
buffer. The reaction is initiated by the addition of peroxide, and the linear
decrease in absorbance is monitored. Another method uses phenol red in
succinate buffer containing H202 MnSO4, gelatin, and lactate. A certain volume
of the reaction mixture is removed at one min intervals and added to an N a O H
solution. Oxidized phenol red is measured at 610 nm [594]. Recently, Pillar
Castillo et al. [593] developed a new method for M n P activity. The assay was
based on the oxidative coupling of MBTH and DMAB, which, in the presence of
H 2 0 2 , Mn z +, and MnP, gives a deep purple-blue colour with a broad absorp-
tion band and a peak at 590 nm. The extinction coefficient is high, which
facilitates detection of even low activities.
The activity of laccase is usually measured by the oxidation of syringal-
dehyde to its quinone form at 525 nm [595]. The molar absorbancy of the color
formed is high. However, syringaldehyde is not very soluble in an aqueous
medium, which necessitates a high alcohol concentration [596]. Laccase activity
is also assayed using the dye ABTS by monitoring the progress of oxidation at
436 nm [-569]. However, if peroxidases and other oxidases are present in addition
to laccase, it would present a problem, since certain oxidases would generate
H 2 0 2 , a substrate for peroxidases [596]. Lonegan and Baker [596] developed
a method for laccase assay using the coupling reaction between 3-methyl-2-
benzothiazoline hydrazone (MBTH) and 3-dimethylaminobenzoic acid (DMAB).
Prior to estimating enzyme activity, samples were pretreated with an active
catalase to destroy hydrogen peroxide and hence minimize peroxidase activity.
CBO and CBQ activities are assayed by measuring their reducing activities
for suitable electron acceptors [263, 264]. Both C D H and CBQ can utilize
D C P I P as an electron acceptor, while only C D H is able to reduce cytochrome c.
D C P I P reducing activity [263] is assayed at 30~ in a reaction mixture
containing cellobiose and DCPIP in a suitable buffer. The decrease in absorb-
ance at 600 nm is monitored, and one unit of activity is defined as the amount of
enzyme necessary to reduce 1 tamol substrate per min. Cytochrome c reducing
activity is determined in a reaction mixture containing cellobiose and cyto-
chrome c in a suitable buffer [264]. The activity is measured at 30 ~ as the
increase in absorbance at 550 nm. One unit of cytochrome c reducing activity is
defined as the amount of enzyme necessary to reduce 1 ~tmol substrate per min.
5. 4 Possibilities for Biotechnology Based on Ligninolytic Enzymes
The ligninolytic enzymes produced by white-rot fungi have broad substrate

specificities and should therefore have potential for use in different areas of
Table 14. Applications of lignin-degrading microorganisms and enzymes
Field Application
Food and feed Mushroom production; improvement in digestibility of wood and straw resi-
dues for use as ruminant feed
Pulp and paper Biopulping and biobleaching; modification of pulp fibers; delignification of
wood chips to reduce energy input in mechanical and chemical pulping
Chemical Modification of isolated lignins to produce useful lignins and chemicals
Environment Treatment of spent kraft bleach liquors and organic pollutants such as PAHs,
PCBs, and dioxins
Biosensors Identification of cellodextrins; measurement of cellulose surfaces of various
pulp fibers using CDH
forest industrial processes, pulp and paper manufacture, by-product utilization,

and waste management (Table 14).
LiP, MnP and laccase, produced by white-rot fungi, have all been studied for
use in pulp bleaching [597]. With mixtures of ligninolytic enzymes, a 20-30%
reduction in kappa number has been observed [598]. LiP has been implicated in
biobleaching with P. chrysosporium [599]. MnP and laccase were detected in
cultures of T. versicolor used for bleaching of wood pulp and also in other
white-rot fungi [600-602]. Paice et al. [602] reported that partially purified
MnP alone demethylated and delignified pulp to almost the same extent as the
complete cell-free culture solution from T. versicolor cultures. More recently,
pure MnP from the fungus SKB-1152, used for pulp bleaching, has been shown
to successfully bleach oxygen-alkali treated hardwood kraft pulp [603]. CBQ
has also been observed in culture solutions of T. versieolor used for pulp
bleaching [602]. Various studies have shown its involvement in the reduction of
oxidation products of laccase or peroxidase such as quinones [260], phenoxy
radicals [589], and Mn III [604].
Different approaches, such as the use of suitable redox mediators for en-
hanced laccase delignification and brightening of kraft pulp have been worked
out [570, 572, 573]. Recently, an efficient laccase mediator system for enzyme
pulp bleaching has been reported from the company Lignozym [605]. The
performance of enzyme mediator systems is now being tried on the pilot plant
scale.
Effective pulp bleaching by cell-free oxidative enzyme such as laccase or
MnP seems to require additional reagents like mediators/activators or other
process controls [606]. Laccase requires a high pressure of Oz and a low
molecular mass electron carrier, while MnPs require a low and controlled
supply of H 2 0 2 along with Mn(II) ions and a suitable chelator such as oxalate
or malonate [606].
The ligninolytic system of P. chrysosporium is also capable of degrading
a number of organic pollutants and xenobiotics. Halogenated phenols too are
oxidized to form oligomeric and polymeric products, usually less toxic than the
original low molecular mass substrates. Among the polyaromatic hydrocarbons
(PAHs) studied for oxidation and degradation by white-rot fungi and their
enzymes are anthracene, methylanthracene, fluorenthrene, biphenyl, phenan-
threne, pyrene, benzopyrene, fluorene, acenaphthene, some monoaromatic com-
pounds, and the PAH component of the anthracene oil from coal tar distillation
1-607, 608]. Because of the high redox potential of lignin peroxidase from
P. chrysosporium, the latter has been suggested for use in bio-remediation of
chlorinated hydrocarbons such as DDT, CC14, TCDD, and PCBs [-609].
Use of free enzymes may have some significant drawbacks such as thermal
instability, susceptibility to protease attack, inhibition of activity, and the
difficulty of separating and reusing them when the reactions are completed. The
phenoloxidases have been successfully immobilized on natural and synthetic
supports such as soils, clays, and porous glass beads to overcome some of these
problems 1-610, 6ll]. Immobilized laccase has been successfully used to remove
color from phenolic industrial effluents 1,612]. Recently, immobilized phen-
oloxidases were found to efficiently remove naturally occurring xenobiotic
aromatic compounds from aqueous solutions 1-613].
LiP produced by P. chrysosporium has also been implicated in the decoloriz-
ation of several dyes 1-614, 615]. The presence of veratryl alcohol stimulates
azodye oxidation. The ability of peroxidases and laccases to decolorize dyes
indicates the possibility of developing processes to reduce color in pulp mill
effluents. Most of the color in chlorine bleach plant effluents emanates from
quinone and conjugated lignin structures, particularly in the effluent from the
first alkali extraction stage (El). The color is mainly associated with the high
molecular mass lignin fractions. Therefore, efficient decolorization and removal
of these high molecular mass compounds are necessary. Among the white-rot
fungi, P. chrysosporium has been tried for bleaching of E1 effluents from bleach
kraft mill processes 1-616]. A continuous process for color removal from bleach
plant effluents with P. chrysosporium was developed to the pilot plant scale
[-617]. The process (mycelial color reduction, MyCoR) utilizes a rotating biolo-
gical contactor on the surface of which the fungus is immobilized. In the MyCoR
process, chlorolignins are somewhat degraded, and low molecular mass chlorin-
ated derivatives are metabolized. Extracellular LiP and MnP may play an
important role in the bleaching process. The potential of the enzymatic pool,
LiP and MnP, in particular, from Pycnoporus sanguineus cultures for decoloriz-
ation of bleach kraft pulp effluents [618] has also been indicated. However,
a recent study [619] has demonstrated the partial decolorization of E1 effluent
by purified MnP, whereas LiP did not appear to have any role in this process.
A CDH/CBO-based biosensor has been developed by Elmgren et al. 1-620].
CDH is immobilized or cross-linked in a redox polymer matrix containing
osmium(III) on a rotating disc electrode functioning as a biosensor. A combina-
tion biosensor, utilizing glucose oxidase in addition to CDH, has also been
developed. The biosensor is coupled to a chromatographic column, and pro-
vides for the identification of cellodextrins 1-621].
R e s e a r c h a n d d e v e l o p m e n t i n v o l v i n g e n z y m e s i n v o l v e d in l i g n i n d e g r a d a -
t i o n is p r o g r e s s i n g w i t h c o n s i d e r a b l e p a c e , a n d o n e c a n see t h e p o t e n t i a l o f
l i g n i n o l y t i c e n z y m e s e i t h e r a l o n e , in m i x t u r e s , o r p e r h a p s p a r t i c u l a r l y in c o m b i -
n a t i o n w i t h r e a d i l y o x i d i z e d s u b s t r a t e s ( m e d i a t o r s ) for t h e b l e a c h i n g of w o o d
pulp.
Acknowledgements. The authors wish to express their appreciation to the Department of Biotech-
nology (Government of India). Ministry of Science and Technology, New Delhi for the DBT
Overseas Associateship award to R.C. Kuhad, and to the University of Delhi for grant of duty leave.
We also extend our sincere thanks to D.E. Akin and G. Henriksson for their suggestions during the
preparation of this chapter. For the drawing of Fig. 6 we are indebted to Paul Simon.
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Lignin
Dimitris S. Argyropoulos and Sam Ben Menachem
Department of Chemistry, McGill University,
Pulp and Paper Research Centre, 3420 University Street, Montreal, Quebec,
Canada, H3A 2A7
1 Occurrence and Role of Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

1.1 W o o d Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
2 Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
2.1 Synthesis of Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
2.2 The Dehydrogenation Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
2.3 The Radical Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
2.4 Lignin - Carbohydrate Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3 Lignin Architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
3.1 The Gel Degradation Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3.2 Possibility of Order in Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4 Solution Properties of Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
4.1 Lignin Associative Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
4.2 Lignin Polydispersity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
5 Lignin Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
5.1 Laboratory Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
5.2 Commercially Produced Lignins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
6 Methods of Lignin Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
6.1 Degradative Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
6.2 Non-Degradative Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
The objective of this chapter is to provide the reader with a general account of various aspects of
lignin chemistry with emphasis on issues that have seen rapid growth during the past two decades.
This is accomplished by describing the research efforts of over 360 literature citations, embarking
from early concepts and concluding with the current views on the subject.
After a general introduction that deals with the occurrence and role of lignin in the cell wall and
within woody tissue, recent advances of lignin biosynthesis are discussed commencing with a de-
scription of the metabolic pathways that determine the synthesis of the various lignin precursors.
The main reactions leading to the various bonding patterns in lignin are then discussed including
accumulating evidence that pertains to the connectivity of lignin to carbohydrates. The overall
architecture of the lignin macromolecule is then dealt with by critically examining various aspects of
the early literature and recent scientific evidence that points to the possibility of order in it. After
a brief description of the m e t h o d s available to isolate lignin, the chapter concludes with an outline of
the various major procedures currently available for its structural determination.
ManagingEditor: Th. Scheper
9 Springer~VerlagBerlinHeidelberg 1997
128 D.S. Argyropoulosand S.B. Menachem
1 Occurrence and Role of Lignin
Second only to cellulose, lignin is amongst the most abundant biopolymers on

earth. It is estimated that the planet currently contains 3 x 1011 metric tons of
lignin with an annual biosynthetic rate of approximately 2 x 10 l~ tons [1, 2].
Lignin constitutes approximately 30% of the dry weight of softwoods and about
20% of the weight of hardwoods [3]. Lignification is associated with the
development of vascular systems in plants, providing resistance to biodegrada-
tion and environmental stresses such as changes in the balance of water and
humidity [4]. Lignin is absent from primitive plants such as algae, and fungi
which lack a vascular system and mechanical reinforcement. The presence of
lignin within the cellulosic fibre wall, mixed with hemicelluloses, creates a nat-
urally occurring composite material which imparts strength and rigidity to trees
and plants. An additional role for lignin has recently been revealed [5-7]
involving complexes of lignin phenolic acids in forage legumes and grasses. The
presence of lignin phenolic acids is thought to inhibit the digestion of potentially
digestible carbohydrates by ruminants.
Industrially one encounters lignin during the process of paper making, which
involves the chemical or mechanical separation of the cellulosic fibres from
woody or other lignified plant material. The chemical separation of lignin from
cellulose has been termed "delignification" and it is one of the complex processes
of the pulp and paper industry [8]. The process of delignification results in the
production of vast amounts of lignin whose properties may vary depending on
which delignification process has been employed and at which stage of delignifi-
cation the lignin was isolated. In general, the lignin by-products resulting from
the process of wood delignification are of a polymeric nature that may serve,
after some modification, as additives in various formulations involving adhes-
ives, thermosets or thermoplastics. Although chemical pulping is a long estab-
lished practice, many controversial questions remain concerning the structure of
native lignin (protolignin), its reactions during delignification and bleaching [9].
To expose the problem further requires an understanding of wood anatomy,
lignin structure and biosynthesis and the way lignin is linked to carbohydrates
in the cell wall. Since wood is the predominant raw material for the pulp and
paper industry the focus of this chapter will be toward understanding wood
derived lignins as opposed to those from annual plants.
1.1 Wood Structure
Figure 1 shows the detailed macroscopic composition of a softwood fibre. The

cellulose microfibrils are arranged characteristically within the various layers in
the cell wall, namely; the primary wall (P), the secondary wall, and the middle
lamella (ML). The secondary wall is further subdivided into three sublayers
($1, $2, and $3) each comprised of cellulose microfibrils in distinct orientations with
reference to the main fibre axes. The $1 and $3 layers are both 0.1~).2 gm in
Lignin 129
Fig. 1. D i a g r a m m a t i c r e p r e s e n t a t i o n o f
a softwood tracheid (Reprinted with permission
from C6te [12])
thickness while the S 2 layer is about 1 10 gm thick containing within it 80-95%

of the cell wall material. The primary wall, (0.1 gm), contains a network of
microfibrils and significantly higher lignin concentration than the secondary
wall, while the middle lamella is composed predominantly of lignin [10].
Despite the fact that the concentration of lignin in the middle lamella is
extremely high, it is the secondary wall that contains about 70% of the overall
lignin present in wood due to its large volume [11].
Chemically, lignin is built from phenylpropane units linked together by
different bonds. A description of how the different bonding patterns emerge will
follow. However, at this point it is essential to mention the fundamentals of
numbering the various carbon atoms in lignin. More specifically, while regular
numbers are used for labelling the aromatic carbons, Greek lettering is used to
label the side chain of the phenylpropanoid. The illustration of Scheme 1 may
assist the reader in comprehending the meaning of the various bonding patterns
described in latter parts of this chapter.
"~C H 2 O H R = another phenyl p r o p a n e unit

I
[3 C H R R " = H or R
I
ct C H R .... R " ' = O H or R
guaiacyl : R' = OCH3, R " = H
R" R' syringyl : R' = R " = O C H 3

OR" para-hydroxyphenyl : R' = R " = H
S c h e m e 1. T h e e l e m e n t a r y p h e n y l p r o p a n e b u i l d i n g b l o c k s o f v a r i o u s lignins
2 Biosynthesis
Lignification is believed to occur in the intracellular layers of the cambium,

where the sapwood and the bark layers of the tree merge [13]. Tracer experi-
ments [14] and UV-microscopic observations [15] have shown that lignifica-
tion is initiated within differentiating wood cells and extends to the intracellular
areas, i.e. primary and secondary cell wall. Terashima [16] has shown that
lignification and cellulose deposition in the plant cell wall proceeds in three
distinct phases. Initially lignification occurs at the cell corner and middle
lamella, after the deposition of pectins is complete and the formation of the
secondary wall $1 has been initiated. During the second phase, an extensive
deposition of cellulose microfibrils, mannan and xylan in the $2 layer takes
place. Yet, the lignification process proceeds very slowly during this stage.
Finally, during the third phase, lignification proceeds extensively. This occurs
after the deposition of cellulose microfibrils in the $3 layer of the secondary wall
has taken place.
2.1 The Synthesis of Lignin Precursors

In 1971, it was demonstrated that lignin is synthesized from/-phenylalanine and
cinnamic acids [15]. These acids are derived from carbohydrates through the
shikimic and cinnamic acid pathways. Supporting evidence for this route was
obtained when radioactive glucose was administered into plants, producing
shikimic acid [17] and radioactive lignins [18-20]. Lignification proceeds with
the conversion of/-phenylalanine to form trans-cinnamic acid (Scheme 2). This
deamination process is catalyzed by l-phenylalanine ammonia lyase (PAL),
a key enzyme found only in plants that can synthesize lignin [21,22] and
some cinnamic acid derivatives [22]. It is worth mentioning that an additional
enzyme, tyrosine ammonia lyase (TAL), which catalyses the formation of p-
cournaric acid from /-tyrosine, is characteristically found only in grasses
[23-25]. The presence of this enzyme may account for the presence of
p-coumaryl alcohol as an additional lignin monomer as well as esterified
p-coumaric acid present mainly in grasses.
As lignification proceeds, cinnamic acid is hydroxylated to p-coumaric and
caffeic acids by specific hydroxylase enzymes [26, 27]. The caffeic acid thus
formed, is then methylated to ferulic acid by O-methyl transferase (OMT)
[28-30]. Up to this point the biosynthetic pathways for softwood and hardwood
lignins is believed to be common [15]. However, they seem to diverge beyond it
[31, 32]. This is because OMT enzymes, of different functionality, possessing
different substrate specificities were found to be present in softwoods and
hardwoods [33, 34]. One of the reasons which accounts for the almost exclusive
presence of guaiacyl lignin in softwoods, is that the monofunctional OMT
enzyme is inhibited competitively by caffeic acid (Scheme 2). For hardwoods, the
Lignin 131
difunctional OMT is inhibited via a feedback mechanism involving 5-hydroxy-

ferulic acid [-34, 35]. This limits the concentration of this intermediate and the
production of ferulic acid, thus restricting the formation of syringyl precursors.
Another key enzyme which is responsible for the differentiation of the G and G,S
pathways, is the ferulic acid-5-hydroxylase.
The absence of this enzyme in gymnosperms also accounts for the near
exclusive formation of guaiacyl precursors in softwoods.
The synthesis of lignin precursors is complete with the reduction of ferulic and
sinapic acids to the corresponding coniferyl and sinapyl alcohols. These reac-
tions are catalysed by three specific enzymes which constitute the reductase
H o OH
H. CH20H H00C_+_NH2 ~OH
OH~ Q shikimicacid CH2
pathway 0 PAL CAH ~ ~ [
O H ~ OH COO~/
H~J~-OH OH
glucose H-H~n~H 1-phenylalanine cinnamicacid p-hydroxycinnamicacid
shikimicacid CMS1
competitiveinhibition oyoH
(in gymnosprems)
OH
~feedback inhibition caffeicacid
( J"- (in angiosperms)

OMT 1
CH2OH o..ro. o.3,-0"

OCH3~OCH 3 q "3COocH3 FA. dOCH
OH
OH OH OH
femlic acid
sinapylalcohol
2
1
sinapic a c i d
1-phenylalanineammonialyase(PAL)
cinnamicacid 4-hydroxylase(CAH)*
5-hydroxyferulic
acid
Rs]
3 4-coumarate3-monooxygenase(CMS)* CH20H
4 o-methyltransferase(OMT) .A
5 ferulicacid 5-hydroxylase(FAH)*
6 reductasesystem(RS)*
d OeH
OH
coniferylalcohol
*These abbreviations have been used for brevity purposes only
Scheme 2. A simplifiedmetabolicpathway of 1-phenylalanineto lignin precursors
system [32, 35]. The enzymes which were recently isolated from various plants
[36, 37] include the hydroxycinnamate-CoA ligase, hydroxycinnamoyl-CoA
reductase and hydroxycinnamyl alcohol dehydrogenase. The ligase and reduc-
tase enzymes of angiosperms and gymnosperms respectively, were found to
show pronounced differences in substrate specificities [30, 38]. In gymnosperms,
both enzymes were found to be inactive with sinapic acid and sinapoyl-CoA
[39,30]. The lack of activation or reduction of sinapate in gymnosperms
contributes toward the exclusive formation of guaiacyl lignins in them.
The lignin precursors formed are of low solubility and are readily oxidized; in
the cell wall they are stabilized as glucosides. The typical glucoside for softwoods
is coniferin and its structure is shown in Scheme 3.
2.2 The Dehydrogenation of the Precursors
Early investigations [40, 41] coupled with the more recent results of Harkin and
Obst [42] have shown that the dehydrogenative polymerization oflignin mono-
mers in plants is caused by a class of enzymes called peroxidases, or the
peroxidase-H202 system. These enzymes, are capable of abstracting a proton
from the phenolic hydroxyl of the precursor molecules, creating resonance
stabilized free radicals. Examples of such radicals, typical of softwood lignin are
depicted in Scheme 4 [42, 43].
More specifically, the dehydrogenation reaction involves hydrogen peroxide
as the electron-accepting substrate for the peroxidase [45, 46]. In addition,
superoxide radicals were suggested to form via the reduction of oxygen by N A D
(nicotinamide adenine dinucleotide) [47]. Recent evidence suggests that hydro-
gen peroxide is produced by the peroxidase enzyme itself, and may play a key
role in the control mechanism [47]. An enzymatically controlled cycle for the
CH20H
H ~ " r - O. w- O ---~, ,~-----CH
H
Scheme 3. Coniferin,a coniferylalcohol
H ()H CH30 glucoside
CHzOH CH2OH CH2OH CH2OH CH2OH

I I I I I
CH
II
CH
II
CH
II
CH 9CH
II I
CH CH CH CH CH
Peroxidase
OCH3 H3
OH O 3 3
Scheme 4. Resonanceforms of softwoodlignin phenoxy radicals (43,44)

Lignin 133
production of hydrogen peroxide, has also been reported [42]. However, excess
peroxide was found to inactivate the peroxidase by the formation of a complex
of an unknown structure [-48]. Recent research has also shown that the presence
of laccase-like phenoloxidase can be correlated to lignin biosynthesis in Zinnia
elegans stem tissues [49]. A review of the recent literature regarding plant
laccases and selected fungal laccases and their role during lignin biosynthesis
has been compiled by Dean and Eriksson [50].
After the oxidation of the monomeric alcohols to phenoxy radicals, the
reaction changes dramatically; the reactions are no longer subjected to
enzymatic control, but to a random polymerization process [51, 52].
2.3 The Radical Polymerization
A major milestone in lignin chemistry was Freundenberg's success in providing

experimental evidence for the enzyme-initiated dehydrogenative polymerization
theory. This became possible by polymerizing coniferyl alcohol to a lignin-like
dehydrogenation polymer (DHP) using liquid squeezed from the mushroom,
Psalliota campestris, known to contain laccase and other oxidative enzymes
[50]. Further attempts to synthesize lignin DHP's using the peroxidase/H202
system were successfully accomplished by Freudenderg [53, 54] and Sarkanen
[55]. Yet, as Sarkanen pointed out, "these lignin polymer models", despite the
fact that structurally were lignin-like, they were not identical to lignins formed in
vivo [55]. When the in vitro polymerization was interrupted, structures of
dimeric and oligomeric intermediates could be isolated and the lignin bonding
patterns identified [56 58].
These experiments and others, involving sinapyl alcohol, support a random
mechanism for the polymerization of phenoxy radicals [59 61]. The reactivity
of such radicals and the probability of a specific mesomeric form to form
a particular linkage depends on /3 electron spin density and steric consider-
ations. Molecular orbital calculations of ~ electron spin densities of lignin model
compounds [62] have shown that free electron spin densities are highest at
specific sites within the phenylpropane unit. These reactive sites (Scheme 4) are
the C1 and C5 positions of the phenylpropane unit, the phenolic hydrogen, and
the aliphatic [3-carbon. Of these reactive sites, those with groups attached at
C1 and C3 in coniferyl alcohol and C1, C3 and Cs in sinapyl alcohol, are less
reactive. Alternatively, the phenoxy oxygen and the [3 carbon are considered as
the most reactive species, readily coupling into aryl ether linkages [63, 64]. This
may account for the high abundance of the [3-0-4 inter-unit linkages in lignin,
estimated to be as high as 50% in softwoods and almost 60% in hardwoods [64].
The polymerization process proceeds with the coupling of the different reson-
ance structures. Amongst the products, highly reactive quinone methides
(Scheme 5) are formed which further react by addition to various nucleophiles.
Free radical exchange reactions, such as hydrogen abstraction from suitable
portions of other molecules, are also possible [65, 66].
134 D.S. Argyropoulos and S.B. Menachem
CIH2OH~__xH3
HC - O X-~.ffx- CH= CH - CH2OH
O'~-CH= CH-CH2OH
With coniferyl alcohol
/ OH
OCH 3
OCH 3
OCH 3 Ct, 13ether structures
.c-o%
H2COH )='xOCH3
[H20]
HC - O _ _ ' ~,,)- CH= CH - CH2OH
O HC- OH
Quinone Methide
~OCH 3
OH
~ - O - 4 structures
H2COH ~OCH3
[glucose]
, HC -O ~ C H = CH-CH2OH
Lignin carbohydrate bond
Scheme 5. Addition reactions to a quinone methide leading to the formation of the various inter-
unit linkages in lignin
The first dimeric lignol to be isolated and identified was a phenylcoumaran

structure analogous to that of dehydrodiconiferyl alcohol [67]. This structure,
which comprises about 10% of the lignin units, is formed by the coupling of two
free coniferyl alcohol radicals centered at C5 and C 0 positions of the phenyl-
propanoid (structures 2 and 4 in Scheme 4), giving rise to a dimeric
quinonemethide. This coupling followed by aromatization of the ring allows
the creation of an a-O-4 linkage (Scheme 6). The C~-C5 bond thus formed is
actually quite stable under pulping conditions, as long as its phenolic group is
etherified.
Other bonding patterns that occur in lignin are the so-called "diaryl propane"
units or 13-1 (Scheme 7). They are present in about 5-10% of the total phenyl-
propane units in lignin and they are thought to be relatively stable under
alkaline pulping conditions.
Ether and ester linkages are also common in lignin. With respect to ether
linkages, several types have been identified to be present, namely: biaryl ethers,
non-cyclic benzyl alkyl ethers, 13-O-4 ethers and diphenyl ethers. Biaryl ethers
Lignin 135
H2COH H2COH
I
HC HC
II II
HC HC
HC ~H"~-"i"f OCH3 . c , / --y "oc.

HC 44.._-.--0 HC 0
Ioc.3
0
~OCH
OH
3
Scheme 6. The phenyl-
coumaran, (C~-C5)interunit
linkage of lignin
o.
HCOH \OCH3 R=H, or OCH3
R~OCH3 Scheme 7. The diaryl propane (13-1)interunit link-

OH ages of lignin
are generated by the combination of an o-methylquinonoid (structure 2 in

Scheme 4) with a phenoxy radical [60, 68, 69]. They comprise only about 6% of
the phenylpropanoid units in spruce lignin [60, 68]. Non-cyclic benzyl alkyl
ethers, present in relatively small amounts in lignin (2-3%), have been cited for
their beneficial effects during pulping [70]. However, recent attempts to detect
them in milled wood lignin, using advanced N M R techniques, have failed [-81].
The combination of C~ and phenoxy radicals (Scheme 5) results in the formation
of 13-O-4linkages [68, 60, 69], linking 3 ~ 5 0 % of the lignin units [64]. Structures
which contain ~, 13ethers are relatively infrequent and have been estimated to
account for less than 1% of the interunit linkages in lignin. However, recent
efforts using advanced N M R techniques have questioned these levels [81,364].
Another lignin structure, containing two ether groups is the pinoresinol
(present in less than 5% of the units) [71]. This structure, is formed by the [3-13
coupling of two coniferyl alcohol radicals followed by double ring closure, as
illustrated in Scheme 8.
Benzyl alcohol groups are also common structures in lignin. These, which
account for more than 30% of the phenylpropane units, are formed by the
addition of a water molecule to a quinonemethide.
Like aliphatic esters, the benzylic structures are also beneficial during alkaline
pulping [72] since they facilitate cleavage of the arylglycerol-[3-aryl ether resi-
dues under soda or kraft pulping conditions with the intermediate formation of
epoxide or episulfide structures. 5-5'biphenyl and diarylmethane structures
which link two aromatic units either through the C5-C5 or Ca-C5 positions,
i~ ~
O
~,,~OCH~I
OH
.fOCH3
CH2OH
.2?
1 '
.,o~"~
HzC CH
I
H C - - CH HC- - CH
I I
I I
CH3Of~[ CH2OH HC~ CH2
I C H 3 0 ~ O/CHz
o
coniferylalcoholradicals
cn30
~o~~ OH
pinoresinol
Scheme 8. Formation of pinoresinol linkages in lignin
CH2OH CH2OH
CH
II
CH
II
CH CH
CH30~OCH3
OH OH Scheme 9. The biphenyl 5 5' interunit linkage of lignin
contain carbon-carbon bonds, which are stable under pulping conditions. While
5-5' biphenyl units are present in the original wood the diarylmethane units are
formed by the condensation of two aromatic rings in lignins during alkaline
and/or kraft pulping (Scheme 9) [73].
As such, their importance becomes of significance in technical lignin prepara-
tions. The frequency of occurrence of such structures is obviously higher in
pulped softwoods, since their lignin is composed almost exclusively of guaiacyl
units which possess aromatic units that contain a free C5 position.
Recently Brunow's group announced the discovery of another bonding pat-
tern present in softwoods lignin [74, 75]. This involves the formation of
a, [3 ethers on the same 5-5' biphenyl structures. The new octagonal moiety has
been identified as the dibenzodioxocin of Scheme 10.
Based on these structural details, a number of lignin models have been
proposed by a variety of investigators. Amongst them one may cite those of
Freudenberg [68], Brauns [76], Erdtman [77], Adler [78], Forss and Fremer
[79], and Glasser and Glasser [80]. Amongst the models proposed for softwood
lignin, Freudenberg's is still the most widely cited [53, 68].
A very significant analytical effort has been carried out through the past
several decades attempting to quantify the various bonding patterns in softwood
and hardwood lignins. The results of some of these efforts are now summarized
in Table 1.
Lignin 137
O O
', (
OH O.~.
R = CH2OH, CH 3 Scheme 10. Dibenzodioxocin present in softwood lignin
Table 1. The frequency of the major linkages in softwood and hardwood lignins* [8, 55, 64, 81-84]
% of Total Phenylpropane Units
Type of Linkage In Softwoods In Hardwoods
[3-Aryl ether ([~-O-4) 45~48 60

a-Aryl ether (a-O-4) 6-8 6-8
Diphenyl ether (4-0-5) 3.5 8 6.5
a-Alkyl ether (c~-O-7) small small
Biphenyl (5-5') 9.5-17 4.5
13-1 7 10 8
Pinoresinol (13-[3) 3
Phenylcoumaran (f3-5) 9 12 6
Lignin-carbohydrate links not determined not determined
Dibenzodioxocin >> >>
* The numbering of the carbons used in the nomenclature of this table is shown in Scheme 1.
2.4 The Lignin-Carbohydrate Connectivity

Detailed studies of wood and grasses point to the likelihood that lignin is not
simply deposited in the cell wall of polysaccharides but is closely linked and
associated in a certain architecture with the carbohydrates [85 88]. A consider-
able research effort has been carried out in studying the nature of the actual
chemical bonds between lignin and carbohydrates [89-91].
Advances in the technology of nuclear magnetic resonance spectroscopy
coupled with a better understanding of the nuclear spin relaxation mechanism in
solids, have provided a powerful tool for probing the molecular motion of
carbohydrates and lignin in solid wood and pulp samples [9~95]. By using
ferric ions as a probe, coupled to proton spin-lattice (T1) and spin-lattice in
a rotating frame (Tlo) relaxation time measurements, Gerasimowitz et al. [94]
derived information related to the structural connectivity of carbohydrates and
lignin in wood pulp. Similar linkages were also invoked by Kolodziejski et al.
who studied the I3C CP/MAS NMR spectra of lodgepole pine wood [96]. By
measuring the proton spin-lattice relaxation times of progressively sulphonated
and methylated wood and pulp samples, Argyropoulos and Morin evaluated the
molecular mobilities of lignin and carbohydrates in the presence of different
counterions [95]. Lignin-carbohydrate associative interactions were invoked,
once again, to rationalize for their observations. On another front, significant
advances in clarifying this issue have been made by carrying out in vitro
lignification experiments in the presence of model compounds. Small sugar
molecules were found to undergo addition reactions resulting in the formation
of ether or ester linkages [97-100]. Early work by Freudenberg demonstrated
that sorbitol or sucrose can readily add to lignin, forming benzyl alkyl ether
linkages [101]. These bonds were found to be further stabilized when the
phenolic hydroxl group of the lignol portion was etherified. Such an etherifica-
tion step is not uncommon to occur via a dehydrogenative mechanism during
lignification. Such an ether linkage, which seems to be considerably more stable
than those of glucosides, is considered to be one of the main reactions leading to
a stable crosslink between lignin and plant polysaccharides. Another lignol-
sugar that has been isolated by Harkin and Freudenberg from in vitro lignifica-
tion experiments, is the guiacylglycerol-[3,y-disucrose ether [98]. The formation
of this ether is believed to follow a radical mechanism which could be associated
with that of lignin biosynthesis. According to this mechanism, coniferyl phenoxy
radicals (catalysed by peroxidases) abstract a hydrogen from a sugar molecule to
generate a sugar radical. This, quickly adds to another lignin C~ radical. The
nature of the resulting bond, (C-C or ether) depends upon the formation of
a carbon radical or an oxygen radical on the sugar molecule. However, the
attachment of two sugar units on a phenyl propane unit is an unlikely event
when one considers that during lignification a variety of competing nucleophiles
are present, including water and other lignols. More recently Leary and co-
workers have shown that sugars will readily add to monomeric lignin
quinonemethides or benzyl alcohols forming predominantly C~ linked carbo-
hydrate benzyl ethers or esters [102].
Ester bonds are also involved in the attachment of lignin to the plant
carbohydrate moiety [103, 113]. Their importance is very significant for plants
and grasses and somewhat less for wood. Ferulic acid in grasses is known to be
esterified with carbohydrates, and etherified with lignin. However, the
topochemistry of its attachment to lignin is not well understood [-104-106].
Similarly, p-coumaric acid is known to be extensively esterified with lignin, but
the regiochemistry of lignin acylation is still a matter of debate [107 109]. The
following description of events represents the state of our knowledge as far as
the biosynthetic pathways leading to such species are concerned. The a-position
of quinone methides, formed during the dehydrogenative polymerization pro-
cess [64], apart from being attacked by water, may also be attacked by free acids
and alcohols, leading to a-esters and a-ethers. This is the case of feruloyl esters
and phenolic glycosides of p-coumaric acid, where the free phenol or the free
Lignin 139
carboxylic acid groups respectively, may trap quinone methides by an addition

to yield a-ethers and a-esters [110, 111]. Alternatively, feruloyl esters can
directly participate in the free radical polymerization process giving rise to
a number of different structures [112]. Furthermore, enzymatically pre-esteri-
fled p-coumaric acid with p-hydroxycinnamyl alcohol monomers, may enable
the formation of p-hydroxycinnamyl p-coumarates which could participate in
the formation of the lignin macromolecule by conventional oxidative coupling
reactions to yield 7-p-coumaroylated lignin [-108].
While ether-linking ferulic acid to the a position of the lignin side chain, via
"opportunistic" quinone methide trapping is still speculative, it has been re-
ported by Ralph et al. [104] that feruloyl esters, if present in the lignifying
matrix, are capable of participating in the free radical lignification process.
Moreover, the identification of new ether-linked ferulic acid-coniferyl alcohol
dimers in grass straws, by Jacquet et al. [106], demonstrates the occurrence of
radical coupling reactions between ferulic acid and coniferyl alcohol to yield
13-aryl ether structures.
As noted above, the lignin-carbohydrate bonds are possible due to the
reaction of quinone methides with various lignols and carbohydrates during
lignin biosynthesis. Despite this there is still controversy whether these bonds
occur between lignin and cellulose or between lignin and hemicelluloses. This is
certainly not an easy issue to resolve but the evidence is more in favour of
a predominant lignin-hemicellulose connection. Since in hardwoods the pre-
dominant hemicellulose is xylan and considering that about 90% of this sugar
can be removed by a mild alkaline treatment, ester links have been proposed to
occur between the lignin and the C6 uronic acid groups of the sugar [85, 114].
3 Lignin Architecture
Amongst the features of any delignification experiment is the observation that

the molecular weights of the solubilized lignin become progressively higher as
delignification proceeds [114 125]. Molecules of relatively small size become
soluble early in the delignification process, while larger fragments appear in
solution in latter stages. This usually occurs when lignin in wood is made soluble
by chemical treatments [115, 116, 126, 127]. Szabo and Goring in 1968 recog-
nized this effect and proposed the gel degradation theory to account for these
observations. However, prior to their proposition a number of other suggestions
were made. These will now be briefly discussed in order to expose some
additional salient features of lignin's polymer chemistry.
The conjugated phenolic nature of the lignin precursors (Scheme 1) allows for
the possibility that condensation reactions occur under delignification condi-
tions. It is thus possible that the molecular weight of soluble lignin may increase
if lignin in wood consists of finite macromolecules with reactive sites that
condense under acidic or basic [128-131] delignification conditions [132-136].

This is generally known as the "condensation theory".
With respect to the condensation theory, a point of major criticism emerges
from the results of Adler et al. [137], Felicetta and McCarthy [138], and Yean
and Goring [116], where a decrease in the molecular weight of soluble lignosul-
fonates was observed upon a second exposure to the reaction conditions. This
contradicts the condensation theory because one would expect, on further
reaction, either an increase or no change in the molecular weight. Nevertheless,
it is now accepted that condensation reactions occur during delignification.
Model compound studies have suggested that such reactions operate to some
degree under both acidic [128] or basic conditions of delignification [129].
During acidolysis, the formaldehyde formed from lignin has been found to be
partly consumed in condensation reactions [139]. Quantitative gel filtration
studies on acidolysis products [128], however, have revealed that the fraction of
"unidentified condensation products" amounts up to 14% by weight of the
isolated lignin. It is thus more prudent to conclude that condensation possibly
occurs during delignification [140]. Its effects, however, are only secondary
contributions to the molecular weight increase phenomenon observed in sul-
fonic acid delignification. Evidence substantiating this suggestion has been given
by Argyropoulos and Bolker [141]. In their communications, Brown et al. [142,
143] concluded that lignin in wood consisted of finite molecules, with two major
components of different molecular weights. Similarly, Obiaga et al. [144] postu-
lated that lignin in wood existed as an assembly of finite entities with a degree of
polymerization equal to 18. Both conclusions were based on the apparent
bimodal distributions observed when lignin samples were chromatographed on
Sephadex gels. These interpretations were later proved to be incorrect by Bolker
et al. [145]. The peak at the high molecular weight end of the chromatograms
was shown to be an artifact, the effect of a high molecular weight tail manifesting
itself at the exclusion volume of the Sephadex gel. Later results of Mbachu [146]
further strengthened the findings of Bolker et al. A low exclusion volume peak has
indeed been reported by Cooper [147] for high molecular weight polystyrene.
Therefore, condensation reactions most likely occur during delignification,
thus producing a network polymer which must then be degraded during delig-
nification. The effects of condensation, however, are secondary contributors to
the molecular weight increase phenomena observed during wood delignification.
Another hypothesis attempting to rationalize the molecular weight increase of
solubilized lignins, is the pre-sieving concept [163]. According to this hypothesis
the lamellar structure of the cell wall or more specifically the pore size, deter-
mines the size of the dissolved lignin macromolecules when wood is delignified.
This is particularly true during the washing of kraft pulp when high molecular
weight lignins diffuse out of the fibres [148, 149]. Lignin fractions, which turned
to be of the order of hundreds of thousands [150], would be too large to pass
through the porous structure of the cell wall. Similarly it was found that when
lignin macromolecules were spread on the surface of a non-solvent [121,152] or
deposited on a carbon coated grid for electron microscopy [153], they assumed
Lignin 141
a thickness of 2 nm, which is considerably larger than the pore size. Only after
the size of the pores becomes larger, as lignification proceeds [148] could these
fractions diffuse out of the cell. This may account for the presence of small
molecules early in the delignification, while larger molecules are released later,
as delignification proceeds. This theory was criticized by Bolker et al. [,145] on
the basis of the work of Bogomolov et al. [123], who found that the molecular
weights of milled wood lignins (see Sect. 5.1) increased with increasing yield.
This could not have occurred if the cell wall pores were responsible for the
sieving action. In milled wood there is no longer need for lignin fragments to
diffuse through any pores because the wood has already fragmented to sizes
much smaller than the size of individual cells.
3.1 The Gel Degradation Theory

The pioneering work of Szabo and Goring [127], which was followed by Bolker
and Brenner [155], and Bolker, Rhodes and Lee [145], dealt with the gel
degradation concept of delignification. In broad terms, the Szabo and Goring
treatment envisages delignification as the reverse of polymerizing a trifunctional
network, while the Bolker and Brenner approach is the reverse of polymerizing
a network formed by the random crosslinking of monodisperse preformed
chains. The difference between the two treatments arises from the assumption of
Szabo and Goring that all ether bonds in lignin are of comparable reactivity
[-145, 156]. Bolker and Brenner, however, proposed that the sites of crosslinking
were the benzyl ether groups, which are expected to be the most reactive of all
the ether linkages found in lignin. They further invoked a monodispersity
approximation for the primary chains of lignin. They based their argument on
Hess' measurements of the molecular weight distribution of Brauns' native
lignin [157]. According to the gel degradation theory, Brauns' native lignin
represents the final sol fraction remaining from the gelation process of lignin.
Accordingly, Bolker and Brenner argued that Brauns' native lignin mainly
comprised primary uncrosslinked chains, with a monodispersity ratio (as they
calculated from Hess) of Mw/M,, = 1.04. Yan [158], not fully convinced of the
validity of the monodispersity approximation, extended the Flory-Stockmayer
theory so as to include the crosslinking of primary chains with any initial size
distribution. In a series of three papers Argyropoulos and Bolker undertook to
experimentally test the reversibility of the Flory-Stockmayer statistics of gela-
tion by using synthetic model networks [159 161]. The results of their efforts
have provided support not only for the gel degradation theory but supplied
information on the solution properties and the molecular weight species distri-
bution expected from the random degradation of network polymers [162].
Evidence of the validity of the network structure of protolignin arrives from the
viscosity, polydispersity, and molecular weight data of soluble lignins discussed
previously. The random degradation of a crosslinked network is expected
(according to the gel degradation theory) to yield polymeric fragments of

spherical configuration whose molecular weights, yields, and polydispersity
ratios will increase as bond fragmentation intensifies within the network. The
network theory of protolignin finds progressively wider acceptance among
researchers in the field as the experimental evidence pointing toward its validity
grows. Although there is still lack of agreement on the precise architecture of
protolignin, major efforts toward solving this controversial issue are emerging
from the application of the gel degradation theory and its various modifications
[158, 164].
3.2 Possibility of Order in Lignin

Despite the fact that a significant compilation of scientific evidence has been
accumulated over the years, pointing to lignin as an amorphous, crosslinked
network polymer [165 167], formed via a random free-radical polymerization
process [169 172], recently discovered aspects of lignin chemistry and details of
its deposition into the cell wall have challenged the universality of this descrip-
tion [168].
Detailed analytical studies of synthetic (DHP's) and milled wood lignins have
documented that such preparations are rather distinct as far as their bonding
patterns and composition is concerned [171]. However, synthetic lignins pre-
pared in the presence of carbohydrates, under conditions that mimic the micro
environment of the cell wall, were found to resemble native lignins [173]. These
observations, coupled with studies of the organization of lignin within the
woody tissue [85-88], point toward the presence of an organizing influence
operating during lignin synthesis and deposition [174, 175]. This dominant
organising influence, according to several researchers [-176 181], can be pro-
vided by strong associative interactions operating between lignin precursors
and the ordered polysaccharidic matrix. Experimental evidence for these
interactions has been provided by studying the adsorption of oligolignols
onto cellulose [182] and by measuring the photoconductivity of wood tissue
[183].
While the cellulose microfibrils are known to be deposited in the cell wall in
complex ordered patterns [,184, 185], the precise deposition patterns of hemi-
celluloses remain unknown. Evidence pointing toward the possibility of hemi-
celluloses being aligned to cellulose [186, 187] and linked to lignin [,188] has
been provided. These observations coupled with the associative interactions to
be discussed later (Section 4.1), and the observation that the cellulose microfib-
rils are closely packed [-189-192], are factors that may indeed contribute to
some organization within lignin. In actual fact, Jurasek in his recent computa-
tional efforts, aimed at modelling lignin, has showed that the spatial constraints
of the cell wall may indeed impose some degree of regularity in lignin [193].
Gravitis and Erins [194] have also examined lignin with various theoretical
Lignin 143
conformational models and have arrived at the conclusion that "under some
conditions quasi-ordered regions of lignin structure can be expected".
On a different front, Atalla has provided evidence indicating that the aromatic
rings of lignin are aligned tangentially to the secondary wall [195, 196]. Based
on these observations, he proposed a new model for the assembly of lignin
[174]. The model suggests that variations in the structure of hemicelluloses, may
result in corresponding systematic changes in the structure of lignin and cellu-
lose [175, 197-199]. While cellulose provides the primary framework, hemicel-
luloses furnish various branches which associate themselves with specific lignin
precursors. More specifically, it would be anticipated that specific monosac-
charide branches are designed to organize the monolignols, while di- and
trisaccharidic branches are designed to selectively bind specific di- and trilignols.
As such, lignin precursors may be subjected to a certain regulatory mechanism
which involves steric factors and sugar binding specificities. Perhaps the most
attractive feature of this model is that it introduces a hierarchic and sequential
control that occurs at multiple levels, in different phases and separate locations
throughout the biosynthetic pathway. This model is different from traditional
models which emphasize the compartmentation of the process [200] involving
different extracellular or membrane bound enzymes [201-203].
Other evidence pointing toward lignin possessing certain orientation with
respect to the cell wall has been provided by its ability to conduct electric charge
as witnessed by photoconductivity measurements of wood [183, 204]. Since
conductance is highly depended on the coherent orientation of the structures
involved in the conduction, the ability of lignins to carry ionic charge has
been taken as proof for the presence of a regular array of similar functionalities
which may become the vehicle for the excitation transfer of electric charge
[-205-207]. A reasonable explanation for the observed photoconductivity in
wood is that the regularity of the surfaces in the polysaccharidic matrix prob-
ably imparts to the lignin a coherence of spatial organization that is sufficient to
facilitate some interactions or coupling between its lowest unoccupied molecu-
lar orbitals.
In 1994 a report of "visual encounter" of order in lignin appeared [208].
Using a scanning tunnelling microscope (STM) images of building units or
modules assembled into larger assemblies were claimed to have been recorded.
During the same year Faulon and Hatcher [209] presented their calculations
that suggested the biopolymer to have a helical structure, characteristic of many
naturally occurring macromolecules.
When one considers most of the evidence presented above, it becomes
apparent that a better paradigm for lignin needs to be developed. As
Goring concluded [210], one should distinguish between lignin in the
middle lamella and that of the secondary wall. Until the lignification process has
been fully understood, the traditional concept of lignin being a uniform,
amorphous, three dimensional network polymer, is probably too simple to
reconcile with all the recent scientific data relating to its native structure and
properties.
4 Solution Properties of Lignin
Early attempts to study the properties of lignin in solution commenced with the
observation that native lignin is insoluble in all good solvents [-211]. In 1956,
Bjorkman [212] discovered that when spruce wood is milled thoroughly, up to
50% of the lignin could be degraded and extracted in aqueous dioxane. During
the milling process, covalent bonds were found to rupture [-213], and low
molecular weight lignin fractions solubilize. This behaviour was also described
by Lindberg [214] and Schuerch [-211]. In 1960, Gupta et al. [215, 217] pointed
out that by invoking a three dimensional polymer network architecture for
lignin, the molecular weight distributions of isolated solubilized lignins could be
accounted for (Section 3). The soluble macromolecules believed to reflect the
properties of the network from which they emerged.
Amongst the most significant of the early observations were the predominant
physicochemical characteristics of lignin molecules in solution. These include
the intrinsic viscosity, branching parameter, and the degree of polydispersity.
Their determination provided useful structural information in relation to the
overall architecture of protolignin. The intrinsic viscosities [-216], at comparable
molecular weights, were found to be 1/40 of those of polysaccharides and about
1/40 of other synthetic polymers [-11, 215, 217]. The low intrinsic viscosities of
dioxane [218], kraft [-219], lignosulfonate [220], and alkali [215, 217] lignins, in
various solvents, led Goring to conclude that these molecules in solution are
compact spherical microgel particles [-11,218]. He also reported that the values
of the Mark-Houwink exponent (a), ranged between 0 and 0.5. This constituted
further confirmation that lignins in solution behave as molecules whose solvated
shape is something between an Einstein sphere (a "ball" impermeable to solvent)
and a non free-draining random coil. Similar conclusions were derived when
other parameters, such as sedimentation coefficients and diffusion constants,
were measured [221,222]. These measurements showed that soluble lignins are
rather compact molecules in solution, though not as compact as simple solid
spheres. In general the chains of the lignin macromolecules in solution are more
densely packed than those of linear flexible polymers such as polystyrene. The
branching parameter, or contraction factor (g) introduced by Zimm and Stock-
mayer [-223], when measured on various alkali lignin fractions, was found to
decrease with increasing molecular weight [-217], as expected for such molecular
configurations. Independently, Alekseev et al. [224] calculated the value of the
Mark-Houwink exponent (~) for lignin solutions using viscosity, sedimentation,
and diffusion data. His calculations supplied further support to the idea that
the lignin macromolecules in solution are of a rigid spherical configuration
[221,222]. More recent results of Pla and Robert [225] showed ~ to be 0.5 for
dioxane lignin solutions. They, too, interpreted their results in terms of the
branched or crosslinked nature of the dissolved lignin macromolecules. Low
Mark-Houwink exponents are not unusual for truly branched macromolecules
in solution. A good example of this can be found in the work of Argyropoulos
Lignin 145
and Bolker who have shown that the soluble fragments isolated from a series of
polyester network polymers beyond the gel point showed an 0~value of only O.15
[226].
4.1 Lignin Associative Interactions

Several authors have raised the possibility that lignin in wood is composed of
low molecular weight molecules which on becoming soluble tend to associate by
non-covalent linkages into complexes of higher molecular weight [227-232]. It
was shown by S. Sarkanen [228, 232] that this process seems to be reversible
and solvent dependent. Low molecular weight kraft lignin fractions generated
higher molecular weight complexes in certain solvents. Such associative interac-
tions among lignin components have been found to occur during gel permeation
chromatography in different mobile phases. Non-aqueous solvents exhibited
multimodal chromatograms, while alkaline aqueous eluents gave rise to broad
chromatographic envelopes with sufficient resolution only in the low molecular
weight region [233, 234]. Connors, Sarkanen and McCarthy [228] have dis-
covered that in non-aqueous solvents, association complexes of kraft lignins
gave rise to apparent molecular weights (as determined by GPC) as much as
three orders of magnitude larger than their constituents. These effects were
eliminated when LiC1 was added to the DMF used as the mobile phase.
Accounts by Ekman and Lindberg [235] and Lindberg [236] show very major
changes occurring in the solubility, hydrogen bond behaviour, and size exclu-
sion chromatograms of lignins after they have been methylated or acetylated.
Conclusions on extensive hydrogen bond formation between free hydroxyl
groups in dioxane lignins have also been reported by Hatakeyama et al. [237]
and Bogomolov et al. [238]. Obviously, any association between lignin molecules
in solution would cause an increase in their apparent molecular weight values.
4.2 Lignin Polydispersity

A considerable amount of scientific data exists supporting the fact that soluble
lignins and derivatives are of high polydispersity [239-246]. The weight to
number average molecular weight ratios for different lignins may vary from
about 3 to over 10-11 [270, 2713, indicating that lignins are extremely polydis-
perse materials. Fractionation studies on lignosulfonates [114] and alkali lig-
nins [241,217] have shown that they contain molecular weights ranging from
1000 to greater than a million (g tool- 1) [247]. The molecular weight distribu-
tion curves for lignins are expected to be unimodal. However, a number of
authors have reported bimodal distributions for soluble lignin [227, 248 249].
Such behaviour was also observed when lignin was isolated from the pulping
liquors of wood [227, 250]. It was only after improvements in chromato-
graphic techniques that these bimodal distributions could be to resolved into
distributions with several maxima [251-253]. More information on molecular

weight distribution within the soluble phase beyond the gel point, which
according to the gel degradation theory should resemble those obtained during
lignin degradation, can be found in the work of Argyropoulos and Bolker [162].
Recently, Glasser has produced a universal plot of polydispersity versus weight
average molecular weight for a series of prototype lignins supplying a linear
expression with some predictive power [270, 271].
5 Lignin Preparations
Generally, there is a lack of agreement on the ultimate structure of lignin and

this may be because, as Pearl wrote [254] "It is practically impossible to isolate
two lignin preparations with identical properties, even by the same procedure".
Although many methods are available for the isolation and purification of
lignin, none provides 100% yield and structural authenticity. To isolate lignin
which closely represents the native material, reactive chemicals and elevated
temperatures must be avoided.
5.1 Laboratory Lignin Preparations
Milled wood lignin (MWL) [255] and cellulase enzyme lignin (CEL) [256] are
good examples of preparations closely resembling the native material. Yet, these
preparations are never free of even minor chemical modifications. For example
several secondary reactions may occur during the milling process, as a result of
free radicals produced during the process [257]. Moreover, CEL preparations
are known to contain protein impurities introduced during the enzymatic
treatment. Despite these shortcomings, MWL and CEL preparations show
minimal structural modification with yields ranging between 25%-66% of
the total lignin and with carbohydrate contents ranging between 2-10%.
The molecular weights (Mw) of these lignin preparations range between
15,000-20,000 and predominantly consist of alkyl-aryl ether linkages [258].
Other techniques for isolating lignin involve treatment of the wood with
organic solvents such as dioxane [294] or ethanol [260, 261], sometimes in the
presence of catalytic amounts of mineral acids (H/SO4), or inorganic Lewis
acids, at elevated temperatures and pressures [262]. The combination of acids
and organic solvents causes the hydrolysis of the ether bonds in lignin and those
between lignin and carbohydrates. Such products are relatively free of carbo-
hydrates, and of low molecular weight fragments [263]. Another technique
involves the degradation of the cellulosic constituents of wood by sodium
paraperiodate [259, 260] or their solubilization by complexation with cupram-
monium hydroxide [261] (cuoxam lignin). The latter gives lignin in high yields
Lignin 147
which is known to retain the morphological features of wood [264, 265] and is
totally insoluble in organic solvents. Amongst various lignin preparations,
Fleming and Bolker found cuoxam lignin to be the most suitable for model
de!ignification experiments [266]. For additional methods of lignin isolation
procedures the reader is referred to more specialized texts [11,255, 263, 267].
Due to the variety of techniques available for isolating lignin, and the
structural variations introduced during the isolation, each lignin preparation is
usually identified by the wood species and the isolation technique, e.g., spruce-
dioxane lignin, or birch-cuoxam lignin. It is important to distinguish that all
these preparations are distinct from protolignin, which is a term used for the
material as it occurs in the plant tissue [268, 269].
5.2 Commercially Produced Lignins

Commercial lignins from hardwood and softwood trees are available from the
two major pulping processes, sulfite pulping and kraft pulping, which uses
sodium sulfide and sodium hydroxide. In addition, pilot plant scale procedures
exist which employ either organic solvents (organosolv pulping) or high pressure
steam followed by alkaline extraction (steam explosion pulping). While the
sulfite pulping process generates water-soluble lignosulfonates as their sodium,
magnesium, or calcium salts, the kraft process produces lignins that are soluble
only in aqueous alkali.
In addition to differences in structure and botanic origin, commercial lignins
also vary in molecular weight. Most such lignins are of weight average molecu-
lar weights (Mw) ranging between 3000 and 20,000 and polydispersity ratios
(Mw/Mn) between 2 and 12 [270,271].
6 Methods of Lignin Analysis
6.1 Degradative Methods

The methods described in this section require the sample to be exposed to
certain reagents and specified conditions that degrade the lignin in a prescribed
manner, in accordance with documented reaction sequences. The products are
then quantitatively analysed by chromatographic techniques yielding structural
information about the building blocks of the lignin sample [272]. Such tech-
niques suffer from being laborious, involving many steps and complex chemical
manipulations, often subjecting the derived quantitative information to large
errors and diminished reproducibility. Despite these limitations these methods
have offered significant advances to our knowledge of lignin structure and
reactivity.
Lignins can be selectively oxidized in alkaline nitrobenzene or permanganate
solutions. The alkaline nitrobenzene oxidation of lignin was first introduced in
1939 by Freudenberg and co-workers [273,274] and was further optimized by

Leopold [275 277]. Traditionally, nitrobenzene oxidation is used for the classi-
fication of wood or plant tissue and of lignin according to its botanical origin.
During this procedure the lignin or the fibrous sample is oxidized in sodium
hydroxide (2N) in the presence of nitrobenzene at 170-180 ~ for 3-4 hours. The
products, mainly aldehydes, originating from the guaiacyl, syringyl and p-
hydroxyphenyl structures in lignin are then subjected to quantitative determination.
The oxidation of lignin in pulps by standard permanganate solutions has been
the basis of the permanganate and kappa number tests, extensively used by the
pulp and paper industry as a means of rapidly determining the lignin content of
pulps. These techniques are based on the fact that potassium permanganate
readily and selectively oxidizes lignin in the presence of carbohydrates. The
permanganate number is conventionally reported to be the number of millilitres
of 0.1 N KMnO4 consumed by 1 g of oven-dry pulp. However, since the size of
the pulp sample and the amount of permanganate applied affected the results it
was modified by Tasman and Berzins [278,279] who proposed the kappa
number. As such, the sample size was adjusted to ensure that approximately half
of the applied permanganate is consumed. This test has been of extreme
significance to the pulp and paper industry in determining the degree of
delignification of a fibrous feedstock during pulping and bleaching operations.
Recent research, however, has brought to light that hexeneuronic acids, formed
during pulping, consume permanganate during the kappa number test leading
to significant errors specially when hardwoods are examined [280].
Analytical protocols involving alkaline permanganate oxidations of fibrous
and lignin samples may provide similar information to that obtained by nitro-
benzene oxidation. Moreover, these techniques offer structural details about the
frequency of the various bonding patterns in lignin [281]. Despite the many
steps involved and the fact that the obtained information is confined to units
possessing free phenolic hydroxyl groups, alkaline permanganate oxidations
have been extremely important in many aspects of lignin chemistry. The signific-
ance of the technique coupled with the complex nature of the various chemical
manipulations have caused the development of a four step standardized proced-
ure [53]. The procedure commences with an alkaline CuO predegradation step,
followed by a methylation step, and ends with two oxidation steps involving
permanganate and hydrogen peroxide [282]. The structure of lignins present in
neutral sulfite [283-285] and in kraft pulps [286-288] has been elucidated using
such techniques.
Another procedure which has significantly contributed to our understanding
of the lignin structure is that of hydrogenolysis [289-291]. The procedure
consists of hydrolytic and thermolytic reactions which cause the partial break-
down and solubilization of lignin. The lignin fragments, diffuse into the liquid
phase where they are rapidly stabilised against condensation reactions by in situ
generated hydrogen atoms on the surface of a catalyst [292]. Despite the fact
that the procedure is not in wide use today the principle may be applicable to
converting lignin into valuable low molecular weight products [293].
Lignin 149
Pepper et al. were the first to propose the principle of acidolysis as a proced-
ure for isolating lignin from plant material [294] and recently from kraft pulps
[-280]. Later the same principle was used as an analytical tool for determining
the occurrence of [3-0-4 and 13-5 structures in spruce lignins [-295]. The proced-
ure calls for refluxing the lignocellulosic material in 0.2 N HC1 in a dioxane:
water mixture (9 : 1, v/v). These conditions have been shown to cleave both a and
[3 aryl ethers linkages in lignin. Such studies have provided evidence for the
existence of additional structural elements in lignin such as 13-13, [3-1, and
quinonoid [69, 296 299]. Acidolysis protocols in the presence of catalysts, other
than hydrogen chloride, have been proposed by Yashuda [300] and Karlsson
[301] and reductive cleavage protocols by Shevchenko et al. [-302]. The main
critique of all methods involving acidolysis has been the fact that almost
invariably the product yields are rather low as a result of condensation reactions
taking place in acidic media [,168]. Consequently efforts have been made to
address these limitations. Since the combination of boron trifluoride and
thioethanol in anhydrous media is known to convert benzylic cation intermedi-
ates to thio-benzyl derivatives, Lapierre et al. have used this principle to develop
the technique of thioacidolysis [-303]. This combination has been shown to
quantitatively and selectively cleave the arylglycerol-[3-aryl ether linkages in
lignin [-303-305]. Recent developments of the technique involving the desulfur-
ization of the products using Raney nickel have been claimed to offer improve-
ments over earlier protocols [-306].
6.2 Non Degradative Methods
Non-degradative methods, involve mostly modern spectroscopic and other non

evasive techniques, yielding structural information about the sample without
the need of subjecting it to harsh chemical environments. Characteristic rc-~z*
transitions of the aromatic nuclei in lignin make it a strong absorber of UV light.
As such, lignin absorbs much more strongly than carbohydrates with an absorp-
tion pattern which has a maximum at approximately 280 nm. Based on detailed
measurements of the UV absorption spectra of model compounds involving
guaiacyl and syringyl moieties [-307], Fergus and Goring pioneered the use of
UV microscopy for studying the distribution of lignin across the cell wall, in
different morphological regions within woody tissue [308, 309] and during
delignification in kraft and neutral sulphite pulping [,310]. Similar efforts by
Boutelje and Eriksson E311] and Yang and Goring [-312] firmly established the
use of UV microscopy for such endeavours. The UV absorption spectra of
lignins may also be used to estimate their free phenolic hydroxyl content [-313].
The method is based on the fact that ionized phenolic groups in lignin absorb at
300 nm fully obeying the Beer-Lambert law. The use of this technique allowed
the determination of the phenolic hydroxyl content in the secondary wall, which
was found to be 50% greater to that of the middle lamella [,314]. The character-
istic UV absorption spectra of lignin have been used for the quantitative and
qualitative determination of native lignins [315, 316], lignosulfonates [317] and

the study of delignification of wood during pulping [318, 319]. Despite its
numerous advantages the method can give erroneous results in the presence of
contaminants that are strong UV absorbers. Its reproducibility has also been
questioned as a consequence of an international round robin study involving
many lignin standards [-320].
In recent years the application of nuclear magnetic resonance spectroscopy to
the characterization, classification and detailed structural elucidation of lignins
has seen widespread utility. The fact that the proton nucleus is of 100% natural
abundance and of high sensitivity made the 1H NMR experiment rather popular
in the early days of the application of this technique to lignin [321 323]. Almost
invariably, 1H-NMR is used on acetylated lignins [322-326] since this affords
better signal resolution. 1H-NMR signal assignment has been based on model
compound [327-329], decoupling [330], and 2D-NMR experiments [331].
Nevertheless, there are some essential limitations to 1H-NMR spectroscopy of
lignins. These include the rather limited range of chemical shifts (12 ppm),
extensive signal overlapping and proton coupling effects. The technique is only
suitable for the qualitative study of the proton distribution in lignins, while
carbon containing groups and labile proton functionalities (OH, COOH, SH)
remain beyond the capabilities of the technique. Recent developments have
claimed that after appropriate derivatization it is possible to quantitatively
determine the phenolic hydroxyl groups in lignin [332].
The early work of Ludemann and Nimz [49] has allowed the use of solution
13C-NMR spectroscopy to become an indispensable tool for the structural
elucidation of the carbon skeleton of lignin. Compared to proton NMR, the
13C_NMR spectra of lignin offer considerably better resolution, with no coup-
ling effects, over a much wider chemical shift range (200 ppm). Convenience,
speed, and a wealth of even quantitative information [333,334,337] have
contributed to the widespread use of ~3C-NMR for the structural analysis of
lignins. Furthermore, a variety of newly developed pulse sequences such as
DEPT [335], INADEQUATE [336], HMQC [81], as well as acquisitions on
13C-enriched lignin samples [337-339] have improved our understanding of
lignin structure and reactivity. Despite all these, some drawbacks remain as far
as the efficient use of ~3C NMR is concerned. These stem from the fact that the
a3C nucleus is not sensitive enough (1/5800 compared to proton) and that it is
only 1.1% naturally abundant. Consequently, relatively large sample sizes and
lengthy acquisition protocols are essential. When quantitation is sought extreme
care is required to allow for complete relaxation of the magnetization which
translates to very lengthy acquisition protocols and the need of external methoxyl
analyses so that the OCH3 signal to be used as internal standard [333, 340].
Advances in instrumentation have made the use of solid-state NMR spectro-
scopy a routine operation [341,342]. As such, various solid-state 13C-NMR
experiments can now be performed routinely on lignin [343, 344] and solid
wood or plant samples [345, 346, 95]. It is thus possible to obtain some informa-
tion about the lignin within a sample without its prior isolation [87]. Dipolar
Lignin 151
dephasing when used as part of the acquisition protocol of solid-state spectra

of wood and pulps has been documented to yield significant information in
relation to the degree of chemical modification occurring in lignin [-347].
Efforts to overcome some of the limitations imposed by proton and 13C_NM R
spectroscopies have prompted the examination of other NMR-active nuclei which
when covalently linked to lignin by appropriate derivatization procedures may
provide additional structural information for these heterogeneous biopolymers.
Early attempts examined the potential of silylation followed by silicon N M R for
the determination of hydroxyl groups in kraft lignin and related model com-
pounds [348, 349]. While the method offers resolved signals for aromatic, phen-
olic and carboxylic acids, large sample concentrations and long acquisition
protocols are essential due to the low natural abundance, low magnetic moments
and high relaxation times of the 298i nuclei. 19F-NMR has also been proposed as
a means of detecting different functional groups in lignins [350-353].
Finally, 31p_NM R spectroscopy has provided a new analytical tool capable
of detecting a variety of functional groups in isolated soluble lignins and within
lignin containing papers in the solid-state [-354]. More specifically, solution
3~p N M R has been used to examine soluble lignins [355] and carbohydrate
[356] samples after phosphitylation with 1,3,2-dioxaphospholanyl chloride and
2-chloro-4,4,5,5-tetramethyl-l,3,2-dioxaphospholane [357, 358]. F r o m a single
quantitative 3aP_NMR experiment it is possible to determine the three principal
forms of phenolic hydroxyls present in lignins i.e. p-hydroxyphenyl, guaiacyl,
and syringyl structures as well as primary hydroxyls, carboxylic acids, and the
two diastereomeric forms of arylglycerol-beta-aryl ether units (13-O-4 structures)
[-359]. Most significantly, however, the technique offers additional rapid (1-2 h
acquisition time) information in relation to the condensed structures in lignin
that can only be obtained by permanganate oxidation [360 364].
Acknowledgments. The authors would like to acknowledge the following indi-

viduals for having critically examined the manuscript prior to dissemination:
Dr. R.M. Berry, Professor K.E. Eriksson, Dr. I. Falkehag, Professor R. Francis,
Dr. G. Leafy, Dr. L. Jurasek, Professor R.H. Marchessault, Mr. Y. Sun and
Dr. S. Shevchenko.
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Fungal Delignification and
Biomechanical Pulping of Wood
M. Akhtar 1, Robert A. Blanchette2, and T. Kent Kirk 3

1 University of Wisconsin Biotechnology Center and Forest Products
Laboratory, Madison, WI, USA
2 Department of Plant Pathology, University of Minnesota, St. Paul, MN, USA
3 Forest Products Laboratory, Madison, WI, USA
1 Fungal Degradation of Wood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
1.2 Wood Decay by White-Rot Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
1.2.1 Microstructural Changes and Delignification . . . . . . . . . . . . . . . . . . . . 161
1.2.2 Lignin-Degrading Enzyme Systems . . . . . . . . . . . . . . . . . . . . . . . . . 164
1.2.3 Enzyme Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
2 Biopulping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2.2 Past Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
2.3 Biomechanical Pulping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
2.3.1 Screening of Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
2.3.2 Evaluation of Selected Fungi in Biopulping Runs . . . . . . . . . . . . . . . . . 174
2.3.3 Optimization of Biopulping Runs . . . . . . . . . . . . . . . . . . . . . . . . . . 177
2.3.4 Microscopy Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
2.3.5 Engineering and Economic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . 185
2.3.6 Prospects for Biopulping Commercialization . . . . . . . . . . . . . . . . . . . . 190
3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
This review article summarizes the results on microstructural changes and delignification,
lignin-degrading enzyme systems, and biopulping of wood with lignin-degrading fungi. Biopulping,
defined as the treatment of wood chips with lignin-degrading fungi prior to pulping, saves substan-
tial amount of electrical energy during mechanical pulping, results in stronger paper, and lowers the
environmental impact of pulping. Optical properties are diminished; however, brightness can be
restored readily with peroxide bleaching. The economics of the process look attractive if the process
can be performed in a chip-pile based system. Past work on biopulping had been minimal, however
a comprehensive evaluation of biopulping at the Forest Products Laboratory suggests that biopulp-
ing has a good chance of commercial success.
9 Springer-VerlagBerlin Heidelberg 1997
160 M. Akhtar et al.
1 Fungal Degradation of Wood
1.1 Introduction
Although wood can be attacked by an array of microorganisms, fungi are the

predominant decomposers in terrestrial ecosystems. Extracellular enzymes pro-
duced by decay fungi and subsequent decomposition processes differ among the
various groups of fungi, resulting in different types of wood degradation. Brown,
soft, and white rot are categories used to separate different forms of decay. These
categories are based on the macroscopic characteristics of the advanced stages
of decay. Brown rot is caused by fungi taxonomically classified in the
Basidiomycotina. These fungi cause rapid and extensive depolymerization of
cellulose early in the decay process [ 1 4 ] . Wood polysaccharides are degraded,
lignin modification occurs, and relatively small amounts of lignin are lost as
decay progresses [-5-7]. In advanced stages of decay, the residue is a brown
mass, which mostly consists of lignin. This decayed wood is sponge-like when
wet, but often cracks and checks into cubical pieces as the wood dries. Brown-
rot fungi are common decomposers in conifer forests and also are responsible for
most decay found in buildings and wood in service.
Soft rot is a term first used to describe decay by fungi attacking wood
surfaces in wet environments [8]. Fungi in the Ascomycotina and Deutero-
mycotina attack wood surfaces resulting in soft, gray to brown decay. Since soft
rot was first reported, knowledge of these organisms has expanded--soft rot
fungi have been found associated with wood in many different situations, such as
in wood treated with preservatives or in wood that receives only intermittent
moisture [6, 9-11]. Two forms of soft rot attack, Type I and Type II, have been
identified [12]. Type I consists of cavities within the secondary wall. Mycelia in
cell lumina produce fine penetrating hyphae that enter the secondary wall, align
growth along the microfibrillar axis of the cell wall, and produce chains of
cavities. The Type II form of attack consists of an erosion of the entire secondary
wall originating from hyphae in cell lumina and progressing toward the middle
lamellae. The entire secondary wall may be degraded, but the middle lamella is
not attacked. Certain fungi, including Hypoxylon spp. and Xylaria spp., were
previously considered to be white-rot fungi, but their mode of attack is more
correctly classified a Type II form of soft rot [6, 13].
Fungi that cause white rot belong to the Basidiomycotina and have the
capacity to degrade all cell wall components, including lignin. The extent of
lignin degradation can vary considerably among species of white-rot fungi [14].
Some species, such as Trametes versieolor, are nonselective in how they degrade
the wood, i.e., they simultaneously degrade lignin, cellulose, and hemicelluloses.
Other species, such as Phellinus pini, Ceriporiopsis subvermispora, and Phlebia
tremellosa, cause preferential degradation of lignin [15]. Some species deplete
lignin, cellulose, and hemicellulose in varying ratios; many species attack both
nonselectively and selectively in different areas of the same substrate [14].
Fungal Delignificationand BiomechanicalPulping of Wood 161
Although white-rot fungi have been categorized by whether they cause selective
lignin degradation or nonselective decay, it is apparent that the degradation
processes of these fungi are extremely variable. Even different strains of one
species of white-rot fungus were recently shown to degrade cell wall components
differentially [16].
1.2 Wood Decay by White-Rot Fungi
1.2.1 Microstructural Changes and Delignification
White-rot fungi enter cell lumina and rapidly colonize ray parenchyma cells that
contain free sugars and other nutrients. The radial arrangement of the ray
parenchyma facilitates access into the wood and allows widespread distribution
of the fungus in the substrate. Access to adjacent cells occurs via pit apertures, or
direct penetration may take place directly through the cell wall [2, 4]. Once
easily-assimilated substances are depleted, degradation of the cell wall is in-
itiated. White-rot fungi that attack all cell wall components simultaneously
cause a localized erosion of all cell wall layers. The attack progresses through the
secondary wall layers and middle lamella (Fig. 1A and B). In advanced stages of
decay, cell walls are eroded extensively, and holes within adjacent cell walls are
frequently observed (Fig. 1B). A different form of cell wall attack occurs in
white-rot fungi that selectively degrade lignin. Hyphae in cell lumina degrade
lignin progressively from the lumen edge of the secondary wall toward the
middle lamella (Fig. 1C and D). Investigations using brominated wood and
X-ray microanalysis of the bromine-lignin complex showed that white-rot fungi
remove lignin from the secondary wall before the middle lamellae between cells
are degraded [17,18]. As the delignification process continues, the middle
lamella is degraded and cells separate from adjacent cells (Fig. 1C and D). The
delignified, cellulose-rich secondary wall remains relatively unaltered (Fig. 1C
and D). The degradation of lignin is extensive throughout the cell walls,
originating from only one or two hyphal filaments within each cell lumen
(Fig. 2A and B).
Wood degradation may be influenced significantly by the ligno-
cellulosic substrate. An important factor that governs the extent and rate
of decay is the amount and type of lignin present in the wood. Wood
from gymnosperms has greater concentrations of lignin than wood from
angiosperms and consists primarily of guaiacylpropyl units. Lignin from
angiosperm wood is composed of varying amounts of syringylpropyl
and guaiacylpropyl units. In studies evaluating decay by white-rot fungi in
different types of wood, angiosperm wood was found to degrade more rapidly
and to a greater extent than gymnosperm wood [1, 15, 19]. Synthetic syringyl
lignin has also been shown to be depolymerized more rapidly than synthetic
guaiacyl lignin in a laboratory investigation using Phaneroehaete chrysosporium
[20].
Fig. 1. Wood decayed by white-rot fungi with simultaneous removal of all cell wall components
(A and B) and preferential degradation of lignin (C and D). A nonselective attack erodes all cell wall
layers, including the compound middle lamellae. Erosion zones coalesce to form holes through cell
walls (arrows).C and D: Lignin in secondary walls and middle lamella regions has been removed by
selective delignification. Cells separate from adjacent cells as a result of the lack of middle lamellae.
The cellulose-rich secondary wall remains. Transverse sections, scanning electron micrographs.
Bar = 10 mm
D e l i g n i f i c a t i o n in a n g i o s p e r m s b y w h i t e - r o t fungi also a p p e a r s to be in-

fluenced b y the syringyl lignin c o n t e n t of the w o o d . Studies of w o o d d e c a y in
Nothofagus dombeyi from the t e m p e r a t e rain forests of s o u t h e r n Chile s h o w e d
extensive delignification b y Ganoderma australe [14, 21]. This is one of the few
l o c a t i o n s in the w o r l d where h u g e logs m a y be c o m p l e t e l y delignified. In
Fig. 2A, B. Selective delignificationin cell walls appears as electron-lucid zones after KMnO4
fixation and transmission electronmicroscopy.A Lignin is progressivelyremovedfrom the second-
ary wall and then from the middle lamella (arrows show extent of delignification).B: Completely
delignifiedcells-detachedcells free of middle lamellae. Bar = 5 mm
temperate forests of North America, Europe, and Japan, delignification usually

occurs in small pockets within the wood or sometimes in large localized zones,
but the delignification of entire logs has not been reported. Nothofagus dombeyi
has an extremely high syringyl lignin content [21J--three to six times that of
Acer, Betula, and other hardwood species--and, worldwide, it appears to be
among woods with the highest content of syringyl lignin. Other angiosperms
growing in southern Chile, such as Laurelia phillipiana, that have low syringyl-
to-guaiacyl lignin ratios are not delignified when decayed by G. australe. In-
stead, a nonselective white-rot type of degradation occurs [21]. These results
strongly suggest that the concentration of syringyl lignin within angiosperm
wood greatly influences the delignification process.
Although white-rot fungi preferentially degrade syringyl lignin in angio-
sperms, some white-rot species extensively degrade guaiacyl lignin in gymno-
sperms. Fungi such as P. pini, Heterobasidion annosum, and P. weirii are usually
found only in coniferous wood and apparently have evolved highly efficient
mechanisms of lignin degradation. Other species of white-rot fungi also do not
appear to be deterred by the guaiacyl lignin of pines and other conifers. Species
such as C. subvermispora can readily delignify the sapwood of loblolly pine,
causing considerable amounts of lignin removal [16]. These species are of
special interest for use in biological pulping processes because they can degrade
lignin in conifers as well as hardwoods.
The process of preferential delignification of woody cell walls by white-rot

fungi has been elucidated using a variety of techniques. Histological stains and
electron-dense compounds that react with lignin can be used to visualize lignin
removal from the cell wall [22, 23]. KMnO4 can be used to fix wood for
transmission electron microscopy and to observe the distribution of lignin in cell
walls. Moderate electron density is evident in the secondary wall where lignin is
found with cellulose and hemicellulose, but intense electron density can be
observed in the highly lignified middle lamellae [17]. As hyphae of white-rot
fungi, located in cell lumina, begin to delignify the secondary wall, an electron-
transparent zone develops (Figs. 2A). This clear zone progressively moves into
the secondary wall. Once lignin is removed from this region, the middle lamella
between cells and, in very advanced stages of decay, the cell corner regions are
degraded and become less electron dense (Fig. 2B) [14].
A light-based microscopic method that employs bright colored stains (astra-
blue and safranin) has recently been used to differentiate zones of delignification
from nondecayed cells that retain lignin [24]. Colloidal gold cytochemistry
using gold-labeled endo-l,4-b-glucanase II, 1-4-b-o-glucan cellobiohydrolase I,
and endo-1,-4-b-xylanase revealed that residual delignified wood contains cry-
stalline and amorphous cellulose but little xylan [25, 26]. Other studies also
have shown that hemicelluloses are usually depleted from wood as lignin is
being degraded [14, 27].
1.2.2 Lignin-Degrading Enzyme Systems
Lignin-degrading enzymes were discovered, and subsequently characterized, in

P. chrysosporium (28 32). Research on the biochemistry of lignin degradation
began in earnest in the 1970s, when 14C-lignins were prepared and used to
determine which groups of microbes are able to mineralize lignin (decompose
it to 14CO2). The higher basidiomycetous fungi were found to be the most
proficient, and P. chrysosporium was chosen for detailed study for a number of
reasons related to ease of experimentation. Subsequent research with this fungus
described the culture parameters important for lignin mineralization and
showed that one or more steps are "secondary metabolic" events triggered by
limitation for certain nutrients. At the same time, the chemical changes that
occur in the lignin polymer during decay by ligninolytic fungi were described
(reviewed by Kirk and Farrell [29]). In the early 1980s, specific degradative
reactions accomplished by ligninolytic cultures of P. chrysosporium were de-
scribed using synthesized "dimeric" lignin model compounds; it was also found
that certain polymeric dyes are decolorized. Cell-free enzyme preparations that
catalyzed dimer cleavage and dye decolorization were reported in 1983, and
subsequently the responsible peroxidase (lignin peroxidase) was described.
Evidence to date indicates that three oxidizing enzymes, lignin peroxidase
(LIP), manganese peroxidase (MnP), and laccase are responsible for the initial
fragmentation of the lignin polymer and production of low molecular mass
breakdown products in white-rot fungi. Not all white-rot fungi apparently

produce all three enzymes, although some, including T. versicolor, do. P.
chrysosporium produces only LiP and MnP (however, laccase has recently been
reported in P. chrysosporiurn [33]), whereas C. subverrnispora produces only
MnP and laccase, and Phlebia ochraceofulva produces only LiP and laccase
[32]. The extracellular H202 required as electron acceptor for the peroxidases is
supplied by glyoxal oxidase, which, with its substrates, is extracellular, and
perhaps also by intracellular sugar and alcohol oxidases. Molecular oxygen is
the electron donor for laccase. Low molecular mass products of lignin degrada-
tion are taken up by the hyphae and further oxidized. The enzymes responsible
for this intracellular oxidation are only beginning to be described [34].
Lignin peroxidase oxidizes aromatic nuclei (phenolic and nonphenolic) by
removal of one electron, generating both phenoxy radicals and cation-radicals.
The latter react spontaneously with nucleophiles (primarily water) and molecu-
lar oxygen. The result is an "enzymatic combustion" in which C-C and C-O
linkages are cleaved, depolymerizing the polymer and opening aromatic rings.
A plethora of aromatic and aliphatic products are thereby formed. In vitro
depolymerization of lignin by pure LiP in the presence of H202 and veratryl
alcohol was recently demonstrated [35]. Veratryl alcohol (3,4-dimethoxybenzyl
alcohol), found in the extracellular milieu of ligninolytic cultures of examined
LiP-producing fungi, is a secondary metabolic product synthesized de novo. It is
a substrate for LiP and stimulates its action, probably not as an electron
mediator as originally thought (see Kirk and Farrell [-29]), but by donating
electrons to LiP so that its catalytic cycle is completed [36]. As mentioned, LiP
is apparently not produced by some white-rot fungi, including C. subvermispora
[37], suggesting that it is not required in all fungi, i.e., that the white-rot fungi
have more than one enzyme system for degrading lignin. Interestingly,
C. subvermispora and certain other LiP-negative fungi do have "LiP-like" genes
[37].
Manganese peroxidase has been found in nearly all studied white-rot fungi.
It catalyzes the oxidation of (complexed) Mn 2 + to Mn 3 +, which in turn oxidizes
lignin. Mn / + is a fairly abundant element in wood. The "physiological" com-
plexes that have been studied, such as the lactate chelate, oxidize only phenolic
units, which constitute only about 10% of the total in lignin in wood [38]. The
phenolic units are oxidized to phenoxy radicals, which can undergo certain
degradative reactions [39]. The MnP/HzO2/Mn 2+ has been shown to de-
polymerize lignin in vitro [40]. The exact function of MnP and the chemistry of
its actual oxidation of lignin are not yet clear.
In a recent study, Bao et al. [41] discovered a lipid peroxidation system
involving MnP. In the presence of Mn(II), MnP promotes the peroxidation of
unsaturated lipids, generating transient lipoyl radical intermediates that are
known to act as potent oxidants of other molecules. This system, unlike MnP
alone, oxidizes and cleaves nonphenolic model compounds via benzylic hydro-
gen abstraction. It also depolymerizes both nonphenolic and phenolic synthetic
lignins, which strongly supports a ligninolytic role for this system in vivo. The
166 M. Akhtar et a].
ligninolytic system of P. chrysosporium is depicted schematically in Fig. 3. We

have included the lipid peroxidation system, even though its role is not yet
established.
Laccase is a blue copper oxidase that is produced (secreted) by most but not
all white-rot fungi. Like LiP, laccase is apparently not required for lignin
degradation in all fungi. Laccase oxidizes phenolic units in lignin to phenoxy
radicals, which is the same process as that brought about by the chelated
Mn(III) produced by MnP (Fig. 3). However, in the presence of appropriate
"primary" substrates (such as ABTS), the effect of laccase apparently can be
enhanced; laccase/primary substrate systems have recently been reported to
degrade lignin in kraft pulp [42] and to oxidize nonphenolic compounds that
otherwise are unattacked by laccase. However, it is not known whether such
"primary" substrates occur and function in vivo, and the actual role of Iaccase,
like MnP, remains unclear. (Note that if "primary" substrates augment laccase
activity in vivo, they can be expected to augment the MnP/Mn 2+ system as well).
The characteristics of LiP, MnP, glyoxal oxidase, and laccase have been
described in review articles [28-31, 39, 43]. General properties of LiPs, common
to all studied LiP-producing fungi, include the following. Lignin peroxidases
consist of acidic isoenzymes encoded by multiple structural genes whose expression
CO2
// / I HO .o \
// / I Glyoxa[oxidase " ~ u k
// / /
H L~ J HO L 0
// I - Lignin peroxidase + H202 / q many
I/ ~ ~ ' ~ ,/> ' prod~,cts
II 1(. )1 I ]~,.+-:C .~o
~/ "/ ~ r/" "OC1"t3 Veratr~lalcohol / ~>~ --OCH3 (sponta. . . . )
~ Manganese peroxidase + HzO2
Manganese peroxidase + MR2. -~-unsaturatedlipid Mn2* ~"~in 3+

~OH
HO. ~ /L HO~ jL..O/Lo.~ 0~L 0/L
many
products 9
"" "T" "OCH3 " ~ "OCH3 /- ~ "OCH3 / ~-- "OCHa
O, OH
0-. L 0"- L
Phenoxy radical
Benzylicradical
Fig. 3. Schemedepicting the lignin-degrading systemof Phanerochaetechrysosporium

Fungal Delignificationand BiomechanicalPulpingof Wood 167
is nutrient-regulated. The extracellular proteins have protoporphyrin IX-type

heme prosthetic groups, are glycosylated, and have molecular weights around
40 kDa. Lignin peroxidases exhibit the common peroxidase catalytic cycle,
being first oxidized by H202 by removal of two electrons to give compound I,
which oxidizes its substrates by removing one electron to give compound II,
which oxidizes its substrate, returning to resting enzyme. The LiP from P.
chrysosporium has been crystallized and its three-dimensional structure resolved
by X-ray crystallography [44, 45].
Manganese peroxidases are like LiP in that they consist of multiple acidic
isoenzymes encoded by multiple structural genes whose expression is nutrient-
regulated; in the case of MnP, regulation by Mn 2+ also occurs. Manganese
peroxidases are slightly larger than LiPs, but exhibit the same basic peroxidase
catalytic cycle. Interestingly, MnP, in the presence of reducing agents such as
glutathione, transfers electrons to molecular oxygen, generating H z O 2. Like
LiP, MnP has been crystallized and its three-dimensional structure determined
[46]. Note that LiP can display MnP activity; in the presence of H202, O2, and
the metabolites veratryl alcohol and oxalate, LiP oxidizes Mn 2 + to Mn 3 + [38].
In at least some white-rot fungi, including T. versicolor, laccases also consist
of acidic isoenzymes encoded by multiple structural genes. The molecular
weights of the laccases of white-rot fungi are in the range 50-65 kDa. Laccases
"store" four electrons from four sequential one-electron oxidations before reduc-
ing molecular oxygen to water.
The molecular genetics of the lignin-biodegrading system have received
attention in the last few years. Such studies have now been done with several
fungi, although P. chrysosporium has been the most thoroughly studied. With
that organism (and with the other studied fungi as well), complex structural gene
families have been found. In P. chrysosporium, 10 lip, 4 mnp, and 1 91ox have
been cloned and sequenced. All the genes contain introns and multiple
glycosylation sites. Further detail is found in recent reviews [31, 43].
1.2.3 Enzyme Localization
Immunological cytochemistry in conjunction with electron microscopy can be

used to localize enzymes within substrates and observe their spatial relationship
within decaying wood. The association of delignified zones with specific en-
zymes would indicate that the enzymes were able to penetrate the cell wall and
accumulate at sites of lignin degradation. Investigations using polyclonal and
monoclonal antibodies to lignin peroxidase and manganese peroxidase have
shown that these enzymes are present in cell walls of wood decayed for 6 to 12
weeks [25, 4749]. High concentrations of enzymes were found in decayed cell
walls that had a loose, modified ultrastructural matrix. Lignin peroxidase and
manganese peroxidase were evident at the edge of electron-dense regions in the
secondary wall or at sites of middle lamella degradation. Significant alteration of
the cell wall was always associated with localization of these enzymes. No
168 M. Akhtaret al.
labeling was evident in nondecayed wood or in wood decayed by a brown-rot

fungus [25].
To more accurately identify areas of the cell wall where enzymes could
penetrate successfully, several ultrastructural studies were completed after infil-
trating decayed wood with extracellular extracts from white-rot fungi or with
purified lignin peroxidase and manganese peroxidase [48, 50]. After impregna-
tion, gold-labeled antibodies were used to determine the extent of penetrability
in the cells. In nondegraded cells, no enzyme preparations were found to enter
the secondary wall. Enzymes were found only on the surface of the cell wall at
the lumen. In samples decayed by white-rot fungi, lignin peroxidase and manga-
nese peroxidase were found within altered cell walls. The enzymes diffused into
the peripheral areas of the secondary wall and into areas where the middle
lamella was becoming less electron-dense. The labeling appeared to be located at
sites where lignin was being degraded. In areas with advanced decay, enzymes were
located within the cell wall at the edges of undegraded cell corner regions [48].
2 Biopulping
2.1 Introduction
The pulp and paper industry is a large and growing portion of the world's
economy. In 1991, paper sales were valued at $122 billion [51], and 267 million
metric tons of paper and paperboard were consumed worldwide. Worldwide
consumption is expected to increase to 300 million metric tons in 1996. A num-
ber of pulping processes have been developed to meet industrial and consumer
needs.
Pulping processes are generally divided into two broad classes, chemical and
mechanical, which produce substantially different fiber characteristics. The
choice of process depends on the end application of the pulp and the raw
material. In many papermaking operations, a combination of chemical and
mechanical pulps is used to obtain the desired paper characteristics.
Chemical pulping involves the use of chemicals to degrade and dissolve the
lignin from the wood cell walls, releasing cellulose fibers. Chemical pulping
processes are low yield (about 40-50%) and require significant waste treatment
and chemical recycling operations; however, the pulps produced have high
strength. Mechanical pulping involves the use of mechanical force to separate
the wood fibers. Mechanical processes are high yield (up to 95%) and give paper
with high bulk, good opacity, and excellent printability. However, these pro-
cesses are energy-intensive and produce paper with lower strength and high
color reversion (tendency to turn yellow with time).
Bleaching of chemical pulps using a combination of chlorination and alka-
line extraction has been used in the pulp and paper industry for many years.
Fungal Deligniflcationand BiomechanicalPulping of Wood 169
Unfortunately, the effluents from chlorination and alkaline extraction stages

cannot be recycled back to the chemical recovery furnace because of their high
level of corrosive chloride. In addition, the effluents contain large amounts of
chlorinated organic compounds, which are known to have toxic, mutagenic, and
carcinogenic effects. In most parts of the world, increased public concern about
the environment is having a large impact on the pulp and paper industry.
Reductions in allowable air and water discharges from pulp and paper mills are
requiring restraint in the use of chlorine and chlorine dioxide for bleaching.
Consequently, the industry is rapidly moving towards alternative technologies
to alleviate this and other problems related to the environment.
Biopulping, defined as the treatment of wood chips with lignin-degrading
fungi prior to pulping, is an experimental process that has been researched
extensively during the past 8 years. It has been studied mainly as a pretreatment
for mechanical pulping. Biopulping reduces electrical energy consumption
(which is the major cost in mechanical pulping), improves paper quality, and
reduces the environmental impact of pulping [52, 53]. The following sections
present a summary of this research and describe key findings.
2.2 Past Work
The use of white-rot fungi for the biological delignification of wood was perhaps
first studied by Lawson and Still [54] at the West Virginia Pulp and Paper
Company research laboratory (non Westvaco Corporation). These researchers
published a survey of the literature (covering 72 lignin-degrading fungi), which
pointed to the dearth of knowledge about the fungal degradation of lignin.
Research was then done at the US Forest Products Laboratory in Madison and
the Swedish Products Laboratory (STFI) in Stockholm. The first published
report on biopulping per se demonstrated that fungal treatment could result in
significant energy savings for mechanical pulping [55]. That research resulted in
a US patent [56], which described a "method for producing cellulose pulp."
Considerable efforts at STFI were directed toward developing cellulase-less
mutants of selected white-rot fungi to improve the selectivity of lignin degrada-
tion and thus the specificity of biopulping [57]. However, the mutant strains
degraded less lignin than did wild-type strains when grown on wood [58] and
did not result in energy savings during subsequent mechanical pulping [59].
Attempts by this group to scale up the biopulping process were not notably
successful [60]. However, subsequent work with Cuban scientists on a pilot
scale with bagasse using mutant strains gave more promising results 1-61].
Eriksson et al. [62] showed that chip colonization is not the rate-limiting step in
biopulping. At the Forest Products Laboratory, Bar-Lev et al. [63] showed that
the treatment of primary thermomechanical pulp with a white-rot fungus prior
to secondary refining reduced energy requirements and increased paper strength
properties. Similar results were obtained in Japan by Akamatsu et al. [64]
during thermomechanical pulping of fungus-treated popular chips. Other
170 M. Akhtaret al.
details on biopulping research were described in two review articles and the
literature cited therein [65, 66].
A comprehensive evaluation of biomechanical pulping was launched in 1987
at the Forest Products Laboratory after the establishment of a Biopulping
Consortium, which involved the Forest Products Laboratory, the Universities
of Wisconsin and Minnesota, and pulp and paper and related companies. The
overall goal was to establish the technical feasibility of using a fungal pretreat-
ment with mechanical pulping to save energy and/or improve paper strength. In
addition, it was assumed that the fungal pretreatment would have less environ-
mental impact than would chemical pretreatments, which turned out to be the
case. The consortium research was conducted by seven closely coordinated
teams: fungal, pulp and paper, enzyme, molecular genetics, economics, engineer-
ing scale-up, and information. However, in this review article, we will focus only
on the work conducted by the fungal pulp and paper, and engineering scale-up
research teams.
2.3 Biomechanical Pulping
2.3.1 Screening of Fungi
There are hundreds of white-rot fungi with varying capacities to degrade lignin,
cellulose, and hemicellulose. We assumed at the outset that the fungi that
degraded lignin selectively would be the best candidates for biopulping. To
ascertain the most appropriate species, a screening program was initiated that
selected fast-growing species that could selectively remove lignin from wood.
2.3.1.1 In Vitro Wood Decay Test

Several methods have been developed to select fungal species with selective
lignin-degrading ability [15, 27, 67-70]. However, one of the most appropriate
methods appeared to be an assessment of decay (chemical analyses of lignin and
wood sugar content) using wood blocks in accelerated decay chambers [15, 71].
Based on this in vitro screening procedure, we selected several species of fungi;
among the best were P. chrysosporium, C. subvermispora, Phlebia brevispora,
Phlebia tremellosa, Dichomitus squalens, and Phellinus pini [15, 71]. Different
strains of these selected species varied in their selectivity towards lignin. Two
fungi were examined in detail: P. chrysosporium and C. subvermispora. As we
tested various strains on different species of wood, it became clear that some
strains are effective with hardwood only, whereas others are effective on both
hardwood and softwood (Tables 1-4). These results clearly showed large differ-
ences among the strains in capacity to degrade lignin and in selectivity [16].
The species or strains selected by this method were then evaluated for their
biopulping efficacy. No apparent relationship was found between the
lignin removal from the wood chips and energy savings or strength improve-
ments during the actual biopulping runs [72, 73]. This suggests that lignin
Fungal Delignification and Biomechanical Pulping of Wood 171
Table 1. Loss of weight, lignin, and wood sugars in aspen wood blocks decayed by different
strains of P. chrysosporium (12-week incubation)
Loss (%)
Glucose Xylose Mannose

Strain Weight Lignin (glucan) (xylan) (mannan)
BKM-F-1767 61.0 80.7 49.9 77.7 61.0

5161 ME-8 53.6 54.4 53.0 61.0 77.6
FP-104297-sp 55.3 47,5 51,3 63,0 81.5
FP-102169 50.2 56.7 51.4 52.0 55.3
HHB-6251-sp 56.2 58.9 55.0 59.4 59.2
5157-A- 1 23.0 30.6 5.2 27.4 60.2
Gold-9-420-1 61.2 73.4 56.1 71.7 67.9
Table 2. Loss of weight, lignin, and wood sugars in loblolly pine wood blocks decayed by
different strains of P. chrysosporium (12-week incubation)
Loss (%)

BKM-F-1767 24.5 20.9 26.1 19.1 31.4

5161ME-8 8.4 3.6 11.2 9.7 14.5
FP-104297-sp 22.7 12.9 30,5 23.8 39.3
FP-102169 19.7 16.9 23.0 17.4 24.3
HHB-6251-sp 11.9 8.3 8.7 6.9 5.3
5157-A- 1 13.6 12.8 10.3 14.8 11.5
Gold-9-420-1 18.7 18.3 17.5 25.7 20.7
Table 3. Loss of weight, lignin, and wood sugars in aspen wood blocks decayed by different
strains of C. subvermispora (12-week incubation)
Loss (%)

ME-485 28.4 61.5 2.5 45.4 72.3

L- 14807-sp 24.4 57.2 6.8 36.9 39.1
L- 15225-sp 25.4 58.8 2.9 40.4 66.3
FP-104027-T 26.4 65.9 2.2 44.6 66.8
L-39292-sp 25.6 63.7 2.1 47.9 66.4
FP- 105752-sp 22.7 55.7 0.6 31.0 30.2
CZ-3 23.8 71.2 6.3 43.8 28.7
L-6133-sp 24.4 70.7 3.4 38.4 29.3
FP-90331-sp 26.5 50.1 7.3 31.5 31.3
Table 4. Loss of weight, lignin, and wood sugars in loblolly pine wood blocks decayed by
different strains of C. subvermispora (12-week incubation)
Loss (%)
ME-485 22.8 31.0 20.3 0 24.2
L-14807-sp 22.5 37.0 14.7 33.2 29.9
L- 15225-sp 23.7 38.2 12.4 27.2 28.1
FP-104027-T 28.3 40.6 18.8 33.8 26.9
L-39292-sp 29.0 42.2 22.4 31.1 26.2
FP- 105752-sp 19.6 33.9 7.1 27.0 10.1
CZ-3 21.3 31.8 14.0 31.0 20.3
L-6133-sp 30.1 34.1 26.2 34.0 18.9
FP-90331-sp 22.7 38.2 14.1 30.0 15.9
modification rather than removal is involved. Screening by way of the in vitro

wood decay test is also time-consuming and labor-intensive. We therefore
sought alternative screening methods.
2.3.1.2 P F I Milling
P F I milling and freeness measurements have previously been used to assess
energy consumption of fungus-treated pulps [74]. We further evaluated this
approach and tried to correlate changes in freeness after P F I mill refining of
coarse aspen pulp treated with selected white-rot fungi with those of paper
strength properties or energy savings obtained during biomechanical pulping of
wood chips with the same fungi. We found that P F I milling and freeness
measurements of pulp can, in themselves, give a good estimate of paper strength
properties [75]. However, follow-up studies suggested that this method can only
be used to evaluate the effect of fungal treatments on energy savings compared
to the control; they cannot be used to discriminate the effect of different fungal
treatments on energy savings [76].
2.3.1.3 Simons Staining

Simons stain [77, 78] had been used previously in various investigations to
evaluate the degree of fibrillation in beaten pulp fibers. The stain consists of
a 1% aqueous solution of Pontamine Sky Blue 6BX and a 1% aqueous solution
of Potamine Fast Orange 6RN mixed in a 1 : 1 ratio. Fibers on microscope slides
are first flooded with stain and then heated at 60 ~ to evaporate the water. The
fibers are rinsed with distilled water to remove excess stain and then covered
with glass. The fibers are then immediately examined under a microscope and
photographed. Fibrillated fiber walls stain orange, and nonfibrillated or un-
damaged fiber walls stain blue.
Our earlier electron microscope studies in connection with biopulping had
shown that pulp fibers obtained from the fungus-treated wood chips had more
uniform fiber length and greater fibrillation, and appeared woolly compared
with control pulps [79], suggesting that extent of fibrillation might be a good
indicator of biopulping efficacy. Therefore, aspen wood chips were treated with
the white-rot fungus C. subvermispora for 4 weeks and then refined through
a single-disk mechanical refiner. Pulp obtained from the fungus-treated chips
had extensively fibrillated fibers that stained a deep orange with Simons stain
(Fig. 4). In contrast, pulp obtained from the untreated control chips exhibited
little fibrillation and stained a deep blue (Fig. 4). This showed that fibers
obtained from the fungus-treated chips could be differentiated from those
obtained from control chips based on the yellowing of fiber ends [80]. We
Fig. 4. Simons staining of control (top) and biopulp (bottom). Pulps from untreated control wood
chips and Ceriporiopsis subvermispora-treated chips (4-weekincubation) were passed through the
refiner only once (Canadian Standard Freeness of pulps at about 700 ml) and then stained [80]
Table 5. Correlation of yellowing of fiber ends with energy savings using selected Ceriporiopsis spp.
on aspen wood chips (2-week incubation)
Yellowing of fiber ends (%)
Species/strain None Slight Intermediate Advanced
Control +
C. rivulosa L-10602-sp. + (0)
C. rivulosa PiiRTO-26K225 + a _1_a (3%)b
C. pannocincta FP-101181-sp. + + " (4%)
C. subversmispora L-3292 + + (7%)
C. subvermispora CZ-3 + (12%)
C. subvermispora L-9186-sp. + + (16%)
Two plus signs for one treatment indicate that the staining pattern was in-between the two
categories
b Values in parentheses represent refining energy savings compared to that of the untreated control
further evaluated this approach to determine whether it can be used to monitor

differences among fungal pretreatments. We found that the intensity of yellow
staining of biopulp fibers obtained under different experimental conditions
correlates very well with energy savings from completed biopulping runs
(Table 5). These results were summarized in a recent publication [81].
2.3.2 Evaluation of Selected Fungi in Biopulping Runs
White-rot fungi screened in the wood decay test or with Simons stain were
evaluated for their performance in refiner mechanical pulping. The process
involved the treatment of wood chips with the fungi in bioreactors on a bench
scale at appropriate temperature and humidity, mechanical pulping of control
and fungus-treated chips through a single-disk atmospheric refiner, preparation
of paper, and testing of the paper for physical properties.
2.3.2.1 Method
Three types of bioreactors were designed and used: a rotating drum bioreactor,
a stationary tray bioreactor, and an aerated static bed bioreactor. Details of the
configuration of each bioreactor have been published (rotating drum bioreactor
[82], stationary tray bioreactor [83], and aerated static bed bioreactor [84]). In
recent studies, we have used the simple and inexpensive aerated static bed
bioreactor (Fig. 5). Chips (1500 g, dry weight basis) are introduced into each 21-1
bioreactor with water (containing nutrients [85] and additive, if any), and the
loaded bioreactors are then usually sterilized by autoclaving. Chip moisture
content is adjusted to 55%-60% on a wet weight basis. The chips are then
inoculated with the fungus and incubated at an appropriate temperature (39 ~
for P. chrysosporium and 27 ~ for other fungi) for 2-4 weeks (in most cases
2 weeks) with humidified air (0.022711-1min-t). Details are described in
previous publications [83, 84, 86].
( ~,u u u u u u (fiG
H~
Fig. 5. Diagram of a static bed bioreactor [84]. The bioreactor was fabricated from a polypropylene
vessel(L). The top of the vesselis sealed with a lid (M), which is vented to the atmosphere through an
exit tube (N). Suspended above the bottom of the reactor (L) is a perforated polypropylene floor (.1),
supported by a stand (K). Air for the bioreactor comes from a regulated supply and passes through
tubing (A) to a fritted glass gas dispenser (B) in a humidification flask (C) containing sterile water.
Humidified air passes through tubing (D) to a rotameter (E) and through tubing (F) to a manifold
(G). Humidified air from manifold(G) is passed through a filter (H) before passing through tubing (1)
to the reactor (L) base
After harvest, the untreated control chips and the fungus-treated chips are
fiberized by multiple passes through a S p r o u t - W a l d r o n Model D 2202 single
rotating 300-mm-diameter disk atmospheric refiner. Energy consumed during
fiberization and subsequent refining is measured using an Ohio Semitronic
Model W H 30-11195 integrating watt-meter attached to the power supply side
of the 44.8-kW electric motor. Pulps are then refined to Canadian Standard
Freeness (CSF) values just above and just below 100 ml. CSF is an arbitrary
measure of the rate of water drainage from a pulp slurry. Handsheets (60 g m - z)
are made with these two pulp samples and tested for physical properties using
Standard T A P P I methods. Energy values and physical properties are regressed
to 100 ml CSF to facilitate comparison. Details of energy measurements,
handsheet preparation, and testing methods have been described
[72, 82, 87, 88].
2.3.2.2 Energy Savings and Physical Properties

In early experiments, several white-rot fungi screened initially by the wood
decay test were further screened for their biomechanical pulping performance
using aspen (hardwood) wood chips [72, 73, 82, 88]. Based on energy savings
and paper strength improvements, six fungi were initially selected: P. chrysos-
porium, P. tremellosa, P. subserialis, P. brevispora, D. squalens, and Poria
medulla-panis. Of these, more emphasis was given to P. chrysosporium because it
is by far the most studied white-rot fungus, grows rapidly, and competes well
with indigenous microorganisms of wood chips. Periodically, additional fungi
Table 6. Energy savings and changes in physical properties from biomechanical pulping of loblolly
pine chips with different white-rot fungi (4-week incubation)
Strength properties Optical properties
Energy Burst Tear Tensile Scattering

savings index index index Brightness Opacity coefficient
Fungus (%) (kN g - i ) (mN m2g - i ) (N mg-1) (%) (%) (m 2 kg- l)
Pc 14 14 1 19 -27 -2.3 -29.8

Hs 26 12 32 - 12 - 21 - 3.6 - 27.6
Pb 28 - 4 21 - 9 - 24 - 2.9 - 28.1
Ps 32 -29 9 -36 -28 -3.7 -34.6
Pt 36 - 18 12 - 17 -24 -4.5 - 32.6
Cs 42 32 67 -1 -22 -4.4 -30.9
Pc = Phanerochaete chrysosporium; Hs = Hyphodontia setulosa; Pb = Phlebia brevispora;

Ps = Phlebia subserialis; Pt = Phlebia tremellosa; Cs = Ceriporiopsis subvermispora
Table 7. Energy savings and changes in physical properties from biomechanical pulping of aspen
wood chips with three strains of C. subvermispora (4-week incubation)

Strain (%) (kN g - i ) (ran m2g -1) (N mg 1) (%) (%) (m 2 k g - i )
FP-90031-sp. 40 23 131 17 - 18 - 2.0 -- 33.8

L-6133-sp. 44 27 137 20 - 21 - 1.3 -- 34.0
CZ-3 48 40 162 27 -- 21 -- 1.3 - 37.1
Table 8. Energy savings and changes in physical properties from biomechanical pulping of loblolly
pine chips with three strains of C. subvermispora (4-week incubation)

Strain (%) (kN g - i ) (raN mZg l) (N mg-1) (%) (%) (m 2 k g - l )
FP-90031-sp. 37 41 54 4 - 21 - 0.94 -- 27.2

L-14807-sp. 30 44 59 3 -- 19 -- 0.63 - 24.9
FP-104027-T 21 45 47 11 - 21 0.0 - 23.1
identified for lignin selectivity on the basis of the wood decay test were also
e v a l u a t e d f o r t h e i r b i o m e c h a n i c a l p u l p i n g efficacy.
As the research progressed, emphasis was given to screening the fungi on
loblolly pine (softwood) chips because this wood, together with other southern
p i n e s , is a m a j o r w o o d s o u r c e f o r m e c h a n i c a l p u l p m i l l s i n t h e U n i t e d S t a t e s .
Some of the fungi selected as being best on aspen wood chips were evaluated on
l o b l o l l y p i n e c h i p s . S o m e o f t h e s e r e s u l t s a r e p r e s e n t e d i n T a b l e 6. All t h e f u n g i
s a v e d e n e r g y a n d s o m e i m p r o v e d p a p e r s t r e n g t h , b u t all a d v e r s e l y a f f e c t e d
paper optical properties. Based on energy savings and strength improvements,

C. subvermispora was identified as the best fungus [86]. Different strains of this
fungus were found to be effective on both aspen (Table 7) and loblolly pine
(Table 8) [84]. A US patent was issued on the use of C. subvermispora for
biomechanical pulping [89].
2.3.3 Optimization of Biopulping Runs
For biopulping, like any industrial microbial process, there is great opportunity
to increase the effectiveness and efficiency and decrease the cost of the process
through optimizing the variables. Our initial work was governed by "best
guesses" for optimal biopulping conditions based on the literature, knowledge of
fungal growth, and past experience. Initial research focused on the fungus
P. chrysosporium and aspen chips; later, C. subvermispora became the focus of
our study. Some of the initial results are in the following text.
2.3.3.1 Wood Batch, Chip Storage, and Chip Movement During Incubation
Some parameters, including wood batch and chip storage conditions (frozen,
fresh, or dried at room temperature) did not seem to affect biopulping perfor-
mance. In early experiments, fungus sensitivity to chip movement was observed
when stationary and rotating drum bioreactors were used. Chip movement
affected the extent of chip degradation, energy consumption during refining, and
paper strength properties [72, 88]. Later studies, however, showed that shaking
the aerated static bed bioreactors once a week during the 4-week incubation
period had no appreciable effect on energy savings or paper strength properties.
2.3.3.2 Inoculum
In any industrial fermentation, the inoculum is of key importance. A number of
variables affect the fermentation, including level, physical form, age, and viabil-
ity. We examined some of these variables in a series of experiments.
The effect of different inoculum levels (2.5%, 5%, 10% and 20%, dry weight
basis), using precolonized chips as inoculum, was studied with P. chrysosporium
on aspen wood chips. The lowest inoculum level (2.5%) gave slightly lower
energy savings than the other three levels.
We postulated that the addition of nutrient nitrogen to the inoculum would
help build fungal biomass and vigor, which in turn would improve biopulping
performance of P. chrysosporium. To test this hypothesis, two concentrations of
glutamic acid (500 and 5000 ppmN) were added to the 5% wood chips inoculum
prior to introducing the fungus. The results suggested that increased inoculum
nitrogen had beneficial effects in terms of energy savings. However, the weight
loss stimulated by the high nitrogen offsets these benefits.
Another approach that was tried to increase the inoculum vigor of
P. chrysosporium was by increasing the age of the inoculum (2, 4, and 6 weeks).
We concluded that inoculum age has little influence under the conditions used.
In all of these experiments, precolonized wood chips were used as an in-

oculum. In other experiments, we evaluated a liquid inoculum (mycelial suspen-
sion) of C. subvermispora on aspen wood chips (0.1% on a dry wood basis).
Results with the liquid inoculum were acceptable but not as good as the results
with the chip inoculum. Because we did not measure the amount of fungus
added with the colonized chips, we do not know whether the increased biopulp-
ing efficacy with chip inoculum was due to increased fungus content or an
inherent advantage of delivering inoculum with wood chips.
2.3.3.3 Nutrients Requirement

The effect of nitrogen and carbon was studied with P. chrysosporium on aspen
chips. Two levels each of nitrogen (25 or 250 ppm N as glutamic acid) and
glucose (4000 or 40 000 ppm) were added to bioreactors. The combination of
25 pprn nitrogen and 40 000 ppm glucose produced the best results in terms of
energy savings. Subsequently, the effect of another nitrogen source (ammonium
tartrate) (0, 36, 108, and 324 ppm N) was examined. We found that 324 ppm
N gave optimum results.
The use of a nonchemically defined nitrogen source, i.e., yeast extract, was
also investigated with both P. chrysosporium and C. subvermispora on aspen
wood chips. With P. chrysosporium, two levels of nitrogen (108 and 324 ppm) on
a dry weight basis were tested with a uniform level of 40000 ppm lactose (dry
weight basis). With C. subvermispora, three levels of nitrogen (108, 324 and
976 ppm) with a uniform level of 4 000 ppm lactose (dry weight basis) were used.
An energy saving of 32% with significant strength improvements was obtained
after 2 weeks, one of the best results ever achieved with P. chrysosporium. With
C. subvermispora, an energy saving of 52% with strength improvements was
obtained for the highest nitrogen level (976 ppm); this result was better than
average for this fungus on aspen chips.
In a few runs made without the addition of nutrients, we noted significant
energy savings and paper strength improvements, although addition of nutrient
nitrogen seemed to enhance the biopulping efficacy of these fungi.
2.3.3.4 Aeration
Solid-substrate fermentations are known to be affected markedly by aeration. In
addition, the ligninolytic activity of fungi depends on oxygen availability.
Consequently, we evaluated the influence of three air flow rates on the biopulp-
ing efficacy of P. chrysosporium on aspen chips: low (0.001 11-1 min 1), medium
(0.02211-1 rain- ~), and high (0.10011- ~min 1). The low flow rate was achieved
using intermittent aeration. The medium and high flow rates gave comparable
energy savings and had similar effects on strength properties. The low flow rate
was suboptimal.
2.3.3.5 Wood Chip Sterilization

Our studies showed that P. chrysosporium could colonize unsterilized wood
chips and perform biopulping when incubated in a bioreactor at its optimum
growth temperature (39 ~ but not at suboptimal growth temperatures; C.

subvermispora was found to be ineffective on unsterilized wood chips even at its
optimum temperature (27-32 ~
2.3.3.6 Bleaching Studies

Fungal pretreatment reduces the brightness of the resulting mechanical pulps by
as much as 15 20 Elrepho brightness points in 4 weeks and 8-10 Elrepho
brightness points in 2 weeks. Consequently, the bleachability and brightness
stability of aspen biorefiner mechanical pulps (BRMPs) were investigated. Com-
parisons were made with untreated aspen refiner mechanical pulp (RMP) and
commercial mechanical pulps: chemithermomechanical pulp (CTMP), thermo-
mechanical pulp (TMP), and groundwood (GW) pulp [90]. P. chrysosporium was
used for the pretreatments reported here; subsequent experiments demonstrated
that aspen chips treated with C. subvermispora respond similarly to bleaching.
Either alkaline hydrogen peroxide or sodium hydrosulfite readily increased
the brightness of aspen BRMP (Table 9). Fungal pretreatment enhanced the
bleachability of the aspen pulps examined. The BRMPs increased by more
brightness points than did corresponding untreated pulps. However, because
the initial brightness values of the BRMPs were lower than those of the
corresponding untreated pulps, the bleached brightness values were not as high
at a given chemical charge as those of untreated mechanical pulps.
Aspen BRMP was readily bleached to 60% Elrepho brightness with 1%
sodium h y d r o s u l f i t ~ a brightness suitable for newsprint; brightness values
approaching 80% were achieved with a two-step bleach sequence. Thus, bleach-
ability of biomechanical pulps appears not to be a serious problem.
Table 9. Bleaching results with aspen pulps (90)
Brightness
Pulp Chemical charge (%)
GW Unbleached 63.1
1.5% H202 80.8
1% Na2S204 71.9
CTMP Unbleached 62.0
3% H2Oz 78.3
1% NazS2O 4 66.3
TMP Unbleached 60.2
3% HzO2 78.6
1% Na2SzO4 66.9
RMP (control) Unbleached 62.2
3% H2O 2 80.0
1% Na28204 77.2
BRMP Unbleached 51.8
3% H202 76.0
1% Na2S204 59.3
Two-step bleach 78.0
3% H202
1% NazSzO 4
Table 10. Effluents from first refiner pass of control and fungus-treated aspen
chips (91)
Treatment BOD COD Toxicity

(g kg-1 pulp) (g kg 1 pulp) (100 EC50-1)
Control 40 74 17
C. subvermispora 36 100 4
Brightness stability was evaluated by subjecting handsheets to accelerated

thermal and photo-aging tests [90]. The stability of BRMP was slightly lower
than that of RMP but slightly higher than that of CTMP.
2.3.3.7 Analysis of Effluents

Samples of the wastewater from the first refiner passes of aspen chips pretreated
with either C. subvermispora or P. chrysosporium were analyzed for biochemical
oxygen demand (BOD), chemical oxygen demand (COD), and Microtox toxic-
ity. Fungal pretreatment of aspen chips with either P. chrysosporium or
C. subvermispora during biopulping substantially decreased effluent toxicity
(Table 10). The BOD values for effluents from fungus-treated pulps were either
slightly lower or higher than those of RMPs, depending upon the fungal species
and whether nutrients were used. The COD values for effluents from fungus-
treated pulps were considerably higher than those from RMP runs, probably
because of the release of lignin-related products. Based on these data, we
conclude that the effluent load from the biopulping runs should be lower and
more benign than that of commercial CTMP mills [91].
2.3.3.8 Global Analysis of Data

The data from all runs, regardless of experimental conditions, were analyzed
statistically in a global manner. The data were plotted as notched box plots [92]
so that any statistically significant differences ( _+ 0.95 level) between the un-
treated control and biopulping runs could be observed. The global analysis
greatly simplified the overall interpretation of a large amount of data. To
illustrate the analysis, a comparison of the efficacy of fungal species in terms of
refiner energy consumption in the biopulping of aspen chips is shown in Fig. 6.
In subsequent analysis, P chrysosporium was found to be less effective than
C. subvermispora on aspen wood chips and relatively ineffective on loblolly
pine chips. The energy required for refining was reduced, in many cases by
40% or more (e.g., Fig. 6) with little loss of wood substance ( < 10%), except
when chemically defined, high nutrient nitrogen supplements were used. Hand-
sheet density was decreased somewhat by treatment of aspen with P. chrysos-
porium, and more with C. subvermispora; with pine, density was unaffected
(Fig. 7).
We treated P. chrysosporium/aspen, C. subvermispora/aspen, and C. subver-
mispora/pine as three separate sets of data, with all runs of these combinations
Wc
Pt
Ps
Pm
Pc 9 "'F~'"
Pb
None ..... ~ ........
Ds
Cv 9 .
Cs - ~ .....
I I I I
1000 1500 2000 2500 3000
LL Wc
Pt
Ps I
Pm I
Pc Fig. 6. Refiner energy consumption of aspen
chips treated with different fungi (top dot-plot,
Pb I bottom box plot) [52]. Wc Wolfiporia cocos (a
None brown-rot fungus), Pt Phlebia tremellosa, Ps
Ds Phlebia subserialis, Pm Pholiota mutabilis, Pc
Cv Phanerochaete chrysosporium, Pb Phlebia
brevispora, Ds Dichomitus squalens, Cv Co-
Cs - C ~ riolus (Trametes) versicolor, Cs Ceriporiopsis
f subvermispora (all white-rot fungi)
1000 1500 I
2000 2;00 3000
Energy
included. Chip pretreatment with either fungus significantly increased burst

index of handsheets from aspen but not that of handsheets from pine (Fig. 8).
Chip pretreatment with either fungus also significantly increased tear index
(single-ply test). Only C. subvermispora was examined with pine; tear index
increased significantly (Fig. 9). Tensile index was increased significantly by chip
pretreatment with either fungus in the case of aspen, but not pine (Fig. 10).
Handsheet brightness was significantly decreased by pretreating chips of either
wood with either fungus (Fig. 11), as was scattering coefficient with C. subvermis-
pora on both woods and P. chrysosporium on aspen (data not shown); opacity
was not significantly affected by either fungus (data not shown).
2.3.4 Microscopy Studies
To gain insight into the mechanism of biopulping, we examined at the micro-

scope level the growth patterns of the fungi in wood, the effects of fungi on wood
cell walls, and the appearance of handsheets made from biopulps.
Pc
None
Cs .@
m
I I I
Q
n
Itl
250 350 450 550
Pc
None
Cs Fig, 7. Density of handsheets made from as-

pen (top) and loblolly (bottom) pine chips
treated with Phanerochaete chrysosporium
! I I (Pc) or Ceriporiopsissubvermispora (Cs) [52]
250 350 450 550
Density
2.3.4.1 Fungal Growth Patterns

Scanning electron microscopy was used to observe fungal growth in and
degradation of nutrient-supplemented aspen chips after a 3-week treatment with
P. chrysosporium. The fungus grew well both across the chip surfaces (Fig. 12)
and throughout the cell walls. The hyphae penetrated the chips through the
lumina of wood vessels and fiber cells as well as through natural wood cell pits
and fungal boreholes. Partial degradation of the cell lumen walls was evident.
Erosion troughs and localized wall fragmentation or thinning were clearly
visible as was a generalized swelling and relaxing of the normally rigid wood cell
wall structure (Fig. 13). Aspen wood chips treated with C. subvermispora showed
packed hyphae within the ray cells. Many crystals of calcium oxalate were found
on the hyphae (Fig. 14) during both the incipient and the advanced stages of
growth. Our observations suggest that the physical basis for the biopulping
efficacy of the fungal treatment is likely to involve an overall softening and
swelling of the wood cell walls as welt as thinning and fragmentation in localized
areas [93].
Pc I ~ ~o o
None
Cs
I I !
CL
r
0 1 2 3
t-
LL
pc ~
None @
Cs
Fig. 8. Burst index for handsheets made from
aspen (top) and loblolly pine (bottom) chips
treated with Phanerochaete chrysosporium (Pc)
I I I or Ceriporiopsis subvermispora (Cs) [52]
0 1 2 3
Burst index
2.3.4.2 Fibers
In another investigation, we compared the microscopic appearance of B R M P
with that of RMP, G W pulp, TMP, C T M P , neutral sulfite semichemical pulp
(NSSC), and kraft pulp. When fiberized, B R M P emerged wool-like, looser, and
bulkier, with fibers rather uniform in length; the pulp also exhibited abundant
fibrillation. In contrast to BRMP, R M P fibers were not as wide, appeared to be
stiffer, were of different lengths, and had only moderate fibrillation. The G W
pulp fibers were stiff and of various lengths, showed reduced fibrillation, and
were accompanied by debris. The T M P and C T M P fibers appeared stiff and
were of various lengths, although longer than R M P fibers, and had moderate
fibrillation. The T M P and C T M P fibers were more twisted than the B R M P
fibers. The NSSC pulp exhibited few stiff fibers and the fibers were of various
lengths; they appeared to be more compressible and conformable when com-
pared to the mechanical pulp fibers. Compared to BRMP, NSSC pulp fibers
were not as compressed and were more variable in length. Kraft pulping
Pc
None
Cs
O~
I I I I I
O. 0 1 2 3 4 5
g
LL
None
Cs
Fig. 9. Tear index (single-ply tear) for hand-
sheets made from aspen (top) and loblollypine
(bottom) chips treated with Phanerochaete
chrysosporium (Pc) or Ceriporiopsis subvermis-
I I I I I pora (Cs) 1-52]
0 1 2 3 4 5
Tear index
produced more uniform, separated, and collapsed fibers, with abundant fibrilla-
tion. The BRMP appeared to be similar to the kraft pulp. These results were
summarized by Sachs et al. [79, 94].
2.3.4.3 H a n d s h e e t s
Aspen BRMP produced a stronger handsheet than did aspen T M P and G W
pulp. Aspen NSSC pulps appeared to be superior to aspen BRMP in handsheet
properties. Of all the pulps, aspen kraft pulp had the highest sheet strength
properties. To gain insight and visually assess how the fiber morphology in these
pulps may have contributed to sheet strength properties, we examined cross
sections of handsheets. Handsheets made from mechanically processed pulps
showed uncollapsed fibers, leading to poor conformability and reduced bond-
ing. The NSSC and kraft pulps gave handsheets that exhibited fibers of en-
hanced compressibility and conformability. Handsheets prepared from B R M P
visually resembled the kraft handsheets, exhibiting good compressibility and
conformability of the fibers [79, 94].
Fungal Deligniflcation and Biomechanical Pulping of Wood 185
Pc 9 ,
None
Cs
I
10 2'o 3'0 ,'o 50 6~0
==
I.L
Pc
I
None
Fig. 10. Tensile index for handsheets made

Cs from aspen (top) and loblolly pine (bottom)
chips treated with Phanerochaete chrysos-
porium (Pc) or Ceriporiopsis subvermispora ( Cs)
[52]
10 '
20 3'0 '
40 50
' '0
6
Tensile index
2.3.5 Engineering and Economic Studies
The engineering and economics studies related to biopulping scale-up are

summarized in the PhD thesis of Wall [95]. The goal of these studies was to
develop the basic scientific and engineering knowledge needed for the commer-
cial utilization of fungi in chip piles. We investigated process scale-up
by obtaining kinetic data, developing process models, and implementing the
processes on a pilot scale [96]. Initial studies were performed using P. chryso-
sporium on aspen wood chips.
Mathematical modeling of biopulping was done to assess the factors that
might kinetically limit fungal growth. Assumptions concerning the
stoichiometry of biological wood degradation were used to predict important
parameters needed to model the growth of P. chrysosporium in wood chips.
Next, the effects of mass transfer on the growth of P. chrysosporium under typical
biopulping conditions were considered. Finally, mass and energy balances were
modeled for an isolated plug of wood chips, and the resulting system of
differential equations was integrated numerically.
Pc o
None ..@
Cs
!
30 5'0 o'o 7O
LL
Pc
None
~ Fig. 11. Brightness of handsheets made from as-

pen (top) and loblolly pine (bottom) chips treated
with Phanerochaete chrysosporium (Pc) or Ce-
I I riporiopsis subvermispora (Cs) [52]
30 ;,o
Brightness
Based on the kinetic and modeling studies, two bioreactor designs were
considered: a packed-bed reactor and a chip-pile-based system (Fig. 15). The
packed-bed bioreactor envisaged for the biopulping process is a bed of wood
chips with controlled air flow. Packed-bed reactors allow better control of
process conditions. The drawbacks to these reactors are that they require much
greater capital expenditure and entail higher operating costs than chip-pile
based systems. A chip-pile based system is defined here as an industrial-size chip
pile modified to increase temperature and moisture control. The advantage of
the chip pile is reduced cost as compared to that of a packed-bed reactor; the
disadvantage is reduced process control.
Early in the biopulping research, Harpole et al. [97] conducted an economic
evaluation based on a thermomechanical process model. Results indicated that
a 25% reduction in pulping energy by fungal treatment would save $21 (US) per
air dry ton (adt) of pulp ($33 with 40% energy savings). The capitalized value of
the energy savings was estimated to be about $250000 for each percentage of
energy saved, at an electricity cost of $0.035/kWh. Thus, a sizeable capital
Fig. 12. Web-like hyphal network on surface of nutrient-supplemented aspen wood chip during
3-week treatment by Phanerochaetechrysosporium[93]. Bar = 1 mm
expenditure for the biotreatment could be accommodated. More recently, an

economic model based on mass and energy balances was made for a controlled
packed-bed reactor process and a chip-pile-based system. The controlled reac-
tor process yields a pretax return on investment (ROI) of 21%, whereas the
chip-pile-based system shows a pretax R O I of 1 0 6 % - 2 1 7 % (depending on the
inoculum costs). The details are summarized in the following text.
2.3.5.1 Economic Analysis of Packed-Bed Reactor

The capital and operating costs were estimated for a 300 metric ton/day
biological pretreatment using P. chrysosporium on aspen and assuming a treat-
ment time of 2 weeks and a dry weight loss of 5%. The air flow rate was assumed
to be 0.59 VVM. The operating costs considered were steam, inoculum, and
electricity for aeration. A reasonable figure for inoculum costs was chosen based
on the information concerning a similar commercial process (see Section 2.3.6).
The most optimistic scenario yields a pretax R O I of 21% (Table 11, Case 1).
2.3.5.2 Economic Analysis of Chip-Pile-Based System

A chip-pile-based system with ductwork to provide aeration, piping to provide
steam, and a sprayer to apply inoculum to chips on a conveyor was chosen for
this analysis. The aeration rate and inoculum level were the same as those used
for the packed-bed reactor; therefore, the operating costs for a chip-pile-based
system are the same as those given for the packed-bed reactor. The capital costs
Fig. 13. The normally rigid cell

wall structure within aspen
chips (top) was modified (bot-
tom) by 3-week treatment with
Phanerochaete chrysosporium
[93]. Modifications included
cell wall swelling (a), enzymatic
softening or relaxing resulting
in the partial collapse of the
tube-like structure (b), and
localized areas of wall thinning
(c) or fragmentation (d).
Bar = 10 gm
considered were those for fans, ductwork, i n o c u l u m tanks, and humidification.

The analysis showed a pretax R O I of 106% 217%, depending on the i n o c u l u m
costs (Table 12).
O u r recent w o r k has focused on C. subvermispora and loblolly pine, with
special attention to those factors m o s t likely to affect the economics of a chip-
pile-based system. These factors are achieving the necessary degree of asepsis,
lowering the cost of fungal inoculum, and maintaining a hospitable e n v i r o n m e n t
in the chip pile.
2.3.5.2.1 Asepsis. In early work, the beneficial effects of biopulping on a labor-

atory scale were seen when w o o d chips were sterilized by autoclaving. C.
subvermispora was not found to be aggressive e n o u g h to compete with indigen-
ous microorganisms in unsterilized w o o d chips. Recently, we discovered that
Fig. 14. Calcium crystals on the surface of hyphae of Ceriporiopsissubvermisporaon aspen wood
chips (4-weektreatment). 1 cm = 5 gm
a brief atmospheric steaming of the chips allowed C. subvermispora to colonize

and be effective [98]. This condition is now being studied from an engineering
standpoint.
2.3.5.2.2 Fungal Inoculum. One of the major costs foreseen during the scale-up
of biopulping is inoculum production. Therefore, several experiments were
aimed at determining the best inoculum level of C. subvermispora for saving
energy and improving paper strength properties in a 2-week incubation. We
found that 0.3% inoculum (dry weight basis) saved 19% energy and improved
paper strength properties, such as tear index, significantly compared to the
control (Table 13). This amount of inoculum is quite high. However, we
discovered that the amount of inoculum can be lowered to 0.0005% (dry weight
basis) or less by adding a cheap and commercially available nutrient source,
corn steep liquor, to the mycelial suspension. This low amount of inoculum is
now well within a commercial range. Subsequent studies have also identified
better strains of C. subvermispora that yield up to 38% energy savings and
improve tear index by 51% [98, 99]. Other nonchemically defined additives,
including yeast extract and molasses, have shown promise in biopulping, but
they have not been found to be as effective as corn steep liquor.
2.3.5.2.3 Chip Pile Environment. Instrumented bioreactors were constructed

and are now in use to mimic chip piles. Current research is aimed at methods for
controlling temperature, aeration, and moisture in chip piles so that the fungal
treatment is effective. These data would support the design of a chip-pile-based
system.
Wood z= Harvesting
Debarking Chips ]=i..................... i I Inoculum I
Chipping train
i (Asep~s) "--~ I Ir~culum
(Nutrient i ! i
components) .e----::-----~ (Nutrients) i ~ :
..................... -]~ IMIxer) :................... ]~, : i I Softened
chips). Mechanical pulping
Bleaching
Biopolping Waste
.[
)
reactor
Bleed air
Air >i'~'l'~"-~i;~r--~.._..T.....~.
[ Humidifier i Air
i Fan2 !
L.......... 1
i F~3i
i Recycle air =
treaV'~nt
Fig. 15. Flowsheets for packed-bed reactor (top) and chip-pile-basedsystem (bottom). Operations in
parentheses are optional [95]
2.3.6 Prospects for Biopulping Commercialization
During the course of the biopulping research, one of the sponsoring companies
developed and commercialized a biotechnological process that is similar to
biopulping in many respects, and it deserves mention here. Cartapip is an
industrial fungal inoculum of Ophiostoma piliferum which was developed by
Sandoz Chemicals Biotech Research Corporation (now Clariant Biotech Re-
search Corporation) and is marketed by the US Sandoz Chemicals Corporation
(now Clariant Corporation). The fungus is a naturally occurring and ubiquitous
"blue stain" organism. It is nonpathogenic. A dilute slurry of the product (a
powder), consisting of fungal spores, is sprayed onto wood chips as they are piled
for storage prior to pulping. The spores germinate and the fungus grows aggres-
sively in the chip piles. Within the wood chips, the fungus grows mainly in ray
Table 11. Economic feasibility of a packed-bed reactor [95]
Case 1 Case 2 Case 3
Installed equipment costs $5 000 000 $10 000 000 $17 000 000
Working capital $206 750 $206 750 $206 750
Total capital investment $5 206 750 $10 206 750 $17 206 750
Utility costs $2.46 $2.46 $2.46
Inoculum costs $3.00 $3.00 $3.00
Labor $0.76 $0.76 $0.76
Yield losses $2.46 $2.46 $2.46
Depreciation $4.96 $9.72 $16.39
Total operating cost $13.64 $18.40 $25.07
Pretreatment value $23.49 $23.49 $23.49
Gross profit $9.85 $5.09 - $1.57
Pretax ROI 21% 5% - 1%
Table 12. Economic feasibility of a chip-pile-based reactor 1-95]
Case 1 Case 2 Case 3
Installed equipment costs $500 000 $500 000 $500 000

Working capital $206 750 $206 750 $206 750
Total capital investment $706 750 $706 750 $706 750
Utility costs $2.46 $2.46 $2.46
Inoculum costs $3,00 $5.00 $10.00
Labor $0.76 $0.76 $0.76
Yield losses $2.46 $2.46 $2.46
Depreciation $0.76 $0.76 $0.76
Total operating cost $9.35 $11.35 $16.35
Pretreatment value $23.49 $23.49 $23.49
Gross profit $14.14 $12.14 $7.14
Pretax ROI 217% 180% 106%
Table 13. Energy savings and strength properties during biomechanical pulping of
loblolly pine chips with C. subvermispora (2-week incubation)
Strength properties
Treatment Energy
(% inoculum on savings~ Burst index Tear index
dry weight basis) (%) (kNg 1) (mN m 2 g 1)
Control 0.62 _+ 0.05 b 1.67 _+ 0.13

0.01 4 0.63 _+ 0.04 1.89 0.09
0.05 11 0.71 _+ 0.04 2.16 _+ 0.20
0.10 12 0.74 +_ 0.03 2.13 _+0.14
0.15 12 0.70 + 0.06 2.04 +_ 0.15
0.30 19 0.70 + 0.05 2.14 _+ 0.15
a Energy savings are calculated on the basis of untreated control values

b Standard deviation
parenchyma cells and resin ducts [100], where its distinctive activity is to degrade
extractives within about 2 weeks during storage [101]. It is not capable of degrading
lignin or cellulose, but it degrades hemicelluloses to a very small extent. Partial
removal of extractives helps solve several problems associated with pitch, including
downtime for cleaning equipment, breakage of paper on the paper machine, de-
creased paper strength, and holes in the paper caused by sticky spots on rolls [102].
The Cartapip process has shown other beneficial effects as well. It improves
incoming chip quality by inhibiting the growth of dark-colored fungi [103, 104].
This in turn increases chip and pulp brightness, and reduces the need for
bleaching chemicals. The Cartapip process also prevents wood losses caused by
wood-degrading microorganisms. During chemical pulping, the process in-
creases yields and reduces rejects because of the improved penetration of
pulping liquors through empty resin canals and ducts of wood chips [105].
The development of the Cartapip process shows that biological treatment of
wood chips can be successfully implemented on an industrial scale. At this point
in the investigations, it would appear that biopulping has a good chance of
commercial success. Four recent developments have led to this optimism: (1) the
discovery of C. subvermispora, which is effective on both hardwood and soft-
wood species, (2) the finding that brief steaming can decontaminate the surfaces
of wood chips so that C. subverrnispora can take over, (3) the use of unsterilized
corn steep liquor to dramatically reduce the inoculum quantity (from 3 kg to
0.25 g fungus per ton of wood [dry wood basis]), and (4) the demonstration of
a successful 1-ton chip pile (green wood) where the fungal pretreatment saved
32% electrical energy. A recently conducted 100-ton (green wood) outdoor chip
pile experiment produced results similar to those obtained using laboratory
scale bioreactors.
Our efforts so far have focused on the use of fungal treatments prior to
refiner mechanical pulping. Recent studies in our laboratory and in others
suggest that the fungal pretreatment is also effective for depitching [106] and
that it gives benefits with thermomechanical pulping, chemithermomechanical
pulping [107], sulfite pulping [106, 108, 109], and kraft pulping [110, 111].
Acknowledgements. We thank the following for their significant contributions to this research:
Michael Attridge, Todd Burnes, Dough Cameron, Kory Cease, Leatha Dameron, Marilyn ElItand,
Mary Greenheck, George Harpole, Eric Horn, Jane Kohlman, John Koning Jr., Gary Leatham,
Michael Lentz, Ed Lightfoot, Efrat Livney, Lou Lunte, Gary Myers, Lewis Otjen, Jean Pierick,
Sanya Reyes-Chapman,Irv Sachs, Gary Scott, Jane Sherwood,Marguerite Sykes,Freya Tan, Ted
Wegner, and Mary Beth Wall. This research was partially funded by the following companies:
Andritz Sprout-Bauer,Boise-CascadeCorporation, Cellulosa Puerto Piray S.A., Champion Inter-
national Corporation, Chimica del Friuli, Consolidated Papers Incorporated, The Dow Chemical
Company, Great Northern Nekoosa Corporation (Georgia Pacific), James River Corporation,
Leykam Miirzataler AG, Mead Corporation, Mets/i Serla Oy, Nelco Chemical Company, Novo
Nordisk A/S, Potlatch Corporation, Procter and Gamble Cellulose Company, Clariant Corpora-
tion, Scott Worldwide, Union Camp Corporation, and WeyerhaeuserPaper Company.
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Solving Pitch Problems in Pulp and Paper
Processes by the Use of Enzymes or Fungi
Roberta L. Farrell 1, Kunio Hata 2 and Mary Beth Wall 3

1 Dept. of Biological Sciences, University of Waikato, Hamilton, New Zealand
2 Nippon Paper Industries Co., Ltd. 5-21-10ji, Kita-ku, Tokyo 114, Japan
3 Clariant Corp, 4000 Monroe Rd, Charlotte, NC 28205, USA
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2 Extractive Degradation During "Natural" Storage . . . . . . . . . . . . . . . . . . . . . . 199
3 Degradation of W o o d Extractives by Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . 200
3.1 Molds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
3.2 Basidiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.3 Sap-Stain Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.4 Industrial Use of Fungi to Solve Pitch Problems . . . . . . . . . . . . . . . . . . . . . 204
4 Enzymatic Pitch Control in the Papermaking Process . . . . . . . . . . . . . . . . . . . . 206
4.1 F u n d a m e n t a l Research and Theory of Lipase Application . . . . . . . . . . . . . . . 206
4.1.1 Identification of C o m p o u n d Causing Pitch Trouble . . . . . . . . . . . . . . . . 206
4.1.2 Application of Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
4.2 Effect of Lipase Treatment on Prevention of Pitch Deposition . . . . . . . . . . . . . 207
4.3 Application to Papermaking Process ........................... 208
4.4 Mill Trial I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
4.5 Mill Trial II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Pitch problems in pulp mills are often caused by the resinous materials (pitch) in wood which
comprise approximately 2 - 8 % of total composition depending upon the species and time of year.
Traditional methods to control pitch problems include natural seasoning of wood before pulping
and/or adsorption and dispersion of the pitch particles with chemicals in the pulping and papermak-
ing processes, accomplished by adding fine talc, dispersants and other kinds of chemicals. Within the
last five years, two new and different methods of combatting pitch, both of which are biotechnologi-
cal in basis, have been developed independently and are now used industrially. H a t a and colleagues
of Nippon Paper Industries developed a pitch control m e t h o d using the enzyme lipase, which
catalyzes the hydrolysis of triglycerides. Farrell and colleagues of Sandoz Chemicals Biotech (now
known as Clariant) developed a method of pitch control and biocontrol using a fungus developed in
the laboratory from the same type of organisms which cause natural aging, the Ascomycetes. This
fungus is non-colored and prevents the staining and decrease of brightness normally associated with
aged wood.
198 R.L. Farrell et al.
1 Introduction
In today's pulp and paper industry, pitch trouble is often caused by the resinous
materials (pitch) in wood. These are some of the materials ("extractives") that are
extracted from wood during the pulping process, and comprise about 2-8% of
its total composition, depending upon the species and time of year. Not all of the
extractives are troublesome, most problems occurring in pulping and paper-
making when there are shifts in pH and/or temperature. During the pulping
process, these resinous materials are released from wood and later stick to the
tile and metal parts including the rolls and wires of the papermaking machines.
The pitch also stains the felts and canvas, and eventually reaches the dryer
section. This pitch accumulation can cause paper spotting and web breaks on
the machine, which are severe problems in production. Pitch content arid
severity of problems from pitch vary with wood species. The pitch of pines,
including loblolly, slash, and red pines is known to cause serious problems.
Hardwood pitch, particularly from tropical hardwood species, and eucalyptus
can also be detrimental.
Traditional methods of controlling pitch problems include seasoning of
wood before pulping. Seasoning requires raw wood logs (roundwood) to be left
outdoors for several months or chips to be piled and left for weeks. It is the most
commonly used method around the world because wood extractives are decom-
posed during the seasoning process. However, accompanying seasoning are
potential losses due to biological deterioration, such as decreased pulp bright-
ness and pulp yield. Moreover, seasoning increases working capital costs due to
high wood inventory and land use. Thus, this method is often unacceptable,
especially in areas where space is limited. Another method used to reduce the
accumulation of pitch is the adsorption and dispersion of the pitch particles with
chemicals in pulping and papermaking processes. This is accomplished by
adding fine talc, dispersants, and other kinds of chemicals.
In Japan, red pine is the most important wood for groundwood pulp. Red
pine groundwood pulp has high opacity and printability. Therefore, it is an
indispensable pulp for the manufacture of newsprint and light weight paper.
However, the red pine groundwood pulp contains a large amount of pitch. Hata
et al. conducted fundamental research and determined that pitch trouble was
caused by triglycerides within the resinous materials in wood [1-1. These trig-
lycerides form a nucleus upon which other resinous materials tend to accumu-
late, causing pitch troubles. Hata et al. developed a new pitch control method
using the enzyme lipase [1 3]. This method was put into practice in a large scale
papermaking process as a routine operation in the early 1990s, and was the first
case in the world in which enzyme was successfully applied in the actual
papermaking process.
In the USA, Farrell and coworkers independently and concurrently were
also studying biotechnological solutions to pitch problems [4, 5]. These studies
were initiated with the goal of solving pitch problems in loblolly pine by the
Solving Pitch Problems in Pulp and Paper Processes 199
application of a fungus developed in the laboratory from the same Class of

organism which causes natural aging, the Ascomycetes [6]. This organism,
Ophiostoma piliferum, belongs to the sap-staining type of organisms, but was
bred in the laboratory to be colorless and non-staining. Thus, the positive
benefits of aging were achieved with increases in pulp brightness. Also, by
directly applying the fungus to the wood chips or logs, the effect of aging was
accelerated. This product, marketed as Cartapip, was the first case in the world
where an organism was commercially successfully applied prior to pulping to
achieve beneficial effect [-7, 8].
2 Extractive Degradation during "Natural" Storage
Living cells are contained in the bark, foliage, and sapwood when the tree is cut.
These cells remain viable for periods of up to six months when the wood is
stored. The living cells in the wood rays (ray parenchyma) respire and release
heat. Bacteria and fungi are provided with good growth conditions during this
heat generation, and the starches and simple sugars of the rays and subsequently
by the extractives of the wood can be metabolized as a source of carbon and
energy. This metabolism results in an overall decrease of pitch with storage of
wood.
The outside storage of pulpwood was introduced in the 1920s as whole logs
(roundwood), and in the early 1950s as chips [9]. This method was the direct
result of the need to stockpile wood as inventory to mills, to handle intermittent
flow of chips to the mill, and to season wood, which resulted in decreased resin
deposition. The reduction in pulp brightness and yield during storage was
shown to be of the same order whether the wood had been stored as chips or as
roundwood [10]. Conditions which affect the wood were shown to be the
following: species of wood, time of cutting, removal of bark, presence of insects,
methods of piling, length of storage time, general housekeeping conditions in the
woodyard, and climatic conditions, especially temperature and moisture. Tem-
perature appeared to be the single most important factor affecting distribution
and prevalence of microorganisms in various sections of the chip pile in one
study on microbiological effects of seasoning on hardwoods 1-11]. Outside
storage of white spruce (Picea glauca) and lodgepole pine (Pinus contorta)
showed decreased wood substances by 3.8% and 4.5% respectively after
6 months. Most of this decrease was attributed to pitch components [12]. This
study also showed that pine kraft pulp yield increased based on seasoned chips,
though spruce kraft pulp yield decreased slightly with time of storage.
Seasoning has been recommended for pine unless the recovery of maximum
tall oil and turpentine yields was desired [13]. Eighty percent losses in tall oil
and turpentine yields resulted after 30 weeks storage. Pulp strength in this and
other studies was shown not to be affected by seasoning, with the possible
200 R.L. FarreU et al.
exception of tear strength. Although seasoning chips reduces pitch troubles, the
negative effects of seasoning on various wood species were also thoroughly
studied in North America and Europe [14, 15]. The single most detrimental
effect was loss of brightness of mechanical and sulphite pulps after storage,
particularly with softwoods [10].
3 Degradation of Wood Extractives by Fungi
A variety of wood-inhabiting fungi including molds, sap-stain, brown-rots, and

white-rots are capable of degrading wood extractives.
3.1 M o l d s
Although molds are capable of degrading wood extractives, the contribu-

tion of molds to extractive degradation is expected to be minimal because
molds grow less prolifically in wood than do other wood-inhabiting fungi.
Molds grow best on wood that is very wet or that has been exposed to very
high humidity for a long time. On softwoods, molds grow mainly on wood
surfaces. On hardwoods, molds can enter the wood at exposed parenchyma,
vessels, and ruptured cells and can move throughout the wood by rupturing
pit membranes [16]. Nilsson and Asserson have shown that the following
molds degrade wood waxes in liquid culture: Penicillium roqueforti, Penicillium
funicolosum, Rhizopus arrhizus, and Trichoderma lignorum [17]. In addi-
tion, they showed that the following molds could reduce the ethanol/benzene
(1:2) extractive content of wood chips: R. arrhizus, Gliocladium viride, Penicil-
lium rubrium, T. lignorum, and Aspergillus fumigatus. The wood chips were
Table 1. DCM extractive content of nonsterile southern yellow pine treated with various
molds
Extractives (%) Extractives (%)

Fungal Species Control 1 Treatment Reduction %
Phlebia roqueforti 3.34 2.16 35

Leptographium 2.27 1.92 15
terrebrantis
Verticicladiella 2.27 1.96 14
truncata
Diplodia pinea 2.27 2.01 11
Codinaea sp. 2.27 2.07 9
Aureobasidium 3.34 3.26 2
pullulans
1 The control was chips that had been frozen at -- 20 ~ since the start of the experiment
Solving Pitch Problemsin Pulp and Paper Processes 201
stored at 35~ and sampled at 30 days. The type of wood chips tested was not
given.
Iverson et al. screened various molds for their ability to degrade
wood extractives 1-18]. Nonsterile southern yellow pine chips were inoculated
with 104 to 108 colony-forming units/g wet weight wood and incubated at
room temperature for 2 weeks. As shown in Table 1, the best fungus tested
was P. roqueforti, which reduced the dichloromethane (DCM) extractive content
by 35%.
3.2 Basidiomycetes
Basidiomycetes, including white-rot and brown-rot fungi, extensively

colonize wood. Brown-rot fungi preferentially degrade wood polysaccharides
including cellulose and cause rapid decreases in its degree of polymerization.
Brown-rotted wood usually shows virtually no decrease in total lignin content.
Rather than degrading lignin, brown-rot fungi modify it by oxidation and de-
methylation of methoxy groups. White-rot fungi are the predominant degraders
of lignin in nature. Some species of white-rot fungi preferentially degrade
lignin to wood polysaccharides, and other species degrade all wood compo-
nents simultaneously. The Basidiomycetes have been shown to degrade pitch
extractives.
Several fungi observed to have "biopulping" activity have also been
shown to degrade wood extractives. Biopulping involves the use of fungally
treated chips to obtain pulping benefits such as reduced energy used during
mechanical pulping or improved chemical pulping efficiency. Lim and Cho
have shown that the ethanol/benzene (1:2) extractive content of oak treated
with Phanerochaete chrysosporium decreased by 46% after twelve weeks [19].
Treatment of sterile southern yellow pine wood chips with P. chrysosporium
for 2 weeks resulted in a 21% reduction in dichloromethane (DCM) extractives
[20]. Fischer et al. have shown that the DCM extractive content of
sterile loblolly pine chips treated with C. subvermispora decreased after
four weeks by 32% [21]. In addition, they showed that both C. subvermispora
and P. chrysosporium decrease the DCM extractive content of sterile spruce
chips [21].
Various Basidiomycetes were screened for their ability to degrade wood
extractives [-18]. Sterile southern yellow pine was inoculated with 104 to l0 s
colony-forming units/100 g wet weight wood and incubated for 2 weeks at room
temperature. As shown in Table 2, P. chrysosporium performed best, reducing
DCM extractives by 51%. Hyphodontia setulosa, Perenniporia subacida,
P. gigantea, and Phlebia tremellosa also performed well, reducing extractives by
about 40%. Poller and Schultze-Dewitz investigated the effect of two brown-rot
fungi, Coniophora puteana and Gloeophyllum saepiarium, and one white-rot
fungus, Phellinus igniarius, on the extractive content of pine [22]. All three
fungal treatments resulted in large reductions in extractive content.
Table 2. Extractive content of sterile southern yellow pine treated with various Basidiomycetes
Control Treatment
Fungal species Extractives (%) Extractives (%) Reduction (%)
Phanerochaete chrysosporium 2.19 1.30 41

P. subacida 3.34 2,01 40
P. gigantea 3.34 2.03 39
P. tremetIosa 1.98 1.21 39
H. setulosa 1.98 1.20 39
Coriolus versicolor 1.98 1,28 36
Inonotus rheades 3.34 2,18 34
Trichaptum abietinium 4.70 3.13 33
C. subvermispora 3.34 2.18 29
Trichaptum biforme 4.70 3.13 24
Schizophylum commune 2.50 2.03 17
Sistotrerma brinkmanii 2.44 2.17 11
Pleurotus ostreatus 2.44 2.30 6
Alurodiscus sp. 2.44 3.70 0
Ganoderma collosum 2.29 1.75 0
PheUinus igniarius 2.44 2.48 0
3.3 Sap-Stain Fungi
Sap-stain fungi rapidly colonize the sapwood of logs and wood chips. These
fungi grow mainly in ray parenchyma cells and are capable of deeply penetrating
logs and wood chips. In addition, these fungi can grow within resin canals,
tracheids, and fiber cells, and penetrate simple and bordered pits, occasionally
forming boreholes through wood cell walls. Sap-stain fungi are not capable of
degrading the major components of the wood cell wall: cellulose and lignin.
Hemicellulose is degraded to a very slight degree. Extractives and simple sugars
found in the parenchymal cells are the major nutrient source for these fungi.
Sap-stain fungi cause a characteristic staining of sapwood, resulting in a blue,
black, grey, or brown discoloration of the wood. Sap-stain causes major eco-
nomic losses in the lumber and mechanical pulping industries. Problems with
sap-stain are most prevalent in warm, humid climates and when wood with
a high sapwood content is used.
Common species of sap-stain on softwoods include: Ophiostoma ips,
O. piliferum, O. piceae, Aureobasidium pullulans, Leptographium lundbergii, Alter-
naria alternata, Cephaloascusfragrans, Cladosporium spp., Lasiodiplodia theob-
romae, and Phiolophora spp. [-23]. Common species of hardwood sap-stain
include: Ophiostoma pluriannulatum, Ceratocystis moniliformis, L. theobromae,
Ceratocystis rigidum [23]. Many of these species are capable of degrading wood
extractives. Extractive degradation by Ophiostoma spp., particularly O. piliferum
and O. piceae, has been most widely studied.
Iverson et al. screened a variety of sap-stain fungi for the ability to degrade
wood extractives [18]. Sterile southern yellow pine was inoculated with the
fungi listed in Table 3 and incubated at room temperature for 2 weeks. The best
Table 3. Extractive degradation by sap-stain fungi on nonsterile southern yellow pine

Control Treated
Fungal Species Extractives (%) Extractives
(%) Reduction
(%)
c. adiposa 2.13 1.26 41
O. piliferum 3.34 2.27 32
C. adjuncti 1.98 1.44 27
C. minor 2.13 1.57 26
O. piceae 2.13 1.57 26
O. populina 2.13 1.62 24
O. abiocarpa 2.13 1.61 24
C. tremuloaurea 2.13 1.65 23
O. fraxinopennsylvanica 2.13 1.71 20
O. plurianulatum 2.13 1.73 19
E. aereum 1.98 1.61 19
C. ponderosa 2.19 1.86 18
C. penicullata 1.98 1.58 15
O. olivaceum 2.27 1.93 15
E. clavigerum 2.13 1.84 14
C. hunti 2.13 1.89 11
C. ambrosia 2.13 1.92 I0
O. distortum 2.27 2.07 9
E. robustum 2.27 2.06 9
C. virescens 2.27 2.11 7
O. 9aleiformis 2.13 1.98 7
C. coerulescens 2.13 2.13 0
O. dryocetidis 2.24 2.27 0
O. stenorcerns 2.13 2.21 0
X. conudamae 2.44 3.16 0
X. hypoxylon 2.44 2.88 0
species for extractive reduction were C e r a t o c y s t i s adiposa, O. piceae, and

O. piliferum. Nine different isolates of O. piliferum were also screened on sterile
southern yellow pine using the same procedure; 22% of the isolates did not
reduce D C M extractives and 55% of the isolates reduced D C M extractives by
25 35%. In addition, 45 different strains of O. piceae were screened on sterile
aspen chips. These strains can be divided into four groups based on their ability
to reduce ethanol/toluene extractives: 24% of the isolates did not degrade
extractives, 46% reduced extractives by 1-15%, 28% reduced extractives by
16 35%, and one isolate reduced extractives by 60%.
Chen et al. studied the effect of five sap-stain fungi on the composition of
aspen and lodgepole pine extractives [24]. Four fungi were selected on the basis
of high lipolytic activity from 100 fungal strains isolated from Canadian lumber
mills and compared with the commercial strain, Cartapip | 97. Analysis of
untreated wood showed that triglycerides were the most abundant component
of both aspen and lodgepole pine sapwood extractives. The wax and steryl ester
content of aspen was about 3 times that of lodgepole pine, and fatty and resin
acids were the second most c o m m o n component of lodgepole pine extractives
but were present in very small amounts in aspen. All five fungi decreased the
total acetone extractive content of aspen and lodgepole pine sapwood to
Table 4. Extractive content of sterile lodgepole pine and aspen

treated with sap-stain fungi
Treatment Aspen Lodgepole pine

Extractives (%) Extractives (%)
Control 3.09 _+ 0.07 2.31 _+ 0.03

Aged Control 2.88 _+ 0.04 2.26 _+ 0.02
Cartapip 97 2.15 _+ 0.01 1.94 _+ 0.03
Strain A 2.13 _+ 0.02 2.08 _+ 0.01
Strain B 2.22 _+ 0.01 NA
Strain C 2.07 _+ 0.04 1.92 _+ 0.11
Strain D 2.08 + 0.05 1.92 + 0.01
a similar degree - 28-33% for aspen and 1 ~ 1 7 % for lodgepole pine (Table 4).
Extractive component analysis showed that all five fungi decreased triglyceride
content and steryl esters/waxes content, and that four of the five fungi increased
free fatty acid content.
3.4 Industrial Use o f Fungi to Solve Pitch Problems
Several studies have shown that wood extractive components such as triglycer-
ides, resin acids, and steryl esters are major components of paper machine pitch
deposits [1, 2, 6]. In addition, pitch outbreaks are more common when resinous
wood species are used and during seasons when wood resin content is parti-
cularly high.
There is a living fungus, marketed to the pulp and paper industry, which
metabolizes and thus removes pitch. This fungus is a colorless strain of
O. piliferum, an Ascomycete of the same species of that often dominates in
naturally seasoned piles. Marketed as Cartapip, with different numbers denot-
ing different strains such as 97 and 58, it is commercialized as a powder
inoculum. Moreover, Cartapip use results in a biocontrol effect, i.e., the presence
of Cartapip reduces growth of other, undesired organisms. One kilogram of the
powder can treat about 1200 tons of wood chips. Industrial use involves
dispersing the powder in mill water and spraying it onto chips as they are
conveyed to a chip pile.
Selective breeding was used to obtain this isolate, which rapidly colonizes
nonsterile wood chips, rapidly degrades extractives, and is colorless and non-
staining. Most O. piliferum strains are a bluish-black color. Growth of pig-
mented fungi on wood chips reduces chip brightness and increases bleach usage
when these chips are used to produce T M P or sulfite pulp. Because Cartapip
outcompetes indigenous microorganisms and maintains chip brightness, use of
this product reduces bleach chemical usage during T M P production, in addition
to reducing the extractive content of chips and pulp, and alleviating pitch
problems. Use of treated chips has also been shown to increase paper strength
[25]. Moreover, treatment of wood chips with Cartapip also results in improved
chemical pulping efficiency [26]. Reductions in kappa number were observed

during laboratory-scale kraft and sulfite pulping. Wall et al. hypothesize that the
improved pulping efficiency observed experimentally is caused by more rapid
and more uniform penetration of steam and cooking chemicals in the fungally
treated chips [27].
Two commercially available strains of O. piliferum, Cartapip 28 and
Cartapip 58, have been shown to degrade the extractives of both hardwoods
and softwoods including aspen, southern yellow pine, red pine, and spruce [27],
Both fungi in two weeks reduced the diethyl ether extractive content of fresh
nonsterile southern yellow pine wood chips by 40%, or 22% if the chips were
aged but not inoculated. In addition, the white strain, Cartapip 58, maintained
chip brightness. This study also showed that Cartapip 28 decreased the D C M
extractive content of sterile southern yellow pine chips by 30% for a 2 week
treatment. O. piliferum has also been shown to reduce more DCM extractives of
nonsterile red pine in 31 days (48% reduction) than in 21 days (32% reduction)
[18]. O. piliferum, strain Cartapip 97, also reduces the DCM extractives of
spruce by 25% in a 2 week treatment, and the DCM extractive content of sterile
loblolly pine chips by up to 35% in a 4 week treatment [21]. Cartapip 97
reduced the acetone extractive content of fresh nonsterile aspen chips by 36%
after a 3 week incubation, or 13% for uninoculated aged chips [27].
Cartapip 28 and Cartapip 58 significantly decreased the fatty acid content
and the unidentified compound content, which includes waxes, alcohols, and
sterols, of southern yellow pine extractives l-6]. In particular, Cartapip 58
decreased esterified fatty acids by 60%. Both esterified fatty acids and non-
saponifiable compounds such as waxes and steryl esters have been shown to be
major components of industrial pitch deposits [6]. Both fungal treatment and
natural microbial activity increased the free fatty acid content of the extractives.
The increase in free fatty acid content results from initial hydrolysis of esterified
fatty acids to free fatty acids. The free fatty acid content of the Cartapip-treated
chips is lower than that of naturally aged chips, indicating further metabolism
and removal of these components by the fungus. In addition, analysis of the
individual fatty acids showed that fungal treatment significantly decreased the
content of the three fatty acids found in highest concentration in the untreated
southern yellow pine chips oleic acid by 44%, linoleic acid by 64%, and
palmitic acid by 45%.
The results of Cartapip treatment such as pitch removal and maintence
of chip brightness and improved paper machine runnability have been
documented by use in mills. In a T M P mill using southern yellow pine, a trial
was performed comparing a two week period using the Cartapip product on
their wood chips to a two week period without product use, and the results are
shown in Table 5 [24]. Reductions in the DCM extractive content of secondary
refiner pulp caused expected reductions in alum, a pitch control chemical.
Because Cartapip 97 is a colorless strain that outcompetes indigenous micro-
organisms, its use results in brighter chips. This effect was observed as a 36.9%
reduction in bleach usage along with increased paper brightness of 0.9%. In
Table 5. Use of a depitching organism in a TMP mill

DCM extractive content of secondary - 37.5%
refiner pulp
Alum usage - 31.7%
Bleach usage -- 36.9%
Brightness + 0.9%
Tensile index + 5.4%
Tear index + 3.4%
Burst index + 3.3%
addition, strength properties were increased, probably due to the lower extrac-
tive content of the paper. Brandal and Lindheim have shown an inverse
relationship between paper strength and pitch content [28]. A two m o n t h
Cartapip 97 trial at a US T M P mill using southern yellow pine showed
significant reductions in the D C M extractives of wood chips and an increase in
burst index [24]. A one week trial was performed at a mill in Northwestern USA
using a blend of 60% lodgepole pine and spruce and 40% fir and hemlock. Only
the pine/spruce mixture was treated because this mixture caused the most
serious pitch problems. Cartapip 97 treatment reduced the averaged D C M
extractive content of the reclaim chips by 25% [25].
4 Enzymatic Pitch Control in the Papermaking Process
4.1 Fundamental Research and Theory of Lipase Application
Initial studies to demonstrate the effect of lipases on pitch were conducted on

Japanese red pine groundwood by the group of H a t a and colleagues at N i p p o n
Paper [1-3]. Subsequent studies have also been carried out on softwood sulfite
pulp and birch sulfate pulp [29-32].
4.1.1 Identificationof Compound CausingPitch Trouble
A new method using an adsorption resin was established, instead of the previous
solvent fractionation method, in order to fractionate red pine pitch and to
determine what were the components that were sticky and causing pitch
troubles [33]. Pitch compounds in red pine as well as deposited pitch were
fractionated using the method and analyzed by gas chromatography. The
changes in pitch compounds during the seasoning period and the contents of
pitch in fresh wood were also investigated in great detail to understand the
seasoning mechanism. These investigations produced the following results:
1. Pitch compounds could be fractionated into polar and nonpolar fractions

[2].
2. Fresh wood contained more nonpolar compounds, especially in winter. The
main component consisted of triglycerides (TG) [2].
3. 96% of the fatty acids that composed TG were oleic and linoleic acids [2].
4. TG was rapidly decomposed and reduced during seasoning [1].
5. Deposited pitch in the papermaking process always contained much TG [2].
Based on these results, TG was estimated to be the key to pitch troubles. In
general, nonpolar compounds such as TG may easily adhere to hydrophobic
surfaces, such as rolls, by Van der Waals forces and build to become pitch
deposits. It was hypothesized by Hata and his coworkers that if TG in pulp
slurry could be converted to less adhesive components, pitch deposits would
decrease. The conversion of TG would enable the use of flesh wood with less
probability of pitch trouble.
4.1.2 Application of Lipase
Lipase specifically hydrolyzes TG, and thus was not expected to affect the
environment or the paper quality. Three kinds of lipase, each produced by
a different microorganism, were used in the original work by Hata and col-
leagues, and their properties are given in Table 6.
4.2 Effect of Lipase Treatment on Prevention of Pitch Deposition
Resinuous materials extracted from red pine wood and groundwood pulp (GP)
were treated with lipase, and their adhesiveness to the hydrophobic surface was
determined [1, 23. As shown in Table 7, the pitch deposits increased when the
ratio of nonpolar compounds to polar compounds increased. Thus, evidently
the nonpolar compounds of the pitch materials had higher adhesiveness to
hydrophobic material and seemed to play an important role in pitch deposition.
TG was shown to be a key material in pitch deposition because the enzymatic
hydrolysis of TG reduced pitch deposition significantly [2]. TG was hydrolyzed
to glycerol and fatty acids with the lipase, and the resulting glycerol dissolved
into water. Fatty acids existed in the form of aluminum salt in the presence of
alum, and were dispersed into the pulp slurry and fixed on the surface of fibers.
CHzOCORi CH2OH R1COOH

~HOCOR2 Lipase ~HOH + R2COOH
~H2OCOR3 ~H2OH R3COOH
Triglycerides Glycerol Fatty acids
(Water-soluble) (Fixed on fibers as A1 salt)
4.3 Application to Papermaking Process
Since the effect of lipase on reducing pitch deposits was confirmed, the techno-
logy was applied to the actual papermaking process [-2,3]. To select optimum
conditions for the lipase treatment in mills, the following factors were investi-
gated: the effects of enzyme concentration, reaction temperature, reaction time,
and agitating mode on the hydrolysis of TG.
The following results were obtained from the investigation:
1. It was necessary to have a strong mixing system to keep contact between
enzyme and TG for the effective reaction by enzyme.
2. Under sufficient mixing conditions, lipase 5 000 U/kgGP (300 ppm Lipase B)
could hydrolyze more than 80% of TG in the surface pitch (n-hexane extract
from GP slurry) within two hours.
3. No effect of the lipase treatment on the brightness and strength of pulp was
observed.
4.4 Mill Trial I
Based on these results, the first long run mill trial was conducted using a large
paper machine [2]. In this mill, the paper machine using red pine GP always had
serious pitch problems because large amounts of red pine were used as raw
materials for GP. Normally, 50% of unseasoned wood and 50% of wood
seasoned for six months were consumed. Therefore, GP had a high content of
pitch and a 30% TG content in the pitch. In order to reduce the pitch problem, it
was in the past necessary to extend the seasoning period of wood to supply the
mill, and the use of fine talc and dispersant was also increased. As an attempt to
solve this problem, lipase was added to the groundwood pulping line just before
the post refiner (Fig. 1). The operating conditions were as follows:
Paper machine: Bel Baie II, wire width 5,080 mm
Paper product: Yellow Telephone Directory paper (YTD) (34 g/m2)
Newsprint (46 g/m2)
Pulp: Red pine groundwood pulp (15-40%), de-inked pulp, soft-
wood semi-bleached kraft pulp
Machine speed: 830 m/rain
Production rate: 220-270 t/d
Enzyme: Lipase A 75-125 ppm GP
Lipase B 50~750 ppm GP
Reaction time: 40-60 rain
An initial test was done in order to understand the proper dosage for a long
run mill trial and to show the hydrolysis rate of TG by lipase in the actual
papermaking process. Lipase A (125-ppm addition) on the first day of the mill
trial reduced the content of TG by 74%.
For a one month lipase trial in the mill, the following parameters
were compared between the usual operation and the lipase treatment opera-
tion in major products, such as newsprint and YTD: Content of the surface
pitch and TG, first pass retention (FPR) of pulp and pitch, pitch deposits on
the wall of the machine chest, amount of wet pitch deposits, number of defects
in paper web, and dynamic friction coefficient (DFC) of paper. For 1.5-2.0%
of oven-dried GP, the content of TG was 16-26% of the surface pitch
[1]. Apparently the lipase hydrolyzed 70% of TG until reaching the
mixing chest inlet. Furthermore, the accumulation of the pitch in the recycled
white water (stock inlet, saveall) decreased to a lower level after the lipase
treatment.
As shown in Fig. 2, the first pass retention (FPR) of pulp did not change with
lipase addition [2]. However, the F P R of the pitch increased from 5-9% to
12-19% in YTD, and from 9 14% to 13-24% in newsprint. As the lipase
hydrolyzed TG, the pitch was dispersed into the pulp slurry and distributed
onto the fibers without unevenness to the surface. Lipase also prevented the
accumulation of pitch in the recycled white water system. As shown in Fig. 3,
pitch deposit was observed as a black piling during the usual operation.
However, pitch deposits could rarely be observed after a 1-month trial with the
addition of lipase. Fig. 4 clearly shows that the lipase prevented the pitch
deposition on the chest wall.
In order to evaluate the pitch deposits in the wire and press sections, pitch
deposit was collected from each section and measured every day. Results
showed a dramatic decrease in the weight of pitch deposit with the lipase
treatment compared to that of pitch in normal operation I-2]. The above results
strongly proved that TG in the pitch was hydrolyzed and then converted to less
sticky compounds. Long term data collected by spot detectors showed that the
number of defects, holes, and spots larger than 1.5 mm was reduced from 61 to
19 as a daily average by the addition of lipase. When comparing long term data
between the normal and the lipase operations, it was clear that the quality of
products improved with lipase use.
When a paper roll is printed on a web offset press, it is very important to
prevent runnability problems such as wrinkling and uneven movement of the
paper roll. This runnability performance is especially a concern in newsprint
rolls. The dynamic friction of paper is thought to be related to these problems,
and dynamic friction coefficient (DFC) is regarded as a quality control para-
meter at some Japanese paper companies. When D F C is low, the web tends to
snake on the printing press. Therefore, when D F C of newsprint drops to a low
level, white carbon (amorphous silica gel SiO2 x H20) is usually added to the
paper furnish to increase DFC.
As lipase treatment was incorporated into the production process, there was
an increase in the newsprint D F C and a decrease in the amount of white carbon
dosage. In order to reach a certain D F C level during newsprint production,
about 2% of white carbon is added in the production process. However, by
incorporating the lipase treatment of GP, there was a decrease in white carbon
dosage to 1%. With the lipase treatment of GP, an increase of DFC was also
confirmed [3].
4.5 Mill Trial H
Another long term mill trial was conducted in March of 1990 [3]. Newsprint
consisting of 15-35% GP was produced from red pine. This wood was normally
seasoned in the mill yard for at least three months after cutting and collecting.
This treatment reduced the TG content in wood and prevented severe pitch
problems in the newsprint production process. However, there were still pitch
deposits on the center roll of the paper machines, especially during winter,
making it necessary to clean the center roll frequently. Lipase was added to
make the operation smooth and to increase the unseasoned wood ratio in its
furnish.
The Machine conditions during the trial were as follows:
Paper machine: Bel Baie II, wire width 3,800 mm
Product: Newsprint (46 g/m2)
Pulp: GP (15 35%), KP, TMP, de-inked pulp
Machine speed: 1,000m/min
Production rate: 200t/d
Enzyme: Lipase A 80-100 ppm GP
Lipase C 400-500 ppm GP
The long term mill trial yielded the following results:
1. The frequency of cleaning was increased during winter (from January to
April). However, the addition of lipase decreased the pitch deposits on the
center roll, and the frequency of cleaning decreased to the level of summer
(Table 8).
2. Increase of pitch deposit was not apparent on using 50% unseasoned wood.
3. Based on the two mill trials, lipase should be added to the pulp slurry before
the addition of alum.
Therefore, with the use of lipase, there was a decrease in TG content in fresh
wood and reduction in the cost of seasoning and bleaching chemicals.
Lipases for hydrolysis of pitch components have subsequently been ap-
plied to other wood species and to chemical pulps with successful results.
Fischer and Messner applied the commercial lipase product Resinase A 2X
(Novo Nordisk A/S) to unbleached softwood sulfite pulp [29-31]. In these
studies, they drew the following conclusions concerning the activity of lipase on
sulfite pulps:
1. Lipase is rapidly absorbed onto pulp fiber within a minute of addition [29].
2. In a pilot mill scale treating 12 tons pulp day, with pulp at 4% consistency,
85 90% of the triglycerides were hydrolyzed, as determined by reduction of
one triglyceride fraction from gas chromotography analysis [30], and the
resin content of the pulp was reduced by 60% [31].
Lipases from Candida cylindracea have been shown to be effective in

hydrolyzing triglycerides in extractives of fresh birch and birch sulfate pulp [32].
The total amount of esterified compounds in fresh birch was decreased by 34%;
50% and 65% respectively of the esterified fatty acids and saturated fatty acids
were also hydrolyzed. For treatment of birch sulfate pulp, the lipases of C.
cylindracea hydrolyzed 30% of the esterified lipids as compared to 40%
hydrolyzed by Resinase A (source organism Aspergillus from Novo Nordisk
A/S). Esters of saturated fatty acids, alcohols and sterols were hydrolyzed by
both lipases, though the C. cylindracea lipase was incapable of degrading esters
of betulaprenols and triterpenoids, whereas the Aspergillus enzyme hydrolyzed
them to some extent.
5 Conclusions
The enzymatic pitch control method using lipase was the first successful
example of the use of an enzyme as a solution to pitch problems and in the
papermaking process. The fungal pitch control method, using the colorless
isolant of Ophiostoma piliferum, was the first successful case of using a live
organism as a solution to pitch problems and in the pulping process. Both
technologies use biotechnology as their basis and have been successfully used in
full scale industrial mills in various parts of the world.
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29. Fischer K, Messner K (1992) Proceedings of the 5th International Conference on Biotechnology
in the Pulp and Paper Industry, p 169
30. Fischer K, Messner K (1992) Enzyme Microb Technol 14:470
31. Fischer K, Puchinger, Schloffer K, Kreiner W, Messner K (1993) J Biotechnol 27:341
32. Mustranta A, Fagernas L, Viikari L, Tappi Journal 78:140
33. Assarssopn A, Akerlund G (1966) Svenks Papperstidn, 69:291
Reduction of Organochlorine Compounds
in Bleach Plant Effluents
Pratima Bajpai and Pramod K. Bajpai

Chemical Engineering Division, Thapar Corporate Research and
Development Centre, Patiala - 147 001, India
List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2 Environmental Impact of Organochlorines . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
3 Reducing the Generation of Organochlorine C o m p o u n d s . . . . . . . . . . . . . . . . . . 226
3.1 Enzymatic Prebleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3.1.1 Hemicellulase Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3.1.2 Ligninolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3.2 Fungal Prebleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
4 Treatment of Bleach Plant Effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
4,1 Biological Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.1.1 Using Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.1.1.1 Aerobic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
4.1.1.2 Anaerobic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
4.1.2 Using Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
4.2 Enzymatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Organochlorines have been a matter of concern in the pulp and paper industry for the last two
decades. These c o m p o u n d s are produced mainly by the reactions between residual lignin present in
wood fibres and the chlorine used for bleaching. Some of the organochlorine c o m p o u n d s are found
to be toxic, mutagenic, persistent, bioaccumulating and cause h a r m in biological systems.
Earlier measures taken by the pulp a n d pape r industry to solve the chlorine problem have
focussed on improving effluent treatment methods. Today, the emphasis of research and develop-
ment work in this area has shifted more towards improving the processes. In the search to produce
pulp with non-polluting chemicals, more efficient pulping m e t h o d s reducing the a m o u n t of residual
lignin passing to the bleaching process and alternative bleaching methods, are being developed.
There are also possibilities for treating effluent with microorganisms and enzymes to remove the
dechlorinated organic material. Each option has inherent advantages and disadvantages with regard
to capital costs, operating costs, ease of retrofit, fabrication a n d installation time. Impact on other
mill unit operations is also considered in choosing the best options. M a n y factors have to be
considered in choosing an effective and economical bleaching/treatment process that meets all the
environmental guidelines.
This review describes the environmental impact of organochlorines, use of enzymatic and
biological bleaching for reducing the generation of organochlorine compounds, and treatment of
bleach plant effluents by biological and enzymatic methods. Advantages and limitations of various
biotechnological methods are discussed.
9 Springer-VerlagBerlinHeidelberg 1997
214 P. Bajpai and P.K. Bajpai
List of Abbreviations
ADI Acceptable daily intake

AOX Adsorbable organic halogens
BOD Biological oxygen demand
COD Chemical oxygen demand
CTMP Chemi-thermomechanical pulp
DDT Dichlorodiphenyltrichloroethane
ECF Elemental chlorine free
EOC1 Extractable organic chlorine
EOX Extractable organic halogen
EPA Environmental protection agency
HC High concentration (consistency)
HRT Hydraulic residence time
ISO International standards organization
LMS Laccase mediator substrate
MBC Modified batch cooking
MCC Modified continuous cooking
MW Molecular weight
OKP Oxygen bleached kraft pulp
PCB Polychlorinated biphenyl
PCDD Polychlorinated dibenzo dioxins
PCP Polychlorophenol
Pow Octanol/water partition coefficient
ppb Parts per billion (1/109)
ppm Parts per million (1/106)
ppt Parts per trillion (1/1012)
RBC Rotating biological contactor
t metric ton
TCDD Tetrachlorinated dibenzodioxins
TCF Totally chlorine free
TCP Trichlorophenol
TEQ Toxicity equivalent
TMP Thermomechanical pulp
TNT 2,4,6-Trinitrotoluene
TOC1 Total organochlorine
Bleachin9 stages
C Chlorination
(CD) Treatment by mixing chlorine and chlorine dioxide simultaneously
(C1 proportion is higher than D)
D Chlorine dioxide treatment
D1 & D2 First and second treatment, respectively with chlorine dioxide
Reductionof OrganochlorineCompoundsin Bleach Plant Effluents 215
(DC) Treatment by mixing chlorine dioxide and chlorine simultaneously

(D proportion is higher than C1)
E Alkaline extraction
E1 & E2 First and second alkaline extraction respectively
Eo Alkaline extraction in presence of oxygen
Eov Alkaline extraction in presence of oxygen and hydrogen peroxide
Ev Alkaline extraction in presence of hydrogen peroxide
F Fungal treatment
H Hypochlorite treatment
L Laccase enzyme treatment
O Oxygen delignification/bleaching
P Hydrogen peroxide treatment
Q Chelation of metal
X Xylanase enzyme treatment
Z Ozone treatment
1 Introduction
The bleaching of pulp became an issue of great concern during the 1980s
primarily because of growing alarm over chlorinated organic compounds,
referred to as adsorbable organic halogen (AOX), in bleach plant effluents.
In particular, the detection of chlorinated dioxins and furans led to strong
reactions.
The paper industry recognized chlorine bleaching to be a potential problem.
As the analytical instruments and methods became available, especially for
chlorinated organic substances, studies were made on the chemical composition
of bleach plant effluents. The new analytical methods and studies also created
a basis for governmental regulations. The focus has been on setting upper limits
for AOX/TOC1. At present, the strictest general emission limit value for AOX is
1 kg/t of pulp. This limit applies to sulphite pulp e.g. in Austria, Germany and
Norway. This is because AOX emissions are easier to reduce with sulphite pulp
than with softwood kraft pulp. However, in Sweden, kraft pulp mills have
individual limits as low as 0.3 kg/t pulp. Some decided or planned regulations
for AOX are shown in Table 1 [1]. The permitted levels are likely to decrease to
about 1 kg/t in the next few years in many countries. In several countries or
provinces, emission limits are decided mill by mill taking into account the local
Table 1. Regulations for discharge of chlorinated organic compounds

measured as AOX (kg/t pulp) from bleaching of chemical pulps
Country 1994 1995-2000 2000-2005
Australia 1.0a
Austria 0.75 1.5 0.5 1.0
Belgium 1.5
Canada
- Alberta 0.29a/1.5
British Columbia 1.5 0
Ontario 1.5 0.8
Quebecb 1.5 (HW) 1.0-2.0
Finlandb 1.0-2.0
Germanyb 1.0
India~ 2.0
Norwayb 1.0-2.0
Japanc 1.5
USAa 0.156
Sweden 1.2-1.5 0.3-1.0 0.3-0.5
"Limits for new mills
bLower limits for hardwood pulps
cGuidelines of Japan Pulp, Paper and Paperboard Association
a Proposed regulation on "Cluster Rules" by EPA
eProposed by Ministry of Environment and Forest, Government of India
Based on data from Ref. [1]
Reduction of OrganochlorineCompoundsin Bleach Plant Effluents 217
factors. Thus, for individual mills stricter emission limits may apply than those
given in the table. According to a decision in 1992 by PARCOM (Paris
Convention for Prevention of Marine Pollution from Land Based Sources and
Rivers) signed by twelve European countries, a general AOX emission limit
would be 1 kg/t from 1995. The limit applies for all types of bleached chemical
pulp and has been accepted by Belgium, Denmark, France, Germany, Great
Britain, Ireland, Luxembourg, the Netherlands, Norway, Portugal, Spain and
Sweden. In the USA, the Environmental Protection Agency (EPA) has proposed
new emission limits for several categories of pulp, the so called cluster rules. The
proposed emission limit for AOX for bleached kraft pulp is 0.156 kg/t. The new
regulations will affect every existing and new facility in the pulp and paper
industry and are expected to be promulgated soon. An AOX limit of 0.156 kg/t
as proposed by the US EPA is very stringent when compared to current
emissions even from Scandinavian mills (Fig. 1). Germany is also discussing
legislation which will not only ban production of pulp using chlorine-containing
chemicals but also consumption of other pulps than totally chlorine free (TCF).
The Ministry of Environment and Forest, Government of India, has categorized
the pulp and paper industry as one of the twenty most polluting industries and
advised the paper industry to self-impose the AOX discharge limit of 2 kg/t of
paper. Some of the state pollution control boards in India have already intro-
duced the AOX limit of 2 kg/t of paper as a controlling parameter.
1.4
1.2 ] Scandinavian mills
II Canadian rnitis
1.0
0.8
v 0.6
x
o
Cluster rulesl
M
0.4
II
0.2
R
3456 10 11 1 2 1 3 1 4 1 5
I I I
161718192021 22,23:425 26 27 28 29 30 31
Mill
Fig. 1. AOX emissionsfrom Scandinavianand someCanadianbleachingkraft pulp millsin 1994

Despite some shortcomings, the kraft process is the most cost effective,
versatile and efficient wood delignification method available. Kraft pulp is
generally more difficult to bleach than sulfite pulp. With softwood pulp, it is
harder to achieve a high brightness product than with hardwood. Chlorine is
one of the popular bleaching chemicals, reacting with most of the lignin remain-
ing in pulp, and degrading and dissolving the lignin as chlorinated organics. The
acidic chlorination is usually followed by alkaline extraction to increase the
solubility of chlorinated lignins. The substitution of organically bound chlorine
by hydroxyl ions takes place under alkaline conditions, eliminating 60-90% of
organically bound chlorine in the pulp I-2]. The stages following the chlorina-
tion and alkaline extraction are known as bleaching stages, and use more
powerful oxidizing chemicals such as chlorine dioxide, hydrogen peroxide and
hypochlorite. The main reaction is to oxidize the chromophoric structures in the
pulp and to increase the pulp brightness. In the production of softwood kraft
bleached pulp by the conventional CE1DIE2D2 sequence, approximately 75%
of the dissolved material (COD and colour) and 95% of the organically bound
chlorine were contained in the C and E1 prebleaching effluents. The major
source was the E1 stage effluent. Lindberg 1-3] reported that the E1 effluent,
which constituted about 12% of the total bleach effluent, accounted for 96% of
the colour, 70% of the COD, and 50% of the BOD in whole bleach plant
effluent.
The amount of TOC1 produced during pulp bleaching varies with wood
species, kappa no. of pulp, bleaching sequence and conditions employed.
Typically, TOC1 in the effluent from a bleached softwood kraft pulp by a con-
ventional sequence is 5-8 kg/t of pulp bleached, representing about 10%o of the
total chlorine charged in the chlorination stage [4-6]. A physical-chemical
classification of this chlorinated organic material, present in spent liquors from
conventionally pulped and bleached softwood kraft pulp, is shown in Fig. 2
[7 9].
About 20% of the organically-bound chlorine found in the bleaching effluent
corresponds to relatively low-molecular-mass (M r < 1000) material. In recent
years, considerable research effort has been directed towards characterizing this
fraction with respect to its individual chlorinated compounds [10-12], as this
fraction is expected to contain those compounds which are potentially toxic to
aquatic organisms because of their ability to penetrate cell membranes or their
propensity to bioaccumulate in the fatty tissues of higher organisms. Some of the
major components of this low-molecular-mass fraction were found to consist of
relatively water-soluble substances such as chlorinated acetic acids or chlorin-
ated acetones, which are easily broken down [8, 9] before or during biotreat-
ment and are thus of minimal environmental significance. The fraction of AOX
which is extractable by a non-polar organic solvent and is referred to as EOX (or
extractable organically-bound halogen) accounts for about 1-3 %0 of the total
organically-bound chlorine. This fraction contains relatively lipophilic (i.e. fat-
soluble) neutral organic compounds, primarily of low molecular weight, and is
therefore of greater environmental significance than the remaining 99 %0 or so of
Reduction of OrganochlorineCompoundsin BleachPlant Effluents 219
8o% I
High M r material I
Relatively hydrophilic (Water soluble)
mainly non-aromatic
Does not permeate cell walls
I AOX < 10% chlorine by weight
100%
~ i R ~19% I
elatively hydrophilic
ncludes compounds
Low M20m~ which can easily be
hydrolyzed or metabolized
e.g. trichloroacetic acid)
Relatively lipophilic Log Pow > 3

(fat soluble) Highly lipophilic
Potentially toxic bioaccumulable
potentially bioaccumulable (e.g. Dioxin - 44%
chlorine by weight)
Fig. 2. The character of AOX in the effluentfrom conventionallypulped and bleached kraft
pulp
the AOX material. The EOX material can be further fractionated according to
its octanol/water partition coefficient (Pow). The fraction having a partition
coefficient greater than 1000 (i.e. log Pow > 3) makes up only 0.1% (or less) of the
AOX and contains those compounds which are most readily bioaccumulable
and considered to be potentially the most toxic and persistent. A component of
particular concern in this fraction is the polychlorinated aromatic material that
has a relatively high level of chlorine substitution, typically three or more
chlorine atoms per aromatic ring.
The major bleaching parameters such as incoming kappa number, C12
dosage, and chlorination and extraction pH and temperature have a significant
effect on the effluent BOD, COD, color loadings and the formation of chlorin-
ated compounds. It has been reported that the pollution load and amount of
chlorinated material produced in the chlorination and extraction stage are
a function of the amount of chlorine applied to the pulp, which is determined by
residual lignin in the pulp. The amount of TOC1 in the C stage spent liquor was
strongly dependent on the C12 dosage and lignin content in the pulp [13-15].
The lipophilic chlorinated organic substances were found to increase with an
increase in the pulp kappa number and C12 dosage [15]. An increase in the
chlorine dosage results in increasing BOD and COD in the chlorination and
extraction effluents [16, 17]. Voss et al. [4, 17] have reported that significant
reductions in the loadings of toxicity and chlorinated phenolics of combined
C and E effluents can be achieved by a higher final pH of chlorination. The
formation of TOC1 and chlorinated phenolics increased with increasing temper-

ature and decreasing pH of chlorination. Alfthan et al. [16] reported that
increase in temperature of the chlorination resulted in increase in pollution
loadings in chlorination and extraction effluents. Crawford et al. [18] reported
that the increase in the end pH and temperature of chlorination and chlorine
charge increases the formation of chloroform, potential mutagen and carcinogen
in the chlorination liquor. The hypochlorite stage is the major source of
chloroform formation. In the C stage, chloroform production is normally much
less, but it increases with increasing pH, temperature and kappa factor. The
higher kappa number also results in an increase in chloroform concentration in
the E stage. Chan and McDonald [19] reported that the use of high C102
substitution in the C stage reduced the chloroform concentration. While C102
substitution reduces the effluent TOC1, color and chloroform, it had little impact
on the production of chlorinated dioxins [20]. The major source of chlorinated
dioxins was found to be the C stage followed by the E stage. The formation of
dioxins is mainly dependent on the brown-stock kappa number, kappa factor,
mixing condition in the chlorination, carry-over and wash water quality.
This article presents a state-of-the-art review of the literature relating to the
environmental impact of the chlorinated organic compounds and the measures
which are used for their reduction. Emphasis is laid upon biotechnical methods
for reducing the discharge of chlorinated organics.
2 Environmental Impact of Chlorinated Organic Compounds
About 300 different chlorinated organic compounds in bleached pulp mill

effluents have been identified to date. About 200 of these are compounds which
include chlorinated resin acids, chlorinated phenolics and dioxins [20-22]. The
main compounds of the general type are listed in Table 2. These compounds,
classified as acidic, phenolic and neutral compounds, are at least partly respon-
sible for the oxygen demand (BOD and COD), effluent color, toxicity,
mutagenicity and carcinogenicity [23-29]. Various chlorinated phenolic, acidic
Table2. Chlorinated organic compounds in bleached pulp mill effluents

Type Number of varieties Amounts(g/t pulp)
Chlorinated phenolics 40 Up to 100
Chlorinated aldehydes,ketones and lactones 45 500
Chlorinated acids 40 Up to 500
Chlorinated hydrocarbons 45
Chlorinated ethers 20
High molecular mass Up to 4 kg C1
Based on data from Refs. [2~22]
Reduction of Organochlorine Compounds in Bleach Plant Effluents 221
and neutral compounds and chlorinated dioxins have been found to be bioac-
cumulative [20, 30]. Up to 2000 ppm organic chlorine has been detected in the
fat of fish from waters receiving bleaching effluent [31]. Untreated pulp and
paper mill effluents can be acutely toxic to fish at concentrations as low as 2%
by volume [26]. Chlorinated phenolics, resin and fatty acids are the principal
contributors to effluent acute toxicity [23, 32-34]. Table 3 lists 12 poly-
chlorinated phenolics selected by the US EPA for possible regulation [35]. The
toxicity of a chlorinated compound increases with increasing number of chlorine
atoms on the organic compounds. Polychlorinated dibenzodioxins (PCDDs)
and 2,3,7,8-tetrachlorinated dibenzodioxin (TCDD) are toxic. It is generally
believed that they are of precisely the right size to fit as the key in the lock in
some vital molecules in living cells and to be firmly attached to this site by the
chlorine atoms at both ends, thus preventing normal functioning of the cells.
T C D D seems to fit perfectly. If, however, more hydrogen atoms in the benzene
rings are substituted with chlorine, the toxicity is reduced, e.g. if all eight
available hydrogen atoms are replaced with chlorine atoms, the toxicity drops to
one per thousandth of that of TCDD. The key does not fit in the lock any more.
Dioxins have received extensive media attention. 2,3,7,8-TCDD is extremely
toxic and bioaccumulative. The toxicity of individual members of the above-
mentioned family of 210 isomers (varieties) of polychlorinated dioxins and
furans differ substantially. The most toxic of them is at least 100000 times as
toxic as the least toxic, so that it is meaningless to judge the quality of an effluent
or pulp by quoting the total dioxin concentration. Dioxins are frequently
reported as toxicity equivalents (TEQ), which is the sum of total chlorinated
dioxins and furans corrected for the toxicity of each relative to 2,3,7,8-TCDD.
For practical purposes, in the pulp industry, the T E Q generally corresponds to
somewhat more than the concentration of 2,3,7,8-TCDD [36, 37].
Table 3. Polychlorinatedphenoliccompoundsproposed for regulation

by the US EPA
Polychlorinatedphenols Minimum level,ppb (gg/1)"
Pentachlorophenol 5.0
2,3,4,6-Tetrachlorophenol 2.5
2,4,5-Trichlorophenol 2.5
2,4,6-Trichlorophenol 2.5
3,4,5-Trichloroguaiacol 2.5
3,4,5-Trichlorosyringol 2.5
3,4,5,6-Tetrachlorocatechol 5.0
3,4,6-Trichlorocatechol 5.0
3,4,5-Trichlorocatechol 5.0
3,4,5,6-Tetrachloroguaiacol 5.0
"The minimum level is defined as the concentration at which the
analytical system gives recognizablemass spectra (correctedfor back-
ground) and acceptable calibration points, using EPA method 1653
Greenpeace claims that there is no safe level of dioxin [36]. On the other
hand, over a billion dollars worth of research has failed to prove any serious
health damage to humans due to dioxin exposure [36].
It is fairly well accepted by scientists in the field that 10 picograms of dioxin
per kg body weight per day is an acceptable daily intake (ADI) for a one in
a million risk of mortality, over and above the other risks of living. However,
a concern about dioxins in effluents is that they bioaccumulate in fish. This
means that they concentrate as they move up the food chain through predator
fish to humans. In Canada, the Federal Government recommends that fish with
over 20 ppt dioxins in the flesh should not be eaten. Occasional fish caught
downstream of mills have concentrations of up to about 100 ppt, but these
incidents can be expected to become things of the past if mills adopt the dioxin
control measures already announced. To help put this all into perspective, it is
worth noting that collection of the ADI includes several safety factors. Extensive
studies of 1200 US soldiers who worked with dioxin-contaminated herbicides in
Vietnam failed to demonstrate any health effect, but there was an out-of-court
settlement of almost $200 million. One might say that legal dangers have been
demonstrated to be associated with dioxin but no serious biochemical danger to
humans.
Recently, the EPA released its 2000-page draft reassessment of the environ-
mental and health effects of dioxins [38]. The report reaffirms the EPA's 1985
conclusion that dioxins and related chemicals are a probable cause of cancer in
humans. It also presents new evidence that dioxins, even in trace amounts, may
cause a wide range of other adverse human health effects including disruption of
regulatory hormones, reproductive and immune system disorders and abnormal
fetal development. The EPA defines dioxins and related compounds as tetra- to
octa-chlorodibenzo-dioxins and furans with chlorine atoms in at least the 2,3,7
and 8 positions, as well as coplanar polychlorinated biphenyls (PCBs). The EPA
expresses the mass of these compounds in terms of their toxicity compared with
the most toxic dioxin congener 2,3,7,8-tetrachlorodibenzo p-dioxin, denoted as
2,3,7,8-TCDD toxicity equivalents (TEQs). In other words, if the mass of
a particular dioxin congener is 100g and it has one tenth the toxicity of
2,3,7,8-TCDD, its mass in TEQs is 10 g.
The reassessment emphasizes dioxin's effects on fetal development, because
these effects have been seen in the offspring of laboratory animals exposed to
very low concentration, and are now being observed in children accidentally
exposed to dioxin-like compounds in the womb [38]. For example, the offspring
of women who in 1979 ate rice oil contaminated with furans and PCBs in the
Yu-Cheng Province of Taiwan exhibit changes in skin pigmentation and hair
growth and are showing signs of abnormal sexual development, says Aronold J.
Schecter, Professor of Preventive Medicine at the State University of New York,
Clinical Campus, Binghamton. Dioxins and dioxin-like compounds apparently
target many sets of genes which encode a variety of proteins, including hor-
mones, enzymes and growth factors. As a consequence, the cell produces an
inappropriate amount of protein. Depending upon the dose, timing and age of
the individual, this cell disruption can lead to diverse biological outcomes,
effects on the reproductive system of the developing fetus, effects on the brain,
disruption of the immune system and cancer. Dioxin's effect has been seen in all
species studied, although they occur at different doses. Humans lie somewhere in
the middle of the sensitivity range, neither extremely responsive nor extremely
resistant, according to the EPA report. Unlike natural hormones, like the
estrogen estradiol, dioxins can be deactivated by binding to sex-hormone-
binding globulin and are not easily metabolized. Dioxins disrupt multiple
endocrine systems, at times behaving as antiandrogens, at others like antiestro-
gens or even estrogens. The half-life of the most studied dioxin 2,3,7,8-TCDD is
10 years in humans [39].
Low molecular mass chlorinated neutral compounds are major contributors
to mutagenicity [31]. The dominant chlorophenolics in C stage effluent are
chlorocatechols, whereas chloroguaiacols are the major species in E stage
effluent. In hardwood bleaching effluent, additional chlorinated compounds
such as chlorinated vanillins, syringealdehydes and syringals were found. But
the concentrations of chlorinated phenolics in the hardwood bleaching effluents
are generally lower than those in softwood bleaching effluent [40]. At least 14
different chlorinated phenolics in conventional C and E1 bleaching effluents
were detected [41].
Sublethal effects of pulp and paper mill effluents are varied. The threshold
concentration for sublethal effects appears to be near 1/10 of the 96 h LCso
concentration [33]. Davis [42] reported the lethal effect at 5 % of the 96 h LCso
concentration of the spent bleaching liquor.
The Swedish pulp and paper industry sponsored studies to determine the
environmental effect of spent bleach liquors and to develop environmentally
compatible bleaching processes [15, 43, 44]. It was concluded that the effects of
bleach plant effluents actually observed in receiving waters were few in number
and limited to receiving waters with poor water exchange or to areas close to the
outlet. Laboratory tests suggested that all sublethal effects on fish of effluents
from the conventional bleaching of softwood kraft pulp disappear after a total
dilution of 70-1000 times [15]. Based on the results of chemical and biological
comparisons of the effluents, various alternative bleaching sequences and treat-
ments were ranked as shown in Table 4. The Swedish project SSVL-85 was
designed to assess long-term large-scale effects of bleach plant effluents and used
model ecosystems to characterize bleach plant effluents [15, 44]. Two concen-
tration levels were used corresponding to dilutions of 400 and 2000 times for
a total effluent volume of 50 m3/t pulp. Five different effluents were tested for
fish and invertebrate toxicity, reproductive disturbances, physiological changes
and disease, parasitic infestation, bioaccumulation of chemicals and genotoxic
effects (mutagenicity and/or carcinogenicity). The ranked sequences did not
change significantly from that in Table 4. However, biological effects were found
at dilutions which were previously thought to render the effluents harmless. The
effluent from a conventional (C95D0s)E1DE2D bleach plant showed strong
biological effects after 166-fold dilutions, with low survival of fish, decreased
Table 4. Bleaching processes in order of decreasing environmental effect
S.no. a Bleaching process Comments
1 (C90DIo) E1HD1E2D2 Traditional technique

2 (CgoDIo) E1HD1E2D2 Improved washing compared to
traditional technique
3 (C9oDlo)E1HD1E2D2 + UfE1 Ultrafiltration of Ex effluent
4 0(C85D15) E1D1E2Dz Oxygen bleaching to kappa no. 20
and washing to 1~12 kg COD/t
5 (C9oDao)EaHD1E2D2 + UfE1 + Z Ozone treatment of permeate and
C-stage effluent
6 0(C85Dxs)E1D1E2D2 Oxygen bleaching to kappa no. 20
and washing to 4-6 kg COD/t
7 (C90Dlo)E1HDaE2D2 + aerated lagoon Total effluent through aerated lagoon
8 (CgoDlo) + Ion exchange Ion exchange of total effluent
9 (D85C15)EID1E2D 2 High substitution of chlorine dioxide
10 0(C85D15)E1D1E2D2 + aerated lagoon Oxygen bleaching to kappa no. 20
11 0(CssD15)E1D1EzD2 Oxygen bleaching to kappa no. 17
aSoftwood Kraft Pulp

UfE~ = Ultrafiltration of E1 effluent
Worst 1
Best 11
Based on data from Ref. 1-15]
invertebrate density and parasitic infestation of stationary fish species. Even at

dilutions over 5000 times, all these effects could be found, although to a substan-
tially decreased degree.
In 1983, the Swedish Government sponsored Project Environment/Cellu-
lose, a three year study of the Gulf of Bothnia, that portion of the Baltic sea
which lies between Sweden and Finland [21]. The study focussed on the area
around the Norrsundet pulp mill, which discharges untreated effluent into the
Gulf and which was regarded as representative of Swedish pulp mills. Near the
discharge point, fish biomass was low, species composition was changed, repro-
duction was reduced and physiological disturbances were seen. The effluent also
affected the diversity, biomass and distribution of invertebrates and plants in the
region [21]. The toxic effects were more pronounced within 4-5 km of the outlet
of the mill, but biological effects were observed in the fish caught 8 to 10 km
from the outlet, where the dilution was estimated to be 5000 times [45].
Bioaccumulating compounds in the fish declined in concentration from the
discharge point to about 5 km out from the shore [21]. The concentration of
AOX in the water, however, showed a larger area of influence. High concentra-
tions of solvent-extractable organically bound chlorine (EOC1) were found in
the sediment 15 km out from the Norrsundet mill and 29 km out from another
mill 75 km north of Norrsundet. Large areas of the Gulf of Bothnia were found
to have higher levels of EOC1 in the sediment. Compared with areas further from
industry, EOC1 levels in sediment 20 to 50 km off the coast of bleached kraft
pulp mills were 10 times the background levels [21]. Between 1979 and 1984, the
Norwegian Centre for Industrial Research studied the content of EOC1 in fish in
relation to the effluent of a CEHD bleached sulphite pulp mill [46]. While the mill
was in operation, the concentration of EOC1 in fish near the outlet was up to 30
times that in fish from a nearby reference area. EOC1 levels in fish did not drop to
the background concentration until 3.5 years after the mill had shut down.
In 1987, Swedish scientists stated that in spite of extensive investigations,
a clear-cut relationship between release of chlorinated organic material and bio-
ecological effects in the receiving waters has not yet been established. Thus, the
TOCI regulations give a general feeling that the release of chlorinated organic
material from bleach plant is critical to the environment [47]. However, concern
has been voiced that the effects found in Sweden are specific to that area,
because the Baltic sea may be a unique ecosystem. It contains a relatively small
number of species and the media is brackish and has a long residence time, of the
order of several decades [483.
Extensive long-term environmental impact studies have also been carried
out in the United States. Since virtually all the mills in the USA have secondary
biological treatment, treated bleach plant effluents were tested. Test work, using
experimental stream channels, has been conducted by National Council of the
Paper Industry for Air and Stream Improvement (NCASI) as part of an aquatic
biology investigation going on since the 1970s. In the latest report, the results of
year-long exposure to treated bleached plant effluent of 20:1 dilution were
reported [49]. Productivity of the rainbow trout population and aquatic flora
was increased compared to that in the control stream, while productivity of the
benthic macroinvertebrate population was unchanged. There was no effect on
the diversity index or histopathology of 20 different fish-tissue types. These
results are clearly contradictory to the general conclusion of the Swedish
investigations. The reason for the differences is not apparent; it may be the effect
of effluent treatment or the sensitivity of the Baltic eco-system.
Another category of compounds which has aroused environmental concern
comprises the high-molecular-mass chlorinated compounds (Mr > 1000). In
bleaching softwood kraft pulp, this fraction of chlorinated compounds accounts
for 50% of dissolved organic material [50] and 70% of TOC1 [20]. Although
these compounds contribute little to BOD5 and acute toxicity due to their
inability to pass the bacterial cell membrane, they are the major contributors to
effluent colour, COD and chronic toxicity. Eriksson and Kolar [51] and
Eriksson et al. [52] found that the high-molecular-mass material was not as
stable as previously thought. High-molecular-mass chlorolignins from pre-
bleaching stages were chemically unstable under conditions that may prevail in
receiving waters. The material was slowly decomposed to various chlorinated
catechols and guaiacols which were methylated in cases where a complete
mixture of bacteria or the white rot fungus alone was used, a condition appear-
ing to be common in nature [15, 53]. The low-molecular-mass phenolics and
their methylated counterparts (more lipophilic) may cause toxicity and bioac-
cumulation in fish.
Chlorate is yet another pollutant recently arousing environmental concern.
The formation of chlorate is largely dependent on the amount of C102 used in
the bleaching. It is a well-known herbicide and biocide, especially for brown

algae [54]. A conventional bleaching sequence produces about 3-6 kg/t of pulp
bleached depending on the level of C102 substitution.
3 Reducing the Generation of Organochlorine Compounds
The emission of chlorinated organic compounds from bleach plants can

be reduced by modifying the pulping and bleaching processes using one or
more of the following strategies: (1) removing more lignin before starting
the chlorination, i.e., reducing the kappa number of unbleached pulp, (2)
modifying the conventional bleaching process, (3) eliminating all chlorine
compounds in bleaching, or (4) recovering bleach plant effluents. These
methods may be physicochemical or biochemical in nature or a combination
thereof.
Lignin from the pulp before chlorination can be removed by extended
delignification of the brown stock pulp using modified continuous cooking
(MCC) [55-58] or modified batch cooking (MBC) [59-61]. The lignin content
can also be removed by oxygen delignification [20, 58, 6~69]. Ozone is also
capable of delignifying an oxygen-treated pulp to a very low kappa number of
5-6 [70, 71]. A lower lignin content in the pulp requires lower chlorine usage
resulting in reduced generation of chlorinated organics. The conventional
bleaching process can be modified by (a) reducing the kappa factor or active
chlorine multiple, i.e., the total active chlorine charge derived by the kappa
number of the pulp entering the chlorination stage, (b) slow and multiple
addition of chlorine [72], (c) chlorine dioxide substitution in the chlorination
stage [4, 13, 17, 72-77], (d) oxidative extraction using oxygen [78-80] and/or
hydrogen peroxide [81-91], or (e) converting to ECF bleaching sequence
[89, 92]. Further decrease in AOX can be achieved by a combination of
extended delignification in the digestor, oxygen delignification, and high chlor-
ine dioxide substitution in the chlorination stage [72]. The trend now is to adopt
a totally chlorine-free (TCF) bleaching sequence in some European countries or
ECF bleaching in North America, which can involve the use of oxygen, enzyme,
hydrosulfite, peroxide, peracetic acid, thiourea dioxide, chelating agents
[72, 93], ozone [70-72, 94-97] etc.
Once chlorine-free bleaching is introduced, the bleaching effluent can be
taken to the black liquor recovery section [98]. A non-polluting bleach plant
has been reported where counter-current washing decolorization and reuse of
the extraction stage effluent as wash water in the chlorination stage is practiced
[99, 100]. Ultrafiltration and reverse osmosis membrane techniques can also be
applied for complete recycling of the effluent [101, 102]. The best-known closed
cycle effluent-free bleaching process was reported in the mid-1960s by Rapson
and Reeve [103-105].
Reduction of Organochlorine Compoundsin Bleach Plant Effluents 227
Apart from the above methods, several biochemical and biological methods
can be used to reduce the generation of organochlorine compounds, as discussed
in the following sections.
3.1 Enzymatic Prebleaching
Pretreatment of pulp with enzymes prior to bleaching helps in substantially

reducing or eliminating the use of chlorine and chlorine compounds. Hemicel-
lulase enzymes and ligninolytic enzymes have been investigated. Hemicellulase
enzymes have no immediate effect on the kappa number and lignin removal of
the treated pulp. However, the consumption of the bleaching chemicals is
reduced and/or brightness ceiling is increased. Ligninolytic enzymes selectively
remove the lignin from the pulp fibre, hence are more useful. The role of different
enzymes on pulp components has been reviewed by Eriksson [106].
3. I. 1 Hemicellulase Enzymes
These enzymes are used commercially in pulp bleaching. The main enzyme
needed to enhance delignification of kraft pulp is reported to be endo-~-
xylanase, but enrichment of xylanase with other hemicellulolytic enzymes has
been shown to improve the effect of enzymatic treatment [107-110]. Xylanases
act mainly on the relocated, reprecipitated xylan on the surface of the pulp
fibres. Enzymatic hydrolysis of this specific type of xylan renders the structure of
the fibre more permeable, allowing lignin and lignin carbohydrates to diffuse
more easily into the bleaching liquor. An alternative explanation is that
xylanases attack the bonds that exist between xylan and lignins, releasing lignins
which can then diffuse more easily into the bleaching liquor [109, 111, 112].
Xylanase treatment has been shown to reduce the requirement of chlorine
for bleaching, while still achieving high brightness and good pulp properties
[113-115]. Results from laboratory study and mill trials show about 3 5 4 1 %
reduction in active chlorine at the chlorination stage for hardwoods and
10-20% for softwoods, whereas the saving in total active chlorine is found to be
20-25% for hardwoods and 10-15% for softwoods [116-123].
Xylanase treatment represents a successful new technology for reducing
chlorine use. More details on xylanase bleaching are available in a recent review
by Bajpai and Bajpai [124] and in another chapter in this volume written by
Viikari et al. [125].
3.1.2 Ligninolytic enzymes
These enzymes, unlike xylanases, attack lignin directly, and hence are more
effective. White-rot fungi are the main producers of ligninolytic enzymes. These
fungi secrete a number of oxidative enzymes and some hitherto unknown

substances (mediators) into their environment, together effecting a slow but
continuous degradation. The most important lignin-degrading enzymes are
lignin peroxidases, manganese peroxidases and laccases. The action of both
lignin peroxidases and manganese peroxidases needs Mn z +, which is oxidized to
Mn 3 +. Mn a + is the real oxidizing agent, attacking the lignin molecule. Laccase
uses molecular oxygen as a cosubstrate. In the absence of the living organism,
the various peroxidases and laccases perform only negligible kappa number
reduction [126, 127]. Egan [128] has reported kappa number reductions of 24
and 26% after treatment with ligninase 118 from P. chrysosporium followed by
alkaline reduction. Viikari et aI. [129] were unable to demonstrate any bleach-
ing effect on pine kraft pulp with ligninases and oxidase from the white-rot
fungus Phlebia radiata or purified ligninase from P. chrysosporium. When the
ligninases were applied after treatment with hemicellulases, there were slight
reductions in kappa number. Arbeloa et al. used lignin peroxidase enzyme from
P. chrysosporium to improve the bleachability of hardwood and softwood kraft
pulps [130]. Enzyme treatment prior to chemical bleaching increased brightness
and decreased lignin content in the pulp. The final brightness of pulp was found
to be higher by about 0.84).9 points than that of the control (Table 5).
Although much of the research has focussed on lignin peroxidases, these
enzymes are not necessarily involved in lignin degradation and may not be
secreted by all lignin-degrading fungi. C. versicolor produces laccases as well as
lignin and manganese-dependent peroxidases [131-134]. However, Archibald
[135] recently found that lignin peroxidases secreted by C. versicolor did not
appear to play an important role in lignin degradation. Dichomitus squalens
[136] and Rigidoporus lignosus [137] produce both laccase and a manganese-
dependent peroxidase but do not produce lignin peroxidase.
It is now known that although lignin peroxidases and laccases play an
important role in degrading the lignin in vivo, in vitro the oxidation reactions
catalyzed by the enzyme result in further polymerization of the lignin [138].
Table 5. Effects of lignin peroxidase on kappa number and bright-

ness in softwood and hardwood kraft pulp
After treatment After bleaching
Kappa Brightness Brightness

number (%) (%)
Softwood
Control 29.7 29.1 88.8
Treated 26.9 24.1 90.7
Hardwood
Control 14.6 31.0 88.6
Treated 11.8 39.0 89.4
Conditions of enzyme treatment: pH 3, temperature 30 ~ veratryl

alcohal 2 mM/l, H202 addition at 100 ppm/h for 3 h
Recently, Hammel and Moen [139] reported depolymerization of a synthetic

lignin by a lignin peroxidase in the presence of H202 and veratryl alcohol, but
this effect has not been demonstrated with lignin in wood or pulp. It is likely that
fungi possess enzyme systems that prevent polymerization [138, 140].
These results imply that single enzymes are not able to mimic the complete
biological system. Small improvements can be achieved by the addition of
low-molecular-mass aromatic compounds like veratryl alcohol or other sub-
stances such as ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate)] and
Remazol blue [126, 127]. In consequence, most scientific and industrial groups
have meanwhile withdrawn from research programs focussing on direct lignin
degradation.
Lignozyme GmbH (Germany) continued work with enzymes plus chemical
mediators which create a redox system throughout the pulp treatment period
[-141-143]. Their idea was to find a system which is a good mimic of the natural
situation. Starting in 1987 with the enzyme mediator concept, Lignozyme has
very recently improved the performance of the mediator system for the laccase of
Coriolus versicolor by changing and further fine-tuning the chemical nature of
the component [144-146]. The treatment of pulp with laccase alone does not
result in any degradation of lignin but merely produces in a structural change or
repolymerization, whereas the laccase mediator system causes a significant
kappa number reduction at reasonable treatment times even if the enzyme
mediator system is applied in several consecutive treatment steps to the same
pulp. In contrast to commonly used pulp-bleaching chemicals (oxidizing and
reducing), no passivation can be observed in enzymatic delignification, i.e.,
efficiency does not drop significantly during consecutive treatment steps. Lac-
case enzyme contains four atoms of copper per molecule and requires oxygen as
a cosubstrate for the oxidation reaction which has to be provided. According to
the present understanding, the laccase, while oxidizing the chemical mediator, is
generating a strongly oxidizing co-mediator which is the real bleaching agent.
Table 6 illustrates the conceptual difference between the indirect and the direct
enzymatic lignin attack. The general technical conditions for enzymatic bleach-
ing with the laccase mediator system are: temperature 40-65~ pH 4-7,
consistency 1-20%, pressure 1-14 bar, duration 1~4 h. The system provides
a broad flexibility with respect to the pulp substrate, the technical requirements
for application, and the final quality of the pulp. The principal applicability has
been demonstrated for softwood and hardwood pulps as well as for annual plant
Table 6. Differencesbetweenxylanaseand laccase/mediatortreatment

Xylanase Laccase/mediator
No or verypoor kappa reduction Very good kappa reduction
Moderate bleachingeffect Good bleachingeffect
Saving of bleachingchemicals Saving of bleachingchemicals
TCF pulp productionpossible
fibres. A repeated enzymatic treatment is possible and results in a 50-70%

kappa number reduction per treatment step. Laccase mediator system (LMS) is
compatible with all other bleaching sequences. The performance of the LMS
(1-hydroxybenzotriazole) has been proven in several pilot trials and is now
ready for large-scale application.
Recently, Kondo et al. [147] examined the bleaching of kraft pulp with Mn
peroxidase (MnP) enzyme, which was isolated from cultures of P. sordid YK-
624. Pulp brightness was increased by about 15 points and kappa number
decreased by about 8 points when unbleached kraft pulp was treated with MnP
in the presence of MnSO4, Tween 80 and sodium malonate with addition of
H 2 O 2 at a rate of 3 ml/h at 45 ~ for 12 h. To establish an absolutely chlorine-
free bleaching process, oxygen-bleached kraft pulp (OKP) was treated with
a four-stage biobleaching process consisting of sequential MnP treatment,
alkaline extraction, MnP treatment and hydrogen peroxide treatment. Fully
bleached kraft pulp (brightness 91%, yield 97%) could be obtained from OKP
by combination of enzyme treatment and hydrogen peroxide bleaching.
MnP from Trametes versicolor was found to delignify softwood kraft pulps
over a wide range of initial lignin contents [148]. Demethylation of residual
lignin in the pulp also occurred. Subsequent treatment of pulp with TCF
chemicals resulted in brighter pulp than predicted from the initial kappa
number. The MnP stage required small doses of hydrogen supplied by the
action of glucose oxidase on glucose. Mn ions already present in the pulp were
chelated by malonate or gluconate during the catalytic cycle. Much of the
bleaching effect was evident after 4 h of treatment with 1 U/ml of MnP, although
bleaching was improved further after 24 h under the conditions used. Laccase,
especially in the presence of the mediator, ABTS, produced a similar effect to
MnP when combined with subsequent TCF bleaching. MnP generates o-
quinones by demethylation of residual lignin, and these structures are suscep-
tible to oxidative ring opening during TCF bleaching, resulting in hydrophilic
soluble lignin.
Ducka and Pekarovicova [149] used crude ligninases from P. chrysosporium
for bleaching of softwood kraft pulp. The pulp, after oxygen bleaching with
kappa number 19.7 and 36.5% MgO brightness, was pretreated with ligninases
(L) or xylanases (X) and bleached in QEopDP sequence. The brightness of pulp
bleached in this sequence was 87.8%, which is about 3.2 points higher than the
control and is approximately the same as that obtained with the use of commer-
cial xylanase.
3.2 Fungal Prebleaching
Pretreatment with fungi has been shown to replace up to 70% of the chemicals
needed to bleach kraft pulp [150]. The usual specificity of biological reactions
and their mild reaction conditions make biological delignification an interesting
alternative to bleaching with chemicals such as pressurized oxygen or ozone.
Only a few white-rot fungi have been tested for their ability to delignify kraft
pulps. Phanerochaete chrysosporium reduced kappa number by about 33% in
hardwood kraft pulp during a 10 day incubation period, whereas Coriolus
versicolor reduced kappa number by about 20 and 33% in a 5 day period
[151-153]. Table 7 shows the performance of various white rot fungi used in
treating hardwood kraft pulps. The largest bleaching effect was noted
with white-rot species IZU-154 [154]. Treatment of hardwood pulps
with alkaline extraction following fungal bleaching did not significantly improve
brightness. Softwood kraft pulps were found to be more resistant to attack by
P. chrysosporium and C. versicolor, possibly because both P. chrysosporium
and C. versicolor tend to attack hardwoods more often than softwoods in
nature [155-157]. Softwood lignin has a different character from hardwood
lignin and is susceptible to blocking reactions that restrict delignification
[158, 159].
On delignification of softwood pulps by P. chrysosporium, kappa number
reductions of 50-70% were achieved [160, 161]. Kirk and Yang [160] reported
a considerable loss in cellulose content during fungal bleaching, which
was attributed to cellulase production in the strain being used. A strain of
P. chrysosporium without cellulase activity has since been developed [162].
Table 7, Change in brightness of hardwood kraft pulp after exposure to various

white-rot fungi
Brightness (%ISO) Incubation
Period
Fungus Increase Final (d)
Phanerochaete 11 39 13
Chrysosporium 0 37.7 14
2.2 35.7 5
P. chrysosporium
(cellulase-free mutant # 431) 2.0 35.5 5
P. chrysosporium
(cellulase-free mutant # 432) 1.9 35.4 5
Coriolus versicolor 8 25 50-67 10
7.1-17.8 38.5 50.3 5
14.5 48.0 5
13.6 50.4 5
13.4a 50.2 5
6 34.0 15
IZU-154 35 63 11
Coriolus hirsutus 23.7 52 5
12 40 11
Phellinus pini 2.3 35.8 5
Pleurotus eryngii 1.9 34.4 5
Pleurotus sajor-caju 2.3 35.8 5
Lentinus edodes 2.4 35.9 5
Aureobasidium puUulans 2.5 36.0 5
"Fungus was immobilized
Based on data from Refs. [152 154,163,195,199]
C. versicolor was found to be ineffective in brightening softwood kraft

pulps despite reductions in kappa number of 8% [163] and 47% [154, 157] over
14 days. However Reid et al. [157] reported that when the pulp was sub-
sequently subjected to alkaline extraction, brightness was increased by 15 points
and kappa number was further reduced. The softwood kraft pulp exhibited
a more pronounced darkening than the hardwood kraft pulp during the first
6 days of treatment, and a longer time lag occurred before kappa number began
to decrease. Direct brightening of softwood kraft pulp was noted when the
C. versicolor was immobilized on polyurethane foam, possibly because of a high-
er fungus/pulp ratio [157].
In Japan, a 5-day fungal (F) treatment of hardwood kraft pulp with IZU-154
replaced a CE1DEzD sequence with an FCED sequence, yielding respective
brightness of 88.8 and 88.1% ISO (84.2 and 85.3% ISO after aging) [154]. The
resulting chlorine saving was 72%, despite a kappa number reduction of less
than 60% in the fungal stage. In another study, a 5-day fungal stage with
C. versicolor essentially replaced the chlorination stage in a conventional bleach-
ing sequence, achieving a brightness of 82% ISO with a FDED sequence
compared to a brightness of 88% ISO with a CE1DEzD sequence [154].
Recently, Kondo et al. have reported biobleaching of hardwood kraft pulp
with lignin-degrading fungi P. sordia YK-624 [147]. A three-stage bleaching
process (5 days fungal treatment, alkaline extraction and again 5 days fungal
treatment) bleached the kraft pulp to about 80% brightness in 10 days, although
brightness increase of the pulp with a one-stage continuous fungal treatment for
10 days leveled off after 5 days incubation.
Wroblewska and Zielinsk [164] examined biodelignification of beech and
birch pulp wood by selected white-rot fungi. One of the strains, designated as
DL-Sth-4, was found to be the best for selective delignifiction of beech wood.
About 25% lignin was lost with very little loss in cellulose content. Pazukhina
et al. used the culture filtrate of several white-rot fungi P. sanguinea, C. ver-
sicolor, Ganoderma applanatum and Trichoptum biforma for bleaching hardwood
kraft pulp [165]. P. sanguinea showed the highest selectivity in lignin degradation.
Nishida et al. [166] investigated the biobleaching of hardwood unbleached
kraft pulp by P. chrysosporium and T. versicolor in the solid state and liquid
state fermentation systems with four different culture media (low nitrogen-high
carbon, low nitrogenqow carbon, high nitrogen-high carbon and high nitro-
gen-low carbon). In the solid state fermentation system with L N - H C culture
medium, pulp brightness increased by 15 and 30 points after 5 days of treatment
with T. versicolor and P. chrysosporium respectively. The pulp kappa number
decreased with the increasing brightness, and a positive correlation between the
kappa number decrease and brightness increase of the fungus-treated pulp was
observed.
Delignification by white-rot fungi is strongly influenced by culture condi-
tions. The major culture parameters affecting lignin degradation are: growth
substrate, nitrogen availability, other culture conditions, oxygen concentration
and mode of cultivation. P. chrysosporium and C. versicolor require a carbon
source such as glucose or cellulose to metabolize lignin to form carbon dioxide

[167], although the role played by the carbon source is not clear. Glucose
supplementation at low concentration (0.47%) was found to stimulate delignifi-
cation in kraft pulps [15!] and lignin in aspen wood [168]. However, there was
no difference in the degree of delignification for thermomechanical pulp (TMP)
at glucose concentrations ranging from 0 to 35% [169]. Glucose was added in
most studies to maintain pulp yield because it is a repressor of endo-l,4,fl-
gluconase, mannanase, xylanase, aryl-/%glucosidase, pectinase and cellulase,
which attack the carbohydrate fraction of the pulp. The white-rot fungus
IZU-154 was able to degrade lignin in hardwood kraft pulp without an
exogenous energy source and without significantly affecting pulp yield. Fusa-
rium solani and Aspergillus japonicus were also able to degrade lignin without
additional carbon source [154, 170-174].
Nutrient depletion, particularly nitrogen depletion, appears to trigger the
development of the ligninolytic system in P. chrysosproium, C. versicolor and
Phlebia brevispora, Phanerochaete sordida and Phlebia radiata with a few excep-
tions [152, 168, 175-179]. The effect of nitrogen depletion does not necessarily
hold true for all white-rot fungi, as the lignin-degrading abilities of P. sajor-caju
and L. edodes do not show strong stimulation by nitrogen depletion [180]. The
ligninolytic system of P. chrysosporium is also triggered by limitations in carbon,
sulphur, Zn z+, Fe 2+ and Mo 4+, but not phosphorus [30, 181, 182]. Other
culture conditions such as temperature and pH have not been studied extens-
ively [181]. Tran and Chambers [151] reported that the optimal temperature
for delignification by P. chrysosporium was 38 ~ but Drew and Kadan [183]
found that the extent of degradation of 14C-kraft lignin in 15 days at 28 ~ was
about 4 times that at 38 ~ Optimum pH values reported for lignin degradation
are different for different fungal strains. Tran and Chambers reported that the
optimal pH of P. chrysosporium was 3.5 for delignification and 4.5 for growth
[151]. Kirk et al. [184] reported an optimum pH of 4 4 . 5 for their strain.
Suppression of delignification was noted at pH less than 3.5 and greater than 5.5
[ 184]. Recently, Royle et al. reported increased lignin degradation by P. chrysos-
porium and L. edodes at pH 3 [180]. o-Phthalate was found to inhibit delignifica-
tion [185]; in one study, lignin degradation rates were doubled by changing the
buffer from o-phthalate to 2,2-dimethylsuccinate (DMS) [176].
Lignin degradation in white-rot fungi is primarily oxidative. It occurred
faster in cultures of P. chrysosporium which had been flushed with pure oxygen
than in those flushed with air [-135, 151,169, 186-188]. Enhanced lignin degra-
dation due to elevated oxygen partial pressures has also been reported for other
white-rot fungi including C. versicolor, Pycnoporus cinnabarinus, Lentinus
edodes, Giifola frondosa, Polyporus burmalus and Merulius tremellosus [189].
However, other species such as Gleoporus dichrous, Pleurotus ostreatus, Bondar-
zewia berkeleyi and IZU-154 were less responsive to increased oxygen concen-
trations [154, 189]. The addition of polydimethylsiloxane (PDMS) oxygen
carriers enhanced lignin degradation by C. versicolor [190]. However, they
alone were not found to appreciably increase the brightness of hardwood kraft
pulp [191]. Together with C. versicolor they acted synergistically, resulting in an

overall increase of approximately 10 brightness points [190].
Lignin degradation is also strongly influenced by the mode of cultivation.
Most researchers have studied stationary cultures, although dependence on
stationary cultures causes difficulties in scale-up. Also, agitation is an important
consideration because of the role of oxygen mass transfer. In a number of
studies, agitation in reciprocating and gyratory shakers caused the formation of
pellets consisting of mycelia entangled with pulp. The formation of mycelial
pellets resulted in suppression of degradation of kraft lignin and lignin model
compounds by P. chrysosporium [-151,156, 169, 192, 193]. Similar delignifica-
tion rates were noted in agitated and stationary cultures and also with the
addition of detergent to the culture [187, 194]. Effective delignification has also
been reported in agitated cultures of C. versicolor in which the formation of
mycelial pellets was prevented and in cultures of P. chrysosporium in the
presence of veratryl alcohol [195, 196, 199].
P. chrysosporium and C. versicolor have been successfully immobilized on
polyurethane foam [153, 157, 190, 197]. Immobilized and free cultures of C.
versicolor have been found to bleach hardwood and softwood kraft pulp at
a comparable rate and to a similar extent [153, 157]. The results showed that
intimate contact between the fungal hyphae and pulp fibres was not required
as long as the media was renewed through contact with the fungus
[132, 153]. Immobilization enabled the pulp to be separated from the mycelia.
Another advantage of immobilization was that the same fungal biomass could
be reused to treat other batches of pulp either immediately or after storage at
4~ [153].
A serious shortcoming of the fungal bleaching process is the long incubation
time required, ranging from 5 to 14 days for both hardwood and softwood pulps.
Softwood incubation periods are likely to be longer than those for hardwood
pulps because softwood pulps require a longer time lag (6 days) before kappa
number decrease or brightness increase can occur [157]. Hardwood pulps
generally have a lag time of 1-2 days [152-154, 160], followed by rapid and then
slower delignification.
Unfortunately, the size of the fungal bioreactor would have to be very large,
considering that daily production could range from 200 to over 1000 air-dried
tonnes of pulps. Most researchers have performed fungal bleaching experiments
at low pulp consistencies of 0.5-2% (w/v) [151 154, 190]. Only Fujita et al.
[154] have investigated fungal bleaching at high consistency (16-24%), which
would allow for a smaller reactor. Both reactor size and incubation period will
have to be reduced in fungal bleaching if it is to become an economical
alternative.
Jurasek and Paice [198] suggested that the lignin may become more flexible
and hydrophilic as a result of fungal enzyme action, resulting in a sorer pulp
with improved bonding and stronger paper characteristics. Reduced colour
reversion was another benefit noted with the fungus IZU-154 [154]. Some
viscosity loss, indicating limited cellulose depolymerization, has been reported
Reductionof OrganochlorineCompoundsin BleachPlant Effluents 235
as a result of fungal bleaching [153, 154, 157, 199]. However, based upon experi-
ments done with free and immobilized cultures, Kirkpatrick et al. [ 153] reported
that up to 25% of the reduction in the pulp viscosity may be due to the presence
of fungal mycelia rather than cellulose cleavage. Although fungal bleaching is
primarily an oxidative process, it appears to be more selective than oxygen
bleaching at high pH and at kappa number less than 17 because there is a better
retention of pulp viscosity.
Only a few researchers have measured the impact of fungal bleaching on
effluent quality. In a Japanese study with FCED bleaching sequence, the COD
and color in the bleach plant waste water were reduced by 50% and 80%
respectively, assuming that the filtrate from the fungal bleaching stage was sent
to a kraft chemical recovery system [1541. Whether this could occur in practice
would depend on the capacity available in the recovery furnace. The authors
suggested that higher reductions could be obtained with an FED or
FE1D1EzDz sequence, although there may be slight loss in pulp yield [1541.
Despite the emphasis on fungal bleaching as a means to reduce the use of
chlorine and the associated formation of chlorinated organics, the effect upon
chlorinated organic discharges has not been reported. As this is an important
factor in the choice of any alternative bleaching sequence, quantitative informa-
tion in this area is needed.
4 Treatment of Bleach Plant Effluents
Numerous physicochemical methods have been used for the treatment of bleach
plant effluents. These treatments include precipitation with lime [200-203],
alum and metal ions [204, 205] and synthetic polymeric coagulants [2061,
adsorption on activated carbon [207], natural clays [208-210] and polymeric
adsorbents [211, 212], membrane techniques [2131, rapid filtration on soil
[214-2161, UV-irradiation [217-2191, and oxidation using oxygen [2201, sul-
furdioxide [2211, hydrogen peroxide and sodium hypochlorite [222-224].
The problems underlying the physicochemical treatments are those asso-
ciated with cost and reliability. Coagulation and precipitation produce a vol-
uminous sludge which is very difficult to dewater. Usually, an extreme pH range
is used for optimum treatment, and the pH needs to be readjusted to neutral
before discharge. Oxidation using ozone and hydrogen peroxide are costly, and
oxidation using chlorine species generates secondary pollutants such as chlorin-
ated organics. The membrane techniques require pretreatment and a large
capital investment. Membrane fouling is also a problem with the membrane
technique.
Biotechnological methods have the potential to eliminate/reduce the prob-
lems associated with physicochemical methods, and are described in the follow-
ing section.
4.1 Biological Treatment
Biological treatment is known to be effective in reducing the BOD and simulta-

neously the toxicity of kraft mill effluents [225, 226]. Some aspects of biological
treatment have been reviewed by Boman et al. [227].
4.1.1 Using Bacteria
4.1.1.1 Aerobic Processes. Biological oxidation is the most widely used tech-
nique to remove BOD and chlorinated organics because of its effectiveness and
low cost. Rogers et al. [228] treated the bleached kraft mill effluent in a bench-
scale aerated lagoon for 29, 58 and 99 h and showed that toxicity, BOD and
resin acids were most consistently reduced during the 99 h treatment. Leach
et al. [229] reported the biodegradation of seven compounds representing the
major categories of toxicants in a laboratory batch aerated lagoon. Resin acids
(major source of acute toxicity) were readily biodegradable, but only part (less
than 30%) of the load of chlorophenolic compounds was removed
[34, 77, 229, 230]. However, Gergov et al. [230] reported that biological treat-
ment, especially the activated sludge process, removed 75-95% of chloro-
phenolics. Chlorinated neutral organic compounds (mutagenicity of the spent
liquor) are removed effectively [231]. Chloroform is stripped off by the air
during biological treatment. COD, TOC1 and high molecular weight material
are reduced to a lesser extent [44]. Eriksson and Kolar [51] have shown that the
high-molecular-mass fraction in bleach effluent cannot be degraded in an
aerated lagoon.
While the Swedish researchers found that the biological treatment is ineffec-
tive in removing TOC1, American mills reported, an average of 5 0 4 0 % TOC1
removal by an aerated lagoon or activated sludge process. Gergov et al. [230]
investigated pollutant removal efficiencies in mill scale biological treatment
systems. They found that 48-65% of AOX was removed in the activated sludge
process. The aerated lagoon was found to be less efficient than the activated
sludge. Recently, Deardorff et al. [232] reported that the efficiency of AOX
removal through biotreatment of combined bleach plant effluent increases with
increasing chlorine dioxide substitution. Biological treatment in an aerated
lagoon reduced the concentration of polychlorinated phenolic compounds by
97%. Jokela et al. [233] reported that aerobic lagoon system removed 58-60%
of the organochlorine compounds from the water phase, and the full scale
activated sludge plants removed 19-55%. Both biotreatments removed all sizes
and classes of organochlorine molecules and slightly shifted the relative size
distribution of the compounds remaining in the water phase towards the higher
molecular masses.
Graves et al. [234] examined the combined effects of oxygen delignification,
C102 substitution and biological treatment on pollutants levels in bleach plant
effluents. Biological treatment reduced COD, BOD, AOX and toxicity, but did
not reduce colour. Lafond and Ferguson [-235] reported that aerobic treatment
in activated sludge reactors removed 14-25% of AOX.
To enhance the efficiency of the biological treatment, Ek and Eriksson [236]
combined ultrafiltration and biological treatment. The result of the process was
better than the sum of the two processes. The detoxification of the effluent
during ultrafiltration was believed to be responsible for the improvement.
The mechanisms leading to the removal of chlorinated organics include
stripping of voltatiles, hydrolysis, chemical oxidation, biological oxidation, foam
separation, adsorption, and precipitation.
4.1.1.2 Anaerobic Processes. Anaerobic biological treatment can also efficiently

destroy chlorophenolic compounds, mutagenicity and acute toxicity [77, 237].
The Enso-Fenox Process was capable of removing 64-94% of the chlorophenol
load and toxicity, mutagenicity and chloroform in the bleaching effluent.
McFarlane and Greenwood [-238] removed 92% of color in kraft bleaching
effluent in a granular activated carbon anaerobic system in a 2-day hydraulic
residence time. The decolorization was decreased to 75% when the retention
time was shortened to 0.85 day. Gunnarsson et al. [239] treated 40% E stage
effluent combined with sulphite evaporator condensate in an anaerobic reactor.
Approximately 60% COD and 90% BOD reductions were achieved. The
methane yield was high, but the reduction of TOC1 was limited, which was still
better than the parallel aerobic treatment of the same effluent.
Many compounds in the bleaching effluent were reported to be toxic to
anaerobic microorganisms. These compounds include residual H202 in CTMP
bleaching effluent, chelating agents, extractives and chlorinated compounds
[240-243]. Adoption of microorganisms to the effluent containing these com-
pounds is necessary to achieve a successful treatment [-240]. Raizer-Neto et al.
[243] studied the efficiency of anaerobic treatment in reducing chlorinated
organic compounds in a fixed bed reactor. AOX removal efficiency was
primarily affected by increasing AOX concentration and was only about
50% for 50 mg/1 COD. BOD 5 removal efficiencies were affected at higher
AOX concentrations of 100 mg/1. AOX loading rate or hydraulic residence
time were found to be more important limiting factors than bleaching
effluent AOX concentration. For AOX loading rates under 40 mg/1 and an
AOX concentration as high as 130 mg/1 in the effluent feed, COD and BOD5
removal were about 80%. Fitzsimons et al. [244] reported 35-40% COD
and 42-45% AOX reductions in a 1.5-day anaerobic treatment. They found
that at least part of the higher molecular mass was dechlorinated. Since
anaerobic microorganisms are believed to be unable to degrade the high-
molecular-mass chlorolignin, the dechlorination of this could be mainly due to
physical and/or chemical action in the anaerobic process. The anaerobic de-
chlorination of chlorolignins is due to a combination of energy metabolism
growth, chemical hydrolysis and probably adsorption and/or insolubilization
[244, 245].
E K and Eriksson proposed a process based on U F and anaerobic and

aerobic biological treatments [236]. The U F was used to separate the high-
molecular-mass material, which is relatively resistant to biological degradation.
Anaerobic microorganisms were believed to be able to remove highly chlorin-
ated substances more efficiently than aerobic microorganisms. The last remain-
ing chlorine atom was removed by aerobic microorganisms. The combined
treatments typically removed 80% of the AOX, C O D and chlorinated phenolics
and completely eliminated chlorate. Lafound and Ferguson [235] reported that
anaerobic treatment in an upflow hybrid reactor removed 17-40% of AOX.
Armentate et al. [246] investigated an integrated anaerobic/aerobic process for
the biodegradation of chlorinated aromatic compounds. The sludge obtained
from the anaerobic digester of a commercial treatment plant was used to obtain
an anaerobic consortium capable of partially dechlorinating 2,4,6-trich-
iorophenol. The clarified and sterilized effluent from the same anaerobic digester
was used as the medium for the anaerobic consortium. During the anaerobic
process, 2,4,6-TCP was first dechlorinated to 2,4-dichlorophenol and then to
4-chlorophenol. The stoichiometric amount of 4-CP was recovered. Similar
results were obtained when the anaerobic microorganisms were immobilized.
After immobilization, the consortium was able to dechlorinate 150 laM of
2,4,6-TCP in four days. Pseudomonas 91athei and an indigenous culture obtained
from same sludge used to produce the anaerobic enrichment culture were shown
to be able to degrade the 4-CP produced from the anaerobic dechlorination of
2,4,6-TCP.
Strehler and Welander [247] investigated the removal of AOX and C O D
from bleached kraft mill effluent in laboratory and pilot scale aerobic suspended
carrier reactors and abiotic thermoalkaline reactors. At pH 7.0, 37 ~ and HRTs
longer than 3.5 hours, a maximum C O D removal of 55% was achieved in the
suspended carrier process. The C O D conversion rate at the minimum H R T was
2.6 kg C O D m 3 d-1. The suspended carrier treatment was operated success-
fully at pH 9.0 and 45 ~ and at pH 7.0 and 50 ~ giving > 50% C O D removal
with an H R T of four hours. The AOX removal achieved at p H 9.0 and 45 ~
(50%) was significantly higher than that at pH 7.0 and 37 ~ (39%) because of an
increased abiotic dechlorination at the higher p H and temperature levels. These
researchers further studied sequential thermoalkaline and biological treatment
on a pilot scale. Thermoalkaline treatment at pH 10.0, 54 ~ and an H R T of two
hours followed by biological treatment at pH 8.0, 35 ~ and an H R T of four
hours removed almost 80% of the AOX and 50% of the C O D from the kraft
mill effluents.
4.1.2 Using Fungi
The reason why bacteria show low efficiency in removing COD, TOC1 and
high-molecular-mass chlorolignins from bleaching effluent is that bacteria de-
grade the substrate by an intracellular enzyme system. The substrate must be
Reduction of Organochlorine Compoundsin Bleach Plant Effluents 239
able to pass the bacterial cell membrane in order to be degraded. In the light of
this, the inefficiency of bacteria in degrading high-molecular-mass chlorolignins
is understandable. Since most TOC1 and COD is due to the high-molecular-
mass chlorolignins, low COD and TOC1 removal efflciencies are also to be
expected.
An approach to degrading high molecular weight chlorolignins is to use
white-rot fungi, the only known microorganisms to efficiently degrade lignin.
They generate a lignolytic activity to degrade lignin in a so-called secondary
metabolism stage when one of several nutrients, nitrogen, phosphorous or
carbon is depleted (unfavorable natural conditions). White-rot fungi excrete an
operating system including extracellular enzymes which can degrade high-
molecular-mass chlorolignins effectively. So far, the widest applications of the
white-rot fungi in waste-water purification have been concentrated on the
decolorization of bleaching or pulp mill effluents [248-250].
The reaction mechanism by which white-rot organisms degrade lignin is in
four steps. Ligninase enzymes catalyze the first two steps. This is followed by an
aromatic hydroxylation step that produces catechol structures in the resulting
fragments [251, 252]. The fourth step is an oxidative ring cleavage catalyzed by
a dioxygenase enzyme. The products of this reaction are converted by the
organism into CO2 and water. Chlorinated aromatics are dealt with by the
organism in a like manner by converting them into catechol structures whose
rings can be cleaved via the same dioxygenase enzyme, the products being CO2,
water and inorganic chlorides. Many other organisms can grow directly on
chlorinated organics, degrading them via the same or similar dioxygenase
pathways or in some cases monooxygenase pathways.
The best known white-rot fungus is Phanerochaete chrysosporium. This
fungus is known to secrete a family of enzymes which degrade both lignin and
modified lignin/-249]. Both high- and low-molecular-mass chlorolignins gener-
ated during the pulp bleaching are significantly degraded [253, 254]. The
fungus reduces the COD by degrading the chlorolignins to CO2 and chloride,
decolorizes the bleaching effluent by destroying both the color bodies and
chromophoric structure, and removes TOC1 by converting it to inorganic
chloride [253-255]. Most of the low-molecular-weight chlorophenolics disap-
pear after 1 day's fungal treatment [256, 257]. This particular fungus is also
known to be able to degrade refractory compounds such as TNT, PCB and
Lindane, DDT, chlorinated dioxin and other difficult biodegradable com-
pounds [258-260].
Decompositions of lignin to CO2 by the lignin-decomposing fungus
P. chrysosporium requires a growth substrate such as cellulose or glucose.
Growth on lignin as a sole carbon source is negligible. In addition to its
requirement for a co-substrate, the ligninolytic system (a) is produced even in the
absence of lignin, (b) is expressed only during secondary metabolism, (c) is
triggered by carbon, sulfur, or nitrogen limitation, (d) is strongly repressed by
glutamate and certain other amino acids, (e) is sensitive to the balance of trace
metals supplied, (f) has a relatively narrow pH optimum (4 5), and (g) is
markedly affected by oxygen concentration [-261-265]. The fungal decoloriz-

ation, like lignin metabolism, is a secondary metabolic event and is probably
attributable to lignin metabolism since lignin and its degradation products are
the main color source in E1 effluent. The optimum conditions favoring fungal
growth are quite different from those favoring decolorization. The pH range for
optimum growth is 4.3~4.8 and decolorization is greatly retarded below pH 4.0
or above 5.0 because of poor growth [261]. The optimum temperature for the
growth of fungus is 40 ~ whereas decolorization is not limited to the same
narrow range of temperature, but takes place with little decrease in rate at
temperatures as low as 25 ~ [261]. The fungal decolorization requires oxygen
and a co-substrate, but, unlike fungal growth, the addition of a nitrogen source
is not necessary. Eaton et al. [266] have outlined a process based on laboratory
and bench scale experiments for decolorization by P. chrysosporium. The fungi
require a growth substrate for decolorization just as they do for lignin degrada-
tion. Investigations have demonstrated that the cellulose-rich primary sludge
serves this purpose well. The fungus requires a fixed surface for pregrowth
without agitation in shallow medium for efficient color reduction. Fixed film
reactors of the rotating biological contactor (RBC) design, which are commer-
cially available and are presently used in waste treatment have been proved to
be effective. Color removal is greatly stimulated by an oxygen-enriched atmo-
sphere. The RBC provides good aeration and can be enclosed for the addition of
supplementary oxygen. A growth stage is necessary before decolorization be-
gins. During this stage, nutrient nitrogen is depleted, and the fungus becomes
ligninolytic and able to decolorize. Based on the above considerations, the
process shown in Fig. 3, which has been termed the FPL/NCSU/MyCoR
process [267], can be outlined. This mycelial color removal process resulted
from the cooperative research between the US Forest Products Laboratory and
North Carolina State University. A fixed film MyCoR reactor is charged with
growth nutrients, which can include primary sludge as the carbon source, and is
inoculated with a suitable fungus. Depending on the mill, the sludge will provide
Influent
uent
Fig. 3. Rotatingbiologicalcontactorfor fungaldecolorizationof pulp mill effluent

some of the required mineral nutrients and trace elements as well as carbon.
Nitrogen-rich secondary sludge can also be used to supply the nitrogen required
for growth. After the mycelium has grown over the reactor surface, it depletes
the available nitrogen and becomes ligninolytic (pregrowth stage 2 to 4 d). The
reactor is then ready for use. Operation for over 60 days has been achieved in
bench reactors in a batch mode. The process converts 70% of the organic
chlorides to inorganic chloride in 48 h while decolorizing the effluent and
reducing both COD and BOD by about half. Huynh et al. [268] used the
MyCoR process for the treatment of the chlorinated low-molecular-mass phen-
ols of the E1 effluent. Their results showed that most of the chlorinated phenols
and other low-molecular-mass components of the effluent were removed during
fungal treatment. Pellinen et al. [269] have reported that the MyCoR process
can be considerably improved in terms of COD removal by simply using less
glucose as the carbon source for the fungi, P. chrysosporium. However, the
dechlorination was reported to be faster at high glucose concentration. The
kinetics of decolorization of E1 effluent with P. chrysosporium in an RBC under
improved conditions have been studied by Yin et al. [270]. The kinetic model
developed for 1 and 2-d retention times showed a characteristic pattern. The
overall decolorization process can be divided into three stages, viz. a rapid color
reduction in the Ist hour of contact between the effluent and the fungus followed
by a zero order reaction and then a 1st order reaction. The color removal rate on
the second day of the 2-d batch treatment was less than that on the first day. The
decolorization in a continuous flow reactor achieved approximately the same
daily color removal rate, but the fungus had a longer working life than when in
the batch reactor, thereby removing more color over the fungal lifetime. Pellinen
et al. [271] studied dechlorination of high-molecular-mass chlorolignin in first
extraction stage effluent with white-rot fungus P. chrysosporium immobilized on
an RBC. The total organic chlorine content of chlorolignin decreased almost by
50% during one day of treatment. Correlation studies suggested that dechlorina-
tion, decolorization and degradation of chlorolignin (as COD decrease) are
metabolically connected, although these processes have different rates. Size exclu-
sion chromatography showed that polymerization took place in the early stage of
the treatment. Low-molecular-mass degradation products were not observed.
A sequential biological treatment using this fungus and these bacteria was
studied to decrease TOC1, color and COD in conventional softwood kraft pulp
bleaching effluent by Yin et al. [272]. In six variations of the white-rot fun-
gus/bacteria system studied (Table 8), only the degree of fungal treatment was
varied. In three of the six variations, ultrafiltration was also used to concentrate
high-molecular-mass chlorolignins and to reduce effluent volume (and thus cost)
prior to fungal treatment. The best sequence, using ultrafiltration/white-rot
fungus/bacteria, removed 71% TOC1, 50% COD and 65% color in the effluent
(Table 8). Fungal treatment enhances the ability of bacteria to degrade and
dechlorinate chlorinated organics in the effluent. The fungus is able to remove
organic chlorine from chlorolignins and to attack the high- and low-molecular-
mass chlorolignins.
~ 2 2 2 2 2
e~
ii! ---- ='" ~
,--M
O
O
i
!
!
.r,
=~
i
! II II ~ - 2 , =
0
Guo et al. [257] investigated degradation of model compounds - chloro-

phenols (pentachlorophenols, 2,4,6-trichlorophenol, and 2,4-dichlorophenol)
and chloroguaiacols (4,5-dichloroguaiacol and 4,5,6-trichloroguaiacol) in pure
water solution by fungal treatment using an RBC. They found that at concentra-
tion of 30 mg/1, 80-85% of chlorophenols and chloroguaiacols could be de-
graded after 3-4 h of treatment. Meanwhile, some of these compounds were
methylated to chloroanisoles and chloroveratrols.
Messner et al. [273] developed a similar process, i.e. the MyCoPOR. In this
process, porous carrier material is inoculated with spores of Phanerochaete
chrysosporium. Shaking-flask tests with and without foam as porus carrier
material showed that the decolorization of an E1 effluent from a sulfite plant was
improved from 50% without foam as supporting material to 70-80% with foam.
Decolorization was accompanied by about 70% AOX reduction when using
foam. If nutrients were replaced and effluent removed daily, the active lifetime of
the mycelium was 6-8 weeks, depending on the fungal strain. A trickling filter
system was found to be the most effective for the degradation of effluents. With
daily replacement of the medium/effluent solution, the decolorization rate of the
E1 effluent was 70% when aerated by air, and AOX reduction was 60%. The
fastest degradation of the effluent took place during the first 3-6 hours, when
about 50% of the color and AOX were reduced.
Another white-rot fungus, which has shown good performance, is Coriolus
versicolor. It requires a growth substrate such as cellulose or glucose for the
decomposition of lignin to carbon dioxide [274]. The culture conditions favor-
ing lignin degradation are similar to those favoring fungal decolorization.
Livernoche et al. [274] showed that C. versicolor in liquid culture removed over
60% of the color of the combined bleach kraft effluent within 6 d in the presence
of sucrose. Decolorization of effluent was more efficient when the concentration
of sucrose (growth substrate) and inoculum was high. Treatment of the same
effluent with this fungus, immobilized in beads of calcium alginate gel, resulted
in 80% decolorization after 3 d in the presence of sucrose. The pH of the effluent
during decolorization decreased from 5.7 to 3.6, probably due to formation of
organic acids resulting from the metabolism of the immobilized fungus. How-
ever, decolorization was not due to lowering of pH. The decolorization process
affected not only the dissolved chromophores but also the suspended solids. The
solids after centrifugation of the zero time samples were dark brown, while the
solids after 4-d incubation were light brown. The beads with the immobilized
mycelium remained light-coloured throughout the experiments with no indica-
tion of accumulation of the effluent chromophores. Decolorization was achieved
more rapidly at pH 5.0 than at pH 7.0. Recycled beads could remove color
efficiently and repeatedly in the presence of air but not under anaerobic condi-
tions.
Biological reactors of the airlift type using calcium alginate beads to immo-
bilize the fungus C. versicolor have been used to study the continuous decoloriz-
ation of kraft mill effluents [275]. The effluent used contained only sucrose and
no other nutrient source. An empirical kinetic model was proposed to describe
the decolorization process caused by this fungus, but it did not shed any light on
the chemical mechanism involved in the decolorization.
Direct use of suspended mycelium of the fungus, C. versicolor, may not be
feasible because of the problem of viscosity, oxygen transfer and recycling of the
fungus. The fungus was, therefore, grown in the form of pellets, thus eliminating
the problems with biomass recycling and making it possible to use a larger
amount [276]. Rate of decolorization with fungal pellets was almost ten times as
high in batch culture as in continuous culture under similar conditions. The
capacity for decolorization decreased markedly with increase of lignin loading
[-276].
Bergbauer and Kraepelin [277] showed that C. versicolor efficiently de-
graded chlorolignins from bleaching effluent. More than 50% of the chlorolig-
nins were degraded in a 9-d incubation period, resulting in a 39% reduction in
AOX and an 84% decrease in effluent color. In a 3-1 laboratory fermenter with
0.8% glucose and 12 m M ammonium sulphate, about 88% color reduction was
achieved within 3 d. Simultaneously, the concentration of AOX dropped from
initially 40 mg/1 to 21.9 mg/1, a 45% reduction within 2 d. With malt extract
instead of glucose, reduction of color unit as well as AOX values were nearly the
same [278].
Bajpai et al. [279] used pellets of T. versicolor strain B-7 for decolorization
of E1 effluent. The mycelial pellets oxidized the chromophores of the effluent in
the presence of any of the cosubstrates sucrose, glucose, starch, ethanol, car-
boxymethylcellulose, microcrystalline cellulose, pulp and malt extract. The
highest decolorization was obtained in the case of glucose. Optimum pH and
temperature were 4.5-5.5 and 30 ~ respectively. In the batch reactor with an
effluent of 7000 color units per litre, the maximum color reduction of 93% was
obtained in 48 h with a C O D reduction of 35%, whereas, in a continuous
reactor, the same level of color and C O D reduction was obtained in 38 h
residence time. No loss in decolorization ability of mycelial pellets was obtained
when the reactor was operated continuously for more than 30 d. They also used
T. versicolor for decolorization of effluent from a pulp mill utilizing agriresidues
[280]. With an effluent of 18 500 color units, a maximum color reduction of 92 %
with a C O D reduction of 69% was obtained.
Royer et al. [281] described the use of the pellets of C. versicolor to
decolorize ultrafiltered kraft liquor in nonsterile conditions with a negligible loss
of activity. The rate of decolorization was observed to be linearly related to the
liquor concentration and was lower than that obtained in the M y C o R process.
This could be due to the lower temperature (22 ~ used in this work and to the
use of pellets with relatively large diameters which could limit the microbial
activity as compared to the free mycelium used in the M y C o R process. An
effective decolorization of effluent having 400-500 color units/1 can be obtained
in presence of a simple carbon source such as glucose. In the repeated batch
culture, the pellets exhibited a loss of activity dependent on the initial color
concentration. The production of the extra cellular enzyme, laccase, was fol-
lowed but could not be used as an indicator of the ligninolytic activity.
Archibald et al. [282] studied the fungal requirements for optimal growth
and decolorization and the mechanism of chromophore degradation by
C. versicolor. Simple carbohydrates were essential for effective decolorization,
and a medium composed of inexpensive industrial by-products provided excel-
lent growth and decolorization. The regulation or control of E1 effluent decolor-
ization appears to differ substantially from that of lignin peroxidase production
and the induction of ligninolytic activity. In particular, modulation of decoloriz-
ation by trace metals, nitrogen and carbon limitation, culture age and veratryl
alcohol was not observed.
Marton et al. [283] reported that C. versicolor reduced the characteristic
dark brown color of diluted alkaline lignin solutions. They found, however,
about half of the color and one fourth of the aromatically absorbing substances
were recovered from the fungal cells by alkaline extraction. Therefore, it was
concluded that adsorption played a major role in lignin degradation. Decoloriz-
ation also proceeded anaerobically but to a lesser extent.
Treatment of E1 stage eff with ozone and the fungus C. versicolor has
also been tried [284]. Both ozone treatment and biological treatment effectively
destroyed effluent chromophores, but the fungal process resulted in greater
degradation as expressed by COD removal. Monoaromatic chlorophenolics
and toxicity were removed partially by ozone and completely by C. versicolor.
Molecular weight distributions showed roughly equal degradation of all sizes of
molecules in both treatments. The combination of a brief ozone treatment with
a subsequent fungal treatment revealed a synergism between the two decoloriz-
ation mechanisms on E1 stage effluent. Effluent was pretreated with
ozone (ll0-160mg/1) or C. versicolor (24h with 2-5g/1 wet weight
fungal biomass). The pretreatment was followed by a 5-d incubation with C.
versicolor. It was noted that partial color removal by ozone pretreat-
ment allowed more effective removal by the fungus than that by fungal pre-
treatment. After 20h, 46-53% decolorization was observed for ozone-
pretreated effluents, compared to 29% for fungal treatment alone. The contribu-
tion of ozone seemed to be most important in the first 24 h following the
pretreatment. Ozone pretreatment also produced a small improvement in the
bioavailability of effluent organics to the fungus. A partial replacement of
chlorine by ozone in the bleach plant (the addition of an ozone bleaching step)
or a brief ozone pretreatment of E1 stage effluent should considerably reduce the
low-molecular-mass toxic chlorophenolics. In addition, the use of ozone should
also improve decolorization by subsequent fungal and possibly bacterial treat-
ments.
Another white-rot fungus, Schizophyllum commune, has also been found to
decolorize and degrade lignin in pulp and paper mill effluent [-285]. The fungus
was able to degrade lignin in the presence of an easily metabolizable carbon
source. The addition of carbon and nitrogen not only improved the decolorizing
efficiency of the fungus but also resulted in reduction of the BOD and COD of
the effluent. Sucrose was found to be the best carbon source for the breakdown
of lignin. A 2 d incubation period was sufficient for lignin degradation by
S. commune. The efficiency of treatment of effluent with this fungus was highest
at pH 4 5 and was further improved by intermittent aeration. Under
optimum conditions, S. commune reduced the colour of the effluent by 90%
and also reduced BOD and COD by 70% and 72% during a 2 d incu-
bation.
A white-rot fungus, Tinctoporia borbonica, has been reported to decolorize
the kraft waste liquor to a light yellow color [286]. About 99% colour reduction
was achieved after 4 d of cultivation. Measurement of the culture filtrate by
ultraviolet spectroscopy showed that the chlorine-oxylignin content also de-
creased with time, and measurement of the culture filtrate plus mycelial extract
after 14 d of cultivation showed the total removal of the chlorine-oxylignin
content.
Addition of a carbon and nitrogen source was found to improve decoloriz-
ation of pulp and paper mill waste water by the fungus Aspergillus niger, leaving
19% of the original color, and reduced about 43% BOD and 41% COD after
2 d of incubation [287]. Tono et al. [288] reported that Aspergillus sp and
Penicillium sp achieved 90% decolorization in one week's treatment at 30 ~ and
at pH 6.8. Later, Milstein et al. [289] reported that these microorganisms
removed appreciable levels of chlorophenols as well as chloroorganics from the
bleach effluent.
Prasad and Joyce [290] used Trichoderma sp., one of the fungi imperfectii, to
decolorize the hardwood E1 stage effluent. Glucose was found to be the most
effective carbohydrate utilized by the fungus, as it stimulated substantial color
reduction without any increase in COD. Addition of nitrogen did not stimulate
the decolorization process, indicating that it is not a rate-limiting factor. The
optimum pH for decolorization and growth was 4.0. Under optimal conditions,
total colour and COD decreased by almost 85% and 25% respectively after
cultivation for 3 d.
Since the majority of TOC1 and color is due to higher molecular
weight chlorolignins, future research should concentrate on the fate of high-
molecular-mass chlorolignins in biological treatment or in the natural environ-
ment. Since bacteria significantly degrade only those chloroorganics of molecu-
lar mass less than 800-1000, research is needed to decrease the chlorolignin
molecular mass or to remove high molecular mass chlorolignins before biolo-
gical treatment is applied, in order to enhance the biotreatability of bleaching
effluent.
Three approaches to decreasing the molecular weight of chlorolignin should
be studied: (1) a modified bleaching process such as oxygen prebleaching and
a high level of chlorine dioxide substitution, (2) a two-stage biological treatment
such as white-rot fungus followed by biological treatment, and (3) physicochemi-
cal treatment followed by biological treatment.
Ultrafiltration can separate high molecular weight material, and biological
treatability of permeate can be enhanced. Another method is precipitation,
which preferentially removes high-molecular-mass chlorolignins. The treated
effluent should be more amenable to biological treatment.
4.2 Enzymatic Treatment
Some enzymes also seem to have the potential to remove color and AOX from
pulp and paper mill effluents. Peroxidase, laccase etc. are the most important of
these. The use of microbial or enzyme-based treatment offers some distinct
advantages over physical and chemical AOX precipitation methods, in that only
catalytic and not stoichiometric amounts of the reagents are needed, and the low
organic concentrations and large volumes typical of bleaching effluents are,
therefore, less of a problem. Also, both complete microbial systems and isolated
enzymes such as peroxidases and laccases have been shown to reduce the acute
toxicity by polymer and thereby rendering less soluble many of the low-
molecular-mass nonchlorinated and polychlorinated phenolics [291 295]. It
has been reported that lignin-type peroxidases secreted by white-rot fungi are
involved in the degradation of a whole range of organic pollutants including
PCB, D D T and Indane, chlorinated anilines, polychlorinated phenolics includ-
ing P C P and chlorolignins, and many more [296-300]. Lyr [301] reported that
laccase of T. versicolor partially dechlorinates PCP, and Hammel and Tardone
[302] reported that peroxidase from P. chrysosporium can partially dechlorinate
P C P and 2,4,6-trichlorophenol. Arcand and Archibald [303] carried out a sys-
tematic study on direct dechlorination of chlorophenolic compounds in pulp
and paper mill effluents by laccase from T. versicolor. It was found that most of
the laccase secreted by T. versicolor could partially dechlorinate a variety of
chlorophenolics. The rate and extent of C1- release were found to be substan-
tially affected by substrate and enzyme concentration and the presence of
multiple laccase substrates. The dechlorination was found to be accompanied by
extensive polymerization of the substrate. Table 9 shows a comparison of the
removal of chlorophenolics from a mixture in E1 effluent by crude laccase with
Table 9. Removalof chlorophenoliccompoundsfrom a mixture in high-molecular-mass

E1 effluent by crude laccase in 30 mina and T. versicolor myceliumin 3 h
Reduction (%)
Chlorophenolic Concentration
Compounds (gM) Laccase Mycelium
2-Chlorophenol 137 35.5 _+1.4 61.5 2.7
3-Chlorophenol 18 51.9 + 4.3 2.0 0.1
4-Chlorophenol 31 48.1 2.9 41.6 1.3
2,4-Dichlorophenol 21 45.0 2.3 64.9 1.8
2,4,6-Trichlorophenol 26 72.4 _+5.2 83.8 6.2
2,3,4,6-Tetrachlorophenol 18 40.7 8.2 71.2 2.7
Pentachlorophenol 24 34.0 8.9 82.1 3.2
4,5-Dichlorognaiacol 52 100 96.8 + 6.1
4,5,6-Trichloroguaiacol 32 100 100
Tetrachloroguaiacol 17 76.5 21.5 95.0 7.6
aReactions were conducted in 20 ml of E1 effluent containing 20 mM D-glucose,pH 4.6,
temp. 25 ~ shaken at 200 rpm in triplicate using either 0.1 U/ml crude T. versicolor
laccase or 0.5 g wet weight (25 mgd.w.) washed T. versicolor mycelium
Based on data from Ref. [3033
that by T. versicolor mycelium, and Table 10 shows the effects of crude laccase
on various chlorophenolics. Arcand and Archibald [303] also studied the
effects of horseradish and P. chrysosporium peroxidases on the mixture of five
chlorophenolics. Both peroxidase enzymes were found to degrade the majority
of all substrates except PCP, whereas the P. chrysosporium peroxidases
was superior to both horseradish peroxidase and laccase in degrading PCP, it
was inferior to horseradish peroxidase in degrading the other four phenolics
(Table 11).
Paice and Jurasek studied the ability of horseradish peroxidase to catalyze
color removal from bleach plant effluents [304]. The color removal from
effluents at neutral pH by low levels of hydrogen peroxide was enhanced by the
addition of peroxidase. No precipitation occurred during the decolorization
process. The catalysis with peroxidase (20 mg/1) was observed over a wide range
of peroxide concentrations (0.1-800 mM) but the largest effect was between
1 mM and 100 mM. The pH optimum for catalysis was around 5.0. Compared
with mycelial color removal by C. versicolor, the rate of color removal by
peroxide plus peroxidase was initially faster (for the first 4 h), but the extent of
color removal after 45 h was higher with the fungal treatment. Further addition
of peroxidase to the enzyme-treated effluents did not produce additional cataly-
sis. Thus, the peroxide/peroxidase system did not fully represent the metabolic
route used by the fungus. One working hypothesis has been proposed to explain
the behaviour of enzymes in the decolorization process [304]. Glucose is used by
the cell to produce peroxidase. One of the extracellular enzymes often found in
white-rot fungi is peroxidase. It seems that this enzyme oxidizes the chromo-
phores and so removes the color from bleaching waste water.
Forss et al. [305] have shown that aeration in the presence oflaccase leads to
a reduction by over 90% in the amount of phenolic compounds in waste water.
They have shown that the amount of chlorophenols is also reduced by 75-99%,
depending on the group of chlorophenols (Table 12). The total reduction was
80%. The sample was aerated in the presence of laccase for 1 h and was
flocculated with aluminium sulfate. Since polymerization by laccase can be
avoided and the degradation of the substrate enhanced by the presence of
appropriate reductant, it may be possible to substantially increase the rate and
extent of laccase-driven dechlorination.
Call (of Lignozym, Germany) has patented a process for decolorization and
decontamination of waste water from pulp and paper mill effluents using
enzyme [306]. In this process, laccase enzyme from C. versicolor is used along
with phenol or nonphenolic aromatic compounds with continuous passing of
oxidation materials (H202, 02 or oxygenated air). Almost complete polymeriz-
ation of the lignin contained in the waste water is achieved, which is 20-50%
above the values attainable with the addition of laccase alone. About 70-90%
lignin is developed into insoluble form, which is removed by flocculation and
filtration.
Ferrer et al. reported that immobilized lignin peroxidase decolorized
kraft effluent [307]. Novel lignin peroxidases called Pulpases produced by
I I I I I
I I I I I '~
2 ,_
+i+i+l+l+l
+i +1 +i +1
~ cM
M ~5~
+1 +1 +1 ~1 ~1
B
"S
b
0 ~0 0 0 ' ~ . ~ ;> ~ ~'~
o 0 0
9~ ~ ~ ~ . ~ = o ~
6
250 p. Bajpai and P.K. Bajpai
.2
"0~ "~
% J
=
8 +1+1 +1+1
vv c:5
o
e.! ,,4 ~
o
o
o o,--- ~ o
O0 r--
o
e~
b
de.!
8
+1+1+1+1+1+1
de.,
o --a
o0_~ U
V t': ~ N , ~
~ . j
m
o " ~ ~ ~ ~..
,.-8 ~ o o ~,
o =.~ = .~ ~ "-6 O.=oO
o No ~
0
l
,It
Table 12. Concentration of chlorinated phenols in bleach plant effluent from a softwood
kraft mill before and after aeration in the presence of laccase and flocculation with alum
Phenolic compound Concentration (rag/l) Reduction (%)
Initial After 1 h aeration
Chlorinated phenols 141 9 93.6

Chlorinated vanillins 813 176 78.3
Chlorinated guaiacols 516 30 94.2
Chlorinated catechols 251 19 92.4
Total 1721 234 86
Based on data from ref. [305]
P. chrysosporium m u t a n t strain SC 26 a n d d e s c r i b e d in 2 p a t e n t s assigned to the

Repligen C o r p o r a t i o n [308, 309] have been c l a i m e d to d e c o l o r i z e b l e a c h i n g
effluents.
5 Conclusions
M a n y process c h a n g e s have been i m p l e m e n t e d o r are being c o n s i d e r e d to

reduce the f o r m a t i o n of A O X a n d c h l o r i n a t e d d i o x i n s from c h e m i c a l p u l p
b l e a c h i n g o p e r a t i o n s . T h e r e are also possibilities for t r e a t m e n t of effluents with
m i c r o o r g a n i s m s a n d enzymes to r e m o v e o r d e c h l o r i n a t e o r g a n i c material. E a c h
o p t i o n discussed has i n h e r e n t a d v a n t a g e s a n d d i s a d v a n t a g e s with r e g a r d to
c a p i t a l costs, o p e r a t i n g costs, ease of retrofit, f a b r i c a t i o n a n d i n s t a l l a t i o n time.
I m p a c t on o t h e r mill unit o p e r a t i o n s is also c o n s i d e r e d in c h o o s i n g the best
options. M a n y factors h a v e to be c o n s i d e r e d in c h o o s i n g an effective a n d
e c o n o m i c a l b l e a c h i n g / t r e a t m e n t process t h a t meets all the e n v i r o n m e n t a l guide-
lines. It a p p e a r s t h a t the o n l y l o n g - t e r m s o l u t i o n will p r o b a b l y be to d e v e l o p the
t e c h n o l o g y which will allow mills to o p e r a t e with zero effluent.
Acknowledgements We dedicate this article to Shri L.M. Thapar (Chairman, Thapar Group of
Industries) on his 65th Birthday, thanking him for his never-failing encouragement. We also thank
Dr. M.P. Kapoor, Director, Thapar Corporate R&D Centre, for his encouragement, Dr. M.B.
Jauhari, General Manager (R&D), Ballarpur Industries Limited, for helpful discussions and Shri S.S.
Gill for excellent typing of the manuscript.
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Hemicellulases in the Bleaching of Chemical
Pulps
A. Suurn/ikki, M. Tenkanen, J. Buchert, and L. Viikari

VTT Biotechnology and Food Research P.O. Box 1501 FIN-02044 VTT
Finland
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
2 Pulping and Bleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
2.1 Wood Hemicelluloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
2.2 Chemical Pulping Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
2.3 Bleaching Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
2.4 Modification of Carbohydrates During Pulping . . . . . . . . . . . . . . . . . . . . . 266
3 Hemicellulases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
3.1 Xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
3.2 Mannanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
3.3 Other Hemicellulases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.4 Enzymatic Accessibility of Pulp Hemicelluloses . . . . . . . . . . . . . . . . . . . . . . 272
4 Hemicellulases in the Bleaching of Kraft Pulps . . . . . . . . . . . . . . . . . . . . . . . . 272
4.1 Proposed Mechanisms of Hemicellulase-Aided Bleaching . . . . . . . . . . . . . . . . 272
4.2 Analysis of the Enzymatic Action in Pulp . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.3 Factors Affecting the Enzymatic Action . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.4 Effects of Different Hemicellulases on Pulp Bleachability . . . . . . . . . . . . . . . . 276
4.5 Action of Enzymes in Pulps Produced by Sulfate Cooking Methods . . . . . . . . . 277
4.6 Action of Enzymes in Sulphite Pulps . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
4.7 Enzymes in Different Bleaching Sequences . . . . . . . . . . . . . . . . . . . . . . . . . 278
4.8 Impact of Enzymes on Pulp Yield and Quality . . . . . . . . . . . . . . . . . . . . . . 279
5 Industrial Enzyme-aided Bleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
5.1 Industrial Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
5.2 Installation of the Enzymatic Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
5.3 Environmental Impacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
5.4 Economics of the Enzymatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . 282
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Hemicellulase-aided bleaching is the first full-scale biotechnical application in the pulp and paper
industry which truly exploits the unique specificity and safety of biocatalysts. Hemicellulases are
used to modify the structure of xylan and glucomannan in pulp fibers in order to enhance the
chemical delignification. This technology can be combined with various types of kraft pulping
processes and bleaching sequences. The aims of the enzymatic treatment depend on the actual mill
conditions, and may be related to environmental demands, reduction of chemical costs, or mainten-
ance or even improvement of product quality. The technology is applied on the mill scale in several
countries. This review describes the principles of the enzyme-aided bleaching, the composition of the
fiber substrates, the basic enzymology involved, and the present knowledge of the mechanisms of the
action of enzymes, as well as the practical results and advantages obtained on the laboratory and
industrial scale.
Advances in BiochemicalEnNneering/
Managing FAitor:T. Scheper
262 A. Suurn/ikkiet al.
1 Introduction
During the past five years, the technology of chemical pulp bleaching has
entered a period of extremely rapid change driven by growing concern for the
environment. Environmental consciousness is evident not only in the market
place, but also in increasingly stringent government regulation of the industry's
waste streams, processes and products. Stringent regulation of chlorinated and
poorly biodegradable unchlorinated organic compounds in mill effluents is
rapidly becoming a universal rule. The industry has assessed various technolo-
gies in order to identify means for achieving an effluent level lower than 0.2 kg of
AOX per ton of pulps. This target has already been achieved and even exceeded
in several mills, e.g. in Scandinavia, using extended cooking, oxygen delignifica-
tion and various ECF (Elemental Chlorine Free) or TCF (Totally Chlorine Free)
bleaching chemicals. Among the new bleaching agents, biotechnical methods
have also been developed and used on the mill scale.
In Europe, the first signs of growing consumer awareness appeared in late
1980s, leading to an almost desperate search for new methods to reduce
environmental impacts. The resulting changes in bleaching technologies occur-
red so rapidly that they may be considered revolutionary. In this transitional
period, the hemicellulase-aided bleaching process was adopted on the industrial
scale. Enzymes are readily biodegradable, and offer an environmentally safe
method for improving pulp bleaching.
The idea of using hemicellulolytic enzymes for increasing the bleachability of
chemical pulps was introduced in the 1980s [1, 2]. Previous reports of applica-
tions of hemicellulases included total hydrolysis of hemicellulosic raw materials
for production of monomeric sugars [3] and the removal of hemicelluloses from
chemical pulps to produce dissolving pulps [4]. The concept of enzyme-aided
bleaching was based on the realization that limited hydrolysis of hemicellulose
in pulps by hemicellulases, mainly xylanases, increases the extractability of
lignin from the kraft pulps in the subsequent bleaching sequences. The xylanase
pretreatment permits the use of lower chlorine charges during the bleaching of
kraft pulps, the bleach-boosting effect being associated with reduced chloro-
organic discharges. The first mill trials were conducted in Finland in 1988 [5],
a remarkably short time after the first reports. As effective and easily applicable
process aids, xylanases are nowadays used in several mills prior to chlorine and
non-chlorine bleaching sequences of kraft pulps (reviewed in [6]).
The hemicellulase treatment, together with a chemical extraction, leads to
a significant reduction of the residual lignin content in the pulps. However, due
to the indirect mode of action, the effect of hemicellulase-aided bleaching is
limited. The development of a directly delignifying biotechnical method is
therefore still the major target for several research groups. The reaction mecha-
nisms of lignin-degrading enzymes on fiber-bound lignin are, however, more
difficult to control, and delignification results with these enzymes have been
rather poor. Recently, however, very promising results have been obtained using
Hemicellulasesin the Bleachingof ChemicalPulps 263
a new concept based on the ability of mediators, oxidized by laccase, to degrade

lignin specifically [7, 8].
The hemicellulase-aided bleaching method represents a new type of generic
technology, already adopted by the pulp and paper industry. Hemicellulases are
the first group of specific enzymes to be used in a large-scale application in this
industrial area. The approach also represents a truly sustainable technology.
Moreover, the technology was shown to be economically competitive a pre-
requisite not always fulfilled by biotechnical applications. The interest in this
type of new technology is also shown by the increasing number of papers
published during recent years describing numerous xylanases from new sources,
as well as bleaching results obtained using various hemicellulases, pulps and
bleaching sequences. Several reviews have also been published [6, 9, 10, 11, 12].
The present review summarizes the major achievements in the field.
2 Pulping and Bleaching
2.1 Wood Hemicelluloses
Wood is mainly composed of cellulose, hemicellulose and lignin. Cellulose is the

most abundant of the components, generally representing 4 ~ 4 5 % of the wood
dry weight [13]. Depending on the wood species, about 20-30% of wood dry
weight is hemicellulose and about 15 25% lignin.
The two main hemicelluloses in wood are xylans and glucomannans
(Table 1). The softwood xylan has a backbone of arabino-4-O-methyl-
glucuronoxylan, which is composed of D-xylopyranose units connected via
[3-1,4-glucosidic linkages. The average molar ratio of arabinose: 4-O-methyl-
glucuronic acid: xylose sugar units in softwood xylan is 1.3:2:10 [13]. Hard-
wood xylan contains 4-O-methylglucuronic acid and acetyl side groups.
Methylglucuronic acids are linked to the xylan backbone by 13-1,2-glucosidic
bonds, and the acetic acids are esterified at the carbon 2 and/or 3 hydroxyl
group. The backbone of softwood glucomannan is composed of [3-1,4-1inked
D-glucopyranose and D-mannopyranose units and is partially substituted by
~-galactose and acetyl units [13]. The two fractions of softwood glucomannan
differ in their average molar ratio of galactose: glucose: mannose. The low
galactose content fraction has a ratio of 0.1:1:4 and is generally called
glucomannan [13]. This is the main fraction of glucomannan in softwoods. The
corresponding ratio in the high-galactose content fraction, galactoglucoman-
nan, is 1:1:3. Hardwoods also contain a small amount of glucomannan. The
ratio of glucose: mannose in this glucomannan varies between 1 : 1 and 1:2,
depending on the wood species. In addition to xylan and glucomannan, both
softwoods and hardwoods contain minor amounts of other hemicelluloses such
as pectin, galactan and arabinan [-13].
264 A. Suurn~ikki et al.
Table 1. Hemicellulose in softwood and hardwood [13]
Content
Wood species Component % of dry weight
Pine Xylan 5-11

Glucomannan 14 20
Birch Xylan 2~30
Glucomannan 1~4
T h e d i s t r i b u t i o n of hemicelluloses in w o o d fibers varies d e p e n d i n g o n the

w o o d species a n d g r o w i n g c o n d i t i o n s [14]. H o w e v e r , in softwoods, the o u t e r
layer of s e c o n d a r y wall ($2) a n d the i n n e r m o s t fiber wall layer, the w a r t y l a y e r
(W), are generally richer in xylan t h a n the m i d d l e of the s e c o n d a r y wall. T h e
g l u c o m a n n a n c o n t e n t of s o f t w o o d fibers increases steadily f r o m the o u t e r p a r t s
to the inner p a r t s of cell walls. In h a r d w o o d s , the o u t e r m o s t layers of s e c o n d a r y
fiber wall are rich in xylan, whereas the c o n t e n t of g l u c o m a n n a n r e m a i n s low
a n d c o n s t a n t t h r o u g h the fiber walls.
2.2 Chemical Pulping Processes
The m a i n a i m in chemical p u l p i n g is to s e p a r a t e the w o o d fibers from each o t h e r

in o r d e r to r e n d e r t h e m suitable for further i n d u s t r i a l processing. T h e delignifi-
c a t i o n of w o o d y m a t e r i a l can be c a r r i e d out, for e x a m p l e , b y sulfate (kraft) o r
Table 2. Annual production of wood-based kraft and sulfite pulp in the

world. Estimate for the year 1996 (FAO survey, 1995)
Pulp productiona
Region Pulping method 10 6 tb/a
Northern America Kraft 62.7

Sulfite 8.2
Scandinavia Kraft 13.3
Sulfite 0.9
Europe Kraft 8.1
Sulfite 2.4
Latin America Kraft 9.1
Sulfite 0.9
Asia, developing Kraft 6.1
Sulfite 0.2
Oceania Kraft 1.2
Sulfite 0.1
Africa, developing Kraft 0.9
Sulfite 0
a Total production (unbleached, bleached)

b Metric tons of air dry pulp
Hemicellulasesin the Bleachingof Chemical Pulps 265
sulfite processes [13]. Today, the predominant pulping method is the kraft
process (Table 2). However, for environmental reasons, sulfite pulping is pre-
ferred in Germany and also in several other countries.
In sulfate, i.e. kraft pulping, the lignified middle lamella located between the
wood fibers is removed in highly alkaline conditions and at a high temperature
[13]. Recently, modified and totally new sulfate pulping processes using altered
cooking conditions such as a relatively even alkali profile and decreased cooking
temperatures have been introduced [15]. The main aim of the pulping methods
such as modified continuous cooking (MCC), superbatch cooking and isother-
mal cooking (ITC) is to produce more easily bleachable pulps having both
relatively low lignin content and acceptable strength properties.
2.3 Bleaching Processes
In kraft pulping, about 90% of wood lignin is solubilized during the cooking
process [13]. The remaining 10% of lignin is mainly responsible for the brown
colour of the kraft pulp and unbleached paper. The primary goal of bleaching is
to remove the residual lignin from the pulp as selectively as possible without
degrading the pulp carbohydrates, especially cellulose, which would lead to
a decrease in viscosity.
Traditionally, the bleaching of chemical pulps has been carried out
with elemental chlorine and chlorine dioxide. However, the chlorinated
organic compounds formed during chlorine bleaching have attracted negative
attention during recent years. Public concern, together with tightened environ-
mental regulations, have driven the pulp and paper industry to seek out and
utilize alternative bleaching processes. Reduction in bleach plant effluents can
be achieved by reducing the lignin content of pulp prior to bleaching by
modified cooking procedures or oxygen delignification, or by replacing the
elemental chlorine used in the bleaching process with other chemicals such as
chlorine dioxide, ozone, oxygen, peroxide and/or peroxyacids [15, 16]. World-
wide, chlorine gas has traditionally been the main chemical used in pulp
bleaching, but the alternative bleaching sequences, e.g. oxidizing agent-
based totally chlorine-free (TCF) and especially the chlorine dioxide-based
elemental chlorine-free (ECF) sequences, are increasingly used in industrial pulp
bleaching [15].
Compared with elemental chlorine or even with chlorine dioxide, the
oxygen-based chemicals are less effective or less selective in reacting with
pulp lignin [13]. To obtain a fully bleached pulp without elemental chlorine,
the lignin content of the pulp entering the bleaching process should be as
low as possible. Oxygen delignification is commonly used as a prebleaching
stage prior to the ECF and TCF bleaching sequences. A reduction of lignin
content by about 50% can be achieved using oxygen, with relatively low
loss of carbohydrate yield and without impairing the strength properties of
pulp [13, 17].
2.4 Modification of Carbohydrates During Pulping

Wood cellulose is rather resistant to the harsh conditions used in chemical
pulp production, because of its crystallinity and linearity. However, hemicel-
lulose components of the wood are heavily modified during pulping processes.
At the beginning of the conventional sulfate process, i.e. kraft cooking, xylan
in wood is partly solubilized by the alkaline cooking liquid, and many of the
side groups and acetic acid residues are cleaved off [18, 19]. Already in the early
phases of the kraft cook, the methylglucuronic acid side groups in both residual
and solubilized xylan are almost completely converted to hexenuronic acid
[20, 213.
As the alkali concentration decreases towards the end of the kraft cook,
dissolved xylan tends to readsorb on the surface of cellulose microfibrils
[22-25]. In addition to free xylan chains, dissolved lignin and covalently bound
xylan and lignin are also believed to relocate to fiber surfaces [26, 27], resulting
in relatively high amounts of lignin on these surfaces [28-30]. The amount of
xylan readsorbed during the cooking depends on the wood species used in
pulping. High amounts of xylan have been found to locate on the surface of
birch kraft fibers, probably partly due to readsorption, whereas in pine kraft
fibers the concentration of xylan on the fiber surfaces has not been observed to
be higher than in the whole fibers [31]. A large part of wood glucomannan is
also dissolved at the beginning of the kraft cook, but owing to their instability in
alkali, the solubilized polymers are completely degraded in the pulping liquor
[23, 32, 33].
As a result of the solubilization of hemicelluloses during cooking,
the distribution and content of xylan and glucomannan in kraft pulp fibers
differs from that in the native wood fibers [14]. In softwood kraft fibers,
the xylan concentration is generally higher in outer layers, and glucomannan
is more concentrated in the middle layers of the fiber. However, due to differ-
ent analysis methods, variations in the distribution of polysaccharides in
softwood kraft fibers have been reported [34-38]. There is, however, general
agreement that the outer surface layer of hardwood kraft fibers is rich in
xylan.
Recently, several modified kraft pulping methods as well as totally new
sulfate pulping methods have been introduced [15]. In the pulps produc-
ed by these methods, little or no reprecipitation of xylan and lignin is expected
to occur owing to the relatively constant alkali concentration throughout
the cooking process. Consequently, the composition of the outer surfaces
of the pulp fibers is probably different from that of the conventional kraft
pulp fibers.
In sulfite cooking, hemicellulose is extensively solubilized to monomeric and
oligomeric compounds, and no reprecipitation occurs [39]. Thus, the distribu-
tion of hemicellulose is relatively constant across the pulp fibers [40]. The
arabinose side-groups of softwood xylan are completely removed in acidic
sulfite cooking [41], although some acetyl groups are present in sulfite pulps.
3 Hemicellulases
Owing to the complex structure of hemicelluloses, several different enzymes are

needed for their enzymatic degradation or modification. The two main
glycanases depolymerizing the hemicellulose backbone are endo-l,4-~-D-
xylanase and endo-l,4-~-D-mannanase. Xylanases and mannanases are produc-
ed by many species of bacteria and fungi (reviewed in [42 46]). Small oligosac-
charides are further hydrolyzed by 1,4-13-D-xylosidase, 1,4-13-D-mannosidase and
1,4-[3-D-glucosidase. The side-groups are removed by 1,3-~-c-arabinosidase,
1,2-a-o-glucuronidase and 1,6-a-D-galactosidase. Esterified side-groups are
liberated by acetylxylan esterase and acetylgalactoglucomannan esterase.
3.1 Xylanases
Since xylan is the most abundant of the hemicelluloses in pulps, a major part
of the published work on hemicellulases deals with the properties, mode
of action and applications of xylanases (reviewed in [42,45-48]). Endo-
Xylanases (1,4-13-D-xylan xylanohydrolases, EC 3.2.1.8) catalyze the random
hydrolysis of 1,4-13-D-xylosidic linkages in xylans (Fig. 1). Most xylanases are
rather small proteins (molecular mass around 20 kDa) with a basic isoelectric
point (pI 8 10). Another group of xylanases has also been identified. These are
somewhat larger (molecular mass > 40 kDa), with acidic isoelectric point
(pI 3-5) [47]. The grouping based on the physico-chemical properties correlates
well with the classification of xylanases into glycosyl hydrolase families 10 and
11 (previously F and G), based on amino acid similarities [49]. The xylanases
belonging to the two xylanase groups so far identified also differ from each other
with respect to their catalytic properties [50]. The family 10 xylanases, with high
Mr and low pI, seem to exhibit greater catalytic versatility than those of family
11, with low Mr and high pI. Thus, they are typically able to hydrolyze highly
substituted xylans more efficiently.
Most of the xylanases characterized are able to hydrolyze different types
of xylans, showing differences only in the spectrum of end products, The
main products formed from the hydrolysis of xylans are xylobiose, xylotriose
and substituted oligomers of two to five xylosyl residues. The chain length
and the structure of the substituted products depend on the mode of action
of the individual xylanase. Some xylanases, however, show rather strict substra-
te specificity. A unique xylanase which requires a glucuronic acid substituent
in the xylan backbone is produced by Bacillus subtilis [51]. At the other
extreme, xylanase produced by Talaromyces emersonii requires at least 24
unsubstituted xylose residues for its action [52]. The three-dimensional
structures of several low-molecular-mass xylanases have recently been deter-
mined [53-56]. The structure of the Trichoderma reesei pI 9 xylanase
MeGIcA Ac Ac Ac
Hardwood I~-- I<--- I~-- I~--
xylan Xyl - Xyl - X y l ~ Xyl - X y l - Xyl ~Xyl - Xyl
Xyl ~ Xyl
Ara MeGIcA MeGIcA
Softwood i~- I<-- I~--
xylan Xyl - Xyl ~ Xyl - Xyl - X y l ~ Xyl - Xyl - Xyl
13-Xylanase ~1~
cz-Glucuronidase --~
c~-Arabinosidase
I"J-Xylosidase
Esterase --~
Fig. 1. Schemeof the enzymaticdegradationof xylan
is represented in Fig. 2. This enzyme is ellipsoidal, having dimensions of 32

by 42 A and, unlike most cellulases, it does not contain any separate sub-
strate binding domain. Some bacterial xylanases, however, have been found
to contain either a cellulose binding domain [-57-59] or a xylan binding
domain [60, 61].
Most xylanases studied are active in slightly acidic conditions between pH
4 and 6 and temperatures below 70~ More thermophilic and alkalophilic
xylanases are of great importance because of the prevailing conditions in
pulp processing. Some of the alkalophilic strains of Bacillus spp. have been
reported to produce xylanases with relatively high activity at alkaline pH
values [62, 63]. Xylanases which are stable and function efficiently at high
temperatures are produced by several thermophilic bacteria [48]. The most
thermophilic xylanases so far described are produced by an extremely ther-
mophilic bacteria Thermotoga sp. [59, 64-67]. T. maritima produces at
least two hyperthermophilic xylanases, of which one has its temperature opti-
mum at 90 ~ and the other at 105 ~ [59]. However, microorganisms living in
extreme conditions are often difficult to grow, and the productivities of
xylanases are usually low. The amino acid homology of xylanases has opened up
the possibility of using novel genetic techniques for screening for better
xylanases for industrial applications. Thus, several xylanase genes encoding
proteins active at temperatures from 75 ~ up to 95 ~ (pH 6 8) have been
isolated from the extremely thermophilic bacteria Thermotoga and Dictyog-
lomus without the laborious production and purification of the respective
enzymes [68].
Hemicellulasesin the Bleaching of Chemical Pulps 269
Fig. 2. Three-dimensional structure of pI 9

xylanase from Trichodermareesei
3.2 M a n n a n a s e s
Endomannanases (1,4-13-D-mannan mannanohydrolase, EC 3.2.1.78) catalyze

the random hydrolysis of 13-D-1,4-mannopyranosyl linkages within the main
chain of mannans and various polysaccharides consisting mainly of mannose,
such as glucomannans, galactomannans and galactoglucomannans (Fig. 3).
Mannanases are generally larger proteins than xylanases (Mr 30-90 kDa) and
have acidic isoelectric points. They also seem to comprise a more heterogeneous
group of enzymes than, e.g., the xylanases. Thus, no clear groups based on
similar biochemical properties have been identified. The main hydrolysis prod-
ucts from galactomannans and glucomannans are mannobiose, mannotriose
and various mixed oligosaccharides. The hydrolysis yield is dependent on the
degree of substitution as well as on the distribution of the substituents [69]. The
hydrolysis of glucomannans is also affected by the glucose/mannose ratio. Some
mannanases are able to hydrolyze not only the 13-1,4-linkage between two
mannose units but also the bond between the adjacent mannose and glucose
units [70]. Recently, the mannanase of T. reesei has been found to have
a multidomain structure similar to that of several cellulolytic enzymes. The
protein contains a catalytic core domain which is associated, surprisingly, by
a linker to a cellulose-binding domain [71, 72]. The mannanase of Caldocel-
lulosiruptor saccharolyticus has also been reported to be part of a multidomain
protein that contains two catalytic domains (one with mannanase and another
270 A. Suurn/ikki et al.
Gal Ac Ac
Softwood 14 I(--- i~---
glucomannan G I c - Man - Man ~ GIc - Man - M a n - Man
t
Man ~ Man
GIc~Man
B-Mannanase BI~
c~-Galactosidase
8-Mannosidase EC>
8-Glucosidase - ~
Esterase
Fig. 3. Schemeof enzymaticdegradation of glucomannan
with endo-glucanase activity) and two substrate binding domains [-73]. In

addition to C. saccarolyticus mannanase, thermophilic mannanase with a tem-
perature optimum of 100~ has been purified from Thermotoga neapolitana
[74]. Mannanases with alkaline pH optima have been detected in an al-
kalophilic Bacillus sp. [75]. As yet, no three-dimensional structures of man-
nanases have been published.
3.30therHemicellulases
Enzymes needed for further hydrolysis of the short oligomeric compounds

produced by endo-enzymes from pulp hemicellulose are [3-xylosidase (1,4-[3-D-
xyloside xylohydrolase, EC 3.2.1.37), [3-mannosidase (1,4-[3-D-mannoside
mannohydrolase, EC 3.1.1.25) and 13-glucosidase. [3-Xylosidases catalyze
the hydrolysis of xylo-oligosaccharides by removing successive xylose residues
from the non-reducing termini (Fig. 1). Correspondingly, 13-mannosidase
catalyzes the hydrolysis of terminal non-reducing mannose residues in mannans
(Fig. 3). Exoglycanases are generally larger proteins than endoglycanases,
having a molecular weight over 100 kDa, and are often composed of two or
more subunits.
The side groups connected to xylan and glucomannan main chains are
split off by a-glucuronidase, a-arabinosidase (a-L-arabinofuranoside ara-
binofuranohydrolase, EC 3.2.1.55) and a-D-galactosidase (~-D-galactoside galac-
tohydrolase, EC 3.2.1.22)(Figs. 1 and 3). Hemicellulose-bound acetyl substitu-
ents are removed by esterases. There are clearly different types of side-group
cleaving enzymes. Some are able to hydrolyze only substituted short-chain

oligomers which must first be produced by the backbone-depolymerizing
hemicellulases (xylanases and mannanases). Others are also capable of attacking
intact polymeric substrates. With regard to applications on fiber-bound
substrates, enzymes of the latter group are more relevant. Most accessory
enzymes of the latter type, however, prefer oligomeric substrates. The synergism
between different hemicellulolytic enzymes is observed as the accelerated action
of endoglycanases in the presence of accessory enzymes. Some accessory
enzymes such as an a-arabinosidase and an esterase of Pseudomonasfluorescens
subs. cellulosa [57] and an acetyl xylan esterase of T. reesei [76] have also
been found to have a multidomain structure with a separate cellulose-binding
domain.
3.4 Enzymatic Accessibility o f Pulp Hemicelluloses
The action of enzymes in pulp is affected by the accessibility of substrates in the

fiber matrix. The main factors limiting the access of enzymes to woody materials
are the specific surface area and the porosity, i.e. the median pore size of fibers
[-77 79]. Other factors such as the molecular organization of the fiber compo-
nents [37, 80, 81] and the linkages between lignin and carbohydrates 1-82-84]
may also have a significant role in the accessibility of fiber-bound substrates,
especially in the case of xylans and glucomannans.
In general, the median size of pores in wood fibers accessible to external
macromolecules is about 1 nm [85]. in chemical pulping, the pore size increases
as a function of the decreasing pulp yield, being 5-6 nm at the yield level of 50%
in the kraft and sulfite processes. At any given yield, the median pore size is
larger in sulfite than in kraft pulps [85]. The molecular size and structure of
enzyme can be expected to be important factors when considering the limited
accessibility of the substrates in fiber matrices. The molecular sizes of enzymes
vary considerably, depending on the molecular weight and steric configuration
of the protein molecules. The molecular size of the xylanase from T. reesei
(20 kDa with a diameter of approximately 3 4 nm [-55]), should allow this
protein to penetrate most of the pores in chemical pulps. Thus, while the native
wood is highly inaccessible to enzymes, chemical pulps can be structurally
modified by enzymes. Using high enzyme dosages, almost 50% of the pulp
hemicelluloses can be removed without hydrolyzing cellulose. In the case of
hemicellulase-aided bleaching, only a minor part, around 10%, of the total
hemicellulose content is usually removed. Considering the different types of
xylans present in pulps, i.e. the residual and readsorbed xylans, it would be
advantageous to remove specifically only those xylans which hinder the extrac-
tion of lignin. However, despite both basic research on substrate specificities of
individual xylanases and applied research on different and modified pulps, "no
evidence of specific enzymatic hydrolysis of the readsorbed xylan has been
obtained".
272 A. Suurn/ikkiel al.
4 Hemicellulases in the Bleaching of Kraft Pulps
It was originally suggested that lignin-carbohydrate complexes may impede the

removal of residual lignin from pulps, and therefore that hemicellulase treat-
ment could improve their bleachability [1]. Positive results were obtained using
unpurified culture filtrates from various sources, having xylanase as the main
activity [1-]. Even with unpurified enzymes, identification of the sugars released
in the enzymatic treatments confirmed that xylanase was the major activity
positively affecting the bleachability [2]. The original aim, total replacement of
chlorine chemicals, was not achieved using hemicellulases and the existing
bleaching technologies based on oxygen-providing chemicals. However, it was
evident that increased extractability of lignin was achieved, which could be
exploited to decrease the bleaching chemical consumption. Subsequently, the
improved chemical extraction of lignin caused by the removal of hemicelluloses
has been studied in a number of combinations of different enzymes, pulps and
bleaching sequences.
4.1 Proposed Mechanisms of Hemicellulase-Aided Bleaching

The effect of hemicellulases in bleaching is not based on direct attack of residual
lignin but on the modification of pulp hemicelluloses, enhancing the removal of
lignin in chemical bleaching. It has been proposed that the action of xylanases is
due to the partial hydrolysis of reprecipitated xylan [86] or to removal of xylan
from the lignin-carbohydrate (LC) complexes [1, 87]. Both of these hypotheses
would allow the enhanced diffusion of entrapped lignin from the fiber wall.
Limited removal of pulp xylan is known to increase the leachability of residual
lignin from kraft pulps [88] and thus also to increase the pulp bleachability
during subsequent bleaching stages. In addition, it has been suggested that the
hemicellulase treatment removes chromophoric groups from the pulp [89, 90].
The role of reprecipitated xylan in the xylanase-aided bleaching of birch
kraft pulps has been confirmed by comparing the effect of xylanase treatment on
bleachability of kraft pulps cooked by a batch method and of pulps produced in
a flow-through digester and therefore containing only traces of reprecipitated
xylan [86]. In birch kraft pulp, the presence of an extensive amount of xylan,
probably partly due to reprecipitation, has later also been observed both in the
primary fines and on the outer surface of pulp fibers [31]. However, in the case
of softwood kraft pulps, the role of reprecipitated xylan is less evident. In a study
of the composition of pulp fiber surfaces [31], no concentration of xylan was
observed in the primary fines or on the outer surface of pine kraft fibers. In
addition, the action of xylanase effective in bleaching was relatively uniformly
distributed on all the surfaces accessible to enzyme [91-]. Xylanase pretreatment
has also been reported to enhance the bleachability of softwood pulps produced
by novel cooking methods with more stable alkali profiles [92 95], and presum-
ably thus containing less reprecipitated xylan than conventional softwood kraft
pulps. These results indicate that mechanisms other than the attack of rep-
recipitated xylan are also involved in xylanase-aided bleaching of softwood kraft
pulps. The role of reprecipitated xylan in xylanase-aided bleaching has been
questioned by Munk et al. [96]. They found that extraction with dimethyl
sulfoxide (DMSO), a chemical which has been claimed to remove reprecipitated
xylan selectively from pulps ]-97], did not improve the bleachability of kraft
pulp, whereas xylanase treatment did. Recently, however, Allison et al. [98]
reported that the removal of pulp xylan by DMSO is dependent on the degree of
polymerization (DP) of xylan. As the D P of reprecipitated xylan is not known,
the role of reprecipitated xylan in xylanase-aided bleaching cannot be conclus-
ively determined by DMSO extractions.
Both softwood and hardwood kraft pulps have been reported to contain LC
complexes in which carbohydrates and lignin may be connected to each other,
for example by ether or glycosidic linkages [82 84]. Increased solubilization of
xylan-lignin complexes, both from model pulps [99] and from kraft pulps
[87, 100], has been observed in xylanase treatment, indicating that LC com-
plexes may also have a role in xylanase-aided bleaching. The action of xylanase
on both reprecipitated and LC xylan in enhancing bleachability suggests that it
is probably not only the type but also the location of the xylan that is important
in the mechanism of xylanase-aided bleaching. The xylanase of T. reesei has
been observed to act rather uniformly in all accessible surfaces of kraft pulps
[101,102], indicating that the effect of xylanase on bleachability is not only an
outer surface phenomenon. The type and location of the enzymatically attacked
xylan, hindering the leaching of pulp lignin, are thus still questions to be
answered.
It has been reported that xylanase treatment also has a slight decreasing
effect on the kappa number [-88, 103]. This has been explained to be
due to removal of lignin or chromophoric structures [99, 103]. However,
the reduction in the kappa number as measured by permanganate oxidation is
partially due to an artifact. The recently discovered hexeneuronic acid [20, 21],
containing a double bond, gives rise to the consumption of permanganate,
increasing the apparent kappa number [105]. Thus, enzymatic removal
of xylan containing hexeneuronic acid groups can lead to a lower kappa
number.
Compared with xylanase-aided bleaching, the mechanism of mannanase-
aided bleaching has attracted only minor interest, probably due to its rather
limited effect in most pulp types. However, the mechanism of mannanase-aided
bleaching has been assumed to differ from that of xylanase-aided bleaching
because of the different distribution of glucomannan and xylan in pulp fibers
[93, 95, 101, 102, 106]. In contrast to the case of xylanases, no correlation
between the amount and composition of enzymatically solubilized glucoman-
nan and the effect on bleachability has been observed [93, 95, 98, 107]. How-
ever, the role of the composition and configuration of the outer surfaces of pulp
274 A. Suurn~ikkiet al.
fibers seems to be important in mannanase-aided bleaching. Mannanase treat-

ment was found to enhance the bleachability of pulps produced by modified or
continuous pulping methods [93, 95], which are generally considered to contain
less reprecipitated xylan and lignin on the fiber surfaces. Furthermore, man-
nanase treatment was effective in enhancing the bleachability of conventional
pine kraft fibers only when the outer surface material of fibers was mechanically
removed prior to treatment [31]. It is possible that underneath the outermost
surface layer of kraft fibers and on the surface of modified pulps the glucoman-
nan is located more closely to lignin, and that the enzymatic removal of
glucomannan therefore increases the leachability of lignin.
4.2 Analysis of the Enzymatic Action in Pulp

The effect of enzymes on pulp bleachability has been studied by different
methods. Enzymes are usually characterized with isolated substrates, which
are used in the determination of their activities. However, these substrates
vary extensively with respect to their origin and composition. Furthermore,
comparison of enzyme activities is complicated by the utilization of different
analysis methods [108]. Hence, in pulp applications, the action of enzymes
must be compared using the actual substrate, i.e. the pulp. In these tests,
the liberation of sugars from the pulp and into solution has usually been
measured either by reducing-sugar analysis or by HPLC. However, the identi-
fication of substituted oligomers may limit the accuracy of the analysis.
Therefore, a secondary hydrolysis, either acidic or enzymatic, is required
[-109, 110]. In addition, increase in the liberation of lignin-derived compounds
after the enzymatic treatment [87] or after alkaline extraction [86, 88, 111] has
been used for the evaluation of different enzymes. The most reliable method to
compare the effects of different enzymes is, however, to bleach the enzymatically
treated pulps. In practice, the brightness increase after one- or two-stage perox-
ide delignification has often been used to predict the effects of enzymatic
treatment on pulp bleachability. This method can generally be used for the
prediction of the effects obtainable using enzymes in conjunction with any
bleaching sequences.
4.3 Factors Affecting the Enzymatic Action

In practical process conditions, the various properties of the enzymes, such as
substrate specificity, pH and temperature optima, and mode of action, are of the
utmost importance. Due to the high temperature and alkalinity of the pulp, it
would be desirable to be able to use enzymes at pH 9-10 at 80-90~ Because
most commercial xylanases of the first generation have not met these require-
ments, adjustment of pH to about 5-7 and cooling of the pulp to 50 60 ~ is
necessary.
Hemicellulasesin the Bleachingof ChemicalPulps 275
The degree of hydrolysis necessary for an optimal result can be adjusted by

choosing the reaction time and enzyme dosage. The amount of enzyme needed is
a key parameter with respect to efficiency, enzyme cost and yield loss. The
degree of hydrolysis has to be tested on the laboratory scale with each pulp and
bleaching sequence used. Generally, it seems that although the degree of hy-
drolysis (solubilization of carbohydrates) increases as a function of the enzyme
dosage used, only minor additional benefit for bleachability can be obtained
beyond a certain enzyme dosage (Fig. 4) [112]. Thus, in order to maximize the
positive effect of the enzyme on the pulp kappa number and brightness and
simultaneously to minimize the yield loss, laboratory scale experiments are
needed to optimize the enzyme dosage.
The action of enzymes is also influenced by the electrochemical interactions
between fibers and enzymes [113]. The carboxyl groups within the fiber cell wall
are mainly responsible for the swelling properties of pulp in water [114]. The
surface charge and swelling of fibers have been reported to affect the action of
xylanases [113]. The more negative the surface charge, the less was the pulp
hydrolyzed. However, the swelling and surface charge may not be the primary
factors affecting the hydrolysis. The type of counter-ions and the degree of
substitution of the carboxyl groups in the pulp were found to have a profound
effect on the action of xylanases in the pulp matrix. Consequently, metal-free
pulps were found to be poorly hydrolyzed by hemicellulases [115]. This ob-
served phenomenon of poor hydrolyzability of metal-free pulp is of practical
importance in the TCF bleaching sequences, in which the metal removal stage is
essential to retain the strength properties of the pulp. However, there appear to
be some variations in the specificities of the enzymes, although systematic
studies have not been carried out.
Brightness Hydrolysis
degree
= yield loss
I I
Enzyme dosage
Fig. 4. The correlationbetweenthe enzymaticsolubilizationof pulp xylanand the effectof xylanase
treatment on pulp bleachability
4.4 Effects o f Different H e m i c e l l u l a s e s on P u l p Bleachability
Crude culture filtrates of hemicellulases produced by different microorganisms

were used in the first delignification experiments [1, 2], and even today most of
the reports published are based on results with unpurified enzymes. The main
enzymes needed to enhance the delignification of both hardwood and softwood
kraft pulp have later been shown to be endo-~-xylanases [116 118]. The effect
achieved by xylanases has in most cases been independent of the origin of the
enzyme, and both fungal and bacterial xylanases have been shown to increase
the bleachability [89]. The role of xylanase activity in the delignification of kraft
pine pulp has been extensively studied with purified xylanases of T. reesei
[112, 119]. The purified xylanases of T. reesei have been shown to have different
pI values, pH optima and substrate specificities [120]. Both pI 9 and pI 5.5
xylanases have been observed to prefer substituted substrates, although the pI
9 xylanase has been shown to be relatively more active against non-substituted
xylans than the pI 5.5 xylanase. However, in the limited hydrolysis of xylans of
pine kraft pulp, both of the purified T. reesei xylanases resulted in approxim-
ately the same kappa number reduction and brightness increase in subsequent
chemical delignification [112].
Glucomannan is the main hemicellulose in native softwood, but in softwood
kraft pulp the relative quantities of xylan and glucomannan are almost equal
[13]. The effects of purified or partially purified endo-acting [3-mannanases from
Bacillus subtilis, Aspergillus niger and Trichoderma reesei on pulp delignification
have been compared in bleaching [117]. Treatment of pine pulp with xylanase
enriched with mannanase of B. subtilis resulted in only a slight increase in
delignification compared with xylanase alone. The mannanase of B. subtilis has
later been shown to be able to solubilize wood mannan, but was totally unable
to solubilize mannan which was bound to kraft pulp [121]. Furthermore, the
efficiency of this enzyme has been shown to be considerably lower in the
hydrolysis of both isolated and fiber-bound mannans than that of T. reesei
mannanase [112]. Clark et al. [107] also compared different enzymes in bleach-
ing and reported that the B. subtilis mannanase was able to enhance the
bleachability of softwood kraft pulps. However, the specificity of action of the
enzyme preparation used can be questioned because, along with solubilized
glucomannan, some oligomeric xylan was also liberated from pulps. The man-
nanases of T. harzianum [122] and T. reesei [93, 95, 106] have been shown to be
effective when used before either peroxide delignification or (D/C) E- or DED-
prebleaching, (These symbols are explained in Table 6). The effect of mannanase
is reported to depend on the pulp used. The mannanase of T. reesei has recently
been observed to have a positive effect on bleachability of softwood pulps
produced by modified and continuous kraft cooking methods, especially after
oxygen delignification [93, 95].
Compared with xylanases and mannanases, the side-group cleaving enzymes
alone have had only minor effects on pulp bleachability [117]. Other purified
enzymes which have been studied for improving the bleachability of pulps
include individual cellulolytic enzymes [123]. Of these, only endoglucanase I from

Trichodermareeseiwas shown to increase the bleachability, but this effect was due
to the non-specificity of this enzyme. The enzyme also possesses xylanase activity,
and acts on the pulp matrix as a xylanase rather than as a cellulase [123].
4.5 Action of Enzymes in Pulps Produced by Sulfate

Cooking Methods
The magnitude of the effect of enzymatic treatment on pulp bleachability
depends on the wood species and the type of cooking process used in pulp
production (Fig. 5). In addition, the residual lignin content (expressed as kappa
number) affects the result of enzyme-aided bleaching. The effect of xylanase
treatment on the bleachability of softwood pulps has been reported to be higher
in conventional kraft pulps having kappa numbers of about 25 than in pulps
cooked to low lignin contents when measured after peroxide or chlorine dioxide
bleaching sequences [92, 93, 98]. However, in contrast to xylanase-aided bleach-
ing, the most pronounced effects of mannanase treatment have been observed in
the low-lignin pulps produced by modified cooking methods [93-95].
4.6 Action of Enzymes in Sulfite Pulps

Xylanases and mannanases have also been tested in the bleaching of sulfite pulps
produced by acid Mg, Mg-bisulphite, two-stage sulfite and alkaline methods
[-124, 125]. Due to the different location and limited accessibility of hemi-
cellulose in sulfite pulps as compared to kraft pulps, lower hydrolysis levels
XYLANASETREATMENT MANNANASE TREATMENT
Kappa
Pulp number Akappa ABrightness Akappa ABrightness
2.5 2.0 1.5 1.0 0.5 0.5 1.0 1.5 2.0 2.5 3.0 2.0 1.5 1.0 0.5 0.5 1.0 1.5 2.0 2.5 3,0
i i I I I I I I i i
m I
Conventional 2e.8 I I
EMCC 18.9 I I
Ext. batch 13.1 I I I
Conv.+ 02 20.3 I I
MCC + 02 13.4 I I I
Superbatch + 6.4
02
Fig. 5. Effects of enzymatic treatment on bleachability of softwood kraft pulps produced by

differentcookingmethods and oxygendelignification(02)
278 A. Suurn~ikki et al.
were obtained with xylanase and mannanase [124]. Furthermore, the enzymatic
pretreatment had no effect on the brightness or the kappa number after one-stage
peroxide bleaching. Although two-stage sulfite pulps contain high amounts of
glucomannan, and reprecipitation of glucomannan is thought to take place
[41], mannanase treatment alone did not improve the bleachability [-124].
4. 7 Enzymes in Different Bleaching Sequences

The use of xylanases in different bleaching sequences of kraft pulp consistently
leads to a reduction in chemical consumption. However, the benefits obtained
by enzymes are dependent not only on the type of pulp, i.e. the origin, produc-
tion method used and residual lignin content, but also on the chemical bleaching
sequence used as well as the final target brightness and environmental goals of
the mill. Originally, xylanases were applied in order to reduce the consumption
of chlorine chemicals, especially chlorine gas. Later, enzymes have been com-
bined with various ECF and T C F bleaching sequences to improve the otherwise
lower final brightness value of pulp or to decrease the bleaching costs.
In chlorine bleaching, an average reduction of 25% in active chlorine
consumption in prebleaching or a reduction of about 10-20% in total chlorine
consumption has been reported in both laboratory scale and mill trials
[1, 2, 118]. As a result of the reduced use of bleaching chemicals, the AOX load
of the bleaching effluent in mill trials has been reported to decrease by 15 20%
[118, 126]. The reduction in the use of chlorine chemicals has important
implications not only for the environment but also for economy as a result of
cost savings or increased pulp production capacity. As a result of the replacement
of chlorine gas by chlorine dioxide, the requirement for chlorine dioxide can
exceed the production capacities of chlorine dioxide at the mill site. In order to
avoid additional investments, xylanase pretreatment allows pulp production with
achievement of target brightness with lower chlorine dioxide consumption. Thus,
the productivity of the mill is increased in addition to the environmental benefits.
The production of T C F pulps has increased dramatically during recent
years. Several alternative new bleaching techniques based on various chemicals
such as oxygen, ozone, peroxide and peroxyacids have been developed. In
addition, an oxygen delignification stage has already been installed at many
kraft mills. In the bleaching sequences in which only oxygen-based chemicals are
used, xylanase pretreatment is generally applied after oxygen delignification to
improve the otherwise lower brightness of the pulp or to decrease bleaching
costs [127-130]. The TCF sequences usually also contain a chelating step in
which the amount of interfering metal ions in pulp is decreased. It has been
observed that the order of the metal removal (Q) and enzymatic (X) stages is
important for an optimal result. When aiming at the maximal benefit of
enzymatic treatment in pulp bleaching, the enzyme stage must be carried out
prior to or simultaneously with the chelating stage [115]. The advantages of an
enzymatic step in different bleaching sequences are summarized in Table 3.
Hemicellulases in the Bleaching of Chemical Pulps 279
Table 3. The advantages obtained by xylanase pretreatment in different

bleaching sequences
Bleaching sequence Benefits
Traditional Reduced C12 consumption
Reduced AOX
ECF Reduced C102 consumption
Reduced AOX
Increased productivity, when C102 limiting
TCF Increased brightness
Reduced chemical consumption
Retained strength properties
4.8 Impact of Enzymes on Pulp Yield and Quality

Partial removal of pulp carbohydrates in enzymatic prebleaching is known to
affect the pulp viscosity. Xylanase treatment improves the pulp viscosity by
hydrolyzing pulp xylan having a relatively low D P and thus lowering the overall
viscosity of the pulp [112, 116, 128, 131]. Treatment of pulps by mannanase
free of cellulase activity has not been observed to affect the pulp viscosity
significantly [93, 106]. Cellulase side activities have generally been considered
to be detrimental in pulp treatments if present in enzyme preparations. This is
especially true when both endoglucanase and cellobiohydrolase activities are
present in crude enzyme preparations. Due to the synergistic action of cellu-
olytic enzymes, a rapid depolymerization of cellulose occurs in this case. H o w -
ever, significant differences in the effects of individual cellulases on the strength
properties of pulp have been observed. The endoglucanases have been found to
be most detrimental due to their action on the a m o r p h o u s regions of pulp
cellulose [-123]. The cellobiohydrolases, on the other hand, did not affect pulp
viscosity significantly even when used in relatively high dosages [-132]. A
summary of the use and the effects of enzymes in prebleaching is presented in
Table 4.
Table 4. Effectof carbohydrate-degradingenzymesfrom T. reesei on bleacha-

bility of kraft pulps [123, 1321
Enzyme Effect on bleachability Effect on viscosity
Xylanase + + + +
Mannanasea + _+
Endoglucanase I + + +
Endoglucanase II 0
Cellobiohydrolase I 0 _+
Cellobiohydrolase II 0 _+
"Depending on the bleaching sequence used
5 Industrial Enzyme-Aided Bleaching
During recent years, the main goal in the enzymatic bleaching of kraft
(sulfate) pulps has been to reduce the consumption of chlorine chemicals in the
bleaching process. However, enzymes can also be used successfully for increas-
ing the brightness of pulp, which is of key importance in the development of
TCF bleaching sequences. Addition of an enzymatic step to any conventional
chemical bleaching sequence results in a higher final brightness value of the
pulp. The Finnish forest companies were the first in the world to start mill scale
trials in 1988. Since 1991 this method has been continuously used on the
industrial scale together with other low-chlorine or chlorine-free bleaching
methods.
In 1992, more than ten mills worldwide were reported to use xylanases
continuously for improved bleaching of kraft pulps. Most of the kraft pulp in
Europe is produced in Scandinavia, where most of the mill trials have also been
performed. Different paper products, including magazine papers (SC, LWC) and
tissue papers, manufactured from enzymatically treated pulps, have been suc-
cessfully introduced to the markets. The results of some published mill trials are
presented in Table 5.
5.1 Industrial Enzymes

Commercial bleaching enzyme preparations consist mainly of endoxylanases.
Most of the enzymes are active at acidic or neutral pH, although some of them
function under alkaline conditions. The first mannanase products emerged
on the market in 1995. Commercial hemicellulases for bleaching are listed in
Table 6.
5.2 Installation of the Enzymatic Step

Detailed laboratory work is generally needed to optimize and adapt the enzy-
matic treatment to individual existing mill conditions. Interestingly, however,
enzyme-aided bleaching has been scaled up directly from the laboratory scale to
the large industrial scale (1000 t pulp/d) without intermediate pilot stages. It has
also frequently been observed (unpublished results) that even higher brightness
values can be reached on the full scale than those attainable in the laboratory,
which is due to, e.g., the more efficient mixing systems and higher pulp consisten-
cies. No expensive capital investments have generally been necessary for full
scale runs. The most significant requirement is the addition of pH adjustment
facilities [-126]. The enzyme solution is delivered by a pump before the high
density storage tower. Enzymes are usually mixed with water before being added
O<DO --~O
<~<< <<<
O
H
9
z
O
~'U-,
0~0~
~ ~++~ ..~ o + ~ ~
e<
'~"~ ,~ ~ ~'~ o
..,-,
N N N N ~ N = N ~ o~
0~
)<
~A
l 0 0
282 A. Suurnfikki et al.
Table 6. Commercial hemicellulases for enzyme-aided bleaching
Name Supplier Enzyme type pH-optimum T optimum ~

Cartazyme SR 10 Sandoz XYL 4-5 60-80
Cartazyme PS 10 Sandoz XYL 7 9 60-70
Cartazyme HS 10 Sandoz XYL 4 5 40-60
Ecopulp X-200 Primalco XYL 5-6 50 55
Ecopulp X-200/4 Primalco XYL 3-4.5 45 55
Ecopulp T X - 1 0 0 Primalco XYL 6 8 50-80
Ecopulp T X - 2 0 0 Primalco XYL 6-8 50 80
Ecopulp XM Primalco XYL + MAN 5-6 50 55
Ecopulp X-100 Primalco MAN 5 6 50 55
Ecozyme Zeneca XYL 7 9.5 65
GS 35 Iogen XYL 5.2 7.8 47 58
HS 70 Iogen XYL 5.3-7.5 45 55
Irgazyme 40 x 4 Genencor XYL 6 7 50-60
International
Irgazyme 40 Genencor XYL 6-7 50 60
International
Irgazyme 10A x 4 Genencor XYL 4 5 50-60
International
Optipulp-L 10000 Solvay XYL 6.5 55
Interox
Pulpzyme HC Novo XYL 6 9.5 60
Nordisk
to the b r o w n s t o c k d e c k e r by a s h o w e r bar, in o r d e r to achieve a m o r e even

d i s t r i b u t i o n of the enzyme s o l u t i o n to the pulp. E n z y m e s are a l l o w e d to react in
the high d e n s i t y s t o r a g e t a n k for up to two h o u r s before the s u b s e q u e n t
chemical b l e a c h i n g steps. T h e e n z y m a t i c p r e t r e a t m e n t has been s h o w n to be
easily a p p l i c a b l e with existing i n d u s t r i a l e q u i p m e n t , which is a c o n s i d e r a b l e
a d v a n t a g e o f this technology.
5.3 Environmental Impacts
Effluent l o a d i n g s of b l e a c h plants, e x p r e s s e d as a d s o r b a b l e o r g a n i c h a l o g e n s
(AOX), c h e m i c a l o x y g e n d e m a n d ( C O D ) a n d color, are d e c r e a s e d b y the use of
x y l a n a s e t r e a t m e n t b e c a u s e of the e n h a n c e d r e m o v a l of lignin w h i c h allows
lower a m o u n t s of chlorine chemicals to be used. In sequences e m p l o y i n g 9 0 %
c h l o r i n e d i o x i d e s u b s t i t u t i o n a n d an a l k a l i n e e x t r a c t i o n after the e n z y m a t i c
t r e a t m e n t , A O X was r e d u c e d to 0.6 k g / t o n of p u l p a n d C O D to 40 k g / t o n of
p u l p , as c o m p a r e d with the reference t r e a t m e n t s in which the A O X a n d C O D
l o a d s were 1 a n d 55 k g / t o n , respectively [103, 139].
5.4 Economy of the Enzymatic Treatment

X y l a n a s e s are sold as c o n c e n t r a t e d liquids, a n d the a m o u n t r e q u i r e d p e r
m e t r i c t o n of p u l p is very low, less t h a n o n e litre. T h e cost of the e n z y m e p e r t o n
of pulp varies and depends on the dosage required and the supplier. The
approximate cost in 1995 was around $2 per ton of pulp [-140]. The prices of
enzymes have decreased due to advances in production strains and technologies.
Due to the low enzyme price and low capital costs of the enzyme stage, the
potential economic benefits of enzyme bleaching are significant. A simple
calculation of relative economic benefits in an ECF sequence reveals that the
reduction of approximately 5 kg C 1 0 2 per ton of pulp, assuming a chlorine
dioxide cost of USD 0.70 per kg, leads to savings of about $2 per ton of pulp in
chlorine dioxide costs alone. The costs of oxygen-based chemicals (ozone,
peroxide) are even higher and the corresponding savings even more pronounced.
Additional savings in alkali can also be expected [-103]. Usually the cost of the
enzyme is only slightly less than that of the competing bleaching chemicals
based only on price. However, other factors, such as decreased AOX loadings
and retention of viscosity or other technical pulp properties may lead to
additional advantages of enzymes which are difficult to specify in terms of direct
costs. In the future, all available technologies will compete with respect to
effectiveness as well as price.
6 Conclusions
The use of hemicellulases in the bleaching of chemical pulps has opened up

a new era of large-scale industrial biotechnical applications in the pulp and
paper industry. The improved bleachability is mainly based on the action of
endo-[3-xylanases, a group of enzymes which can be efficiently produced on an
industrial scale. The partial hydrolysis of xylan facilitates the extraction of lignin
from pulp in higher amounts and with higher molecular mass.
The enzymatic pretreatment method is applicable to any traditional or
modern bleaching sequence at existing plants without significant investments.
The primary goals of the enzymatic treatment have been to decrease the
chemical consumption, to reduce environmental loadings, and to increase the
final brightness of the pulp. In the totally chlorine-free (TCF or NCC, non-
chlorine chemical) bleaching sequences, enzymes can improve the brightness,
which may otherwise remain below the acceptable level, without loss of viscos-
ity. Enzyme-aided bleaching is thus both environmentally and economically
advantageous.
Hemicellulase-aided bleaching has been adopted for continuous use world-
wide in several mills and is growing in usage. The fact that the technology has
taken some years to gain acceptance is not surprising. Biotechnology represents
a new type of generic technology for the pulp and paper industry, and requires
broad-mindedness and special interdisciplinary knowledge to be fully exploited.
The properties of the first generation enzymes were not fully optimal to mill
operations with respect to pH and temperature. Furthermore, the indirect
hemicellulase treatment is limited in its benefits compared with oxidizing chem-

icals such as oxygen or ozone. However, remarkable progress in the general
knowledge and attitude towards biotechnical methods has been made, and the
expectations for direct delignifying enzymes are high. The mode of action of
hemicellulases also points to the applicability and usefulness of these enzymes in
combination with new delignifying enzyme systems. The method has clear
environmental benefits and furthermore is aIready economically attractive.
Acknowledgement We thank J. Rouvinen of the University of Joensuu for providing the three-
dimensional structure shown in Fig. 2
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Using Enzymes in Pulp Bleaching:
Mill Applications
J.S. Tolan and M. Guenette

Research Manager and late Research Associate, logen Corporation,
400 Hunt Club Road, Ottawa, Ontario K1V 1C1, Canada
This chapter is dedicated to the memory of the co-author of this manuscript,

Maryse Guenette, who died August 11, 1995.
1 Introduction ............................................. 291

1.1 X y l a n a s e E n z y m e s in B l e a c h i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
1.2 X y l a n a s e A c t i o n o n P u l p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
2 Benefits w i t h X y l a n a s e T r e a t m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
2.1 C 1 0 2 ( C h l o r i n e D i o x i d e ) B l e a c h i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
2.2 T C F B l e a c h i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
2.3 P u l p Q u a l i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
2.4 Effluent Q u a l i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
3 Mill I m p l e m e n t a t i o n o f E n z y m e T r e a t m e n t .......................... 295
3.1 C h o i c e o f E n z y m e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
3.1.1 B l e a c h i n g Benefit u n d e r I d e a l C o n d i t i o n s . . . . . . . . . . . . . . . . . . . . . . 295
3.1.2 C o m p a t i b i l i t y w i t h Mill C o n d i t i o n s . . . . . . . . . . . . . . . . ......... 297
3.1.3 Q u a l i t y C o n t r o l for P u l p D a m a g i n g C o m p o n e n t s . . . . . . . . . . . . . . . . . 298
3.1.4 P u l p Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
3.1.5 E n z y m e S t o r a g e S t a b i l i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
3.1.6 R e g u l a t o r y C o m p l i a n c e ................................ 300
3.1.7 C o s t / B e n e f i t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
3.2 Mill O p e r a t i o n s ........................................ 301
3.2.1 X y l a n C o n t e n t o f the P u l p .............................. 301
3.2.2 I n h i b i t o r s in the P u l p ................................. 302
3.2.3 B l e a c h i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
3.3 A p p l i c a t i o n of E n z y m e to the P u l p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
3.3.1 L o c a t i o n o f E n z y m e T r e a t m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
3.3.2 D i s p e r s i o n o f E n z y m e a n d A c i d i n t o the P u l p . . . . . . . . . . . . . . . . . . . 304
3.3.3 p H A d j u s t m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
3.3.4 T e m p e r a t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
3.3.5 R e a c t i o n T i m e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
4 M a i n t a i n i n g a n d M a x i m i z i n g Benefits o f E n z y m e T r e a t m e n t . . . . . . . . . . . . . . . . 306
4.1 E n s u r i n g Successful E n z y m e T r e a t m e n t .......................... 307
4.2 B l e a c h P l a n t C o n t r o l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
5 F u t u r e D i r e c t i o n s in X y l a n a s e B l e a c h i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
6 Conclusion .............................................. 309
References ................................................ 309
Advances in Biochemical Engineering/

Biotechnology, Vol. 57
9 Springer-Verlag Berlin Heidelberg 1997
290 J.S. Tolan and M. Guenette
Xylanase enzymes have proven to be a cost-effective way for mills to realize a variety of bleaching
benefits, including: reducing or eliminating C12 use, decreasing AOX discharges, freeing up chlorine
dioxide generating capacity, or increasing the bleached brightness ceiling- without expensive capital
investments. These benefits are achieved over the long term when the enzymes are selected and
applied properly in the mill. Chapter 7 described the chemistry of hemicellulase action on pulp. This
chapter describes the industrial benefits of xylanase enzyme treatment and the issues that must be
considered to get the most benefit from using xylanase treatment in a mill.
Using Enzymesin Pulp Bleaching 291
1 Introduction
1.1 Xylanase Enzymes in Bleaching

The use of xylanase enzymes to enhance the bleaching of Kraft pulp was first
discovered in 1986 [-19] and has advanced to the point of ongoing mill usage
today. Xylanase enzymes are now being produced for the pulp industry by
several companies around the world. Many mills worldwide have experience in
using enzymatic bleaching enhancement, and a CPPA survey showed that as of
early 1995, about 1 million tonnes per year, or 10% of the bleached Kraft pulp
made in Canada, is enzyme-treated [17].
1.2 Xylanase Action on Pulp
Chapter 7 provides an extensive discussion of the properties of the enzymes used

in pulp bleaching, and only a few points will be highlighted here. Xylanase
enzymes partially hydrolyse the hemicellulose portion of pulp. Unlike the conven-
tional bleaching chemicals, these enzymes do not brighten or delignify the pulp.
Rather, these enzymes act on the pulp to make the subsequent bleaching by the
oxidative chemicals more efficient. The precise mechanism of this xylanase action
on the pulp is not known. There is evidence that xylanase allows larger molecular
weight lignin to be removed from the pulp in the alkali extraction stages, so it is
postulated that removal of a portion of the xylan either releases xylan-bound
lignin or increases the ability of high molecular weight lignin to diffuse through
the surface of the fibre. In either case, removing a portion of the xylan from the
pulp increases the efficiency of the oxidative chemicals.
The xylanase enzymes used commercially are made in several-day batches of
submerged liquid culture fermentation by Bacillus bacteria, Trichoderma fungi,
or other microbes. The enzymes are essentially free of living cells. Xylanase
enzymes have been used previously in the food industry (to improve the yield of
starch in corn and wheat processing) and the animal feed industry (to improve
the digestibility of wheat to chickens and pigs). The xylanase enzyme formula-
tions for pulp bleaching contain little of the activity for degrading the cellulose
and lignin fractions of the pulp (i.e. cellulase and ligninase).
2 Benefits with Xylanase Treatment
2.1 ClO 2 (Chlorine Dioxide) Bleaching
Historically, pulp has been bleached with chlorine and chlorine dioxide. Much
of the motivation to install xylanase treatment systems has been from the
pressure on the mills to decrease the use of these chlorine-based chemicals for
environmental, regulatory, and market reasons. F o r this reason, a large amount
of work has been carried out on the use ofxylanase enzymes to decrease chlorine
and chlorine dioxide usage. This discussion focuses on chlorine dioxide which is
preferred over chlorine in modern mills, although the effects with chlorine are
very similar. In either case, xylanase is usually added onto brownstock at the
final washer prior to the bleach plant (see Fig. 1).
Figure 2 shows data that is typical of that generated in laboratory studies of
enzyme treatment with chlorine dioxide bleaching. In this case, softwood brow-
nstock was treated with xylanase and then bleached in five separate bleaching
Fig. 1. Xylanase is added to brownstock

prior to the bleach plant and acts on the
pulp in the brownstock storage tower
Enzyme Benefit in Bleaching

Softwood Kappa Number 27.3
92
J
f =
c/l
U)
f
J
84
28 30 32 34 36 38 40
Total Chlorine Dioxide (Kg/t)
9 Untreated 9 Enzyme
Fig. 2. Xylanase treatment increases the brightness of the softwood pulp by 1.5 points after ClOg
bleaching, or decreases the C10 2 usage to reach 90 Brightness by 14.5%
Using Enzymes in Pulp Bleaching 293
stages with C102 and no chlorine. F o r a given usage of chlorine dioxide, the
enzyme treated pulp is brighter than the untreated pulp. In Fig. 2, the enzyme
treated pulp is bleached to 91 Brightness at a C102 usage that bleaches the
untreated pulp to 89.5 Brightness. Alternatively, to reach a given target bright-
ness with enzyme treated pulp requires less C102 than the untreated pulp. In
Fig. 2, enzyme treated pulp requires 32,5 kg/t of C102 to reach 90 Brightness,
while the untreated pulp requires 38 kg/t. Enzyme treatment saves 5.5 kg/t of
C102, or 14.5% of the total across the bleach plant.
Figure 3 illustrates various advantages that can be obtained by using
xylanase treatment in a mill bleaching with C102. Figure 3 shows the total C102
required across a five-stage bleach plant to reach 90 Brightness as a function of
the K a p p a factor (which is a measure of the a m o u n t of C102 used) of the first
stage. In the absence of xylanase treatment, the o p t i m u m K a p p a factor to bleach
this pulp (softwood, K a p p a number 27.3) to 90 Brightness is 0.24. This is
operating Point A indicated on the figure. Xylanase treatment can be used to
accomplish several objectives, the choice of which depends on the needs of
a given mill. These objectives are represented by operating points on the figure,
as follows:
1) Debottleneck C102 generator capacity. With xylanase treatment, the lowest
C102 usage is indicated by Point B, a K a p p a factor in this case of 0.22. This
corresponds to the optimum from the point of view of bleaching costs and of
Enzyme Benefit in D,= Bleaching

Softwood Kappa Number 27.3
70
60
O~
o2
tO 5O
\
i r
40 D~
30 E (
0,18 0.2 0.22 0.24 0.26 0.28 0.3 0.32
Kappa Factor
9 Untreated 9 Enzyme
Fig. 3. Enzyme treatment can be used to improve the operation of the bleach plant in several ways,
as compared to that without enzyme treatment (Point A). Enzyme treatment can be used to obtain
the lowest CIO2 usage (Point B) or the lowest AOX or TOX (Point D) or to decrease the back-end
C10z (Point C) or to increase the brightness ceiling (Point D or Point A, with enzyme treatment)
debottlenecking the C102 generator capacity. It is interesting to note that the

optima both with and without enzyme treatment occur with 65% to 70% of
the C102 in the first stage and the remainder in the later stages.
2) Decrease AOX discharges and TOX content. Although the organochlorine
level in the effluent (AOX) will decrease with decreases in the total C102
usage, the maximum decrease in AOX and TOX (organochlorine level in
the pulp) is obtained by decreasing the Kappa factor as much as possible
(Point D).
3) Decrease later stages C102 usage. Sometimes control of the later stages of the
bleach plant is easier than the first stage. Point C indicates the corresponding
operation with xylanase treatment at the same Kappa factor, 0.24, as for the
untreated pulp. The total C102 savings in this case is significant but not as
great as at the optimum Kappa factor of 0.22.
4) Increase the brightness ceiling. This is achieved by using the same amount of
chemicals with xylanase treatment as without (Point D or Point A with
enzyme treatment).
2.2 TCF Bleaching

There has been increasing interest in the use of enzyme treatment to enhance
T C F bleaching. Most of this effort has focused on enzyme treatment of oxygen
delignified pulp, followed by multiple stages of hydrogen peroxide. Moncho AB
in Aspa Bruk, Sweden and Metsa Bothnia of Finland have reported mill
operation in such sequences with 1-2 point brightness gains by enzyme treat-
ment [3, 8]. There is a less comprehensive sense of the impact of enzyme
treatment in these systems than in chlorine compound bleaching. Nonetheless,
the mill experience confirms lab data which shows that increases in the bright-
ness ceiling or savings of 5 to 10 kg/t of peroxide can be obtained by enzyme
treatment. In these cases, enzyme treatment can be (and has been) run
simultaneously with the additional chelation of the pulp necessary in T C F
bleaching. In fact, the neutral pH of enzyme treatment is optimum in many cases
for chelation of the magnesium, iron, and manganese ions that must be removed
before bleaching with hydrogen peroxide.
2.3 Pulp Quality

In the 1995 CPPA survey, most mills reported no change in pulp strength with
enzyme treatment, confirming laboratory findings, Some mills, however, noted
an increased unbeaten tear strength on enzyme treated pulp, which is consistent
with the notion that hemicellulose removal would decrease interfiber bonding
El7].
Senior et al. [ l l ] reported that enzyme treatment decreases the total
organochlorine (TOX) of the pulp, but this was not observed by [10] at a mill.
The source of the conflicting results might be the difficulties in measuring low
levels of TOX and the effects of brightness target on TOX [4]. The rationale that
xylanase could decrease TOX is that (1) xylanase decreases chemical usage, and
(2) chlorinated lignin can bind to xylan.
2.4 Effluent Quality
In terms of enzyme effects on mill effluent, the decrease of AOX by enzyme

treatment is the best known and the most widely reported in the CPPA survey.
Enzyme treatment appears to decrease the AOX in proportion to the decrease in
chlorine compound usage, i.e. a 15% decrease in C102 will decrease the bleach
plant AOX discharge by 15% [2]. This data is supported by mill experience.
Because the bleach plant effluent colour is dependent on the chlorine usage,
a decrease in chlorine compounds also decreases the effluent colour [2]. There is
an increase in the bleach plant effluent B O D of 2 to 5 kg/t resulting from the
release of low molecular weight xylan from the pulp. This BOD is of an easily
degraded nature in secondary treatment systems, and no change in the treated
effluent BOD has been observed with enzyme treatment.
3 Mill Implementation of Enzyme Treatment
This section describes the areas that should be considered for long term enzyme
usage in a mill. An understanding of the factors which influence the following is
essential: the choice of enzyme, mill operations, and reaction of the enzyme with
the pulp.
3.1 Choice of Enzyme
In choosing an enzyme, one should consider the bleaching benefit delivered

under ideal conditions, the compatibility of the enzyme treatment with mill
conditions, the quality control for pulp-damaging components, the storage
stability, regulatory compliance, and the cost/benefit.
3.1.1 Bleaching Benefit under Ideal Conditions
The first two points that a mill usually needs to address regarding enzyme
treatment are whether xylanase treatment can meet the mill's objectives, and the
comparative performance of the various commercial xylanases. These questions
are related and can be best approached as a first step by carrying out a
laboratory study under ideal conditions. A laboratory study is preferred over

a mill trial at this point because of the superior control of the conditions and the
pulp and the ability to make more rapid evaluations. If the laboratory study is
carried out with proper technique (which is not trivial), the results predict mill
performance well.
Ideal conditions are usually used in an initial study because they place an
upper limit on the benefit of enzyme treatment, and establish the goals to be met
in ongoing mill-scale treatment. "Ideal" conditions are usually chosen as the
o p t i m u m p H and temperature for enzyme treatment, and with a treatment time
of two hours or more. The pulp and bleaching sequence are chosen as "typical"
mill pulp in terms of K a p p a number, chemical usage, brightness targets, and
other measures; sometimes a representative range of pulps and bleaching se-
quences should be tested. The enzyme dosage should also be in the expected
operating range.
All of the commercial enzymes contain the same generic xylanase activity,
but beyond this they are quite different. In addition, the bleaching benefit varies
a m o n g enzymes due to the differences in the enzyme structure and the mecha-
nism of the xylanase action on pulp. These points are illustrated in Fig. 4 for two
enzymes with similar xylanase acitivity but different bleaching benefit. This
demonstrates that laboratory evaluation of bleaching benefit on pulp is not
necessarily related to the enzyme activity against xylan substrate.
Effect of Enzyme Dosage

9o
89.5
89
r
t" 88.5
0l
't"
m 88
Y
87.5
0 0.2 0.4 0.6 0.8 12 14

Enzyme Dosage (litres/t pulp)
9 Enzyme 1 9 Enzyme 2
Fig. 4. The benefit of enzymetreatment is different for different xylanase enzymesdue to differences
in the type of action of the enzyme. In this example, two enzymes with the same xylanase activity
(3000 units/ml) used at optimum conditions have different bleaching enhancement of the pulp.
Enzyme 1 is from Bacillus bacteria and Enzyme 2 is from Trichoderma fungus
3.1.2 Compatibility with Mill Conditions
The range and variability of the enzyme treatment conditions during actual mill
operation must be taken into account to obtain benefits in ongoing enzyme
treatment. Like all bleaching chemicals, the effectiveness of xylanase depends on
the pH, temperature, and length of time for the chemical to act. Section 3.3
describes in detail how to maintain the conditions in the brownstock storage
tower in a mill. This section describes the differences in operating conditions
among enzymes.
The optimum pH for enzyme treatment varies, depending on the enzyme,
from about pH 3 to about pH 9. The optimum pH is important because it
influences the amount of corrosion, pitch deposition, and other operating
problems for the mill. The breadth of pH range also varies among enzymes from
about 0.5 to 2.0 units, and this determines the degree of difficulty in controlling
the pH. It is important to note that, for a given enzyme, the optimum pH varies
among pulps by up to 2 pH units [12] (see Fig. 5). The optimum pH must be
determined for each enzyme on each pulp.
The desired temperature for enzyme treatment varies from about 30 to about
60 ~ depending on the enzyme. The compatibility of these temperatures with
the C-stage temperature, the cooling of the brown system, etc. must be ad-
dressed. The effective temperature range spans 5 to 10 ~ above and below the
optimum temperature. The minimum reaction time required for enzyme treat-
ment varies from about 10 minutes to about 2 hours depending on the enzyme
(see Fig. 6). The compatibility of these brownstock storage times with the
brownstock level and swings in grades must be addressed.
Fig. 5. For a given enzyme,the optimum pH varies from pulp to pulp. From Tolan (1992a)
Time Course of Xylanose Treatment

84
.....----I
83
82 f
f
8O
70J,
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Time (hrs)
9 Enzyme 2 9 Enzyme I
Fig. 6. The treatment time required to enhance the bleachingvaries among enzymes.In this case,
Enzyme 1 requires l hour while Enzyme2 requires 10-20 minutes
3.1.3 Quality Controlfor Pulp Damaging Components
The majority of mills responding to the 1995 CPPA survey indicated that the
bleaching benefits of xylanase treatment are obtained with no compromise in
pulp quality or properties [17]. This confirmed laboratory work and included
essentially no change in the beating curves while maintaining tear, tensile, and
burst strength, bulk, opacity, fibre length, and other more specialized properties.
However, some mills observed problems with pulp quality, including loss of tear
strength (2 mills) and pitch deposition (2 mills). These are not caused by the
xylanase, but rather by impurities in the enzyme. The quality control procedures
used in the enzyme manufacturing are an essential element in preventing these
problems.
The presence of cellulase enzyme impurities can cause degradation of pulp
strength. The levels of cellulase that can decrease pulp strength have been
characterized by [16]. Pulp strength loss results from two types of cellulase
activity, carboxymethylcellulase (CMCase) and Filter Paper activity (FPA) (see
Fig. 7). The levels of CMCase and FPA that can damage pulp are much lower
than can be detected in the standard assays and can vary from batch to batch in
xylanase production. Therefore, the assurance of pulp quality requires setting
a specification for the maximum allowable cellulase activity (with the appropri-
ate margin for safety) and modification of the standard cellulase assays to ensure
detection of the relevant levels of cellulase activity.
10000
A
x 1000
E
,~ lOO
oz.
r
9~ lO
0.001 0.01 0.t 1 10 t00 1000
FPA (lU/kg)
Fig. 7. Combinations of Filter Paper Cellulase and CMCase that weaken pulp. The lines indicate
the treated pulp strength relative to the untreated. From Tolan (1995)
The action of xylanase itself does not cause pitch to form or deposit on pulp.
However, in the C P P A survey, two mills reported pitch deposition associated
with non-protein matter in the xylanase enzymes [17]. This non-protein matter
would most likely be an organic stabilizer added to the enzyme or impurities
from the fermentation broth. The likelihood of pitch deposition increases if
a batch of enzyme is of very different colour or odour than previous batches or if
there is a large amount of sedimentation.
3.1.4 Pulp Yield
L a b o r a t o r y data suggests that enzyme treatment decreases the pulp yield across
the bleach plant. The yield loss varies a m o n g enzymes and depends on the
dosage of enzyme and the xylan content of the pulp. See Fig. 8.
The putative yield losses must be viewed with caution for several reasons
[14]. First of all, they are too small to be observed in a mill and are near the limit
of detection of the lab procedures. Second, there are significant differences
between laboratory tests and mill operation. There is no loss of fines in the lab
but there is in a mill; if the enzyme merely solubilizes fines, there would be no
resulting loss of yield in a mill. In addition, the manner of drying pulp in the lab
of using a 105 ~ oven overnight might misrepresent the water holding capacity
of pulp dried on a pulp machine. Research is under way to understand further
the enzyme's effects on yield.
Yield Lossby Two Enzymes

5
C 3
0 0.2 0.4 0.6 0.8

Yield Loss (%)
9 Enzyme 2 9 Enzyme 1
Fig. 8. There is a small yield loss associated with xylanase treatment. The yield loss depends on the
enzyme dosage and the enzyme used.
3. t.5 Enzyme Storage Stability
Typically, one to two weeks of enzyme inventory is maintained on site. The

storage must be within the temperature range to maintain enzyme activity while
limiting microbial contamination. This temperature varies a m o n g enzymes. In
some cases, refrigerated storage will be required.
3.1.6 Regulatory Compliance
The regulatory status of xylanases depends on the microbial origin of the

enzyme; some xylanases are from microbes that are F D A approved, in the
United States, for example, while others are not. The regulations also vary from
one agency to another, such as F D A versus its C a n a d i a n counterpart, H P B .
There is also variability in I S O compliance and other certifications.
3.1.7 Cost~Benefit
As with any other specialty chemical, the mill should evaluate the cost in terms
of price and value.
3.2 Mill Operations
Mill operations exert an important influence on the benefits xylanase enzymes

can provide. Three aspects of a mill's operations which influence the benefits
attainable with enzyme treatment are: 1) the xylan content, which depends on
the chip furnish and the digester operation, 2) inhibitors in the pulp, which
depends on the brownstock washing, and 3) the subsequent bleaching of the
pulp, as characterized by the bleaching sequence. The effects of each in the
context of xylanase treatment are discussed below.
3.2.1 Xylan Content of the Pulp
The higher the xylan content of the pulp, the greater the bleaching benefit by
xylanase. The xylan content of the pulp depends on the xylan content of the chip
furnish and the pulping operation.
Among chip furnishes, the important distinction is between hardwood and
softwood; hardwood contains more xylan than softwood. The percentage of the
bleaching chemicals saved by xylanase treatment is thus greater on hardwood
than on softwood; at good treatment conditions, the decrease in chlorine
chemicals is about 20% on hardwood and 15% on softwood.
The variation in xylan content among hardwood and softwood species used
in Kraft pulping is not significant. Most of the mill experience has been with
softwood because of its higher chemical requirements for bleaching.
The digester operation affects the xylan content of the pulp significantly [9].
For example, sulfite pulping destroys most of the xylan and thus sulfite pulp is
not suitable for enhanced bleaching by enzyme treatment.
In conventional Kraft pulping, the xylan content depends strongly on the
effective alkalinity. The lower the alkalinity, the higher the xylan content and the
benefit of using xylanase enzymes. At high (19-22%) alkalinity much of the
xylan is solubilized, which decreases the benefit of xylanase treatment. This is
often the case with pulp cooked to below the target Kappa number. At low (less
than about 18%) alkalinity, the xylan structure is more stable, and the bleaching
enhancement by xylanase is greater by up to 2- to 3-fold over the high alkalinity
pulp. This is often the case for pulp cooked to a higher Kappa number.
The relation between alkalinity, xylan content, and bleaching enhancement
is applicable for hardwood and softwood over a range of Kappa numbers and
for MCC (modified continuous cooking) and oxygen delignified pulps. This
relationship is understood qualitatively and allows one to assess roughly the
benefit of xylanase treatment based on the pulping operation. For example,
softwood at Kappa number 30 has a fairly high xylan content if it is cooked with
15% alkalinity. Such a pulp will respond well to xylanase treatment. On the
other hand, the pulp will have a lower xylan content and xylanase response if it
is cooked to a 20 Kappa number by using higher alkalinity. In this case, there
will be an increasing enzyme benefit with increasing Kappa number, which can
120
I Untreate
110 / ~1
----
9 ~11~09 nzyme
9 Untreated i
~'m-1008090 /~.~lel~ ~~:~ )9149149
~"
I--Om 9 49r~iud
i'i~~ 9 Enzyme Treated
70
20 22 24 26 28 30
Kp#
Fig. 9. Mill data showing increasing enzyme benefit with increasing Kappa number. This is
consistent with a higher xylan content at higher Kappa number.
act to make the bleach plant chemical usage more consistent. Figure 9 shows
mill data demonstrating this point by illustrating the total active chlorine (TaC1)
as a function of the Kappa number for enzyme treated and untreated pulp.
By the same rationale, the pulp cooked to Kappa number 20 might have
a higher xylan content if it is MCC than if it is a conventional cook, because the
alkalinity is lower for an MCC pulp cooked to a given Kappa number. Thus, the
MCC pulp at Kappa number 20 will respond better to enzyme treatment than
the conventionally cooked pulp at Kappa number 20 (and maybe even that at
Kappa number 30). The ability to maintain low alkalinity is the reason that
xylanase benefits have been achieved in mills with conventional, MCC, and
oxygen delignification systems.
Mill experience has confirmed laboratory data that indicates that an-
thraquinone (AQ) itself has little effect on xylan content. The use of AQ affects
xylan content only in that it can change mill pulping practice. For example, if
a mill using AQ decreases its alkalinity (to increase yield), it will increase its
xylan content and benefit due to xylanase. If the mill increases alkalinity (to
decrease Kappa number), it will decrease xylan content and enzyme benefit.
3.2.2 Inhibitors in the Pulp
Mill experience has shown that the day-to-day variation in the extent of
brownstock washing has little impact on enzyme performance. This confirms
laboratory data, and we conclude that inhibitors are not routinely present in
pulp. However, poor washing (greater than 25 kg/t of soda carry over) can
decrease the maximum enzyme treatment temperature by 5 ~ which is impor-
tant in mills that want to run as hot as possible [6]. These results are very
enzyme dependent, and probably vary among mills [13].
3.2.3 Bleaching
The bleaching sequence and brightness target influences the enzyme's benefit to
the mill. As a basis of comparison, about 15% of the C102 is saved by using
enzyme treatment on softwood with C102 bleaching to 90 Brightness, with the
chemical usages optimized both with and without enzyme treatment. The
enzyme benefit is greater at higher brightness targets, especially near the bright-
ness ceiling, and lower at lower brightness targets (see Fig. 2, brightness targets
of 91 and 88 respectively). In shorter sequences, the same effects are observed,
but at lower brightness targets, as the brightness ceilings are lower. For example,
in a three-stage sequence, the enzyme benefit can be greater than 15% chemical
savings at 88 Brightness, and the benefit is decreased at, say, 83 Brightness.
The other aspect of the bleaching sequence that can effect the enzyme benefit
is the use of hydrogen peroxide. Usages of 6 to 9 kg/t of peroxide in the
extraction stages with enzyme treatment results in about 80% of the total C102
savings that would result if the enzyme and peroxide benefits are added separ-
ately. The reason for the overlap in enzyme and peroxide benefits is not known,
but might relate to either an increased brightness ceiling in high peroxide
sequences, or the possibility that peroxide removes a portion of the hemicel-
lulose from the pulp. At low peroxide usages (less than 3 kg/t), the enzyme and
peroxide benefits are additive.
3.3 Application o f Enzyme to the Pulp
As with any bleaching agent, the effectiveness of enzyme treatment depends on

maintaining the appropriate pH, temperature, and reaction time. Where
xylanase differs from the other chemicals is that these properties vary between
enzymes and that the conditions must be controlled on the brownstock storage
tower, which was not originally designed for enzyme treatment. Several factors
must be taken into account to ensure that the enzyme is applied to the pulp
properly without causing any long term problems for the mill.
3.3.1 Location of Enzyme Treatment
Enzyme is added to the pulp prior to the bleach plant (see Fig. 1). The majority
of enzyme installations have been with enzyme added to the brownstock before
the high-density storage tower. The enzyme then acts on the pulp in the storage
tower and the treated pulp is ready for bleaching in the chlorination stage. The
brownstock storage tower often has a retention time of at least 30 minutes, and
the enzyme effects after treatment of unbleached brownstock are well character-
ized.
There has been little application of enzyme treatment upstream of the final
brownstock washer. In this case, the acid usage would be higher than down-
stream. Further, the ability to wash after enzyme treatment is not advantageous
because the enzyme does not release a significant amount of lignin from the
pulp.
In mills with oxygen delignification, enzyme treatment is after oxygen to
avoid cooling and neutralizing the pulp around the oxygen stage and upsetting
the mill's sulfur balance. In lab studies, the savings in chemicals by enzyme
treatment are no greater before oxygen delignification than after [20].
3.3.2 Dispersion of Enzyme and Acid into the Pulp
The adequate dispersion of enzyme and acid into the pulp is important to
enzyme performance. Some of the hardware configurations that have been used
include: a shower bar for acid, then one for enzyme on the back of the decker
[18]; separate hoses for acid and enzyme into the repulper; injection of the
additives into the chute above the stock pump; or, injection of enzyme at the
suction side of an MC (medium consistency) pump. The degree of mixing that is
achieved by any of these equipment configurations depends on the absorbency
of the brownstock as well as the equipment used. Therefore, it is difficult to state
flatly that any one set-up is best for a given mill. A mixing test can and should be
carried out on any given system to evaluate its degree of mixing and enzyme
performance [12].
That having been said, medium consistency pumps usually provide adequate
mixing of the enzyme into the pulp. The results with thick stock pumps are
highly variable. Thick stock pump systems can, however, be configured to
approach the mixing performance of a medium-consistency pump.
3.3.3 pH Adjustment
As stated in Sect. 3.1.2, the pH optimum and operating range for enzyme
treatment varies among enzymes but generally falls between pH 3 to 9. This
typically requires acidification of the brownstock, because the stock, though
washed, is alkaline. The stock is typically 10% consistency, so the pH control
requires some care. With the appropriate pH control system, the pH is main-
tained within 0.2 units, which is adequate for the purposes of enzyme
treatment.
Sulfuric acid has been used for pH adjustment of the brownstock at almost
all of the enzyme installations, with some mills using SO2, hydrochloric, and
C-stage acid filtrates [17]. In any case, one must evaluate the corrosive tenden-
cies on the equipment, which has been a problem at some mills [5].
Typically 2 6 kg/t of acid is used, so an inexpensive source of acid is
preferred.
Prior to acidifying the brownstock on a long term basis, one should charac-
terize the soda carry-over and whether any tendency to form HzS exists. In no
case should the pulp be acidified if HzS is expected in a routine operation.
Fortunately, the quality of the washing is usually adequate to eliminate the
sulfides that can lead to HzS emission.
The issue of whether acidification of the brownstock changes the amount of
chemical required to bleach the pulp must be taken into account in assessing the
benefit of enzyme treatment. In chlorine bleaching, sufficient acid is present in
the chemical and pulp filtrate such that the additional acid with xylanase
treatment has no effect on the bleaching chemical usage. Chlorine dioxide is less
acidic than chlorine, so the issue of the effect of the acid alone on chemical
usage must be looked at more closely when bleaching with high C102 substitu-
tion.
The efficiency of 100% C102 bleaching is at an optimum below pH 4 [7].
Therefore, if the vat pH on the chlorination tower is at or below pH 4, the
additional acid with enzyme treatment will have no effect on the efficiency of the
first stage. In practice, pH 3.5 is used to offer a better margin for error. Many
mills already have an existing pH control at the bottom of the chlorination stage
to maintain this pH. If not, the combinations of C102 usage and soda carry over
that result in a pH greater than 4 are calculated from the acidity of the C102
(which varies among generators) and the alkalinity of the pulp. For example, the
vat pH is typically below pH 4 if a mill is using more than about 15 kg/t C102
(from an R8 generator) in the first stage and the soda carry-over is 10 kg/t (this is
Kappa number 22, Kappa factor 0.18, average washing).
If the vat pH is greater than 4, then the acidification for enzyme treatment is
likely to be beneficial for the efficiency of the C102 bleaching. This will occur
with poor washing or low C102 use in the first stage. In this case, the mill should
evaluate the effect of the acidification before running the xylanase trial.
3.3.4 Temperature
As stated above, the temperature optimum and operating range for enzyme
treatment varies among enzymes but is between 30 and 60 ~ Running at cooler
temperatures results in similar effects, but over longer treatment times. For
a given enzyme, the maximum operating temperature varies among mills, due
mostly to differences in the extent of brownstock washing [6, 12]. The temper-
ature is typically controlled by the use of cold or hot shower water on the washer
preceding enzyme treatment.
Brownstock Retention Time

100
80
v
60
E
F 40
c
0
9 9
C 20
r
n, 0
0 20 40 60 80 100
Tower Level (%)
Fig. 10. The brownstock retention time can vary greatly, even when the level is maintained. Data
from tracer studies at a softwood mill
3.3.5 Reaction Time
Depending on the enzyme used, a minimum of 0.2 to 2 hours of residence time is

required for the enzyme treatment [15] (see Fig. 6). There is little enzyme action
on the pulp beyond 4 to 6 hours. Note that channelling is common in brown-
stock storage towers, so the actual retention time does not always correspond
to the nominal plug flow time [1]. The results of brownstock retention
time measurements in a mill are shown in Fig. 10. In this case, the retention
time for a given tower level varies significantly. This demonstrates the desir-
ability of carefully monitoring the retention time in the brownstock storage
tower.
4 Maintaining and Maximizing Benefits of Enzyme Treatment
Enzyme treatment trials of a few days duration are readily carried out in a mill
because of the low capital cost and safety associated with enzymes. During such
trials, the high level of staffing and mill interest often lead to successful results.
However, it is a greater challenge to maintain the benefits of enzyme treatment
during ongoing operation, where far less attention is paid to the enzyme
treatment. This section addresses the issues that are important in ongoing
enzyme treatment.
Fig. ll. Data of monthly chemicalusage with and without enzymetreatment. The fluctuations in
chemical usage make the evaluation of enzymebenefit difficult
The benefits of xylanase treatment are often difficult to observe or quantify

because of the subtle nature of the enzyme's action: it neither brightens nor
delignifies the pulp. Furthermore, the chemical savings by xylanase treatment is
roughly equivalent to the monthly fluctuation in bleach plant chemical usage
(see Fig. 11).
Maintaining the benefits of enzyme treatment requires (1) maintaining "suc-
cessful" enzyme treatment, that is, effective xylanase action on the pulp, and (2)
obtaining tangible benefit in the bleach plant.
4.1 Ensuring Successful Enzyme Treatment

As with any bleaching chemical, ensuring successful treatment requires an active
chemical, maintaining the treatment conditions, and measuring effectiveness of
treatment.
The verification of xylanase activity in the enzyme tote is carried out with
on-site test kits using a xylan substrate.
The treatment conditions and other aspects of the application of enzyme to
the pulp are described in the previous sections. It is important that these
are maintained in the appropriate ranges. This includes the monitoring of
pH, temperature, retention time, enzyme flow, acid flow, stock flow, and water
flow.
The effectiveness of xylanase treatment on pulp can be assessed on samples
taken before or after brownstock storage.
90
88
(0')
(Y)
o)
E
9 Untreated i
86
'E
9 9 T~ n o ~ - 9 Enzyme Treated
m
n
121
84
82
0.25 0.3 0.35 0.4 0.45 0.5
TKf (up to D1)
Fig. 12. The D1 brightness increasing about 1 point due to enzyme treatment. If the set point on the
controller is not changed, the a m o u n t of chemical saved will be low
4.2 Bleach Plant Control
Once successful enzyme treatment is obtained, the bleach plant must be run in
such a manner as to achieve the benefits desired. This often requires careful
attention to the control of the bleach plant, because the action of xylanase does
not register on the chlorination stage brightness sensors. The bleach plant
control strategy to use with enzyme treatment therefore depends on the mill's
objectives with enzyme use. As shown in Fig. 3, in some cases a mill will use the
same amount of bleaching chemicals with enzymes as without, and simply
obtain a higher brightness. In other cases, a mill will desire to decrease the
Kappa factor and/or the later stage chemical charges. The appropriate adjust-
ments to the bleach plant sensors' set points are required in these cases.
Figure 12 shows the D1 brightness increasing with enzyme treatment.
Without adjusting the D1 set point, the mill obtains little chemical savings.
It is also important to maintain pulp quality while using xylanase to decrease
the quantity of bleaching chemicals. In the vast majority of mill operations, the
decreased usage of chlorine-based chemicals has been achieved without an
increase in the shives, pitch, or dirt counts [16]. This is a key operating point in
many mills, in which the bleaching strategy is limited by these factors as much as
or more than brightness. In some cases the pulp quality has been maintained by
decreasing the chlorination temperature by 10~ or so when the chemical
addition is decreased, in order to maintain the slowly-acting residual that i s
essential to bleaching shives.
5 Future Directions in Xylanase Bleaching
T h e d e v e l o p m e n t in x y l a n a s e b l e a c h i n g is focusing on i m p r o v e d e n z y m e p r o p e r -
ties a n d i m p r o v e d e n z y m e p e r f o r m a n c e . I m p r o v e d p r o p e r t i e s includes h i g h e r
p H a n d t e m p e r a t u r e t o l e r a n c e of the enzymes, to m a k e the e n z y m e t r e a t m e n t
o p e r a t i o n s m o r e c o m p a t i b l e with existing mill o p e r a t i o n s . I m p r o v e d e n z y m e
p e r f o r m a n c e is being a p p r o a c h e d by t a i l o r i n g the e n z y m e a c t i o n m o r e closely to
the hemicellulose structure of the pulp, to result in a g r e a t e r b l e a c h i n g benefit o r
a higher p u l p yield.
6 Conclusion
X y l a n a s e enzymes have p r o v e n to be a cost-effective w a y for mills to realize

a variety of b l e a c h i n g benefits, including: r e d u c i n g of e l i m i n a t i n g c h l o r i n e use,
decreasing A O X discharges, freeing u p c h l o r i n e d i o x i d e g e n e r a t i n g c a p a c i t y , o r
increasing the brightness ceiling- w i t h o u t expensive c a p i t a l investments. These
benefits are achieved over the l o n g t e r m when the e n z y m e s are selected a n d
a p p l i e d p r o p e r l y in the mill.
7 References
1. Bodenheimer VB (1969) In: Southern Pulp and Paper Manufacturer, Sep: 42-45
2. Elm DD, Choma P, Strunk WG, Klein RJ, Sundaram VSM (1993) In: Proc 79th Annual Meeting
CPPA, Jan: 25-29
3. Fasten H (1993) In: Proc Non-Chlorine Bleaching Conf, Hilton Head, South Carolina, Mar:
1~17
4. Histed JA, Vega Canovas R, Ruscitti G (1991) In: Proc TAPPI Pulping Conf, Orlando, Florida
Nov: 2-6
5. Hitzroth A (1992) In: Proc CPPA Spring Conference, Jasper, Alberta
6. Luers M (1992) In: Proc CPPA Spring Conference, Jasper, Alberta
7. Reeve DW, Weishar KM (1991) In: TAPPI 74(6): 164
8. Reilama I, Kukkonen K (1993) In: Proc Non-Chlorine Bleaching Conf, Hilton Head, South
Carolina, Mar: 12-17
9. Rydholm SA (1965) Pulping Processes, Wiley, New York, Robert E. Krieger Publ Co, Malabar,
Florida
10. Scott B, Young F, Paice M (1992) In: Proc CPPA Spring Conference, Jasper, Alberta
11. Senior DJ, Hamilton J (1992) In: J Pulp and Paper Sci 18(5): J165-J169
12. Tolan JS (1992a) In: Proceedings 78th CPPA Annual Mtg, Montreal, Quebec, Jan
13. Tolan JS (1992b) In: Proc TAPPI Pulping Conf, Boston, Massachusetts, Nov: 6 10
14. Tolan JS (1993) In: Proc Non-chlorine Bleaching Conf, Hilton Head, S.C. Mar: 1~17
15. Tolan JS, Vega Canovas, R (1992) In: Pulp and Paper Canada 93(5): 31-36
16. Tolan JS (1995) In: J Pulp and Paper Sci 21(4): J13~J137
17. Tolan JS, Olson D, Dines RE (1995) In: Proc 81st CPPA Annual Mtg, Montreal, Quebec
18. Turner JC, Skerker PS, Burns BJ, Howard JC, Alonso MA, Andres JL (1992) In: TAPPI 75(12):
83 89
19. Viikari L, Ranua M, Kantelinen A, Sundquist J, Linko M (1986) In: Proc Int Conf Biotechnol
Pulp and Paper Ind, Stockholm, Sweden, p 67-69
20. Yang JL, Ericksson KE (1992) In: Proc Kyoto Conf, Uni Publishers Co, Tokyo, Japan
Biotechnology for Solving Slime Problems
in the Pulp and Paper Industry
S.C. Johnsrud
STFI, Swedish Pulp and Paper Research Institute, Stockholm, Sweden
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
2 Biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
2.1 Glycocalyx . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
2.2 Microbial Extracellular Polysaecharides . . . . . . . . . . . . . . . . . . . . . . . . . . 313
2.3 Biofilm Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
2.4 Organisms within the Slime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
3 Slime Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
3.1 Biocides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
3.2 Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
4 New Research Solving Slime Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
4.1 Surface-Active Agents in Slime Control . . . . . . . . . . . . . . . . . . . . . . . . . . 318
4.1.1 Synthetic Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
4.1.2 Biosurfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
4.2 Alternative Biocide-Free Growth Control Methods . . . . . . . . . . . . . . . . . . . 320
4.2.1 Enzymatic Slime Deposit Control . . . . . . . . . . . . . . . . . . . . . . . . . . 320
4.2.2 Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
4.2.3 Introduction of Competing Micro-organisms . . . . . . . . . . . . . . . . . . . . 323
4.3 Biological Equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
4.4 Biodispersants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
5 Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
Biotechnology for Solving Slime Problems in the Pulp and Paper Industry includes the following
topics: biofilms, glycocalyx; microbial extraceUular polysaccharides; organisms within the slime;
slime control, biocides, cleaning; surface-active agents in slime control, synthetic surfactants,
biosurfactants, alternative biocide-free growth control methods, enzymatic slime deposit control,
bacteriophages, introduction of competing micro-organisms; biological equilibrium; and biodisper-
sants.
9 Springer-VerlagBerlinHeidelberg1997
312 S.C. Johnsrud
1 Introduction
The selection, evaluation, and development of biocide-free systems for

application in pulp and paper mills to eliminate the proliferation of biological
slimes is necessary for environmental, health and safety, and economic
reasons.
Alternatives to the conventional biocidal methods are under development.
These methods involve the use of specific enzymes, bacteriophages, competing
organisms, biological complex formers and biodispersants. At present, however,
none of the methods mentioned is sufficiently well developed to provide a full-
scale alternative to chemical biocides.
An understanding of biofilm ecology requires the integration of concepts
from microbiology, engineering, and chemistry, and encompasses features
ranging from microbial growth to papermaking. The prevention of biofilm
build-up is especially interesting in that the microflora in biofilms are univer-
sally found to be more resistant than their planktonic counterparts to the action
of antimicrobial agents. The development of a deep understanding of the
underlying basis for this resistance is the salient point to be tackled before
adequate slime control systems can be developed.
2 Biofilm
A well-known and long-standing problem in paper manufacture is the prolifer-

ation of biological slimes on machinery. The slime seeds into process water,
clogs wires, and contaminates the product itself- sometimes to the point of
discolouration.
Virtually every surface examined in natural, industrial, and pathogenic
ecosystems is colonized by biofilms consisting of adherent (sessile) bacterial
populations enmeshed within a glycocalyx matrix [1].
Although many studies on free-living bacteria (planktonic) most commonly
isolated from process waters have been reported [2-6], relatively few of these
have been concerned with the biofilm environment.
Biofilm build-up is characterized by an increased resistance to biocides, by
the development of anaerobic zones, by structural heterogeneity, and by protec-
tion from predative grazing micro-organisms and from desiccation [7]. Some
recent reports relate to the formation of biofilm in pulp and paper mills [8 11].
A few studies have been made on biofilms in the paper machine environment
[8, 12-14]. Most investigators of biofilm development in engineered systems
have concentrated on enumerating the bacterial population, with relatively
fewer studies considering the possible importance of filamentous fungi in such
systems [15-16].
Biotechnologyfor SolvingSlime Problems in the Pulp and Paper Industry 313
Today, much effort is being directed towards elucidating those features that
determine the resistance of bacterial biofilms towards antibacterial agents
[1, 17-21]. An understanding of the behaviour of the biofilm glycocalyx and the
physiology of the sessile bacteria in relevant, defined environments would
greatly assist the development of effective treatment strategies.
2.1 Glycocalyx
A biofilm is a gelatinous matrix consisting of bacterial cells and their secreted

extracellular polymers.
Many procaryotic organisms secrete slimy or gummy materials on their
surface. A variety of structures consist of polysaccharides. The terms capsule and
slime layer are sometimes used, but the more general term glycocalyx is also
applied. Glycocalyx is defined as the polysaccharide-containing material lying
outside the cell. The glycocalyx varies in different organisms, but usually
contains glycoproteins and a large number of different polysaccharides, includ-
ing polyalcohols and amino-sugars. The glycocalyx may be thick or thin, rigid
or flexible, depending on its chemical nature in a specific organism. The rigid
layers are organized in a tight matrix which excludes particles. This form is
referred to as a capsule. If the glycocalyx is more easily deformed, it does not
exclude particles and is more difficult to see. This is referred to as a slime layer
[22].
Glycocalyx layers serve several functions in bacteria. The outer polysacchar-
ide layers play an important role in the attachment of micro-organisms to
surfaces [23-27]. They concentrate nutrients and protect microbes from toxic
heavy metals and antibacterial agents often used in antifouling treatments
[1, 18]. In addition, since outer polysaccharide layers probably bind a signifi-
cant amount of water, there is reason to believe that a glycocalyx layer plays
some role in the resistance to desiccation.
2.2 Microbial Extracellular Polysaccharides
The troublesome slime deposits are mainly composed of microbial extracellular

polysaccharides. The extracellular polysaccharide structures may conveniently
be divided into homopolysaccharides, i.e. polymers of a single sugar or amino-
sugar residue, and heteropolysaccharides, where more than one type of residue
is present. Examples of homopolysaccharides are the glucans and levans.
Levans, polymers of fructose, have been isolated from Bacillus subtilis [28, 29],
Bacillus polymyxa [30, 31], Streptococcus sp [32], Corynebacterium laevaniJbr-
roans [33], Aerobacter levanicum [34], Arthrobacter sp [35], Acetobacter
acetigenus [35], and Pseudomonas sp [353.
However, the majority of bacterial extracellular polysaccharides are made of
more then one type of sugar residue, and they often contain uronic acids [36].
314 s.c. Johnsrud
Many bacteria secrete organic polymers with limited solubility in water, which
tend to accumulate as loose, confluent layers in the immediate neighbourhood
of the cell, just outside the wall. Unlike the cell wall, the capsule or slime layer
seems to have no important direct role in the maintenance of cellular function.
Some bacteria form a slime layer only when growing at the expense of a specific
substrate which is a direct biochemical precursor of the slime substance in
question. This behaviour is characteristic of certain streptococci, bacilli,
pseudomonads and xanthomonads, which form copious quantities of either
dextrans or levans when growing at the expense of the disaccharide sucrose. No
other metabolizable sugar, including glucose and fructose themselves, can serve
as a substrate for the synthesis of these polysaccharides. Consequently, dextran-
and levan-forming bacteria produce these capsular materials only when growing
on a sucrose-containing medium. Certain kinds of capsular substances can be
removed from the cells by treatment with specific hydrolytic enzymes. Such
enzymatic treatment leaves the cell unharmed [37]. Effluents rich in sucrose
specifically encourage the growth of dextran- and levan-producing organisms.
2.3 Biofilm Structure
Although biofilm formation has been studied extensively in natural aquatic

systems, waste water treatment systems, and medical appliances [19, 38-43],
only a few studies have been made on biofilms in the paper machine environ-
ment [12-14].
Recent work by V/iisanen et al. [8] has shown that paper machine biofilm
has a complicated biological and chemical structure, which morphologically
resembles those of waste water biofilms, biofilms in industrial cooling towers,
and natural river ecosystems.
2.4 Organisms within the Slime
The close microbial association of bacteria, fungi, algae, and protozoa in paper
mill slimes appears to depend upon a delicate balance between environmental
(physical and nutritional) and interbiotic factors. The microbial habitat in paper
mills acts in the same way as a chemostat, growth being controlled by a small
but continuous supply of nutrients. General physical conditions (temperature,
oxygen, pH) basically control the types of microbial flora developing in process
water systems. Of these factors, only the seasonal temperature (3-25 ~ varies to
any significant degree, and then only in open or semi-open mill systems. Closed
(recirculatory) systems maintain relatively high temperatures (30-50 ~ in the
process water systems. Thus, a flora develops depending upon the quantitative
distribution of the organisms and also upon specific factors which facilitate
deposition, development, and rapidity of growth [44]. Many microbial
Biotechnologyfor Solving Slime Problems in the Pulp and Paper Industry 315
processes occurring in the environment are not possible with single-species

populations, but require consortial activities [-45].
Within the slime, microbial dominance is dynamic. The microbial composi-
tion of the slime is dependent on its origin and subsequent development in the
mill system. Slime formation depends both on a mechanism of inception and on
the subsequent conditions for growth of the organisms. The relative importance
of a specific species is difficult to assess, as many factors such as the influence of
nutrients [16,46-49], the presence of specific inhibitors and stimulants in
woods, and physical conditions can control both its amount and type of growth.
Seasonal changes, slime treatment variations, fresh water recirculation vs non-
recirculation, type of pulp stock, and cleanliness also cause variations in micro-
flora [16, 5, 50].
Cell concentrations in biofilms are three to four orders of magnitude higher
than those of planktonic cells. Concentrations of 1012 cells/cm 3 are commonly
found within biofilms, whereas the maximum concentration for cells in suspen-
sion is l0 s 10 9 cells/cm 3 [51].
A wide variety of micro-organisms have been implicated in slime/biofilm
formation. Some reports emphasize the role of bacteria in slimes (in particular
locations) [52, 53], while others emphasize their predominately fungal nature
[-54-56].
The types of micro-organisms that cause slime in industrial environments
are typically the heavily encapsulated fast growing bacteria, such as species of
Pseudomonas, Aerobacter, Alcaligenes, Arthrobacter, Proteus, Bacillus, and
others [37, 57 59]. In addition, bacteria found most often in paper and board
machine slime include species of Flavobacterium, Clavibacter, Sphaerotilus, and
Leptothrix [-60, 2].
Common fungal species encountered in slimes are Aspergillus, Penicillium,
and Cephalosporium [5,46,61 63]. In closed process water systems where
temperatures are often maintained above 30 ~ the thermotolerant fungi play
an important role in slime formation in paper mills [58]. Several mould species
are also known to be synergistic with bacterial species, forming tough slimes
that are resistant to biocides and difficult to control. These include Clados-
porium, Geotrichum, and Mucor [64].
The extremely varied group of micro-organisms produces an equally varied
number of extracellular polysaccharides, reserve materials, and other excretory
products that make up the biofilm.
Biofilm systems may provide an anaerobic zone enabling the sulfate-reduc-
ing bacteria (Desulfotomaculum, Desulfovibrio) [65] to grow and metabolize even
when the bulk water contains oxygen close to saturation. The outer layer of this
film will consist of heterotrophic bacteria depleting oxygen and developing an
environment suitable for sulfate-reducing bacteria to grow and metabolize
if sulfate and excess organic carbon are present. Sulfate reduction in bio-
film systems is of concern in many water systems [66-69]. Sulfate-reducing
bacteria produce hydrogen sulfide, which can deteriorate concrete or corrode
metals [70, 71]. A general hierarchy of heterotrophic organisms, denitrifiers,
316 s.c. Johnsrud
iron-reducing bacteria, sulfate-reducing bacteria, and methanogens may be

found in thick microbial films where excess organic material, nitrate, iron, and
sulfate are present.
3 Slime Control
Adequate control of microbial-induced slime deposits is an essential part of

modern pulp and paper manufacture. The complex interactions of fibres, water,
extractives, air, micro-organisms, and chemical additives can produce a wide
variety of deposits, to which additional modifications may be made by the
mechanical processing involved in various stages of the papermaking process. In
addition to the adverse influence on product quality, such deposits increase the
difficulty of operation of paper machines, cause production losses, and increase
the cost of maintenance. These problems can be avoided by using slimicides and
other microbicides.
3.1 Biocides
Microbicides have become an indispensable part of paper production, parti-

cularly because modern processes and the water supply conditions in most
plants greatly promote the growth of micro-organisms. In many factories,
trouble-free working with largely closed water circuits and continuous produc-
tion of coated papers and boards has been made possible only by the systematic
use of slimicides and preservatives. Since the different types of micro-organism
have varying degrees of resistance to microbicides, there is no single preparation
which can solve all the preservation problems occurring in the paper industry.
The properties demanded of microbicides, in addition to high activity and
cost-effectiveness, also vary according to their specific field of application.
Slimicides must be used in concentrations which do not disturb the papermak-
ing processes, even if they are added in massive doses. The products must be
compatible with the many auxiliaries used in papermaking. Furthermore,
slimicides must have low toxicity and, in particular, low ecotoxicity, and no
appreciable amounts must be present in the finished product, so that the paper
can also be used in food packaging. Perservatives for coating mixes, filler
suspensions and sizes, and active ingredients for the antimicrobial finish of
paper and board have to meet stringent requirements with regard to the absence
of colour and odour, compatibility, and physiological harmlessness in their use
concentrations. Most chemical companies have developed chemicals that can be
classified as either oxidizing biocides, non-oxidizing biocides, or biodispersants.
The application of biocides and their mode of action are well documented
1-72-74]. Antimicrobial agents have been used in the pulp and paper industry
since the 1800s. Many of the early active ingredients were used indiscriminately,
with little regard for workers' health and safety or environmental concerns. In
recent years, registration of slimicides has become more difficult, since increased
emphasis is now placed on characteristics such as slimicide handling, health and
safety issues, and fate in the environment.
In Sweden, slimicides used in the paper industry are covered by the Ordi-
nance on Pesticides [1985: 836], which means that they may not be imported or
handled without approval from the National Chemicals Inspectorate (KEMI).
Recently, K E M I has published a report on the risk assessments of slimicides
used in the Swedish paper industry from a health and environmental protec-
tion standpoint. At the beginning of 1990, there were 21 active ingredients
and 37 products approved for use as slimicides in the paper industry in Sweden.
The number of slimicides on the Swedish market was significantly reduced as
a result of the re-registration in 1994. All documentation dossiers for the 40
approved products with a total of 11 active ingredients were evaluated and
followed by toxicological and ecotoxicological hazard and risk assessments
[75].
The active ingredients presented in the report are:
CAS No. 32718-18-6 Bromo-chloro-5,5-dimethylhydantoin
CAS No. 52-51-7 2-Bromo-2-nitropropane-l,3-diol (Bronopol)
CAS No. 126-11-4 2-Hydroxymethyl-2-nitropropane-l,3-diol
CAS No. 2491-38-5 2-Bromo-4'-hydroxyacetophenone
CAS No. 10222-01-2 2,2-Dibromo-2-cyanoacetamide
CAS No. 1192-52-5 3,4-Dichloro-5-oxo-l,2-dithiol
CAS No. 111-30-8 Glutardialdehyde
CAS No. 26192-55-4 5-Chloro-2-methyl-4-isothiazolin-3-one
CAS No. 2682-20-4 2-Methyl-4-isothiazolin-3-one
CAS No. 31075-24-8 Poly[oxyethylene-bis(dimethyliminoethyl)dichloride]
CAS No. 21564-17-0 2-(Thiocyanomethylthio)benzothiazole (TCMTB).
The health risks when handling stimicide products are mainly connected
with their corrosive, strongly irritative, and sensitizing properties. Almost all
products have shown severe effects at very low concentrations in both animals
and humans, primarily by dermal and/or inhalation exposure. No particular
differences in the health risk assessment among the substances could be seen.
Exceptions are poly [oxyethylene-bis(dimethyliminoethyl)dichloride] (POIDC)
and bromo-chloro-5,5-dimethylhydantoin (BCDMH), which have not shown all
these hazardous properties.
The report concludes that the benefit of T C M T B as a stimicide does not
outweigh its risks, and that it should therefore be phased out of the Swedish
market during 1995. Bronopol should be used only at paper mills with advanced
waste water treatment plants containing aerated basins or other types of
biological treatment steps. Under such conditions, the benefit of Bronopol as
a slimicide outweights its risks. The benefits of using the other active ingredients
as slimicides are today considered to exceed the risks.
318 S.C. Johnsrud
3.2 Cleaning
Other components in a control program are mechanical cleaning and boil-out at
extreme pH levels and high temperatures. Cleaning is often done with high
pressure water flushing and flushing of the system with lye and tensides.
Pollution and deposits, e.g. of organic matter and barium sulphate, which
promote growth and slime formation, are thus eliminated. A meticulous clean-
ing is important and may minimize the need for chemical biocides. Mechanical
cleaning and boil-out are often impracticable, and can be costly because they
usually involve equipment down time. In addition, the harsh chemicals used in
a boil-out can challenge what is often an already stressed waste water treatment
system.
Supplementary measures, i.e. in addition to mechanical cleaning, involve the
use of surfactant, film-forming, complex-forming and fixation chemicals and, to
a certain extent, beneficial bacteria and enzymes in treatment programs.
Unintentional slime control with other chemicals such as preservatives and
bleaching chemicals occurs to some extent. Bleaching chemicals such as chlorine
dioxide, ozone, hydrogen peroxide, and a system using peracetic acid/hydrogen
peroxide act as biocides in those processes or process steps in which they are
used. Minor contributions fi'om other preservatives have no actual role in the
slime control program. Slime control exclusively with bleaching chemicals is not
at present an economically feasible approach.
4 New Research- Solving Slime Problems
Current research in both medical and industrial biofilm prevention and destruc-
tion has concentrated on dispersants. These can be of several different types, but
the most common are based on surfactant chemistry.
4.1 Surface-Active Agents in Slime Control
4.1.1 Synthetic Surfactants
Surface-active agents are often called by other names, including: surfactants

(particularly in the USA), association colloids, colloidal electrolytes, paraffin
chain salts, amphiphatic compounds, and tensides (particularly in Europe). It
should be noted, however, that whereas most detergents (from the Latin deter-
gens, cleansing) are surface-active agents, not all surface-active agents are
detergents. A surfactant is a molecule which has both a water-soluble and
a water-insoluble (usually hydrocarbon) portion [76]. This balance of hy-
drophilic and hydrophobic moieties in the same molecule imparts unusual
properties, including an ability to lower the surface tension of water. Chemically

synthesised surfactants are usually classified according to the nature of the
head-group, and accordingly can be of anionic, cationic, nonionic, or zwit-
terionic types [77]. Surfactants are used widely in industry, agriculture, and
medicine. The materials currently in use commercially as surfactants are mainly
produced by chemical synthesis or as relatively inexpensive by-products of
industrial processes, e.g. lignosulfonates from papermaking.
Surfactants can cause papermaking process problems, including decreased
sizing in paper, increased moisture holding of paper resulting in high energy
usage, and foaming. Surfactants are usually readily biodegradable and could be
a cause of increased microbiological growth [78]. Some dispersants are also
phosphate-based, thus providing an essential nutrient that is often a limiting
factor for biological growth.
4.1.2 Biosurfactants
Micro-organisms are also producers of dispersants. Microbiologically derived

surfactants (biosurfactants) are more conveniently discussed in terms of (1) the
biochemical nature of the surfactant, e.g. protein, polysaccharide, lipid, or
a complex containing two or more types of biomolecule, and (2) the producing
microbial species [79].
The term biosurfactant has generally been used very loosely to refer to any
compound which has some influence on interfaces. For example, it is often
applied to biopolymers which have emulsifying properties but do not lower the
surface tension of water appreciably or demonstrate other characteristics of
a classical surfactant.
Surfactin, produced by Bacillus subtilis ATTC 21332 is one of the most
effective biosurfactants known. It reduces the surface tension of water from
72 m N / m to 28 m N / m at a concentration as low as 0.005% [-80].
Polysaccharides occur as microbial energy reserves and as structural mater-
ial in cell walls and extracellular capsular layers. A large number of extracellular
polymers have gained commercial importance due to their ability to alter, at low
concentrations, the rheological properties of aqueous solutions. Such polysac-
charidic hydrocolloids are used in the food, pharmaceutical, cosmetic, oil, paper
and textile industries as thickening agents and viscosity modifiers.
The best characterized lipopeptide surfactants are surfactin, which has been
isolated from several strains of Bacillus subtilis and Bacillus pumilis [81, 82],
viscosin, produced by Pseudomonasfluorescens [83], and arthrofactin, produced
by Arthrobacter sp. strain MIS38 [84].
Antibiotics are relatively low-molecular-mass molecules produced by bac-
teria or fungi, which kill or inhibit the growth of other micro-organisms in
a selective manner. Many of the antibiotics are amphiphiles and would be
expected to exhibit surface active properties. Surfactin (subtilisin) produced by
various strains of Bacillus subtilis, has antifungal properties and anti-tumour
320 S.C. Johnsrud
activity [85]. Antibacterial activity of lichenysin A, produced by Bacillus

licheniformis, has also been demonstrated [86].
Biosurfactants are involved in cell adhesion, emulsification, dispersion, ftoc-
culation, cell aggregation, and desorption phenomena. Acinetobacter calco-
aceticus RAG-l, whose ability to degrade and disperse crude oil [87] was shown
to be due to the emulsification of the oil by an extracellular polyanionic
amphipathic heteropolysaccharide, emulsan, has been used in low concentra-
tions to inhibit the adhesion of bacteria to hydrophobic surfaces. It was sugges-
ted that the anti-adherence properties of emulsan are due to the formation of
surface films which render hydrophobic surfaces hydrophilic, thus preventing
the attachment of bacteria through hydrophobic interactions [79]. Biodispersan
is yet another extraceUular anionic heteropolysaccharide produced by A. calco-
aceticus A2 that changes the surface properties of limestone. It acts both as
a grinding aid and dispersant for limestone, which is used as a filler in paper
[88, 89], and has been reported to be used to disperse calcium carbonate
suspensions [90]. Pullulan from Aureobasidium puIlulans is used as a flocculat-
ing agent in packaging [91].
Conventional surfactants are currently used for a broad range of purposes in
a large variety of different applications [92]. Most requirements for a conven-
tional surfactant can be met by a biosurfactant. Biosurfactants are biodegrad-
able, potentially less toxic than the synthetic compounds currently used, and can
be produced from a variety of substrates. To justify the replacement of a syn-
thetic surfactant with a biological compound, it is necessary to find a more
effective agent for a given application, and/or one that can be produced more
cheaply [76].
4.2 Alternative Biocide-free Growth Control Methods
Alternatives to the conventional biocidal methods are under development.

These involve the use of specific enzymes, bacteriophages, competing organisms,
biological complex formers, and biodispersants. At present, none of these
methods are sufficiently well developed to provide a full-scale alternative to the
use of chemical biocides.
4.2.1 Enzymatic Slime Deposit Control
Today's research is focused on both the prevention and the disruption of biofilm
build-up caused by slime-forming species of bacteria, yeasts and fungi. Many of
the approaches include the use of enzymes. These are occasionally of only one
type, but more frequently several enzymes are used in combination. The appro-
priate enzyme or combination of enzymes is determined by the extracellular
polysaccharides contained in the biofilm. Several inventions and patents have
been reported in this field in recent years [93-99].
Certain kinds of polysaccharide substances can be removed from the cells by

treatment with specific hydrolytic enzymes. Such enzymatic treatment leaves the
cells unharmed [37]. The stripping of the extracellular polysaccharide layer
prevents the cells from attaching to other surfaces and makes them more
accessible to conventional biocide treatment.
The use of enzymes in the control of microbiological slime deposits, particu-
lar EDC-1 (enzymatic deposit control) developed by Hatcher [100, 101] has
proved useful under modern mill conditions [102]. EDC-1 is an enzyme that
hydrolyses and depolymerizes the fructose polysaccharide levan, which has been
identified in paper mill slime [103]. EDC-1 is a patented enzyme product [93]
obtained by aerobic fermentation of a common non-pathogenic, non-spore-
forming soil bacterium, which has been tailored to suit the specific needs of the
paper industry. It is non-toxic, does not promote resistance, does not accumu-
late in the system as it is inactivated at 80 100 ~ and is safe to handle. The
product can be dosed solely or in conjunction with conventional biocides [104].
Simultaneous application of EDC-1 and biocide allows the amount of the latter
to be reduced. EDC-1 is used by paper mills in the USA, Scandinavia, the UK,
and Japan.
It has been pointed out that it is in practice not always possible to achieve
satisfactory slime control by dosing EDC-1 solely to the process waters. The
reason is that EDC-1 is a specific levan-hydrolysing enzyme, i.e., it is restricted
to levan-producing bacteria, which have been shown to be a secondary factor in
many process waters. The bacterial number may increase during enzymatic
treatment of the extracellular polysaccharides, since the carbohydrates released
serve as food for the bacteria.
Despite the strong indications that exo-polysaccharides are involved in
adhesion, enzyme digestion has tended to show that proteases are more effective
than carbohydrate-degrading enzymes in removing bacteria from surfaces
[105].
Micro-organisms have cell walls which confer rigidity, and, if the cell wall is
removed in some manner, the cell usually dies because of lysis resulting from
osmotic imbalance. Cell walls in micro-organisms are composed of substances
such as cellulose, chitin, mucopeptide, and [3-1,3-glucan. In addition, many
organisms have capsules, slime layers, or other surface components which are
polysaccharides or proteins attached to the outside of the rigid layer or inter-
digitated with it, conferring additional rigidity and/or protection. Enzymes are
known which will attack all of these polymers [106].
The application of lytic enzymes with glucanase and protease activity in
paper machine process water to destroy microbes, causing precipitates and/or
forming slime and/or adversely affecting product quality, has been advocated
[96]. Enzymatic dispersion of biological slimes has been claimed for a pento-
sanase-hexosanase enzyme, Rhodozyme HP-150 [98]. The invention is parti-
cularly effective in treating industrial waters used in the operation of cooling
towers to disperse slimes and slime-forming masses within such waters to
prevent the deposit of such slimes on the heat exchange surfaces of cooling
322 S.C. Johnsrud
towers and surfaces associated with such units. Orndorff in his patent [97]
describes a method of killing and inhibiting the growth of microorganisms in
industrial process streams using peroxidase- or laccase-catalysed oxidation of
various phenolic compounds to generate microbicidal oxidation products, e.g.
poisonous quinones.
The use of a blend of enzymes has been shown to be more effective in treating
microbially produced extracellular polysaccharides in cooling waters and in
papermaking broke water than the use of a single enzyme. The composite
enzyme system tested consisted of cellulase, a-amylase and protease [99]. None
of these methods is sufficiently well developed to constitute a full-scale alterna-
tive to chemical biocides, with the exception of the use of EDC-1.
Some manufacturers and service companies assert that the enzymes avail-
able are too species-sPecific and can be used only on a smaller scale or as
a supplement to conventional biocidal methods
4.2.2 Bacteriophages
Bacteriophages are viruses that infect and destroy bacteria. Viruses are classified
initially on the basis of the hosts they infect. Thus we have animal viruses, plant
viruses, fungal viruses, and bacterial viruses. Bacterial viruses, sometimes called
bacteriophages (or phage for short, from the Greek phago meaning to eat), have
been studied primarily as convenient model systems for research into the
molecular biology and genetics of virus reproduction. However, the develop-
ment of industrial applications has been slow.
Viruses lack an independent metabolism. They multiply only inside living
cells, using the metabolic machinery of the host cell [-107]. A bacteriophage can
proliferate only by coming into contact with its specific host bacterium. When
this occurs, the phage lyses the bacterium completely.
Several reports and patents have been published which describe attempts to
use bacteriophages to control biofouling related mainly to the use of seawater
[108, 109]. The application of bacteriophages in paper mill white-water systems
was reported by Vfi/itanen and Harjy-Jeanty in 1986. Their concept was based
on the idea of isolating harmful bacterial strains of process waters and thereafter
searching for virulent, lytic bacteriophages for these bacteria. They tested
bacteriophages lytic for the bacteria Enterobacter and Klebsiella in model
systems and found that phage activity against E. agglomerans prevented its
growth for more than 19 hours. When K. pneumoniae was dosed with phage at
3-h intervals, bacterial growth was no more curtailed than when the culture was
infected only at the beginning of the test. They concluded that further studies to
determine the efficacy of bacterial control in process waters must take into
consideration the expected development of bacterial resistance to phage attack
and the variation in bacterial strains among paper mills. The main advantages of
using phages in combating bacteria is their bactericidal (killing) nature, selectiv-
ity, and non-toxicity to man and the environment. The main drawback is that
Biotechnologyfor SolvingSlimeProblemsin the Pulp and Paper Industry 323
the phages have to be isolated for each harmful bacterium, the type of which
may vary between paper mills [110].
Similar investigations have been reported by Araki and Hosomi [111] using
Pseudomonas sp (S-l) and its corresponding bacteriophage (pS-1). Their results
demonstrated that the proliferation of the slime-forming bacteria was retarded
by the corresponding bacteriophage by up to 10 hours. They concluded that
further fundamental studies of the bacteria-phage ecosystem within the white-
water system will be required before the technique can be applied on a commer-
cial scale.
Unlike conventional biocides, bacteriophages will not impair the activity of
the sludge used in waste treatment systems.
4.2.3 Introduction of Competing Micro-organisms
Competition Antagonism Populations can be limited in two ways: (1) by the

exhaustion of an essential nutrient, and (2) by the production of toxic substan-
ces. Organisms adapt not only to their environment but also to the presence and
competitive influence of other organisms. Strict competition means that both
organisms feed upon some common resource which is present in limited
amounts. The outcome will depend on the relative growth rates; the organism
which grows the faster will eventually replace the other, if a sufficient number of
generations is available. The environmental requirements for both organisms
may be identical, but the growth rates may still differ, because growth rates are
influenced by various intrinsic factors in the cell.
It is common experience that antagonistic organisms occur frequently in
natural habitats. The alteration of the redox potential by one organism may
inhibit the growth of another. Obligate aerobes may be inhibited by the growth
of facultative organisms which consume all the oxygen. Antibiotics are chemical
substances which are produced by living organisms and which are able to kill or
inhibit the growth of other living organisms. Antagonism has been found
between pairs of the following organisms in all possible combinations with each
other and with themselves: bacteria, fungi, algae, and protozoa [112].
During the last decade, some adventurous approaches have been adopted in
solving slime problems. The inoculation of non-slime-forming organisms to
outcompete the slime-formers has been advocated [113, 114]. The slime treat-
ment invention patented by Oberkofler et al. [114 3 is based on the introduction
of a consortium of bacteria, commercially available in freeze-dried or liquid
form. The bacteria are pregrown prior to inoculation of the circuit water, and
the amount added is calculated on the basis of the total organic carbon (TOC)
present. It is also suggested that additives may be introduced together with the
bacteria, thus favouring their proliferation. Compounds mentioned include
tensides to prevent the adhesion of bacteria, lignosulfonates to increase the
nutrients, and enzymes to catalyse the breakdown of organic substances. Aeration
of the circulating water with oxygen or air, or the addition oxygen-releasing
324 s.c. Johnsrud
compounds such as hydrogen peroxide are also suggested [115]. The invention
is not restricted to bacteria. Fungi alone or in a blend with bacteria may be used.
The patent outlines an undiscriminated use of bacteria and fungi, of which the
mixed culture of freeze-dried bacteria used contains many of the genera asso-
ciated not only with slime formation in pulp and paper mills but also such
genera undesirable for paper and board intended to come into contact with
foodstuffs.
Mill trials, run continuously for more than nine months, have nevertheless
shown that the addition of the selected micro-organisms, e.g. bacteria, to the
circulating water reduced the build-up of slime on solid surfaces and in the
liquid phase [113].
4.3 Biological Equilibrium

The Biochem Process was invented as an alternative to the application of
biocides. It aims at reaching a biological equilibrium between the microflora
and the available nutrients in closed white-water systems [-116].
The process uses modified lignosulfonates and a non-toxic chelating agent
different from those commonly used in papermaking. The objective of the
lignosulfonate treatment is to maintain the organisms and the suspended mater-
ial in the white-water in a colloidal state thereby preventing the bacteria from
agglomerating and forming deposits. The bacteria, which carry a net negative
charge, adhere to the modified lignosulfonate. As the bacteria digest the organic
substances within the colloids, the material becomes more flocculent. The
lignosulfonate, which is water soluble, is drained off on the wirepress, whereas
the bacteria enter the finished paper. The process allows biocide-free operation
and is applicable to all paper grades. Counter-indications are open water
systems, unbleached kraft pulp, use of recycled fibres, and an increase in organic
matter.
The Bimogard system [117] consists of non-toxic blends of surface-active
agents and a derivative of a sugar-free lignin polymers. The blends are tailored
specifically to each application. Bimogard is designed to reduce the number of
slime-forming bacteria only. Thus a total bacterial count cannot always reveal
its effectiveness.
The Bimogard system, which resembles the Biochem process, is claimed to
be the first completely biocide-free slime control system in Europe. So far,
nothing has been published about how this system operates, but more than 22
Bimogard systems are in operation on paper machines for newsprint, light
weight coated (LWC) fine paper, tissue, greaseproof paper, and paperboard.
Reports from the mills indicate that the system has brought about significant
improvements in efficiency. For example, the hole frequency drops, the time
between wash-ups increases, cleaning is easier, and there are fewer wet-end
breaks and therefore lower broke production.
4.4 Biodispersants
Biodispersants play an essential role in modern treatment programmes to
control biofilms caused by excessive growth of micro-organisms such as bac-
teria, yeast, and moulds.
Biodispersant technology is based on non-ionic polymers, which are non-
toxic, non-foaming, colourless, and free of organic solvents. Because of their
non-ionic character, they will neither increase the system anionicity nor interfere
with other papermaking chemicals. Biodispersants have no pH limitations and
are suitable for use in both acid and alkaline papermaking [118-120].
The non-ionic products which have seen large-scale commercial use fall into
four general types, i.e. alcohol ethoxylates, alkylphenol ethoxylates, poly-
oxyethylene esters, and polyoxyethylene-polyoxypropylenederivatives. The lat-
ter are mixed polymers with hydrophobic groups derived from propylene oxide,
further reacted with ethylene oxide until the desired properties are attained.
Ordinarily they are of rather high molecular mass, often with much more than
the usual 8 to 15 molecules of ethylene oxide characteristic of the other
nonionics [78].
The product can be added continuously or intermittently, the dose rate
being dependent on the problem to be solved. It is preferable to add the products
to the short circuit before the headbox or clarified water. The use of biodisper-
sants is recommended in cases where the amount of biocides has to be reduced,
the efficacy of the biocide has to be increased, or the biocides have to be totally
replaced. Because biodispersants can neither kill micro-organisms nor inhibit
their growth, the use of biocides may still be needed, even in cases where the
ultimate goal is to avoid the addition of biocides altogether. However, the use
of biodispersants can enhance biocide effectiveness by eliminating biofilm,
inorganics, and organics which can inhibit penetration of the biocide into the
organism.
5 Future Prospects
It is conceivable that an increased understanding of the behaviour of the biofilm

glycocalyx and of the physiology of the sessile bacteria in a relevant, defined
environment would greatly assist the development of effective treatment strat-
egies by means of enzyme engineering and surfactant technology. These would
then replace chemical biocides.
Nevertheless, whatever the nature of the new applications, the eventual
changeover to biocide-free methods will be possible only when the new tech-
nologies become beneficial and realistic replacements.
At least in the near future, the great challenge is to realise the possibility of
running paper machines on a totally ecological basis.
326 S.C. Johnsrud
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Advances in Biochemical Engineering

With 41 Figures and 52 Tables

Library of Congress Catalog Card Number 72-152360

Springer-Verlag Berlin Heidelberg 1997

Typesetting: Macmillan India Ltd., Bangalore-25

Professor Dr. T. Scheper

Professor Dr. K.-E. L. Eriksson

Prof. Dr. W. Babel Center of Environmental Research

Prof. Dr. S.-O. Enfors Department of Biochemistry and Biotechnology

Prof. Dr. K.-E. L. Eriksson Center for Biological Resource Recovery

Prof. Dr. B. Mattiasson Department of Biotechnology

Prof. Dr. H. d. Rehm Westf'alische Wilhelms Universit/it

Prof. Dr. P. L. Rogers Department of Biotechnology

Prof. Dr. H. Sahm Institute of Biotechnology

Prof. Dr. K. Schi~gerl Institute of Technical Chemistry

Prof. Dr. G. T. Tsao Director, Lab. of Renewable Resources Eng.

Prof. Dr. K. Venkat Phyton Inc., 125 Langmuir Lab.

Prof. Dr. John Villadsen Department of Biotechnology

Prof. Dr. U. yon Stockar Swiss Federal Institute of Technology Lausanne

Prof. Dr. C. Wandrey Institute of Biotechnology

One of natures most important biological processes is the conversion of wood

September, 1996 Karl-Erik L. Eriksson

Forest Tree Biotechnology

Microorganisms and Enzymes Involved

Fungal Delignification and Biomechanical Pulping of Wood

Solving Pitch Problems in Pulp and Paper Processes

Reduction of Organochlorine Compounds in Bleach

Hemicellulases in the Bleaching of Chemical Pulps

Using Enzymes in Pulp Bleaching: Mill Applications

Biotechnology for Solving Slime Problems in the Pulp

Author Index Volumes 51 - 57 . . . . . . . . . . . . . . . . . . . . . . . . . 329

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

A file with the complete volume indexes Vols. 1 through 11 in

This file distributed without any expressed or implied warranty.

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HELP returns a detailed instruction set for the

Jeffrey F.D. Dean 1, Peter R. LaFayette 2, Karl-Erik L. Eriksson z,

Advances in Biochemical Engineering/

5.3 Other Applications of D N A Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

List of Symbols and Abbreviations

2.1 Wood and Paper Products

2.2 Breeding and Life-Cycle

2.3 Man-Made and Environmental

3.1 Cell and Tissue Culture o f Forest Trees

replicates the phenotype of the source tree. However, unlike macropropagation

Production of plantlets from axillary shoots is similar to vegetative propagation

Organogenesis is the de novo production of plant organs (buds, shoots, and

systems, organogenic cultures are usually stimulated to produce buds by addi-

3.5 Somatic Embryogenesis

Somatic embryogenesis is the de novo production of structures resembling

3.6 Protoplast Culture

Another potential application of in vitro culture which may eventually succeed

3.7 In Vitro Screening and Somaclonal Variation

As mentioned earlier, organogenic and embryogenic regeneration systems may

Another technique that is likely to come into widespread use in operational

3.9 Artificial Seeds

sustain ex vitro post-germinative growth. These features would facilitate mech-

As illustrated by the wide variety of specialized cultivars available within

Species Method Reference

4.1 Tissue Culture Considerations

All of the considerations previously discussed for the regeneration of intact

4.2 Biological Gene Transfer (Agrobacteria-Mediated