(Birgitte K. Ahring (Editor), B.K. Ahring, I. Ang (BookFi) PDF

Preface
Preface
In November 1776, Alessandro Volta performed his classic experiment disturb-

ing the sediment of a shallow lake, collecting the gas and demonstrating that this
gas was flammable. The science of Biomethanation was born and, ever since, sci-
entists and engineers have worked at understanding this complex anaerobic bio-
logical process and harvesting the valuable methane gas produced during anaer-
obic decomposition. Two lines of exploitation have developed mainly during the
last century: the use of anaerobic digestion for stabilization of sewage sludge,
and biogas production from animal manure and/or household waste. Lately, the
emphasis has been on the hygienic benefit of anaerobic treatment and its effect
on pathogens or other infectious elements. The importance of producing a safe
effluent suitable for recirculation to agricultural land has become a task just as
important as producing the maximum yield of biogas from a given type of
waste. Therefore, anaerobic digestion at elevated temperatures has become the
main area of interest and has been growing during the last few years.
Anaerobic digestion demands the concerted action of many groups of
microbes each performing their special role in the overall degradation process.
Both Bacteria and Archaea are involved in the anaerobic process while the
importance, if any, of eukaryotic microorganisms outside the rumen environ-
ment is still unknown. The basic understanding of the dynamics of the complex
microflora was elucidated during the latter part of the last century where the
concept of inter-species hydrogen transfer was introduced and tested. The isola-
tion of syntrophic bacteria specialized in oxidation of intermediates such as
volatile fatty acids gave strength to the theories. Lately the use of molecular tech-
niques has provided tools for studying the microflora during the biomethana-
tion process in situ. However, until now the main focus has been on probing the
dynamic changes of specific groups of microorganisms in anaerobic bioreactors
and less emphasis has been devoted to evaluating the specific activities of the
different groups of microbes during biomethanation. In the future we can expect
that the molecular techniques will be developed to allow more dynamic studies
of the action of specific microbes in the over-all process. From the present
studies we know that many unknown microbes are found in anaerobic bio-
reactors. Especially within the domain of Archaea, there are whole phyla of
microbes such as the Crenarchaeota, which make up significant fractions of
microbes in a reactor but without cultured representatives. Improving the tech-
niques for the isolation of presently unculturable microbes is a major task for
the future.
X Preface
Anaerobic digestion of waste has been implemented throughout the world for
treatment of wastewater, manure and solid waste and most countries have sci-
entists, engineers and companies engaged in various aspects of this technology.
Even though the implementation of anaerobic digestion has moved out of the
experimental phase, there is still plenty of room for improvements. The basic
understanding of the granulation process, the basis for the immobilization of
anaerobic microbes to each other without support material in UASB reactors, is
still lacking. Like any other bioprocess, anaerobic digestion needs further con-
trol and regulation for optimization. However, until now suitable sensors for
direct evaluation of the biological process have been lacking and anaerobic
bioreactors have generally been controlled by indirect measurements of biogas
or methane production along with measurements of pH and temperature. The
newly development of an on-line monitoring system for volatile fatty acids could
be a major step in the right direction and the use of infra-red monitoring sys-
tems could bring the price down to a reasonable level. A better performance of
large-scale anaerobic bioreactor systems for treatment of complex mixtures of
waste can be expected to be based on on-line monitoring of the process in the
future along with controlling software for qualified management of these plants.
Besides treatment of waste, anaerobic digestion possesses a major potential
for adding value to other biomass converting processes such as gasification,
bioethanol or hydrogen from ligno-cellulosic materials. Conversion of ligno-cel-
lulosic biomass will often leave a large fraction of the raw material untouched
which will be a burden for the over-all economy of the process and will demand
further treatment.Anaerobic digestion will on the other hand be capable of con-
verting the residues from the primary conversion into valuable methane, which
will decrease the cost and the environmental burden of the primary production.
Biomethanation is an area in which both basic and applied research is
involved. Major new developments will demand that both disciplines work
together closely and take advantage of each other’s field of competence. The two
volumes on Biomethanation within the series of Advances in Biochemical Engi-
neering and Biotechnology have been constructed with this basic idea in mind
and, therefore, both angles have been combined to give a full picture of the area.
The first volume is devoted to giving an overview of the more fundamental
aspects of anaerobic digestion while the second volume concentrates on some
major applications and the potential of using anaerobic processes. The two vol-
umes will therefore be of value for both scientists and practitioners within the
field of environmental microbiology, anaerobic biotechnology, and environ-
mental engineering. The general nature of most of the chapters along with the
unique combination of new basic knowledge and practical experiences should,
in addition, make the books valuable for teaching purposes.
The volume editor is indebted to all the authors for their excellent contribu-
tions and their devotion and cooperation in preparing these two volumes on
Biomethanation.
Lyngby, January 2003 Birgitte K. Ahring

CHAPTER 6
Perspectives for Anaerobic Digestion

Birgitte K. Ahring
University of California, Los Angeles (UCLA), School of Engineering and Applied Science,
Civil and Environmental Engineering Dept., 5732 Boelter Hall, Box 951593, Los Angeles,
California 90095-1593, USA
Present address: Biocentrum, The Technical University of Denmark, DTU, Block 227,
2800 Lyngby, Denmark. E-mail: bka@biocentrum.dtu.dk
The modern society generates large amounts of waste that represent a tremendous threat to
the environment and human and animal health. To prevent and control this, a range of differ-
ent waste treatment and disposal methods are used. The choice of method must always be
based on maximum safety, minimum environmental impact and, as far as possible, on val-
orization of the waste and final recycling of the end products. One of the main trends of
today’s waste management policies is to reduce the stream of waste going to landfills and to
recycle the organic material and the plant nutrients back to the soil.Anaerobic digestion (AD)
is one way of achieving this goal and it will, furthermore, reduce energy consumption or may
even be net energy producing. This chapter aims at provide a basic understanding of the world
in which anaerobic digestion is operating today. The newest process developments as well as
future perspectives will be discussed.
Keywords. Anaerobic digestion, Carbon-flow, Microbiology, Antimization, Gas yild, Effluent

quality
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Microbiology of Anaerobic Digestion . . . . . . . . . . . . . . . . 3

2.1 General Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Syntrophic Acetate Conversion . . . . . . . . . . . . . . . . . . . . 5
2.3 Microbiology of Thermophilic Digestion . . . . . . . . . . . . . . 7
2.4 Establishing a Stable Microflora in Thermophilic Reactors . . . . 8
3 Anaerobic Digestion Plants . . . . . . . . . . . . . . . . . . . . . 12
4 Anaerobic Digestion as a Way to Add Extra Value . . . . . . . . . 14
5 Optimization of Anaerobic Digestion . . . . . . . . . . . . . . . . 15

5.1 Increasing the Digestibility of the Waste . . . . . . . . . . . . . . 15
5.2 Optimization of Reactor Configuration . . . . . . . . . . . . . . . 17
5.3 Optimizing Process Control and Stability . . . . . . . . . . . . . . 19
5.4 Improving the Microbial Process and its Efficiency . . . . . . . . . 22
Advances in Biochemical Engineering/

Biotechnology, Vol. 81
Series Editor: T. Scheper
© Springer-Verlag Berlin Heidelberg 2003
2 B. K. Ahring
6 Optimization of Effluent Quality . . . . . . . . . . . . . . . . . . 23

6.1 Inactivation of Pathogens and Other Biological Hazards . . . . . . 23
6.2 Control of Chemical Pollutants . . . . . . . . . . . . . . . . . . . 25
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
1
Introduction
The modern society generates large amounts of waste that represent a tremen-
dous threat to the environment and human and animal health. To prevent and
control this, a range of different waste treatment and disposal methods is used.
The choice of method must always be based on maximum safety, minimum
environmental impact and, as far as possible, on valorization of the waste and
final recycling of the end products. One of the main trends of today’s waste man-
agement policies is to reduce the stream of waste going to landfills and to recy-
cle the organic material and the plant nutrients back to soil. Waste is increasing-
ly becoming a problem and secure recirculation is gaining more and more atten-
tion. Anaerobic digestion (AD) is one way of achieving this goal and it will, fur-
thermore, reduce energy consumption, or may even be energy producing, which
is of major importance to the global environment. Anaerobic digestion has been
implemented for years as a means for the stabilization of sewage sludge; how-
ever, during the past years anaerobic digestion technologies have been expand-
ed to emphasize treatment and energy recovery from many other types of
wastes including animal wastes, source-sorted household wastes, organic indus-
trial wastes and industrial wastewater. Compared to incineration, anaerobic
digestion creates more energy during the treatment of wastes, which normally
have high water content. During incineration the nutrients are lost. Following
the increasing interest in implementation of anaerobic digestion, optimization
of this process is becoming increasingly more important. Despite the increased
efforts spent on waste reduction, the amounts of waste are increasing through-
out the world. This has led to ideas for a total removal of waste through injection
into the deep underground (below 2 km) into old oil wells far below any the
groundwater level [1]. The recovery of methane will, however, be of importance
for the feasibility and economy of this technique and methane development at
these high temperatures, pressures and salinity is now under investigation.
This chapter focuses on the perspectives for optimization of anaerobic diges-
tion after a brief introduction to the microbiology of anaerobic digestion. Opti-
mization is a double-sided task: it involves both an increase of the biogas yield,
which again implies an increased removal of the organic material in the waste,
as well as ensuring an effluent with a sufficiently high quality to allow for recy-
cling of the material as a fertilizer. A number of areas for improving the biogas
yield will be discussed such as, for example, increasing the digestibility of the
Perspectives for Anaerobic Digestion 3
waste, optimizing process control, and improving the microbial process. With
respect to effluent quality, emphasis will be on inactivation of pathogens and
control of chemical pollutants.
2
Microbiology of Anaerobic Digestion
2.1
General Scheme
A major value of anaerobic digestion is linked to the production of biogas

(methane and carbon dioxide) formed as the end product during degradation of
organic material without oxygen. This energy is renewable and CO2 neutral and
can be used for production of electricity and heat. Many different consortia of
microorganisms with different roles in the overall process scheme are needed
for the AD process, which occurs naturally in anaerobic ecosystems such as sed-
iments, paddy fields, water-logged soils and in the rumen [2].
Three major groups of microorganisms have been identified with different
functions in the overall degradation process [3] (Fig. 1):
1. The hydrolyzing and fermenting microorganisms are responsible for the ini-
tial attack on polymers and monomers found in the waste material and pro-
duce mainly acetate and hydrogen, but also varying amounts of volatile fatty
acids (VFA) such as propionate and butyrate as well as some alcohols.
Fig. 1. The anaerobic degradation process

4 B. K. Ahring
Fig. 2. Carbon flow in anaerobic environments with active methanogens
Fig. 3. Carbon flow in anaerobic environments without active methanogens
2. The obligate hydrogen-producing acetogenic bacteria convert propionate

and butyrate into acetate and hydrogen.
3. Two groups of methanogenic Archaea produce methane from acetate or
hydrogen, respectively.
The major part of the carbon flow in a well-operating anaerobic reactor occurs
between the fermentative microorganisms and the methanogens. Only between
20 and 30 % of the carbon is transformed into intermediary products before
these are metabolized to methane and carbon dioxide (Fig. 2) [4].
A balanced anaerobic digestion process demands that the products from the
first two groups of microbes responsible for hydrolyzing and fermenting the
material to hydrogen and acetate, simultaneously are used by the third group of
microbes for the production of methane and carbon dioxide. The first group of
microorganisms can survive without the presence of methanogens but will,
under these conditions, form an increased amount of reduced products such as
VFA (Fig. 3).
The second group does, however, rely on the activity of the methanogens for
removing hydrogen to make their metabolism thermodynamically possible as
their reactions are endergonic under standard conditions and only occur when
hydrogen is kept below a certain concentration. The relationship between the
VFA-degrading bacteria and the hydrogen-utilizing methanogens is defined as
syntrophic due to the dependent nature of this relationship and the process is
Fig. 4. Interspecies hydrogen transfer
called interspecies hydrogen transfer (Fig. 4) [3]. The lower the hydrogen con-
centration the better are the thermodynamics of the VFA degradation. The dis-
tance between the VFA degrader and the hydrogen utilizer does, therefore, affect
the concentration of hydrogen in the liquid phase, which again affects the ther-
modynamics of the process. Therefore, the conversion is improved in granules
and flocks compared to a situation where the microbes are distributed freely in
a liquid solution [5]. The two partners have to share a very small amount of
energy and the conditions for ensuring energy for both microbes is very strict
and can only be met within a narrow range of hydrogen concentrations [6].
2.2
Syntrophic Acetate Conversion
Syntrophic relationships have also been found to be of importance for conver-

sion of acetate when the acetate-degrading methanogens are inhibited by high
concentrations of ammonia [7, 8] or sulfite (unpublished). Under these condi-
tions the acetate-utilizing methanogens are inhibited and other groups of
microbes replace them to obtain energy from the oxidation of acetate to hydro-
gen and carbon dioxide (Fig. 5).
Due to thermodynamic constrains this reaction proceeds much better at
increased temperatures and is the way of acetate transformation when the tem-
perature is higher than 60°C, close to the upper temperature limit of ther-
mophilic acetate-utilizing methanogens [9, 10]. In accordance with this, the pop-
ulation of Methanosarcina species disappeared more or less instantaneously
from a biogas reactor operated on manure, when the temperature was increased
from 55 to 65°C [11]. Concurrently, the acetate concentration first increased and
6 B. K. Ahring
Fig. 5. Modified anaerobic degradation process with syntrophic acetate conversion
then stabilized at a level somewhat higher than that found at 60°C [12]. This
coincided with a significant increase in the population of hydrogen-utilizing
methanogens [11] indicating that this group had become dominant in the over-
all conversion. Both syntrophic acetate oxidation and methanogenesis from
acetate can be simultaneously active in a reactor system as indicated by several
isotope studies often showing that less than 95 % of the methane produced from
acetate is derived from the methyl group. Isotope experiments with biomass
from thermophilic reactors have further shown that the concentration of acetate
affects the competition between the two processes. When the concentration of
acetate is low, syntrophic acetate conversion is the major process for acetate
transformation [13, 14]. However, when the concentration of acetate is above the
threshold level [15] for the specific population of acetate-utilizing methanogens
in the reactor, these will be the major group active in the system. These findings
further explain why the numbers of hydrogen-utilizing methanogens are high in
thermophilic granules, which have exclusively been fed with acetate for a long
period [16]. Furthermore, the numbers of acetate-utilizing methanogens are
highest close to the surface of the granules, where the concentration of acetate is
highest, while the populations of hydrogen-utilizing methanogens increased
towards the center of the granules [16].
The first microbe found to perform acetate oxidation was a thermophilic bac-
terium belonging to the group of homo-acetogenic bacteria capable of reversing
the acetate-forming reaction from hydrogen and carbon dioxide [17]. This bac-
terium used a very limited range of substrates all related to its homo-acetogenic
nature [17]. Over time more microbes have been identified as being capable of
carrying out this reaction. Some of these microbes have been found to use a
large variety of substrates [18] and, furthermore, to be normal members of the
populations of fermentative microbes in thermophilic reactors. This indicates
that, at least in thermophilic reactors, syntrophilic acetate oxidation could be
performed by a variety of the fermentative bacteria in the reactor when no
other substrates are available. This needs, however, further verification.
2.3
Microbiology of Thermophilic Digestion
Microbes thriving at high temperatures have been known for years [19]. The
reaction rate of many chemical reactions will double by an increase of 10°C
according to the Arrhenius equation. The same is, however, not always the case
for microbial reactions where the temperature response is specific for the par-
ticular microbe. Different groups of microbes have been identified where the
ones of interest for anaerobic digestion are mesophilic strains with an optimum
between 30 and 40°C, and thermophilic strains with an optimum between 50
and 60 °C [20]. The mixed microflora found in an anaerobic bioreactor general-
ly shows an increasing rate from a temperature of 20 to 60 °C and the theoreti-
cal temperature gap between mesophilic and thermophilic strains is not appar-
ent when viewing the process as a whole [21]. Anaerobic digestion at a temper-
ature below 20°C, or at a temperature above 60 °C, generally shows a lower
methane yield than within these limits. However, anaerobic digestion has been
shown to be possible even at extreme thermophilic conditions of 70 °C and more
[28 – 30]. Experiments with high temperature digestion of manure showed that
major changes occurred in the microbial populations of the anaerobic reactor
when the temperature was increased from 55 to 65 °C [12]. Besides a significant
increase in the population of Archaea compared to Bacteria, also the popula-
tions of methanogens underwent large changes over time. The population of
hydrogen-utilizing methanogens did, for example, change from a major popula-
tion belonging to the genus Methanobacterium to another belonging to
Methanococcus over a 3-month period [11]. Such results clearly demonstrate
that reactors operated at extreme conditions can take months before a stable
microflora has established. With this in mind it is difficult to guess if the
methane yield actually will be lower after an extended period of many months.
Within the normal temperature range the general carbon flow of ther-
mophilic reactors was found to be very similar to that of mesophilic reactors
[31]. A slightly higher amount of the carbon was channeled directly into acetate
and a slightly smaller amount of carbon was turned over via the pool of VFA [4].
Many extreme thermophilic Bacteria or Archaea have been found to produce
mainly acetate and hydrogen as their end products [32]. Therefore, less butyrate
and propionate can be expected at these high temperatures. Different maximum
temperatures were found for the different microbial groups in a thermophilic
anaerobic reactor treating manure [33]. For instance, among the methanogens,
the acetate-utilizing methanogens have a much lower temperature maximum
(ca. 62 °C) compared to the hydrogen-utilizing methanogens (ca. 75 °C) [33].
However, the actual temperature of the reactor affects the specific populations
8 B. K. Ahring
which are active in the reactor. Therefore, a higher temperature optimum and
maximum is found for the main metabolic groups in extreme thermophilic reac-
tors compared to thermophilic reactors [28]. Methane production was found in
microbial mat samples taken from a slightly alkaline hot spring at 80 °C [34].
This demonstrates that methanogenesis is possible even at this very high tem-
perature.
2.4
Establishing a Stable Microflora in Thermophilic Reactors
Waste such as sewage sludge, manure or household waste contains many differ-
ent populations of anaerobic or facultative anaerobic microorganisms. Most of
these microbes are mesophilic and only a very small number of true ther-
mophiles is present. The number of microbes in raw sewage sludge utilizing sub-
strates such as acetate or cellulose at 60 °C is extremely low (ca. 100 per g) [35].
The numbers are somewhat higher at 55 °C but still much lower than the num-
bers at 37 °C [35]. These facts clearly show the problems of establishing stable
reactors at higher temperatures. Where the microflora of mesophilic reactors
can be established directly based on the raw material fed to the reactor, the
microflora of the thermophilic reactor has to be propagated from small minor-
ity populations found in the raw materials [36]. Many thermophilic full-scale
reactors have failed through history, especially within the area of sewage sludge
treatment. The reason is basically a lack of understanding of the principles for
establishing a stable thermophilic microflora in the reactor. The same also
applies to the literature, which is full of experiments with unstable thermophilic
laboratory reactors often performing poorly compared to mesophilic reactors.
When reviewing the literature describing these experiments, Wiegant [37] con-
cluded that process stability is lower in thermophilic reactors and that ther-
mophilic reactors generally have higher concentrations of volatile fatty acids in
the effluent compared to mesophilic reactors. During recent years where more
thermophilic reactors have been implemented, it has been shown that this con-
clusion it not correct and that stable thermophilic reactors with a balanced
thermophilic microflora perform just as well as stable mesophilic reactors [33,
38, 39].
The key to obtain a balanced thermophilic microflora is to give optimal
growth conditions to the small numbers of thermophilic populations found in
the raw material during start-up of the bioreactor [36]. If sufficient thermophilic
seed material is available, it is possible to carry out a rapid start-up of a ther-
mophilic reactor [33]. The seed material should be evaluated before use with
respect to the destruction of volatile solids in the reactor from which the seed is
obtained as well as the concentration of VFA. If possible, it will be beneficial to
perform a methanogenic activity testing of the seed material to establish the
potential of this seed for transforming extra loads of methanogenic substrates
(acetate and hydrogen) [40, 41]. After addition of the seed to an empty reactor it
should be allowed to equilibrate for 1 day before feeding is initiated at the
desired thermophilic temperature. A slow and graduate change of the tempera-
ture only prolongs the start-up phase and does not select for true thermophiles
Fig. 6. Start-up using thermophilic digested seed material

9
10 B. K. Ahring
24 h
t d = 24 h
24 h
td = 8 h
Fig. 7. Unbalanced growth of methanogens (dots) and fermentative bacteria (rods)
possessing an optimum growth rate at thermophilic temperatures. Probing of

methanogens in bioreactors has clearly demonstrated that mesophilic meth-
anogens are present in thermophilic reactors and vice versa [42]. However, the
major populations are those with an optimum temperature close to the reactor
temperature [43]. After equilibration, feeding should then be initiated corre-
sponding to a hydraulic retention time approx. 25 % higher than the retention
time of the reactor where the seed came from. Normally the reactor is only part-
ly full at this stage and, therefore, the reactor is operated in a fed-batch mode
during this period of time. During start-up, the VFA concentration should be
monitored on a daily basis. If the VFA concentrations continue to decrease after
approx. 3 days of feeding or remain at a stable low level, the hydraulic retention
time can be lowered. By repeating this pattern and, at the same time, keeping a
tight eye on the concentration of VFA – especially the isoacids [44, 45] – it is pos-
sible to reach the desired final retention time in approx. 1 month. A schematic
drawing of the expectable feeding pattern and the expectable response in VFA is
shown in Fig. 6. For sewage sludge it is possible to obtain a stable process with a
high reduction of volatile solids at a hydraulic retention time as low as 6 days at
thermophilic conditions.
If no seed is available, it is even more important to plan the start-up in a con-
trolled condition. It is important to avoid over-loading and build-up of VFA.
Therefore, a start-up material containing only small amounts of organic mater-
ial should be chosen. Mesophilic digested waste material has a much lower
organic content than raw waste material and has at least as many thermophilic
microorganisms as found in this material [46]. Immediately upon an increase of
the temperature to the thermophilic region, these thermophilic microbes will
start growing. As the acid-producing microbes grow much faster than the
Fig. 8. Start-up using mesophilic digested seed material. Arrows indicate feeding of the reactor
11
12 B. K. Ahring
methanogens, the first reaction is often an increase in the concentration of VFA

[47] (Fig. 7).
Due to the limited amount of undigested material present, this increase is
only relatively small and does not affect the over-all digestion process. Immedi-
ately after a decreasing trend is seen in the volatile fatty acids corresponding to
growth of the population of VFA-degrading and methanogenic microbes, it is
appropriate to start feeding.A portion corresponding to approximately the dou-
ble of the desired hydraulic retention time is appropriate. Depending on the
response in VFA concentration, this trend can be continued every day unless the
VFA starts to increase. After 3 to 5 days of continuous feeding it is time to lower
the hydraulic retention time again and this pattern can be continued until the
reactor has reached the desired hydraulic retention time. This is normally
reached within a period of 2 months. A schematic drawing of the expectable
feeding pattern and the expectable response in VFA is shown in Fig. 8.
The time needed for performing the start-up with a small amount of ther-
mophilic seed material can further be reduced by addition of mesophilic-digest-
ed material in addition to the daily feeding with raw waste material. This was
used with success for start-up of the new thermophilic sewage sludge digester
at Western Lake Superior Sanitary District in Duluth, Minnesota during the
summer of 2001 [48].
3
Anaerobic Digestion Plants
A large number of different AD-technologies and AD plants are found today
throughout the world. The largest number of AD plants in the modern society
treats primary and secondary sludge (biosolid) in municipal wastewater treat-
ment plants. These plants basically stabilize the waste material and the biogas
produced is often of minor importance. For some of the large wastewater plants,
the biogas produced is used for electricity production and the idea of improving
the biogas yield is attracting increased interest [49, 50]. A large number of sin-
gle household biogas units have further been implemented in developing coun-
tries such as China, India and Africa [51, 52]. These units will normally provide
gas for cooking and lighting in the households.
Another major field for anaerobic digestion is the industrial wastewater from,
especially, food processing industries where the wastewater is heavily polluted
with easily degradable organic carbon [53]. Treatment of municipal wastewater
has further been implemented in developing countries such as India especially
where the average temperature is rather constant [54]. Anaerobic treatment is
basically a way to reduce BOD while the nutrients such as nitrogen and phosphor
are left untouched [50]. Recent studies have, however, shown that nitrogen can be
denitrified in a chemoautotrophic anaerobic process using nitrite as an electron
acceptor [55, 56]. A better way to implement anaerobic treatment of municipal
wastewater would be to recover all nutrients and heavy metals from the waste-
water after the anaerobic treatment using membrane technology. In this way, the
benefits of anaerobic process with respect to space, speed, low sludge production
and cost can be fully exploited and the valuable nutrients can be reused.
A large-scale biogas facility for treating manure from several farms in combi-
nation with other organic wastes such as food wastes and source-sorted house-
hold wastes – the so-called co-digestion – was launched in Denmark at the end
of the 1980s [57, 58]. Addition of even small amounts of organic industrial
wastes increases the gas production significantly (Fig. 9). Especially fatty or oily
wastes have a much higher gas potential than manure and a much higher con-
centration of organic material (higher dry matter content) – but also wastes rich
in carbohydrates and proteins will improve the gas yield per unit of reactor vol-
ume. Digestion of sewage sludge or manure yields from about 1 – 2 cubic meter
biogas per cubic meter reactor volume per day while the reactors will produce
between 4 and 10 cubic meter biogas per cubic meter reactor volume with addi-
tion of ca. 20 % fatty waste.
Today around 22 large-scale AD plants have been built in Denmark mainly in
the regions with high manure production and all of these plants are co-digest-
ing many types of raw materials [33, 59]. The idea of large scale centralized AD
plants treating mixtures of waste have spread throughout Europe and to the rest
of the world especially during the last ten years [60]. Besides common biogas
plants, the numbers of farm biogas plants for large pig farms have steadily
increased in many European countries [61]. Many of these plants further sup-
plement the manure with raw materials with a higher gas potential. Recently, a
large number of biogas projects are on their way in USA [62] and especially in
California due to the energy crisis starting at the end of year 2000, which again
Fig. 9. Addition of 5 % fish oil will double the biogas production

14 B. K. Ahring
has resulted in higher prices on electricity. Biogas plants in USA are often much
larger compared to the so-called “large-scale” biogas plants in Denmark and
treat manure from diaries or feeding lots having up to hundred thousand cows
or from major pig or chicken production. The AD technology is, therefore, suit-
able both for small and large-scale applications. The economics depend upon
the scale, and larger plants will in general have a better economy than smaller
plants. Raw sewage sludge has a very low dry matter content and, therefore, the
potential for treating waste in these plants is only used to a low degree. Addition
of food waste, restaurant waste or organic industrial waste could be a good way
to make use of this potential. Several concepts are based on treatment of mix-
tures of sewage sludge in combination with household waste such as the Finish
Waasa process [63]. Very good results have been obtained in Grindsted, Den-
mark with co-digestion of source-sorted food waste together with sewage
sludge. The food waste was collected in paper bags and only a small amount was
removed before the digestion process [65].
4
Anaerobic Digestion as a Way to Add Extra Value
Production of biofuels from biomass such as bioethanol or gasification of bio-
mass only makes use of a fraction of the biomass. The same is true for many oth-
er biomass-based productions of non-food products. The biomass fraction left
is, however, often a good substrate for methane production (Fig. 10). In this way
biogas production can add approximately 30 % more value to the production of
bioethanol from biomass such as wet straw or corn stovers [66].
The AD process will further purify the process water allowing for recircula-
tion within the system, which will further decrease the cost of ethanol produc-
tion. In the future it is expected that more valuable products than methane will
be sought from waste. However, these niche productions will as a rule only use a
part of the waste and methane production from the final residues can add
further value to the production and will decrease the pollution load of the end
products before their final disposal.
Fig. 10. Simultaneous production of bioethanol and biogas

5
Optimization of Anaerobic Digestion
The economy of a biogas plant is directly linked to the amount of biogas pro-
duced per unit of raw material treated in the plant. Some costs are fixed such as
the cost of transporting the material to the AD plant and back again to the end
user or the end destination, while others are variable such as construction costs.
Lowering the water content of the raw material and running the process with
higher dry matter content can significantly decrease the cost of treatment.
This is of major importance when the raw material has to be transported to
a centralized biogas facility and in this case it is often beneficial to separate
the manure into a solid fraction and a liquid fraction, which is left behind at
the farm. The liquid fraction can be used as a nitrogen-rich fertilizer at the
form.
The potential for increasing the biogas yield of manure or sewage sludge is
larger as only approximately half of the organic material is converted in this type
of material. This is, however, not the case for most organic industrial wastes or
source-sorted household waste, which have been found to be more easily
degradable, and approximately 80 % of the organic material is converted to bio-
gas [67, 68]. Manure is, however, the major raw material available for a large-
scale use of AD technology in most of the world and a large-scale implementa-
tion of AD will have to be based on this raw material. AD will further improve
the quality of manure by making a more stable material with fewer pathogens
and less odor. For wastewater sludge the interest in increasing the conversion of
the organic material is further linked to the reduction in the final amount of
biosolid, which has to be disposed after the treatment. Suitable end-use of
digested sewage sludge or biosolids is becoming an increasing problem for many
communities throughout the world. Some major ways to improve the gas yield
in AD plants will be by (Fig. 11):
1. Increasing the digestibility of the waste,
2. Optimizing the reactor configuration,
3. Optimizing process control and stability, and
4. Improving the microbial process and its efficiency
5.1
Increasing the Digestibility of the Waste
Several methods have been discovered to increase the digestibility of manure or

sewage sludge ranging from mechanical, chemical to biological methods such as
enzyme treatment. Chemical treatment with bases or acids or treatment with
mixtures of enzymes have generally been found to increase the accessibility to
microbial conversion into biogas – but these processes have all been found to be
too expensive for practical implementation [69]. Decreasing the particle size
was found to increase the gas production from manure and the increase in gas
production exceeded by far the extra costs of implementing a macerating unit
with several knives [70].
16 B. K. Ahring
More gas
Sewage
Anaerobic
digestion
Animal wastes A. Optimization of
Biogenic biogas production
wastes • Increasing the
digestibility
• Optimizing reactor
Source-sorted configuration
household wastes • Optimizing process
control
• Improving microbial
process
Food-processing
wastes
B. Optimization of
effluent quality
• Inactivation of
pathogens
Crops Fertilizer • Control of chemical
pollutants
Fig. 11. Major goals for anaerobic digestion of today
A handful of wastewater plants in the world have further implemented the

use of thermal hydrolysis where concentrated sludge is treated by a combination
of high temperature (133 – 180 °C) and pressure (3 –10 bar) with the aim of
improving the digestibility of the sludge (The Cambi Process) [71]. However, the
influence of this process on gas production per unit sludge treated is still not ful-
ly documented, and the amount of sludge ending as carbon dioxide due to the
treatment is unknown. The wet oxidation process using alkaline conditions and
oxygen in addition to high temperature and pressure has been found to be supe-
rior for breaking the lignin associated to hemicellulose and cellulose as ligno-
celluloses [72]. The products of the lignin-oxidation (carboxylic acids and phe-
nolics) are further found to have highly convertible to methane and carbon
dioxide (approx. 80 % COD removal) [73]. The pure cellulose and hemicellulose
found in the hydrolysate is expected to give a methane yield corresponding to
the methane potential of the mannouronic sugars. Due to the hydrolytic capa-
bility of microbes in the AD process, it is expected that enzyme addition is not
needed for conversion of hydrolysates produced by wet oxidation. However, this
still needs to be verified along with the optimal way of implementing wet oxida-
tion as part of the AD process for materials such as manure containing a high
fraction of lignocellulosic material. Furthermore, the economics of this extra
step needs to be evaluated.
Another way to enhance the digestibility of the raw material is by Pulse
PowerTM technology developed by Scientific Utilization Inc. in Decatur, AL [74].
This equipment incorporates rapid-pulse high-power electric technology ori-
ginally developed for antimissile laser and particle-beam devices, which pro-
duces disruptive shock waves in the raw material. The shocks are expected to
break large molecules into shorter fractions and have been claimed to enhance
destruction of volatile solids by 50 to 100 %. However, a study of the efficiency of
this method in a full-scale system showed no improvements.
5.2
Optimization of Reactor Configuration
The AD process can be conducted in a single-step or multi-step process [47].

Continuous processes are generally most favorable when treating large amounts
of waste and thermophilic temperatures have the largest potential due to higher
reaction rates which corresponds to smaller reactor volumes. Separation of the
solid phase from the liquid phase of manure or sewage sludge is a technical solu-
tion which has been well documented during the last years and which can be
implemented both before and after the anaerobic reactor [75 – 77]. Separation
will further allow for optimal design of the process so that the liquid fraction can
be left at the farm, or treated locally in small compact plants, while the solid fac-
tion can be transported to a centralized plant for treatment. Especially pig
manure contains very high concentrations of phosphorus and, therefore, large
land areas are needed to use the manure as a fertilizer afterwards. If the solid
fraction is removed from the manure, the farm is able to use the liquid fraction
on a much smaller land area and pipes can be used for the spreading. After
digestion the phosphorus-rich solid fraction is an excellent fertilizer [78]. If the
separation is carried out on fresh manure approximately 70 % of the gas poten-
tial will remain in the solids [70].
Recently, we demonstrated that the conversion of organic material in manure
could be increased along with an increase in the over-all biogas yield by using a
two-phase system combining a short hydrolysis step performed at 70 °C fol-
lowed by a methane-producing step at 55 °C, both done in continuous stirred
tank reactors (CSTRs) (Fig. 12). The performance was compared to a single-
phase process in a CSTR reactor with the same over-all retention time, and the
first estimation showed that the extra gas produced was sufficient to justify the
implementation of an additional reactor and the need for extra heating energy
(unpublished).
The possibility of using an immobilized reactor system after a short hydrolyt-
ic step during a two-phase conversion of waste such as manure, sewage sludge
and household waste does possess a potential, which needs more attention
(Fig. 11).A number of systems such as the up-flow anaerobic sludge blanket reac-
tor are in use throughout the world for treatment of industrial wastewaters. How-
ever, only limited experience has been obtained from full-scale use of this reactor
for the treatment of solids [79, 80]. Having retention times in the range of hours,
the potential is apparent, as much smaller reactors are needed to treat the same
amount of waste. Furthermore, the immobilized system has lower construction
and running costs, no stirring system will be needed. The sludge produced in the
system can be recirculated back to the hydrolysis step decreasing the final sludge
production from the system, and again the phosphorus is concentrated in the sol-
18
B. K. Ahring
Fig. 12. Different reactor configurations for anaerobic digestion of waste

id fertilizer while the liquid fertilizer is rich in nitrogen. The over-all economy is,
therefore, improved although a separation is needed between the hydrolysis reac-
tor and the immobilized reactor to ensure that the amount of suspended solids in
the influent to the immobilized system is acceptable.
5.3
Optimizing Process Control and Stability
Process stability is important for the operation and economy of any AD plant.
Imbalance often affects the methanogens in the anaerobic process and leads to
a VFA accumulation [44]. It is important to note that some inhibitory com-
pounds equally affect all the major groups in the anaerobic digestion process.
This is the case for long-chain fatty acids [81, 82] or for phthalate esters such as
DEHP [83]. No VFA accumulation was observed when reactors were inhibited
with DEHP. Inhibitory compounds in waste are, however, generally either
ammonia or sulfide, which are found in high concentrations in some types of
waste [84–87]. Furthermore, high concentrations of proteins in the incoming
waste can lead to the development of inhibitory concentrations of ammonia and
sulfide. For both of these compounds, the toxic effect is dependent on pH and
temperature – the higher the temperature and pH, the higher the toxic effect
[88]. Due to the high ammonia concentration, thermophilic digestion of swine
manure has been found to be difficult [89]. Adaptation to an inhibitory com-
pound is, however, possible over time and the anaerobic process can work with
stable performance but with a lower gas yield as long as the concentration of the
toxic compound is kept relatively constant. Process stability is, however, lost if
the concentration of the inhibitor is fluctuating as seen in the large-scale biogas
plants when treating many types of wastes in different ratios. In immobilized
anaerobic systems, the biomass has generally been found capable of withstand-
ing much higher concentrations of inhibitory compounds [90]. This is probably
due to concentration gradients in the biofilm creating niches where the
microbes are protected from toxic concentrations of the inhibitory compounds.
The use of a two-phase digestion system is, therefore, expected to show superi-
or performance by compared to a one-phase system for waste containing high
concentrations of inhibitors. Increasing the biomass concentration in a biogas
reactor by recirculation of the biomass was found to increase the gas yield dur-
ing anaerobic digestion of swine waste [91, 92]. In accordance with this, the
inhibitory effect of swine manure can be counter-acted by addition of other
wastes such as lipid-containing wastes, which result in a higher biomass con-
centration in the reactor besides a dilution of the manure.
Process problems in AD plants are generally difficult to detect before the
process is severely affected and the gas production decreases. In general the
plant operator has very little information about the condition of the process and
no instruments inform him when the process is becoming unstable (Fig. 13).
As a result, the plant is often operated with a very low organic loading to pre-
vent problems from occurring. A good sensor would, however, make it possible
to optimize the operation and to ensure maximum use of the reactor space with-
out having any process failures (Fig. 14).
20
Gas
Brave
Optimum
Cautious
Suppliers
Time
Storage Tank
Storage Tank
Operator
Fig. 13. Operation of today’s biogas plant

B. K. Ahring
Gas
Optimum
Suppliers
Time
Storage Tank
Storage Tank
Registration
of data
PLC
21
Fig. 14. Future operation Operator

22 B. K. Ahring
Recently, a sensor has been developed that can measure VFA on-line in bio-
gas reactors [93]. This development allows a continuous monitoring of the
anaerobic process and with the development of logic control systems it should
be possible to improve the economy of AD plants through a more stable and
optimized operation in the future. A number of kinetic models have been
developed for anaerobic waste reactors but so far no control algorithm has
been developed and tested based on VFA data in addition to the normal data
available at the AD plant (flow rates, amount of raw materials, gas production,
temperature etc.). A combined sensor and control system can be expected in the
future.
5.4
Improving the Microbial Process and its Efficiency
Improving bioprocesses by implementation of microbial populations with

improved degradation abilities (bioaugmentation) has been known for years.
However, only few studies have been done on bioaugmentation and the results
are inconsistent. To obtain a clear picture of the potential to use specific
microbes for improvement of the process, it is necessary to follow the fate of the
microbes added to the reactor system over time. Only microbes with the abili-
ty to thrive and proliferate in the reactor will be of importance in a longer-term
prospective. Molecular techniques are available today for studies of popula-
tions in reactor systems and using such techniques we demonstrated that a spe-
cific cellulolytic bacterium, present in manure inhabited the reactor [94]. The
same technique could be used to test for specific added microbes. Compared to
controls without any pretreatment a more than 20 % increase in the methane
yield was found by incubating separated fibers from cow manure with specific
extreme thermophilic xylanolytic microbes for 2 days before the material was
resuspended in the liquid and digested [69]. This finding seems promising in
the context of the two-phase system described above and deserves further
examination.
Isolation and characterization of the acetate-utilizing methanogens from
thermophilic manure plants in Denmark showed important differences between
the different isolates of Methanosarcina species with respect to temperature
optimum and growth rates [95]. The strain derived from the best performing
thermophilic biogas plant was the acetate-utilizing methanogen with the high-
est growth rate and highest temperature optimum.When using this methanogen
to seed reactors where the organic loading was increased by a sudden addition
of lipids to the feed of manure, the seeded reactor was found to be superior to
overcome the changes compared to the unseeded reactor, which was inhibited
severely and accumulated VFA. No major accumulation of VFA was found in the
seeded reactor compared to the unseeded reactor, and biomass from this reac-
tor had a much higher specific methanogenic activity on acetate than for hydro-
gen and formate, which was almost the same in both reactors. The fact that seed-
ing had an effect even after several retention times indicates that the added
methanogen grew in the reactor as further demonstrated using a probe specific
to this strain (unpublished). These findings have practical implications and
show that better performance can be obtained when lipid-containing waste is

introduced into a biogas reactor operated on manure if the reactor is seeded
with a robust acetate-utiling methanogenic strain with a higher growth rate
than the native strain in the system.
6
Optimization of Effluent Quality
Besides production of biogas the AD plant produces a residue or effluent with a
potential market value. Until now, no applicable standards for these products
have been available and recycling of AD-residues has generally been poorly reg-
ulated in most countries. The main issues related to quality management when
recycling AD-residues is
1. to break the chain of disease transmission by inactivation of pathogens and
other biological hazards and
2. to control chemical pollutants (organic and inorganic).
Inactivation of pathogens is increased with increasing temperatures and, there-
fore, thermophilic digestion has a much high sanitary effect than mesophilic
digestion.
6.1
Inactivation of Pathogens and Other Biological Hazards
Sewage sludge and segregated household wastes are both high-risk wastes that
can be heavily contaminated with pathogens [96]. Several reviews on pathogens
in livestock waste, factors influencing microbial movement and methods for
inactivating pathogens have been published [97 – 99] New regulations with
respect to concentrations of pathogens and organic pollutants could potential-
ly be threatening to land-disposal of digested material. The regulations made
both by the US EPA and the European Union demand specific treatment
processes before the use of sewage sludge on agricultural land [1, 100]. How-
ever, for unrestricted use of digested sewage sludge a further reduction of
pathogens will be required. Several studies found anaerobic digestion to be
superior to aerobic digestion in reducing the density level of pathogens [101].
Conventional mesophilic digestion was found to be insufficient for meeting the
new requirements for unrestricted land-use [101, 102]. An increased elimina-
tion of pathogens can be achieved by using different treatment processes
including composting of sewage sludge at high temperatures, exposing the
material to radiation or specific temperatures for a defined period. Thermo-
philic digestion is still not included as a mean for producing a clean effluent.
Biosolid A demands that the number of coliforms is less than 1000 per g dry
solid, the number of Salmonella is less than 3 per 4 g dry solid, enteric virus and
helmic ova should not be detectable. These restrictions are based on logarith-
mic reduction experiments performed in buffer with the respective microbes.
However, experience obtained under anaerobic digestion showed that other
parameters than time and temperature are of importance for reduction of
24 B. K. Ahring
pathogens. The anaerobic environment seems to have an additional effect,

which is not accounted for in the biosolid classification [102]. It was demon-
strated that the high level of VFA [103], ammonia and sulfide [104] and alkaline
pH enhance inactivation of pathogens [105]. Several reports on thermophilic
AD of sewage sludge showed that thermophilic digestion is more efficient in
reducing the pathogens and pathogen indicators than mesophilic digestion
[102, 106 –108]. Further improvement of the process by extension of one
digester into a series of several reactors operating at 55 °C [110], application
of a two-stage process with an acidogenic reactor operating at 55 °C and 60 °C
coupled to a 37 °C methanogenic reactor [111], or combinations of thermo-
philic pretreatment or posttreatment at 62 °C with conventional mesophilic
digestion [109] all showed that it is possible to make a stabilized sludge fulfill-
ing the requirements for Biosolid A. Recent experiments done at Terminal
Island Treatment Plant in Los Angeles clearly showed that Class A biosolid can
be produced by switching the process from mesophilic digestion at around
35 °C to thermophilic digestion temperatures at 55 °C [112]. However, for
unrestricted use of the effluent a required holding period of one day is needed
at 55 °C, which is difficult to meet in a conventional sewage sludge treatment
system. By testing the effluent quality and demonstrating that the solid meets
all requirements it is possible to obtain a Class A biosolid classification. The
testing program, however, has to be repeated on a regular basis to maintain the
classification.
Pathogens are further of major interest when manure from several farms is
treated in centralized large-scale biogas plants. When the Danish Action Pro-
gram for Large Scale Biogas Plants was implemented more than 10 years ago,
hygienic aspects were central as a consequence of transperation of manure from
several farms. Therefore, a veterinary program was initiated and this led to
implementation of a number of control functions, which have gained major
interest and respect throughout the world [96, 113]. In this program the fecal
Streptococci (enterococci) were found to be excellent indicator organisms
instead of coliforms (the FS method) during digestion at temperatures up to
60 °C. These microbes are present in manure or other materials of intestinal ori-
gin as the coliforms, but in contrast to coliforms, they are much more resistant
to high temperatures and the anaerobic environments and, therefore, most path-
ogenic bacteria, viruses and parasitic eggs will be inactivated long before these
microbes. An FS log10 reduction of around 4 and 5 was needed to give an accept-
able effluent quality, which basically implies that the AD process is operated at
thermophilic conditions or that a high-temperature step is added to a mesophilic
reactor [96].
Pathogenic viruses have been identified in sewage sludge, segregated house-
hold waste and manure [96]. The absence of enteric viruses showed no correla-
tion with porcine parvovirus in a previous study of thermophilic anaerobic
digestion of manure [102]. This indicates that this virus could be a poor indica-
tor for human pathogenic viruses. The effect of the AD process on viruses fur-
ther depends on the way the virus is found the environment. For instance, it was
found that a virus in tissue was less sensitive than a free-living virus [114]. Much
more work is, however, needed to understand the fate of a virus during anaero-
bic digestion including the possibility to remove a resistant virus by pretreat-

ment such as thermal hydrolysis or wet oxidation.
Besides the potential for pathogens, material of animal origin such as waste
from slaughterhouses and milled bones from cows and sheep can contain infec-
tious elements resulting in transmittable spongiform encephalopathy (TSE). To
reduce the risk of spreading of these diseases the European Commission has
very recently defined special methods for slaughtering of animals to ensure that
all risk material is removed from the feed-chain [115]. This regulation defines
how the different types of wastes (animal by-products) not used for human con-
sumption have to be treated. All waste of animal origin is divided in three cate-
gories with different demands. The new regulation demands an approval of all
plants treating animal waste including quality control by the plant and society.
The banning of meat and bone meal as fodder for animals intended for human
consumption following the increased number of European cases of bovine
spongiform encephalopathy (BSE) has led to investigations of possible and safe
disposal methods of the meal. During the discussion of disposal methods,
anaerobic digestion followed by utilization of the fertilizer value by spreading
the digested sludge on arable land was suggested. The idea was, however,
abandoned, and instead, a large part of the Danish meat and bone meal is
utilized in cement production and is thereby lost from cycling of nutrients.
BSE and the variant of Creutzfeldt-Jakob disease (vCJD) are generally consid-
ered to be transmitted by the ingestion of proteinaceous agents (prions), which
accumulate in the brain and spinal cord of infected animals and humans.
The disease-causing protein (PrPSc) is an abnormal isomer of a host-encoded
protein (PrP) that has the ability to change the conformation of normal
PrP to PrPSc. The infectious PrPSc is, however, considered to be extremely resis-
tant to enzymatic degradation, heat, and chemical treatment. Proteases are
ineffective in inactivating PrPSc, and bioassays have shown that protein
remained infectious after autoclaving at temperatures up to 138 °C for 60 min
[116]. Among the different chemical inactivation methods tested, alkaline treat-
ments have so far shown most promise, although they are not completely effec-
tive. Complete inactivation might, however, be achieved by combination of
methods. Based upon one study, in which scrapie-infected hamster brain
homogenate remained infectious after 3 years incubation in soil [117], we
assume that only a minor reduction of prions will occur during the AD process
and that sufficient pretreatment will be necessary to eliminate prions before the
anaerobic reactor.
6.2
Control of Chemical Pollutants
Among the chemical pollutants, heavy metals are mainly problematic in wastes
of industrial origin and are found in high concentrations in some organic waste
and in sewage sludge from wastewater treatment plants with certain industrial
influents. Through source reduction and elimination of specific types of wastes,
it is generally possible to meet the standards regarding heavy metals for use of
residues produced by AD.
26 B. K. Ahring
Agricultural waste can contain persistent organic contaminants such as pes-

ticides, antibiotics, and other medicine residues. Industrial wastes, sewage
sludge, and household wastes can contain aromatics, aliphatic and halogenated
hydrocarbons, organochlorine pesticides, PCBs, PAHs, phthalates, linear alkyl
benzenesulfonates (LAS), nonylphenol and nonylphenol ethoxylates. During AD
most of the water-soluble organic contaminants are degraded to various
degrees. However, hydrophobic compounds such as high molecular phthalates,
PAH, and LAS are tightly bound to the particulate phase and are partly unavail-
able for biological conversion [118]. The potential to remove organic pollutants
by pretreatment of sewage sludge by wet oxidation was studied very recently.
Unfortunately, these results showed that the conditions suitable for keeping a
biogas potential in the waste material resulted in production of high amounts
of organic pollutants with a smaller molecular weight than the initial pollutants
industries (unpublished). Effectively, a complete decontamination demands
incubation at very high temperatures (more than 250 °C) and pressure, which
implies that the final gas potential is marginal and that the costs are very high.
When comparing full-scale mesophilic and thermophilic AD-reactors operated
on the same sewage sludge, it was found that the thermophilic process delivered
an effluent with significant lower concentrations of organic pollutants than the
effluent from the mesophilic reactor [119]. A higher bioavailability due to a
higher solubility of the hydrophobic elements could explain the differences
observed. Recent experiments indicated that extreme thermophilic processes
improve this reaction further but this needs to be further investigated before any
conclusions can be drawn.
7
Conclusions
Aaerobic digestion is an important way of handling waste in society. While the
emphasis previously was focused on stabilization of sewage sludge, emphasis
today is focusing on creation of an effluent, which safely can be used as a fertil-
izer on farmland. Production of biogas is furthermore gaining more attention,
especially for treatment of manure from large-scale animal production. In this
picture other types of organic waste such as wastes from food processing or
from households will be interesting as a mean of boosting the gas production
and, thereby, the economy of the AD plant. Anaerobic digestion can further add
value during use of waste or other biomasses for the production of chemicals
and energy and this synergy is expected to be further exploited in the future.
Anaerobic digestion is a mature technology today. However, as demonstrated
in this chapter, there is plenty of room for optimization and improvements. The
standardized CSTR reactor has its limitations and implementation of more effi-
cient reactor types such as the immobilized reactor systems has a major poten-
tial for treatment of solid waste. Process control on the current AD plant is still
relying on in- and output data and no information is available on-line for check-
ing the state of the process and its performance. The microbiology in AD plants
is normally regarded as a big black box and very few attempts have been made
to control the actual microflora in bioreactors treating waste. Recent research
has demonstrated that AD plants within close distance of each other can possess
different microfloras with different characteristics. Some microbial strains will
add superior characteristics to the reactor system and this has major implica-
tions for the future of AD plants. Most waste is only partly degraded in the AD
plant. Improving the digestibility of waste by using physical or chemical pre-
treatment methods, which will make the waste more accessible for anaerobic
degradation, is another area with major perspectives.
One of the most promising areas for the future is the use of extreme ther-
mophilic digestion within the AD plant. The high temperature process will allow
for better hydrolysis of the solids, for better sanitation and for better removal of
xenobiotics during the treatment process.
Acknowledgement. I would like to thank Zuzana Mladenovska, Hinnerk Hartmann, Thomas

Ishøy and Peter Westermann for valuable input to this chapter.
8
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Received: March 2002

CHAPTER 6
Metabolic Interactions Between Methanogenic

Consortia and Anaerobic Respiring Bacteria
A.J.M. Stams 1 · S.J.W.H. Oude Elferink 2 · P. Westermann 3
1 Wageningen University and Research Centre, Laboratory of Microbiology,
Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands.
E-mail: fons.stams@algemeen.micr.wag-ur.nl
2 ID TNO Animal Nutrition, Edelhertweg 15, P.O. Box 65, 8200 AB, The Netherlands.
E-mail: s.j.w.h.oudeelferink@idtno.nl
3 Department of Environmental Microbiology and Biotechnology, The Technical University
of Denmark, Building 227, 2800 Lyngby, Denmark. E-mail: pw@biocentrum.dtu.dk
Most types of anaerobic respiration are able to outcompete methanogenic consortia for com-
mon substrates if the respective electron acceptors are present in sufficient amounts. Further-
more, several products or intermediate compounds formed by anaerobic respiring bacteria
are toxic to methanogenic consortia. Despite the potentially adverse effects, only few inorgan-
ic electron acceptors potentially utilizable for anaerobic respiration have been investigated
with respect to negative interactions in anaerobic digesters. In this chapter we review com-
petitive and inhibitory interactions between anaerobic respiring populations and methano-
genic consortia in bioreactors. Due to the few studies in anaerobic digesters, many of our dis-
cussions are based upon studies of defined cultures or natural ecosystems.
Keywords. Competition, Sulfate reduction, Denitrification, Acetogenesis, Inhibition
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2 Metabolic Interactions in Methanogenic Bioreactors . . . . . . . . 32

2.1 Competitive Interactions . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.1 Kinetic Competition . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1.2 Thermodynamic Competition . . . . . . . . . . . . . . . . . . . . . 36
2.2 Inhibitory Interactions . . . . . . . . . . . . . . . . . . . . . . . . . 36
3 Competition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.1 Competition in the Presence of Oxygen . . . . . . . . . . . . . . . . 37
3.2 Competition Between Nitrogen Reducers and Methanogenic
Consortia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3 Competition Between Manganese and Iron Reducers and
Methanogenic Consortia . . . . . . . . . . . . . . . . . . . . . . . . 39
3.4 Competition Between Sulfate-Reducing and Acetogenic Bacteria
and Methanogenic Consortia . . . . . . . . . . . . . . . . . . . . . 40
3.4.1 Competition for Hydrogen . . . . . . . . . . . . . . . . . . . . . . . 41
3.4.2 Competition for Acetate . . . . . . . . . . . . . . . . . . . . . . . . 43
3.4.3 Competition for Methanol . . . . . . . . . . . . . . . . . . . . . . . 45

32 A.J.M. Stams et al.
3.4.4 Competition for Organic Acids and Ethanol . . . . . . . . . . . . . 46

3.4.5 Competition for Sulfate . . . . . . . . . . . . . . . . . . . . . . . . 48
3.5 Competition Between Sulfate-Reducers and Acetogens
in the Absence of Sulfate . . . . . . . . . . . . . . . . . . . . . . . . 49
4 Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
1
Introduction
Very few environments exist in which only one population of microorganisms
thrives or where populations of microorganisms do not affect each other either
positively or negatively. As discussed in Chap. 1, anaerobic ecosystems such
as methanogenic bioreactors are characteristic by their complex food-chains
and the close symbiotic relationship between the different links in the chain,
and are often exemplified as classical symbiotic ecosystems in which organisms
consume the products of the preceding link in the chain, rather than consum-
ing each other. The symbiosis between hydrogen-producing and hydrogen-
consuming microorganisms is confined to a narrow range of hydrogen partial
pressures outside which the reactions become thermodynamically unfavorable
for one or the other part of the relationship. This can be caused by overloading
with easily degradable compounds or by unintentional influence of inhibitory
compounds. Compounds inhibiting methane production in a digester might
exert their action either by direct inhibition of microbes in the anaerobic degra-
dation chain or by stimulating microorganisms present in the digester to com-
pete with methanogens or preceding links leading to reduced methane produc-
tion and other unfavorable effects such as corrosion [1]. In this chapter we
will discuss various types of direct and indirect competitive interactions be-
tween methanogenic consortia and anaerobic respiring bacteria in anaerobic
bioreactors.
2
Metabolic Interactions in Methanogenic Bioreactors
2.1
Competitive Interactions
Competition between two or more populations of microorganisms is a negative

relationship in which the different populations often are adversely affected with
respect to their survival and growth. Also competition is considered the most
important interaction among organisms, and is one of the major responsible
causes of the selection pressure leading to the evolution of species.
Metabolic Interactions Between Methanogenic Consortia and Anaerobic Respiring Bacteria 33
Fig. 1. Model of kinetic and thermodynamic competition among sulfate-reducing bacteria and
methanogenic Archaea
The competitive interactions among anaerobic microorganisms can be roughly

divided into kinetic competition and thermodynamic competition (Fig. 1).
Kinetic competition refers to the determination of competitive capabilities by
kinetic measurements of microbial growth, although the underlying mechanism
for the observed effects might be thermodynamic. Thermodynamic competi-
tion means that one organism is capable of growing at and maintaining a sub-
strate concentration below the minimum concentration for uptake (threshold
concentration) of other organisms due to a higher energy yield in the conver-
sion of the compound.
In anaerobic fermentation of organic compounds, numerous pathways and
combinations of pathways are used leading to different energy yields. However,
since anaerobic fermentation is internally optimized in the cells to gain a maxi-
mum energy yield and an optimal redox balance [2, 3] the energetic outcome is
often the same. This has the consequence that fermentative competitive interac-
tions are mainly of kinetic character. Most of the studies which have examined
competition between anaerobic fermenting bacteria have focused on gastro-
intestinal systems [4] and very little is known on this type of competitive inter-
action in anaerobic digestion processes. Apart from interactions between fer-
menting sulfate-reducing bacteria and acetogenic bacteria, we will not discuss
this topic in this chapter.
Table 1. The respiration hierarchy
Acceptor Product E0¢ (V)
Oxygen O2 Water H2O +0.82

Manganic ion Mn4+ Manganous ion Mn2+ +0,80
Ferric ion Fe3+ Ferrous ion Fe2+ +0.77
Nitrate NO3– Nitrogen N2 +0.76
Selenate SeO42– Selenite SeO32– +0.48
Arsenate AsO43– Arsenite AsO33– +0.14
Sulfate SO42– Sulfide HS – –0.22
Carbon dioxide CO2 Methane CH4 –0.24
Carbon dioxide CO2 Acetate CH3COO – –0.29
In contrast to aerobic conditions where most heterotrophic microorganisms

utilize oxygen as a terminal electron acceptor and in most cases follow the same
metabolic pathway ending in complete mineralization of the organic com-
pounds into CO2 and H2O, the biochemical diversity of anaerobic microbial
communities is huge. A large number of electron acceptors can be used by dif-
ferent anaerobic organisms in anaerobic respiration processes (Table 1). The
most important inorganic electron acceptors are Mn4+, Fe3+, NO3– , SO42– and CO2 .
The respiration processes where these acceptors are used are normally separat-
ed either in space or time. This is due to a different energy outcome of the
processes according to the Gibbs equation: DG0¢ = –n · F · DE0¢ in which DG0¢ is the
Gibbs free energy at pH = 7; n is the number of electrons transferred in the oxi-
dation-reduction reaction; F is Faraday’s constant (96.490 kJ/V) and DE0¢ is the
redox potential (E0¢) of the electron-accepting reaction minus the redox poten-
tial of the electron-donating reaction. From this equation it is obvious that the
larger the difference is between the redox potentials of the half-reactions, the
larger is the amount of energy available to the organism performing the reac-
tion. The consequence is a hierarchy, which often resembles the order seen in
Table 1.
In most environments, some of the respiration processes do not occur, or only
occur to a minor extent, due to the lack or exhaustion of available electron
acceptors. The energy available to a respiring organism is not only dependent
upon the difference in redox potential between electron donor and acceptor.
Also concentrations of the reactants and temperatures deviating from standard
conditions affect the energy outcome according to the Nernst equation DG =
DG0 + RT · ln [B]/[A] in which DG0 is the change in Gibbs free energy under
standard conditions, R is the gas constant, T is temperature and [B] and [A] are
the concentrations of the two components of the reaction A ¤ B. According to
the respiration hierarchy, sulfate reduction excludes methanogenic utilization
of common substrates, which is verified in high-sulfate environments such as
marine sediments [5]. However in, e.g., freshwater sediments, the two processes
can coexist or even be dominated by methanogenesis due to equilibrium dis-
placements caused by low sulfate concentrations making sulfate reduction ther-
modynamically less favorable than methane production [6].
2.1.1
Kinetic Competition
This is the classical competitive interaction, the theory of which has been estab-
lished in studies of defined cultures in chemostats [7, 8]. According to kine-
tically-based competition models, the outcome of interactions between two
microorganisms competing for the same growth-limiting substrate can be pre-
dicted from the relationship between substrate concentration and the specific
growth rate (µ) according to the Monod equation: µ = µmax ¥ S/Ks + S. Two
typical relationships can be observed in studies of competitive interactions
(Fig. 2 a, b).
In Fig. 2a, organism I will grow faster than organism II at any substrate con-
centration, while the outcome in Fig. 2b is dependent upon the substrate con-
centration. The pattern seen in Fig. 2a is typical of organisms utilizing different
electron acceptors with different energy yields for the oxidation of a common
substrate, since the energy yield is higher for the electron acceptor with the
highest redox potential at all electron donor concentrations. The pattern seen in
Fig. 2b is typical for organisms utilizing the same metabolism but having differ-
ent ecological strategies. In natural ecosystems, such as sediments, the concen-
tration of nutrients needed to support growth is often very low. Among the
organisms using the same type of metabolism under these conditions, type II in
Fig. 2b having a high substrate affinity (low Ks) and a relatively low maximal
growth rate (µmax) will normally dominate. This group is assigned to “K selec-
a
Growth rate
Substrate concentration
Fig. 2. Growth rate as a function of substrate concentration in two different scenarios (a and
b). a represents two organisms with different energy metabolism, I having the highest energy
yield. b represents two organisms with the same energy metabolism, but with different eco-
logical strategies. I is assigned to “r” selection while II is assigned to “K” selection
tion” which refers to organisms that can most effectively utilize the resources
available [9]. In gastrointestinal environments and anaerobic bioreactors, oppor-
tunistic types of organisms (type I) will normally dominate, since type II has a
longer doubling time than the retention time of the system. This group is
assigned to “r selection” referring to a high potential r value (rate of population
growth/individual) [9].
2.1.2
Thermodynamic Competition
In natural environments, the substrate concentration for most organisms is

normally well below Ks . For all organisms, there is a specific minimum concen-
tration of substrate necessary to gain conservable energy. This minimal “quan-
tum” of energy, which can be conserved, corresponds to the energy needed for
translocation of 1 proton. The phosphorylation of ATP to ADP has a DG¢ of
+49 kJ/mol corresponding to 60–70 kJ/mol when compensating for energy con-
servation efficiency [10]. Since 3 protons are needed in the phosphorylation of
ADP to ATP, we can assume that the smallest amount of energy which can be
conserved is 1/3 of the phosphorylation energy, corresponding to a minimum
DG¢ of –20 kJ/mol. Inserting this value and DG¢0 for different respiration process-
es in the Nernst equation, the substrate concentration yielding the minimum
amount of energy (the threshold concentration) can be calculated for each process
under the prevailing conditions of the specific ecosystem. Several authors have
shown that organisms utilizing electron acceptors with higher redox potentials
can maintain electron donor concentrations below the threshold for uptake
of organisms utilizing electron acceptors with lower redox potentials [11–13].
Other studies have shown that significant differences in threshold values for
common substrates also can be found among species utilizing the same type of
metabolism [14].
2.2
Inhibitory Interactions
Several compounds, which serve as electron donors to respiring bacteria, might

inhibit members of the methanogenic consortia. Also some products from
anaerobic respiration might affect the activity of these consortia. The modes of
action can be indirect by increasing the redox potential to levels that interfere
with the biochemistry of the anaerobic microorganisms, or direct by chemical
reaction with proteins or other cell constituents.
It has been assumed that many anaerobic microorganisms have specific
demands for low redox potentials in their environment to make their energy
metabolism thermodynamically possible [15]. This conception has since been
moderated and several reports have shown that the parameters controlling
growth of most anaerobes is the oxygen concentration and only to a lesser
degree the redox potential of the environment. This has been demonstrated in
studies of fermentative rumen bacteria, but also in studies of microorganisms
considered extremely sensitive to aerobic conditions [16]. Fetzer and Conrad
[17] have, for instance, demonstrated that methane production in axenic cul-
tures of Methanosarcina barkeri proceeded at normal rates in oxygen-free
media where the redox potential was elevated to +420 mV.
The direct inhibition of methanogenic consortia by electron acceptors is
mediated by several mechanisms. Oxygen is toxic to all obligatory anaerobic
microorganisms. Many anaerobes are rich in flavine enzymes, and may also con-
tain quinones and iron-sulfur proteins, which can react spontaneously with oxy-
gen to yield hydrogen peroxide, superoxide and hydroxyl radicals. Since most
anaerobes lack peroxidase, catalase and superoxide dismutases, which destroy
the reactive oxygen species, damage of essential cell components can occur upon
oxygen exposure. Superoxide dismutase has, however, been demonstrated in
some anaerobic microorganisms. Kirby et al. [18] have, for instance, character-
ized a superoxide dismutase from the obligatory anaerobe Methanobacterium
bryantii. Other electron acceptors, such as oxidized nitrogen and sulfur species,
have also been shown inhibitory to anaerobic microorganisms.
Although the metabolism of these electron acceptors is competitive to anaer-
obes utilizing electron acceptors with a more negative redox potential, the reduc-
tion of the inhibitory compounds might lead to the production of less inhibitory
compounds and, hence, relieve the inhibition. In some cases, however, the prod-
ucts of anaerobic respiration are more toxic than the parent compounds. This
will be discussed in details in the next chapters.
3
Competition
3.1
Competition in the Presence of Oxygen
Although oxygen is the naturally occurring electron acceptor yielding the high-
est amount of energy leading to effective outcompetition of anaerobic microor-
ganisms, oxygen respiration and anaerobic metabolism are mutually exclusive
processes mainly due to the toxicity of oxygen, which can be observed in all
aerobic environments. Most facultatively aerobic microorganisms capable of
anaerobic respiration suppress these processes in favor of oxygen respiration
when oxygen is present. Only environments in which rapid changes between
oxic and anoxic conditions occur, such as alternating sludge treatment basins,
favor constitutively anaerobic respiring bacteria [19]. In true oxic environments,
anaerobic processes are normally only occurring in organic-rich micro- and
macro-niches, where oxygen is depleted at a higher rate than it diffuses into the
niche.
Oxygen is normally excluded in anaerobic digestion processes, and only small
amounts might enter the reactors together with, e.g., strongly aerated substrates
[20]. Due to the low solubility of oxygen, this does normally not pose a problem
to the anaerobic microorganisms in the digester and is rapidly scavenged by
facultative bacteria. Kato et al. [21] demonstrated a high oxygen tolerance of
methanogens in granular sludge due to mainly oxygen consumption by faculta-
tively anaerobic bacteria metabolizing easily degradable substrates.
3.2
Competition Between Nitrogen Reducers and Methanogenic Consortia
From an immediate evaluation of redox potentials of methanogenesis and nitrate

reduction, it is obvious that nitrate reducers should outcompete methanogens
due to the much higher energy yield of nitrate respiration. This has been veri-
fied in a few natural environments [22]. Under most circumstances, however, the
effects of nitrogen oxides to anaerobic digestion are ambiguous and very com-
plex, and to our knowledge no certain verification of competition in anaerobic
digesters in which inhibition has been excluded has been published so far.
Denitrification and methanogenesis are performed by microbial populations
each requiring their distinct environmental conditions. Most true denitrifiers
are facultatively anaerobic bacteria utilizing either oxygen respiration or deni-
trification as sole energy source. If none of these metabolisms are possible due
to the lack of appropriate electron acceptors, the bacteria will probably not
thrive in the digester. Instead, fermentative bacteria reduce oxidized nitrogen
species for dissimilatory electron dissipation. The product is either nitrite or
ammonia, and only the reduction of nitrate to nitrite is energy yielding. The fur-
ther reduction to ammonia is considered non-energy yielding and hence with-
out competitive value. Several authors have shown that high carbon to nitrogen
ratios which are normally found in anaerobic digesters favor dissimilatory
nitrate reduction to ammonia [23], while others [24] found that a high COD/NO3–
did not favor dissimilatory reduction of nitrate to ammonia. The nature of the
carbon source has also been shown to influence whether nitrate is reduced to
ammonia or dinitrogen [25]. When glucose or glycerol was added as carbon
source, 50% of the nitrate was reduced to ammonium, while 100% was denitri-
fied completely in the presence of acetate or lactate.
Several authors have demonstrated that denitrification and methanogenesis
can proceed in the same reactor as long as the two processes are spatially sepa-
rated. Hendriksen and Ahring [26] found that denitrification took place in the
bottom of an upflow anaerobic sludge blanket reactor utilizing all available
nitrate. Methanogenensis occurred in the uppermost part of the reactor, which
was depleted from nitrogen oxides. In a mixed culture system of denitrifying
and methanogenic sludge in a digester enriched with methanol, Chen and
Lin [27] observed no competitive interactions between the two communities.
Methanogenesis was, however, inhibited as long as nitrate or nitrite was present
in the reactor. Percheron et al. [24] studied methanogenesis and nitrate reduc-
tion in an anaerobic digester fed with sulfate-rich wastewater. Sulfate reduction
was inhibited by the presence of nitrate while methanogenesis proceeded until
the onset of denitrification and production of nitrite after which it also was
inhibited. Sulfide served as electron donor for some of the denitrifying bacteria.
When sulfide was precipitated by ferrous iron, only dissimilatory nitrate reduc-
tion occurred with no nitrite production. This led to a stimulation of metha-
nogenesis compared to a control digester, probably due to extensive acetate
production by the dissimilatory nitrate reducers. No specific competitive inter-
actions between methanogens and denitrifiers were verified in this study either.
Clarens et al. [28] studied the effects of nitrogen oxides and denitrification on a
pure culture of an aceticlastic methanogen (Methanosarcina mazei) and mixed

cultures of M. mazei and a denitrifying bacterium (Pseudomonas stutzeri). It
was demonstrated that the observed cessation of methanogenesis upon nitrate
addition was a consequence of inhibition by denitrification products (NO2– ,
N2O) rather than competition by the denitrifying bacterium. The authors found
that 50 mM NO3– inhibited methanogenesis from acetate by 65% while 0.18 mM
NO2– and 0.32 mM N2O were almost completely inhibitory.
In a study of the effects of nitrogen oxides on methanogenesis and other
metabolic activities in an anoxic rice-field soil, Klüber and Conrad [22] tried to
resolve inhibition and competition among nitrogen-respiring bacteria and
methanogens. The addition of nitrate, nitrite, nitrous oxide and nitric oxide all
resulted in an immediate arrest of methanogenesis until the nitrogen oxides
were consumed. Methanogenesis then resumed at a similar or lower rate. None
of the nitrogen oxides affected acetate concentrations negatively while nitrate,
nitrite and nitrous oxide additions temporarily reduced hydrogen partial pres-
sures to low exergonic or even endergonic values for methanogenesis. Since
more than 70% of the methane produced was derived from acetate, Klüber and
Conrad’s results indicate that toxicity rather than competition is responsible
for the inhibition observed. When nitrate, nitrite or nitrous oxide were added,
sulfate or/and ferric iron concentrations increased, probably as respiration
products of sulfide and ferrous iron oxidation coupled to denitrification. The
maintenance of low hydrogen partial pressures might, therefore, be due to the
activity of iron or sulfate reducing bacteria rather than denitrifying bacteria.
The mechanisms of nitrogen oxide inhibition of methanogenesis in anaero-
bic digesters can be considered far from solved, and is probably a complex
mechanism composed of toxicity, competition, and indirect stimulation of other
respiring bacteria by oxidation of reduced electron acceptors such as ferrous
iron and sulfide.
3.3
Competition Between Manganese and Iron Reducers and Methanogenic Consortia
Both manganic ions [Mn(IV)] and ferric ions [Fe(III)] can act as potent electron
acceptors in anaerobic respiratory processes carried out by a variety of microor-
ganisms coupled to the oxidation of organic and inorganic compounds. Mn(IV)
and Fe(III) can also be reduced in non-enzymatic chemical reactions under
anaerobic conditions, and much of the effort in earlier studies of respiration of
the two compounds was devoted to the separation of non-biological from bio-
logical reductions [29]. The isolation of numerous bacteria capable of Fe(III)
and Mn(IV) reduction and properly designed experiments with environmental
samples have, however, unambiguously verified this type of bacterial respira-
tion. Several authors have shown that methanogenesis and other terminal
anaerobic processes can be outcompeted by ferric- and manganic-reducing bac-
teria due to their maintenance of acetate concentrations and H2 partial pressures
below the threshold of methanogenic Archaea and sulfate-reducing bacteria
[12]. Although respiration with Mn(IV) or Fe(III) is thermodynamically more
favorable than sulfate reduction or methanogenesis, several authors have shown
that Mn(IV) and Fe(III) respiration is less efficient with crystalline than with
amorphous forms of the two electron acceptors [30, 31]. Lovley and Phillips [12]
showed that sulfate reduction and methanogenesis are only inhibited by Fe(III)-
reducing bacteria when Fe(III) is in an amorphous form. In a study of anaerobic
respiration processes in flooded soils, Peters and Conrad [32] found that Mn(IV),
Fe(III), and sulfate reduction proceeded simultaneously, possibly due to the
crystalline structures of the Mn and Fe minerals in the soils.
Since oxidation of short-chain fatty acids and H2 are the main electron-
donating processes of both Fe(III) and Mn(IV) reduction, one could expect that
these two electron acceptors could play a significant role in anaerobic digestion
when present in high concentrations. Besides direct competitive interactions,
Mn(IV) has been shown to act as an electron acceptor in the oxidation of ele-
mental sulfur (S0) to sulfate catalyzed by sulfate-reducing bacteria [33]. This
could lead to the stimulation of sulfate reduction upon exhaustion of Mn(IV).
Very few investigations have, however, been carried out regarding the effects
of Fe(III) and Mn(IV) on anaerobic digestion. One major reason for this could
be the very low contents of iron and manganese normally found in wastewater.
In average wastewater with a BOD of 290 g O2/m3, the typical iron and manganese
concentrations have been estimated to 3.5 mg/g BOD and 0.35 mg/g BOD,
respectively [34]. In a study of the effect of ferric chloride addition to anaerobic
sludge digesters to precipitate struvite (MgNH4PO4 · 6 H2O), Mamais et al. [35]
added FeCl3 at doses ranging from 0 to 20.5 mM Fe/L. A slight increase in gas
production was observed upon FeCl3 addition, but no other effects were found.
Further investigations are needed with respect to these two electron accep-
tors to clarify their actual and potential effects on different anaerobic digestion
processes.
3.4
Competition Between Sulfate-Reducing and Acetogenic Bacteria
and Methanogenic Consortia
In environments where sulfate is present, sulfate-reducing bacteria will compete

with methanogenic consortia for common substrates. Direct competition will
occur for substrates like hydrogen, acetate and methanol. Compared with metha-
nogens, sulfate-reducing bacteria are much more versatile than methanogens.
Compounds like propionate and butyrate, which require syntrophic consortia in
methanogenic environments, are degraded directly by single species of sulfate-
reducing bacteria. The physiology of sulfate-reducing bacteria has been reviewed
before by Widdel [36],Widdel and Hansen [37] and Colleran et al. [38], while the
physiology of methanogenic consortia was reviewed by Stams [3], Schink [39]
and Verstraete et al. [40]. Some key reactions in anaerobic environments are list-
ed in Table 2.
Kinetic properties of sulfate-reducers, methanogens, and acetogens can be
used to predict the outcome of the competition for these common substrates [6,
41–44]. For bacteria growing in suspension, Monod kinetic parameters such as
the half-saturation constant (Ks) and the specific growth rate (µmax) can be used.
When bacterial growth is negligible, as is often the case in reactors with a dense
Table 2. Acetogenic and methanogenic reactions, and sulfate-reducing reactions involved in

the degradation of organic matter in methanogenic bioreactors, and sulfate-reducing biore-
actors, respectively
Reaction DG0¢ a
[kJ/reaction]
Syntrophic Acetogenic reactions

Propionate– + 3 H2O Æ Acetate– + HCO3– + H+ + 3 H2 +76.1
Butyrate– + 2 H2O Æ 2 Acetate– + H+ + 2 H2 +48.3
Lactate– + 2 H2O Æ Acetate– + HCO3– + H+ + 2 H2 –4.2
Ethanol + H2O Æ Acetate– + H+ + 2 H2 +9.6
Methanol + 2 H2O Æ HCO3– + H+ + 3 H2 +23.5
Methanogenic reactions
4 H2 + HCO3– + H+ Æ CH4 + 3 H2O –135.6
Acetate– + H2O Æ CH4 + HCO3– –31.0
Methanol Æ 3/4 CH4 + 1/4 HCO3– + 1/4 H+ + 1/4 H2O – 78.2
Sulfate-reducing reactions
4 H2 + SO42– + H+ Æ HS– + 4 H2O –151.9
Acetate– + SO42– Æ 2 HCO3– + HS– –47.6
Propionate– + 3/4 SO42– Æ Acetate– + HCO3– + 3/4 HS– + 1/4 H+ –37.7
Butyrate– + 1/2 SO42– Æ 2 Acetate– + 1/2 HS– + 1/2 H+ –27.8
Lactate– + 1/2 SO42– Æ Acetate– + HCO3– + 1/2 HS– + 1/2 H+ –80.0
Ethanol + 1/2 SO42– Æ Acetate– + 1/2 HS– + 1/2 H+ + H2O –66.4
Methanol + 3/4 SO42– + 1/4 H+ Æ HCO3– + 3/4 HS– –90.4
Homoacetogenic reactions
Lactate– Æ 11/2 Acetate– + 1/2 H+ –56.6
Ethanol + HCO3– Æ 11/2 Acetate– + H2O + 1/2 H+ –42.6
Methanol + 1/2 HCO3– Æ 3/ Acetate– + H O
4 2 –55.0
4 H2 + 2 HCO3– + H+ Æ Acetate– + 4 H2O –104.6
a DG0¢-values are taken from Thauer et al. (1977) [2].
biomass concentration, Michaelis-Menten kinetics may be used to predict which

type of organism has the most appropriate enzyme systems to degrade sub-
strates. Therefore, both the Vmax/Km and the µmax/Ks ratio gives an indication of
the outcome of competition at low substrate concentrations [42].
3.4.1
Competition for Hydrogen
In anaerobic environments methanogens, homoacetogens and sulfate-reducers
will compete for hydrogen. Thermodynamically, homoacetogenesis is less favor-
able than methanogenesis and sulfate reduction. Homoacetogens are very poor
hydrogen-utilizing organisms [13]. When grown on organic substrates like
ethanol and lactate in the presence of hydrogenotrophic methanogens, they
even produce hydrogen. In the absence of methanogens 1.5 acetate is produced
per lactate or ethanol that is degraded. However, in the presence of methanogens
only 1 acetate per lactate or ethanol is produced, while reducing equivalents are
disposed of as hydrogen.
Table 3. Selected growth kinetic data of hydrogenotrophic sulfate-reducing bacteria and

methanogens. For references see Oude Elferink [81] and Oude Elferink et al. [149]
Microorganism Ks µmax Yield a Km Vmax

(µM) (1/day) (g/mol H2) (µM) (µmol/min · g)
Sulfate reducers
Desulfovibrio
desulfuricans b 1.6–4.3 1.9 1.8–4.0 88
vulgaris b 0.7–5.5 0.6–3.1 1.3–4.0 30
Desulfovibrio G11 2.4–4.2 1.2–1.6 1.4–2.0 1.1 65
Desulfobacter hydrogenophilus 1.0
Desulfobacterium autotrophicum 0.7–1.1
Desulfobulbus propionicus b 0.2–1.7
Desufomicrobium escambium 1.4
Methanogens
Methanobacterium
bryantii 0.3–1.9 0.6
formicicum b 1.2–3.1 0.9 2
ivanovii 0.8–1.7 1.1 14
Methanobrevibacter
arboriphilus b 0.7–3.4 0.6–1.3 6.6
smithii 4.1
Methanococcus vannielii 4.1
Methanospirillum hungatei 5.8–7.3 1.2–1.8 0.3–0.5 5.0 70
strain BD 2.4–2.8
Methanosarcina
barkeri b 1.4–1.8 1.6–2.2 13 110
mazei 1.4–1.7
a The yield is given in gram cell dry weight per mol.
b Several strains.
Studies with sediments and sludge from bioreactors have indicated that at an
excess of sulfate hydrogen is mainly consumed by sulfate reducers [6, 45–49]. In
reactors with immobilized biomass the activity of hydrogenotrophic methano-
gens is completely suppressed within a few weeks when sulfate is added [50]. As
hydrogenotrophic methanogens are still present in high numbers in such reac-
tors, this effect cannot simply be explained by Michaelis-Menten or Monod
kinetic data (Table 3). In methanogenic environments the hydrogen partial pres-
sure is low. However, by addition of sulfate the hydrogen partial pressure may
even become lower. The hydrogen partial pressure becomes so low that thermo-
dynamically hydrogenotrophic methanogenesis is not possible any more (Fig. 1).
In freshwater sediments a threshold hydrogen concentration of 1.1 Pa has been
measured; this value was lowered to 0.2 Pa by the addition of sulfate [6].
An additional effect of the addition of sulfate is that hydrogen formation
becomes less important. In the absence of sulfate, hydrogen has to be formed by
acetogenic bacteria in the oxidation of compounds like lactate, alcohols, propi-
onate and butyrate. However, in the presence of sulfate, all these compounds
can be oxidized directly by sulfate-reducers without the intermediate formation
of hydrogen. However, this explanation cannot be the only one because fer-
mentative glucose- and amino acid-degrading bacteria will always form some
hydrogen.
Methanogens, which grow on H2/CO2 , are autotrophic [51].Among the hydro-
gen-utilizing sulfate-reducing bacteria both autotrophic and heterotrophic
species have been isolated [37]. The classical Desulfovibrio species require
acetate and carbon dioxide or another organic carbon source for growth where-
as, e.g., Desulfobacterium sp. can use CO2 as the sole source of carbon [37, 52, 53].
An interesting observation has been made by Brysch et al. [54]. Enrichments in
media with H2 and sulfate as energy substrates and carbon dioxide as the sole
carbon substrate resulted in stable cultures of Desulfovibrio and Acetobacteri-
um, in a cell ratio of about 20 to 1. The Desulfovibrio species required acetate for
growth, which was provided by the homoacetogenic Acetobacterium species.
Sulfate-reducing bacteria have a higher affinity for hydrogen than homoaceto-
gens, but apparently the sulfate-reducers are dependent on the homoacetogens
for synthesis of their carbon source acetate. It can be speculated that under these
conditions the kinetic properties of homoacetogens determine the kinetic
properties of the sulfate-reducers. In that case, methanogens would win the
competition for hydrogen from the sulfate-reducers even at an excess of sulfate.
Unfortunately, an experiment which could demonstrate this has never been
performed. Van Houten et al. [55, 56] started up bioreactors at high hydrogen
partial pressures with solely bicarbonate as carbon source. This led to the coex-
istence of sulfate-reducers and homoacetogens.
3.4.2
Competition for Acetate
It has been shown that in marine and freshwater sediments acetate is mainly
consumed by sulfate-reducers when sufficient sulfate is present [45, 46, 49, 57].
However, for anaerobic digesters it is less clear how acetate is degraded. A com-
plete conversion of acetate by methanogens, even at an excess of sulfate, has
been reported [46–48, 50, 58–61]. However, in some studies a predominance of
acetate-degrading sulfate-reducers was found [62–64]. Some factors which may
affect the competition between sulfate-reducers and methanogens are discussed
below.
The work of Schönheit et al. [43] has indicated that the predominance of
Desulfobacter postgatei in marine sediments could be explained by its higher
affinity for acetate than Methanosarcina barkeri. The Km values were 0.2 and
3.0 mM, respectively (Table 4). However, in bioreactors Methanosarcina sp. are
only present in high numbers when the reactors are operated at a high acetate
concentration or operated at a low pH [65]. Generally, Methanosaeta (former
Methanothrix, [66]) sp. are the most important aceticlastic methanogens in
anaerobic bioreactors [65, 67–69]. Also in freshwater sediments Methanosaeta
seems to be the most numerous acetoclastic methanogen [70]. Methanosaeta sp.
have a higher affinity for acetate than Methanosarcina sp.; their Ks is about
0.4 mM [71]. In addition, D. postgatei and other Desulfobacter species are typi-
cal marine bacteria, which have not yet been isolated in freshwater media [72].
Table 4. Selected growth kinetic data of acetotrophic sulfate-reducing bacteria and methano-
genic bacteria. For references see Oude Elferink [81] and Oude Elferink et al. [149]
Microorganism Ks µmax Yield a Km Vmax

(µM) (1/day) (g/mol ac.) (mM) (µmol/min · g)
Sulfate reducers
Desulfobacter
curvatus 0.79
hydrogenophilus 0.92
latus 0.79
postgatei b 0.72–1.11 4.3–4.8 0.07–0.23 53
Desulfotomaculum acetoxidans 0.65–1.39 5.6
Desulforhabdus amnigenus 0.14–0.20 0.6 28
Desulfobacca acetoxidans 0.31–0.41 0.6 43
Methanogens
Methanosarcina barkeri b 5.0 0.46–0.69 1.6–3.4 3.0
mazei b 0.49–0.53 1.9
Methanosaeta
soehngenii b 0.5 0.08–0.29 1.1–1.4 0.39–0.7 38
concilii 0.21–0.69 1.1–1.2 0.84–1.2 16
a The yield is given in gram cell dry weight per mol.
b Several strains.
The aceticlastic sulfate-reducers that prefer freshwater conditions, such as

Desulfoarculus baarsii [73], Desulfobacterium catecholicum [74], and Desulfo-
coccus biacutus [75], show very poor growth with acetate. Only Desulfobacteri-
um strain AcKo and Desulfotomaculum acetoxidans show good growth with
acetate under mesophilic conditions (see Table 4). Unfortunately no Ks or Km
values are available for these bacteria.
Two abundant acetate-degrading sulfate-reducers, Desulforhabdus amnigenus
and Desulfobacca acetoxidans, were isolated from sulfate-reducing bioreactors
[76, 77]. The Michaelis-Menten parameters for D. amnigenus (KM = 0.2–1 mM,
Vmax = 21–35 µmol · min–1 · g protein–1) and D. acetoxidans (KM = 0.1–1 mM,
Vmax = 29–57 µmol · min–1 · g protein–1) were in the same range as or slightly
better than those of most Methanosaeta species (KM = 0.4–1.2 mM, Vmax =
32–170 µmol · min–1 · g protein–1). This was also the case for the specific growth
rate and the threshold value for acetate, which were 0.14–0.20 day –1 and <15 µM
for D. amnigenus and 0.31–041 day–1 and <15 µM for D. acetoxidans. Reported
values for Methanosaeta species are 0.08–0.69 day–1 and 7–69 µM, respectively.
Putting all kinetic information together, it seems that the growth kinetic prop-
erties of acetate-degrading sulfate-reducers are only slightly better than those of
Methanosaeta.
When the growth kinetic properties of the sulfate-reducers are only slightly
better than those of the methanogens it can be expected that the initial relative
cell numbers affect the outcome of competition experiments. This is in particu-
lar the case for methanogenic sludge from bioreactors where a major part of the
microbial biomass may consist of Methanosaeta. When methanogenic bioreac-
tors are fed with sulfate, the few initial acetate-degrading sulfate-reducers have
to compete with huge numbers of aceticlastic Methanosaeta species. In UASB
reactors the sludge age can be as high as 0.5–1 year [78]. Visser et al. [79] have
simulated the competition between sulfate-reducing bacteria and methanogens
using a biomass retention time in the reactor of 0.02 day–1, a maximum specific
growth rate of 0.055 and 0.07 day–1 for the methanogen and sulfate-reduc-
ing bacterium, respectively, a Ks value for acetate of 0.08 and 0.4 mM acetate,
respectively, and different initial ratios of bacteria. Starting with a ratio of
methanogens/sulfate reducers of 104, it will take already one year before the
numbers of acetate-degrading sulfate-reducing bacteria and acetate-degrading
methanogens are equal. Nevertheless, long-term UASB reactor experiments of
Visser [65] showed that sulfate-reducers are able to outcompete methanogens
for acetate, even if the seed sludge initially only contains low numbers of aceti-
clastic sulfate-reducers. In his acetate- and sulfate-fed UASB reactor it took
50 days before acetate degradation via sulfate reduction was observed, and
another 50 days to increase it to 10%. The shift from 50 to 90% of acetate degra-
dation via sulfate reduction took approximately 400 days.
Methanosaeta can only grow on acetate, whereas Methanosarcina can use a
few other substrates besides acetate, like hydrogen, methanol and methylated
amines [71, 79]. Aceticlastic Desulfobacter sp. also use a limited range of sub-
strates; solely hydrogen, acetate and ethanol provide good growth [72]. Desul-
fobacca acetoxidans is also a true specialist. It only showed growth on acetate
[76]. However, Desulfotomaculum acetoxidans and Desulforhabdus amnigenus
use a wide range of the common substrates for sulfate-reducers for growth [77,
80]. It is not clear to which extent these bacteria can grow mixotrophically.
During growth on, e.g., butyrate or ethanol acetate is even excreted [80, 81].
However, if low concentrations of acetate and other substrates are used at the
same time the outcome of the competition between Methanosaeta and these sul-
fate-reducers will be affected. Gottschal and Thingstad [82] described a model
in which it is shown that during competition on mixtures of substrates in con-
tinuous cultures not only the specific growth rate determines the outcome of a
competition, but also the yield on the different substrates.
3.4.3
Competition for Methanol
Methanol is an excellent substrate for mesophilic methanogens and homoaceto-

gens. Methanosarcina species, Acetobacterium woodii, Eubacterium limosum and
Butyribacterium methylotrophicum show very fast growth on methanol [83–88]
(Table 5). The homoacetogens require externally supplied bicarbonate for
growth, while the methanogens do not. Remarkably, only a very few mesophilic
species of sulfate-reducing bacteria can grow on methanol [89–91]. The maxi-
mum specific growth rates of these sulfate-reducers are much lower than those
of the methanogens and homoacetogens. This suggests that sulfate-reducers are
poor competitors for methanol.
The competition between methanogens and homoacetogens in bioreactors
has been studied by Florencio [92] it appears that the Ks value of methanogens
Table 5. Specific growth rates and growth yields (g dry weight · mol–1 ) of methanol-utilizing
anaerobic bacteria. For references see Florencio [92], and Nanninga and Gottschal [90]
Microorganism µmax (1/day) Yield (g/mol methanol)
Methanogens
Methanosarcina barkeri
strain MS 2.35 3.5
strain 227 1.85 3.8
Methanosarcina mazei 3.24
Methanosarcina acetivorans 3.20
Homoacetogens
Acetobacterium woodii 5.3–8.2
Eubacterium limosum 2.38 7.1
Butyribacterium methylotrophicum 1.85 8.2
Sulfate reducers
Desulfovibrio carbinolicum 0.22
for methanol is 0.25 mM, while that of the homoacetogens is much higher
(16 mM). This indicates that at a low methanol concentration methanol is main-
ly used by methanogens. Only at a high methanol concentration, and addition-
ally a high bicarbonate concentration, was a substantial part of the methanol
consumed by homoacetogens.
During growth on methanol methanogens and homoacetogens produce
some hydrogen. The amount of hydrogen which is produced is affected by the
presence of sulfate-reducers. This results in the coexistence of methanol-utiliz-
ing and hydrogen-utilizing anaerobes [84, 93–95]. Thus, it seems that in mixed
communities growing on methanol there is an indirect competition between
methanogens and sulfate-reducers as well.
We have studied methanol conversion in mesophilic and thermophilic sulfate-
reducing bioreactors at high sulfate concentrations.At low temperature methano-
genesis became the dominant process, indicating that methanol is mainly
consumed by methanogens (Weijma, unpublished results). However, at a high
temperature (65 °C) sulfate reduction became the dominant process [96]. Some
thermophilic Desulfotomaculum species show excellent growth with methanol.
3.4.4
Competition for Organic Acids and Ethanol
In anaerobic environments with high sulfate concentrations, sulfate-reducing

bacteria compete with acetogenic bacteria for substrates like lactate, ethanol,
propionate and butyrate. Little is known about this competition.
The fate of ethanol and lactate in anaerobic environments is not completely
clear. A few methanogens are able to oxidize ethanol and other alcohols [97, 98].
In the presence of sulfate they can be oxidized by, e.g., Desulfovibrio species.
However, lactate and ethanol (+CO2) can also be fermented by bacteria in a pro-
pionic acid or homoacetogenic fermentation. In addition, lactate (+acetate) and

ethanol (+acetate) can be fermented in a butyric acid fermentation by Clostrid-
ium kluyveri. Chemostat experiments have indicated that at low concentrations
lactate and probably also ethanol are mainly consumed by sulfate-reducers.
Desulfomicrobium outcompeted Veillonella and Acetobacterium at low acetate
concentration. However, it appeared that the Veillonella sp. had a much higher
specific growth rate than the sulfate-reducer, 0.30 and 0.17 h–1, respectively.
Interestingly, sulfate-reducers are also able to ferment lactate and ethanol. Lac-
tate and ethanol can be oxidized to acetate and hydrogen, provided that the
hydrogen partial pressure is kept low by methanogens [99], while Desulfobulbus
species are able to ferment lactate and ethanol in a propionic acid fermentation
[37, 100, 101].
For wastewater with an excess of sulfate it is to be expected that sulfate-reduc-
ing bacteria become predominant over syntrophic fatty acid-degrading consor-
tia, because of their better growth kinetic properties (Table 6). It is obvious that
at high sulfate concentrations, sulfate-reducing bacteria grow much faster than
the syntrophic consortia.Almost no Ks and Km values for propionate and butyrate
Table 6. Specific growth rates (1/day) of sulfate-reducing bacteria and of acetogenic bacteria
in co-cultures with hydrogenotrophic methanogens/sulfate reducers, growing on butyrate or
propionate. For references see Oude Elferink [81] and Oude Elferink et al. [149]
Sulfate-reducing Syntrophic
culture co-culture
– sulfate + sulfate
Butyrate-degrading strains
Desulfoarculus baarsii 0.4
Desulfobacterium autotrophicum 0.67–1.11 27
Desulfococcus multivorans 0.17–0.23
Desulfotomaculum acetoxidans 1.11
Desulfotomaculum strain Gro111 1.2–1.3
Syntrophomonas sapovorans 0.6
Syntrophomonas wolfei 0.2 0.3
Syntrophospora (Clostridium) bryantii 0.25
sporeforming strain FMS2 0.31
sporeforming strain FSS7 0.34
non-sporeforming strain FM4 0.24
non-sporeforming strain B1 0.1
Propionate-degrading strains
Desulfobulbus elongatus 1.39
Desulfobulbus propionicus a 0.89–2.64
Desulfococcus multivorans 0.17–0.23
Syntrophobacter fumaroxidans 0.02 0.15–0.17
Syntrophobacter pfennigii 0.07 0.07
Syntrophobacter wolinii 0.06 0.02–0.10 0.18–0.21
culture PT 0.1
culture PW 0.23 0.14
a Several strains.
degradation have been reported. Therefore, a comparison of the growth of

syntrophic cultures and sulfate-reducers at low substrate concentrations is not
possible. The existence of two subpopulations of propionate-oxidizers in metha-
nogenic sludge was reported [102], a fast-growing one with a µmax of 1.2 day–1
and a Ks of 4.5 mM, and a slow-growing one with a higher affinity (µmax of
0.13 day–1 and a Ks of 0.15 mM).
Several researchers investigated the competition for propionate and butyrate
between sulfate-reducers and acetogens in anaerobic reactors and in sediment
slurries. In most cases syntrophic consortia are easily outcompeted by sulfate-
reducers [48, 50, 60, 103]. However, in some of these studies no distinction
can be made between a direct oxidation of propionate and butyrate by sulfate-
reducers and an indirect conversion whereby the fatty acids are oxidized to
acetate and hydrogen by the acetogenic bacteria followed by hydrogen con-
version via sulfate reduction. In this respect it is important to note that sulfate-
reducers keep the hydrogen partial pressure lower than methanogens, and
that propionate- and butyrate-degrading acetogens grow much faster in co-
culture with hydrogen-consuming sulfate-reducers than with hydrogen-con-
suming methanogens [104, 105]. Therefore, the reported critical role of sulfate-
reducers in mediating propionate and butyrate degradation [48, 50, 106, 108]
may be that of a hydrogen-consumer or that of a direct propionate or butyrate-
oxidizer.
Findings of Harmsen [108] and Raskin et al. [109] seem to support the direct
propionate oxidation by sulfate-reducers. The population dynamics of propi-
onate-oxidizing bacteria in two UASB reactors, one fed with propionate and
sulfate and the other with only propionate were studied. In the first reactor the
number of Desulfobulbus sp. increased rapidly, and in the second reactor the
number of syntrophic propionate oxidizers increased. It seems unlikely that
Desulfobulbus acted as a hydrogen scavenger in the first reactor, although Desul-
fobulbus sp. are able to use H2 as well as propionate, because no syntrophic pro-
pionate-oxidizers were enriched in this reactor, and all Desulfobulbus cells were
localized on the outside of the granule, not intertwined with other bacteria.
Remarkably, Syntrophobacter species are also able to grow on propionate and
sulfate [110–113]. The importance of the sulfate-dependent growth of these bac-
teria is not fully understood
3.4.5
Competition for Sulfate
At low sulfate concentrations the growth of the sulfate-reducing bacteria will be

sulfate-limited.Also under conditions of high sulfate concentrations, sulfate lim-
itation may occur due to mass transfer limitation of sulfate into the biofilm.
Nielsen [114] reported that sulfate limitation could already occur in a biofilm of
a few hundred µm thick when the sulfate concentration in the bulk solution was
below 0.5 mM.
Under sulfate-limiting conditions aceticlastic sulfate-reducers will have to
compete with other sulfate-reducers for the available sulfate. Laanbroek et al.
[115] experimented with three bacterial strains, Desulfobacter postgatei, Desul-
fobulbus propionicus and Desulfomicrobium baculatum in sulfate-limited

chemostats. They found that D. baculatum was the most successful competitor
for limiting amounts of sulfate, followed by D. propionicus and then by D. post-
gatei. The Km for sulfate of D. postgatei is 200 µM [116], a value which is much
higher than the reported Ks and Km values for several Desulfovibrio strains
(5–77 µM) [117–119]. The affinities for sulfate of Desulfobacter strain AcKo,
Desulfotomaculum acetoxidans, Desulforhabdus amnigenus and Desulfobacca
acetoxidans are not known. However, if these species have a higher Ks value than
other sulfate-reducers, one might speculate that limiting amounts of sulfate
would result in an oxidation of compounds like hydrogen, formate and butyrate
by sulfate-reducing bacteria, while acetate is used by the aceticlastic metha-
nogens.
Competition for sulfate between sulfate-reducing bacteria could explain the
results obtained in studies with sulfate-limited reactors, where acetate seemed to
be the least favored substrate for sulfate reduction, compared to propionate,
butyrate and hydrogen [50, 107, 120]
When hydrogen-utilizing sulfate-reducers have the highest affinity for sulfate
this would indicate that under sulfate-limiting conditions fatty acids are oxi-
dized in syntrophy with hydrogen-utilizing sulfate-reducers and not directly by
Desulfobulbus species.
3.5
Competition Between Sulfate-Reducers and Acetogens in the Absence of Sulfate
The role of sulfate-reducing bacteria in the anaerobic digestion in the absence of

sulfate has hardly been investigated. Yet, recent studies showed that sulfate-
reducing bacteria can be present in methanogenic sludge to upto 15% of the
total biomass [109]. It is known that several types of sulfate-reducing bacteria
have fermentative or syntrophic capacities. Widdel and Hansen [37] gave an
overview of the fermentative and syntrophic growth of sulfate-reducing bacte-
ria. Growth of sulfate-reducers in the absence of sulfate could explain the fast
response of methanogenic ecosystems to the addition of sulfate. Some substrates
which can be fermented by sulfate-reducers are pyruvate, lactate, ethanol,
fumarate and malate, fructose, serine, choline, acetoin and S-1,2-propanediol
and propanol + acetate. Sulfate-reducers can also grow as acetogens in the
absence of sulfate. Desulfovibrio sp. oxidize ethanol or lactate to acetate when
co-cultured with methanogens [99, 121–124]. It has been reported that Desul-
fovibrio sp. were the main lactate- and ethanol-degrading bacteria in a reactor
treating whey in the absence of sulfate [125, 126]. However, others reported
that only in the presence of sulfate were Desulfovibrio sp. the dominant lactate
degraders, while in the absence of sulfate lactate was fermented according to
the usual fermentation pattern of Propionibacterium [48]. Syntrophic formate
degradation has been reported for Desulfovibrio vulgaris in association with
Methanobacterium bryantii [127], and a Desulfovibrio-like organism could
syntrophically degrade alcohols like 1,3-butanediol, 1,4-butanediol, 1-butanol
and 1-propanol in the presence of 10 mM acetate and Methanospirillum hun-
gatei [128].
The role of sulfate-reducing bacteria in propionate degradation becomes

more intricate by the work of Wu et al. [129, 130]. They were the first to report
that the syntrophic conversion of propionate was mainly performed by sulfate-
reducing bacteria, and they were able to isolate such an organism. This indicates
that in the absence of sulfate certain propionate-degrading sulfate-reducing bac-
teria are able to oxidize propionate in syntrophic association with H2-consum-
ing anaerobes, while in the presence of sulfate they couple propionate oxidation
to sulfate reduction. This represents a considerable ecological advantage of this
type of sulfate-reducing bacteria over obligate syntrophic propionate-degraders
in ecosystems where sulfate is continuously or intermittently available.
Interestingly, as mentioned before, several Syntrophobacter species, including
S. wolinii [111], S. pfennigii [112], S. fumaroxidans [110, 131], strain HP1.1 [113],
were shown to grow on propionate with sulfate. For S. wolinii this finding was
very remarkable because S. wolinii grows as an acetogen in the presence of
Desulfovibrio G11 [104]. Phylogenetic research, based on 16S rRNA sequences,
showed that Syntrophobacter species belong to the Gram-negative sulfate-reduc-
ers [108, 132].
Thus far, growth of sulfate-reducers on butyrate in the absence of sulfate but
in the presence of methanogens was not yet demonstrated. However, Desul-
fovibrio sp. were detected in a fixed-bed reactor fed with butyrate without sul-
fate [133, 134].
4
Inhibition
As discussed earlier in this chapter, several substrates and products of anaerobic
respiration might have inhibitory effects on the methanogenic consortia in
anaerobic digesters.
Much of the decrease in methane production caused by intermediate nitro-
gen oxides of the denitrification process (NO2–, NO and N2O) is due to toxicity of
these compounds rather than competition and unfavorable redox conditions.
The inhibition mechanism of nitrate and its denitrification products is still
largely unknown. The reduction of oxidized nitrogen species for dissimilatory
electron dissipation by fermentative bacteria yields ammonia which numerous
authors have demonstrated to be toxic to methanogenic consortia. Ammonia
is mainly toxic in its un-ionized form (NH3) while the ammonium ion (NH 4+)
is much less toxic, and toxicity is therefore dependent upon pH and tempera-
ture of the reactor. Fig. 3 shows the effect of temperature and pH on the per-
centage of total ammonium (NH 4+ + NH3) which appears as NH3 . It is obvious
that increasing temperature and pH leads to increased NH3 concentrations in a
reactor.
If the sludge fed to the reactor simultaneously contains high amounts of pro-
teinaceous material or/and pig manure, large amounts of ammonia are released
from the fermentation of amino acids and other nitrogen-rich compounds
[135]. Ammonia has been shown to mainly affect acetate-utilizing methano-
genic Archaea, and to a lesser degree, hydrogen-utilizing methanogens and syn-
trophic bacteria [136]. A decrease in pH and an increase in the concentration of
Metabolic Interactions Between Methanogenic Consortia and Anaerobic Respiring Bacteria
Fig. 3. The effect of pH and temperature on the dissociation of H2S and NH3 . DH of the two reactions (H2S ´ HS– + H+, and
NH3 + H+ ´ NH+4 ) were calculated from enthalpies of formation [150]. Equilibrium constants at the three temperatures were then
calculated from Van’t Hoof ’s equation (ln K2/K1 = DH/R(1/T1 –1/T2). Finally, the undissociated fractions at different pH values (f)
51
were calculated from the equation f = 100 ¥ (1+K/10–pH)–1. (---)25 °C; (–––) 37 °C; (––––) 60 °C
volatile fatty acids observed in ammonia-inhibited reactors, however, point

towards an inhibition of all terminal microorganisms of the anaerobic degrada-
tion chain [137]. In two studies on the effects of high ammonia concentrations
(7 g NH 4+ – N/L) on methanogenesis from acetate, Blomgren et al. [138] and
Schnürer et al. [139] demonstrated that aceticlastic methanogenesis was dis-
placed in favor of syntrophic acetate oxidation in enriched and defined cultures
growing with acetate as the only substrate. When the anaerobic processes are
inhibited by ammonia, the decrease in pH will counteract the effect of ammonia
due to a decrease in the free ammonia concentration.
Since anaerobic reactors used in different ammonia toxicity studies have
often been operated at different pH values, it is difficult to generalize about the
inhibitory concentration as different concentrations of NH3 ammonia are pre-
sent. In most reactor studies, however, inhibitory concentrations are in the range
1.7–5 g total ammonia-N/L, corresponding to 0.4–1 g NH3-ammonia/L [135,
140, 141]. Several authors have also shown that the biogas process can be adapt-
ed to ammonia concentrations above 4 g total ammonia/L without any reduction
of the methane yield [135, 142, 143].
Sulfide produced by sulfate-reducing bacteria and by fermentation of sulfur-
containing amino acids has been shown to be inhibitory to the biogas process by
several authors [144, 145]. Similar to ammonia, it is generally assumed that the
neutral undissociated sulfide is the agent of toxicity since it is only membrane
permeable in this form [146]. The pH is therefore also an important determinant
of the toxicity, but contrary to ammonia, low pH values and low temperatures
favor the undissociated sulfide (Fig. 3) . Much of the published literature on sul-
fide toxicity does not take pH into consideration, which makes general conclu-
sions about toxicity levels difficult. Since sulfide readily reacts with most metals
to form insoluble metal sulfides, the toxicity of sulfide is also related to metal
concentrations in the sludge. However, several authors have found that sulfide
inhibits the biogas process at concentrations around 50 mg/L [144, 147]. Sulfide
and ammonia have been shown to inhibit methanogenesis in thermophilic
anaerobic digesters synergistically. A sulfide concentration of only 23 mg/L led
to an approximately 40% decrease of the methane production in a digester
treating material with a high ammonium concentration [140]. From Fig. 3 it
is obvious that optimal conditions for maintaining a low concentration of un-
dissociated H2S and NH3 are occurring at lower pH values for thermophilic
digesters than for mesophilic digesters.
5
Conclusion
Compared to many other anaerobic environments, anaerobic digesters receiving
municipal sludge or animal wastes are generally sparsely exposed to inorganic
electron acceptors. Of the large amounts of easily degradable carbon only a tiny
fraction is consumed by respiring bacteria. In digesters receiving industrial
wastes, however, significant amounts of electron acceptors stimulating anaero-
bic respiration other than methanogenesis might occur and pose a problem as
described in this chapter. In most natural environments, inorganic electron
acceptors and the corresponding respiration types are confined to distinct zones
in a stratified system. A similar zonation can be established in sludge blanket
reactors such as UASB reactors. In stirred reactors, however, the maintenance of
gradients outside particles is difficult and probably only sporadically occurring;
thus, in principle, several types of anaerobic respiration might proceed simulta-
neously given sufficient amounts of organic electron donors. The interactive
pattern of electron acceptors, intermediate products and respiring microorgan-
isms is therefore very complex under these conditions and only partly under-
stood as discussed in this chapter. The role of electron acceptors such as ferric
iron and manganese has only been very sparsely studied in anaerobic digesters.
Also the role of newly described respiration systems such as humic acid respira-
tion [148] awaits thorough investigation in anaerobic digesters.
6
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Received: January 2002

CHAPTER 6
Kinetics and Modeling of Anaerobic Digestion Process

Hariklia N. Gavala 1 · Irini Angelidaki 2 · Birgitte K. Ahring 1
1 The Environmental Microbiology and Biotechnology Group (EMB), Biocentrum-DTU,
bldg 227, The Technical University of Denmark, 2800 Lyngby, Denmark.
E-mail: hng@biocentrum.dtu.dk
2 Environment and Resources DTU, Bldg 115, The Technical University of Denmark,
2800 Lyngby, Denmark
Anaerobic digestion modeling started in the early 1970s when the need for design and effi-
cient operation of anaerobic systems became evident.At that time not only was the knowledge
about the complex process of anaerobic digestion inadequate but also there were computa-
tional limitations. Thus, the first models were very simple and consisted of a limited number
of equations. During the past thirty years much research has been conducted on the peculiar-
ities of the process and on the factors that influence it on the one hand while an enormous
progress took place in computer science on the other. The combination of both parameters
resulted in the development of more and more concise and complex models. In this chapter
the most important models found in the literature are described starting from the simplest
and oldest to the more recent and complex ones.
Keywords. Anaerobic digestion, Kinetics, Modeling
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2 Kinetics of Anaerobic Digestion . . . . . . . . . . . . . . . . . . . . . 60
2.1 Microbial Growth Kinetics . . . . . . . . . . . . . . . . . . . . . . . . 60
2.2 Hydrolysis of Biopolymers . . . . . . . . . . . . . . . . . . . . . . . . 62
2.3 Acidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
2.4 Acetogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
2.5 Methanogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3 Modeling of Anaerobic Digestion . . . . . . . . . . . . . . . . . . . . 68
3.1 Models Using Un-Ionized VFA Inhibition as the Primary Key
Parameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.2 Models Using Total VFA Inhibition as the Primary Key Parameter . . 73
3.3 Models Considering the Different Composition of Wastewater . . . . 75
3.4 Models Using H2 as the Primary Key Parameter . . . . . . . . . . . . 77
3.5 Models Using NH3 as the Primary Key Parameter . . . . . . . . . . . 84
3.6 Recent Developments on Anaerobic Digestion Modeling:
Anaerobic Modeling Task Group Work Presentation . . . . . . . . . . 89
4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

58 H.N. Gavala et al.
Abbreviations
ATP adenosine 5-triphosphate
COD chemical oxygen demand
CSTR continuous stirred-tank reactor
FBR fluidized sand-bed reactor
LCFA long-chain fatty acids
NADH reduced form of nicotinamide adenine dinucleotide
NAD+ oxidized form of nicotinamide adenine dinucleotide
VFA volatile fatty acids
1
Introduction
Anaerobic digestion is one of the main processes used for sludge stabilization.
Furthermore, anaerobic digestion is widely used for the treatment of manure,
industrial wastewaters and the organic fraction of municipal solid waste. The
microbiology of anaerobic digestion is complicated, since it involves several
bacterial groups, each performing a separate task of the overall degradation
process. So far, up to nine steps have been identified during the anaerobic con-
version of organic matter. However, one can distinguish four main steps and
three major bacterial groups (Fig. 1): the hydrolytic-fermentative bacteria that
hydrolyze and convert the organic compounds to volatile fatty acids with the
simultaneous production of hydrogen (H2) and carbon dioxide (CO2), the
acetogenic bacteria that convert the above-mentioned acids to acetic acid and
finally the methanogenic bacteria that produce methane, either from acetate or
from H2 and CO2 .
Anaerobic digestion has the advantages of producing small amounts of
sludge, requiring less nutrients and energy than an aerobic treatment process
whereas the generated biogas can be used as an energy source. Unfortunately,
anaerobic systems can be unstable and this instability is usually caused by feed
overload or by the presence of an inhibitor or even by inadequate temperature
control. This is certainly a factor that limits the applicability of anaerobic diges-
tion. Therefore, appropriate mathematical models need to be developed in order
to overcome this problem and also to design and operate efficiently anaerobic
systems.
Several models have been developed during the last 35 years. In the first stud-
ies on anaerobic process modeling, special attention was paid to the description
of the final stage of the anaerobic digestion, methanogenesis, which was consid-
ered also as the most important step of the overall process. These models were
very simple and consisted of a limited number of equations. More complicated
models describing two or even more bacterial groups and also including inhibi-
tion kinetics, pH calculations and gas-phase dynamics came later. Also much
attention has been paid to the modeling of the anaerobic degradation of “syn-
thetic substrates” such as glucose. On the other hand, and despite the difficulties,
many successful attempts exist on the modeling of the anaerobic degradation of
Kinetics and Modeling of Anaerobic Digestion Process 59
Fig. 1. Bioconversion of organic matter to methane during the anaerobic digestion process
real and complex wastewater. Nowadays, a large number of models can be found
in the literature each of them having its own potential and worth. No unified
modeling framework for the anaerobic digestion process exists so far. However,
an international anaerobic modeling task group was established in Japan in
1997. This group has now formulated a common platform for the establishment
of an anaerobic model.
In the following sections, kinetics and existing models on anaerobic sus-
pended growth systems are discussed. At first, general microbial growth kinet-
ics is presented and a discussion on hydrolytic kinetics during anaerobic diges-
tion process follows. Only some representative kinetics on acidogenesis, aceto-
genesis and methanogenesis are included in this chapter since some excellent
reviews on this subject have already been published [1–3]. Recently, a review of
some of the important models for the anaerobic digestion has been published as
well [4].
In this chapter and in order to facilitate the study of the numerous models
found in the literature, a classification has been attempted according to the pri-
mary key parameter used. Thus five major categories are distinguished: models
considering (a) the non-ionized VFA inhibition, b) the total VFA inhibition,
c) the different composition of the wastewaters, d) the H2 as regulator of the
volatile fatty acids production and e) the un-ionized ammonia inhibition. Due
to the complexity of the existing models it could be that some of the models
described and especially the recent ones use more than one key parameter. As a
rule of thumb the first two categories are mostly referring to older models that
use only one key parameter and consider the organic content of a wastewater as
a whole whereas the third category refers to models that gave much attention to
the different composition of wastewaters. On the other hand, most of the mod-
els described in fourth and fifth category are more complex and consider more
than one key parameter. Finally, a description of the common platform for an
anaerobic model that has been established so far by the international anaerobic
modeling task group is given.
2
Kinetics of Anaerobic Digestion
2.1
Microbial Growth Kinetics
Cell growth generally involves a respiratory (electron transport phosphoryla-

tion, [5]) or a fermentative (substrate-level phosphorylation, [5]) conversion of
the substrate to products (catabolism) which releases energy in the form of
adenosine 5-triphosphate (ATP). The energy obtained from the catabolic reac-
tions is used for both the synthesis of new cells and the maintenance of old ones
(anabolism).
Catabolism Substrate Æ Microbial products + Energy
Anabolism Substrate + Energy Æ Microorganisms
Metabolism Substrate Æ Microbial products + Microorganisms
In general, the metabolism of the microorganisms is coupled with the produc-
tion of ATP. The ratio of the ATP mass produced per substrate mass consumed
is defined as the ATP yield factor, YATP [6]. Accordingly, the biomass yield factor
and the product yield factor are expressed as follows:
Biomass yield factor:
DX
YX/S = 6 (1)
DS
Product yield factor:
DP
YP/S = 6 (2)
DS
where X, S, P symbolize the amounts of the biomass, the substrate and the prod-
uct, respectively.
Anaerobic degradation gives low biomass yield factors compared to aerobic;
this is due to the low energy (ATP) yield of anaerobic metabolism. In particular,
the anaerobic biomass yield factor usually lies between 0.05–0.2 g of biomass
produced per g of substrate consumed whereas the aerobic one could be as high
as 0.5 g of biomass produced per g of substrate consumed. The yield factors
could be determined either experimentally or theoretically from the stoichiom-
etry of the biochemical reactions.
The bacterial growth is often described by a series of mathematical expres-

sions according to the following Eq. (3)
rX = µ (S, X) · X (3)
where rX is the bacterial growth rate and µ symbolizes the specific growth rate of
the microorganisms.
Monod [7] suggested the following Eq. (4) for the specific growth rate:
µmax · S
µ = 03 (4)
KS + S
where µmax is the maximum specific growth rate achievable when S KS and KS
symbolizes the saturation constant, meaning the value of the limiting nutrient
(substrate) concentration at which the specific growth rate is half its maximum
value.
By combining the Eqs. (3) and (4) the microbial growth rate is as follows:
µmax · S
rX = 03 · X (5)
KS + S
whereas the substrate consumption rate, rS , follows Eq. (6).
1 µmax · S
rS = 7 · 03 · X (6)
YX/S KS + S
However, Monod’s equation is incapable of predicting the decrease of the bio-
mass concentration that is due to the endogenous respiration and the cell lysis.
McCarty [8] developed the following modified Monod equation taking into con-
sideration the endogenous respiration and the cell lysis [Eq. (7)]:
µmax · S
rX = 03 · X – b · X (7)
KS + S
where b is the specific decay rate (or decay coefficient).
In general, the decay coefficient lies around 5% of the maximum specific
growth rate. Nevertheless, the methanogens have a relatively low decay coeffi-
cient (almost 1% of their maximum specific growth rate) and thus it can be
ignored during modeling and simulations of the anaerobic process.
Other expressions of the microbial growth rate as a function of the substrate
concentration are presented in Table 1.
The above expressions are incapable of describing the bacterial growth when
an inhibitory factor is present. In anaerobic digestion many factors could inhib-
it the whole process and especially the methanogenesis step. Intermediate prod-
ucts such as volatile fatty acids and even compounds that are used as substrate
could be inhibitory at high concentration. Also hydrogen sulfide (H2S), ammo-
nia (NH3), chlorinated hydrocarbons, aromatic compounds, fatty acids and
heavy metals among other compounds are either inhibitory or toxic – depend-
ing on their concentration [13]. The most common inhibition types used in
anaerobic models are expressed according to Eqs. (8) and (9) and are those of
Table 1. Expressions of the microbial growth rate as a function of the substrate concentration
Equation Specific growth rate, µ
Sn
Moser [9] µmax · 02
S n + KS
um · S
Contois [10]
B·x+S
04
µmax · S
Grau et al. [11]
S0
02
µmax · S
Chen and Hashimoto [12]
K · S0 + (1 – K) · S
0208
Haldane [14] first used by Andrews [15] and the non-competitive inhibition
type, first introduced by Ierusalimsky [16], respectively.
1
µ = µmax · 001 (8)
KS I
5+5+1
S KI
S KI
µ = µmax · 01 · 0 (9)
KS + S KI + I
where KI is the inhibition constant and I symbolizes the concentration of the
inhibitor.
Haldane inhibition has been used by several researchers for describing the
inhibition caused by either the un-ionized volatile fatty acids (butyrate, propi-
onate, acetate) or the total volatile acids concentration. Other investigators have
used the non-competitive inhibition type in order to describe the inhibition
caused by either the volatile fatty acids or other toxic substances, e.g., ammonia.
Dinopolou et al. [17] studied the inhibition of acidogenesis by volatile fatty acids
and they concluded that the non-competitive type describes it better. Mösche
and Jördening [18] studied the inhibition of acetate and propionate degradation
by propionate (substrate inhibition) and the inhibition of propionate degrada-
tion by acetate (product inhibition). They concluded that the substrate inhibi-
tion is best described by the Haldane equation whereas the non-competitive
type of inhibition describes the product inhibition best.
2.2
Hydrolysis of Biopolymers
Hydrolysis means both the solubilization of insoluble particulate matter and the
biological decomposition of organic polymers to monomers or dimers, which
can pass the cell membrane. Hydrolysis of organic polymers is usually carried
out by extracellular enzymes (hydrolases) and it may or may not be the rate-lim-
iting step of their bioconversion under anaerobic conditions. However, solubi-

lization is not necessarily an enzymatic process catalyzed by biologically pro-
duced enzymes but could take place due to physicochemical reactions as well. It
is very difficult to describe the whole process by reliable kinetics since hydroly-
sis of a complex, insoluble substrate depends on many different parameters such
as the particle size, pH, production of enzymes, diffusion and adsorption of
enzymes to particles.
Hydrolysis of organic polymers is often described by a first-order kinetic
model [Eq. (10)] as the enzymatic activity is not directly coupled to the bacteri-
al growth [1, 2]. Nevertheless, it has been reported that a first-order function
may be most appropriate for complex, heterogeneous substrates, while other
hydrolysis functions may be more appropriate for single homogeneous sub-
strates [19]. However, McCarty and Mosey [20] claimed that hydrolysis could
be considered as a microbial process and, besides first-order kinetics, they sug-
gested a “pseudo-Monod” equation with a high saturation constant (5000–
10000 mg/L).
rS = Kh · S (10)
where Kh is the hydrolytic constant.
A really interesting approach is described in the study of Vavilin et al. [21]
where hydrolysis of complex organic matter is considered as a two-phase
process. The first phase is a bacterial colonization, during which the hydrolytic
bacteria cover the surface of the solids and its rate depends on the contact area
available. Hydrolytic enzymes degrade the solid surface at a constant depth per
unit of time during the second phase.According to the aforementioned study the
hydrolysis rate is given by Eq. (11).
rS = Kh · SF1/3 · S2/3 (11)
where SF is the concentration of influent biodegradable organic matter. The
hydrolytic constant Kh of the two-phase model is a function of the ratio between
the characteristic sizes of bacteria and particles hydrolyzed according to
Eq. (12).
ÇB d
Kh = 6rmS · 5 · 4 (12)
ÇS dS
where rmS is the maximum specific hydrolysis rate, ÇB and ÇS are the bacterial and
particles densities, respectively, d denotes the depth of the bacterial layer and dS
is the current diameter of particles. This approach may be reduced, in some cas-
es, to the Contois kinetics [10] (Table 1) as it predicts exponential growth of the
hydrolytic biomass at a high solids-to-biomass ratio and first-order kinetics at a
low solids-to-biomass ratio.
Information about the hydrolysis of the undissolved part of different waste-
waters is presented in Table 2. In all studies first-order hydrolysis kinetics were
assumed. However, in the study of Miron et al. [22] on hydrolysis of the compo-
nents of primary sludge, it was reported that none of them followed first-order
hydrolysis. They concluded that hydrolysis still remains the less defined step in
the anaerobic digestion process. Furthermore, Schober et al. [23] concluded that
Table 2. Kinetic constant (d–1) values for the hydrolysis of the undissolved part of different
wastewaters
Substrate k h (d–1 ) Temperature (°C) Reference
Mixture of primary and 0.077 25 calculations from [1]

secondary sludge 0.150 35 experimental
results from [25]
Primary sludge (from a domestic 0.007–0.990 35–60 [26]
wastewater treatment plant)
Algae biomass 0.11–0.032 20 [27]
Primary sludge (from a domestic 0.4–1.2 35 [28]
wastewater treatment plant)
Secondary sludge 0.168–0.6 35 [29]
zero-order kinetics describes better the hydrolysis in the acidogenic reactor dur-
ing the two-stage anaerobic digestion of municipal solid organic wastes. On the
other hand,Vavilin et al. [24] reported that Contois kinetics (Table 1) are prefer-
able to the traditional first-order kinetics when considering the optimal design
of a two-stage anaerobic digestion system.
A wide range of hydrolysis rate constants concerning the hydrolysis of carbo-
hydrates, proteins and lipids has been reported assuming first-order hydrolysis.
Some representative values coming from different studies are presented in the
following paragraphs. However, one should take into account that the substrate
hydrolysis rate depends very much on the origin and the previous acclimation
of the anaerobic culture [30–32]. The dependency of the hydrolytic constant on
the previous acclimation of the anaerobic culture coming from the study of
Gavala et al. [31] is presented in Table 3.
Many studies on the hydrolysis of carbohydrates under anaerobic conditions
have been made while much attention was paid to the hydrolysis of cellulose in
the rumen [33–36]. The study of O’Rourke [28] on the hydrolysis of cellulose in
a continuous, lab-scale reactor gives interesting information on the factors that
influence the hydrolytic constant assuming first-order hydrolysis. The results of
the aforementioned study are presented in Table 4.
Proteins are hydrolyzed by extracellular enzymes, the proteases, into
polypeptides and amino acids. It has been reported in the past that the protein
hydrolysis is a slower process than the carbohydrate hydrolysis [37]. However,
Table 3. Dependence of the hydrolytic constant on the previous acclimation of the anaerobic
culture [31]. The substrate was a mixture of piggery, olive mill and dairy wastewater
Inoculum Hydrolysis constant (d–1)
Undissolved proteins Undissolved carbohydrates
Digested piggery wastewater 0.68 0.28

Digested olive-mill wastewater 0.35 0.19
Digested dairy wastewater 0.24 0.13
Table 4. Hydrolytic constants (d–1) for cellulose hydrolysis as a function of temperature and
solids retention time [1, 28]
Temperature (°C) Solids retention time (days)
5 10 15 30 60
35 1.95 1.21 0.62 0.38 0.21

25 0.29 0.27 0.27 0.34 0.16
20 0.09 0.14 0.13 0.14 0.10
15 – 0.05 0.03 0.10 0.08
Table 5. Representative values of the hydrolytic constant, Kh , for different proteins hydrolysis
Substrate Kh (d–1) Reference
Casein 0.35 [38]

Gelatin 0.60 [38]
Corn-protein 0.04 [39]
the hydrolysis rate depends very much on the solubility of the protein, the pH
and the origin of the anaerobic culture. Some representative values of the
hydrolytic constant, Kh , (assuming first-order hydrolysis) for the hydrolysis of
casein, gelatin and corn-protein under anaerobic conditions, are presented in
Table 5.
The term “lipids” includes a heterogeneous group of biomolecules which are
soluble in organic solvents of low polarity and not in water. The first step of
lipids biodegradation under anaerobic conditions is their hydrolysis by a group
of esterases, the lipases. For example, hydrolysis of one molecule of a phospho-
glyceride results in one molecule each of glycerin and phosphoric acid and two
molecules of fatty acids. In the literature, not much information exists on the
anaerobic biodegradation of specific lipids. On the contrary, many studies exist
on the hydrolysis of lipids when considering them as a homogeneous part of the
organic load of a wastewater. The study of O’Rourke [28] on the hydrolysis of the
lipid part of the primary sludge in a continuous, lab-scale reactor gives interest-
ing information on the factors that influence the hydrolytic constant assuming
first-order hydrolysis. The results are presented in Table 6.
Christ et al. [40] studied the hydrolysis of carbohydrate, protein and lipid
fractions in different organic waste. The ranges of the hydrolysis constant values
are presented in Table 7. For comparison purposes, the results of the literature
study of Gujer and Zehnder [1] on the first-order hydrolysis of complex bio-
molecules are presented in Table 7 as well.
Table 6. Hydrolytic constants (d–1) for the hydrolysis of the lipid part of the primary sludge as
a function of temperature and solids retention time [1, 28]
Temperature (°C) Solids retention time (days)
5 10 15 30 60
35 0.01 0.17 0.11 0.06 0.04

25 0 0.01 0.09 0.07 0.03
20 0 0 0.02 0.05 0.03
15 – 0 0 0 0
Table 7. Kinetic constants (d–1) for carbohydrate, protein and lipid hydrolysis
Substrate Reference
[40] [1]
Carbohydrates 0.025–0.2 –
Cellulose – 0.04–0.13
Proteins 0.015–0.075 0.02–0.03
Lipids 0.005–0.010 0.08–1.7
2.3
Acidogenesis
During the acidogenesis step the dissolved organic matter is biodegraded main-
ly to volatile fatty acids and alcohols by a heterogeneous microbial population.
The dominant species in anaerobic digesters is the bacteria while small popula-
tions of protozoa, fungi and yeasts have been reported as well [41]. It is mainly
the obligatory and facultative anaerobic bacteria that carry out fermentative
conversion of the substrate to products. Much attention was paid to the acido-
genesis of carbohydrates during the last decades and in almost all cases Monod
kinetics was assumed. On the contrary, limited data exist on the kinetics of
anaerobic degradation of amino acids despite the fact that the pathways of their
anaerobic biodegradation and their corresponding products have been exten-
sively studied. In Table 8 representative kinetics concerning the anaerobic
biodegradation of glucose, cellulose and starch are reported. Many studies also
exist on the production stoichiometry of the various products coming from car-
bohydrates and/or proteins metabolism. Most important factors that influence
the production stoichiometry are the interspecies hydrogen transfer [42–45], the
pH [46], the dilution rate [46, 47] and the previous acclimation of the anaerobic
culture [32].
Table 8. Representative values of kinetic constants concerning the acidogenesis of carbohydrates
Culture acclimated in Substrate µmax (h–1) YX/ S (mgVSS/mgCOD) KS (g/L) Doubling time Temperature Reference
(h) (°C)
Primary sludge glucose 0.3 0.15 0.4 2.3 36.5 [48]

cellulose 0.071 0.16 0.0368 9.8
Synthetic substrate glucose 1.25 0.27 0.0225 37 [49]
coming from agri-

cultural products
dextrose glucose 1.25 0.162 0.0225 0.5 37 [50]
Primary sludge glucose 2.76 0.0706 35 [51]
starch 1.56 0.591
glucose 0.323 0.494 37 [52]
various 0.3–1.25 0.14–0.17 24–672 37 [2]
carbohydrates
67
2.4
Acetogenesis
In general, two different types of acetogenic mechanisms can be distinguished:

(a) acetogenic hydrogenations and (b) acetogenic dehydrogenations.Acetogenic
hydrogenations include the production of acetate as a sole end product either
from fermentation of hexoses or from CO2 and H2 . Usually the step of acetoge-
nesis in anaerobic digestion refers to acetogenic dehydrogenations and specifi-
cally to the anaerobic oxidation of long and short (volatile) chain fatty acids.
Obligate proton-reducing or obligate hydrogen-producing bacteria carry out
anaerobic oxidation of fatty acids. They are inhibited by even low hydrogen par-
tial pressures and consequently they can survive only in syntrophic association
with microorganisms that consume hydrogen such as the acetoclastic methano-
gens. Many studies have been performed so far on the anaerobic oxidation of
long- and short-chain fatty acids. Table 9 includes representative kinetics con-
cerning the anaerobic biodegradation of some long-chain fatty acids while
kinetics concerning the bioconversion of propionate and butyrate to acetate are
reported in Table 10.
2.5
Methanogenesis
A very limited number of organic compounds are used as carbon and energy
sources supporting growth of methanogenic bacteria. So far, CO2 , CO, formic
and acetic acid, methanol, methylamines and dimethyl sulfide have been identi-
fied as substrates for methanogenesis. Almost the 65–70% of the methane pro-
duced in the anaerobic digesters comes from acetate. On the other hand
methanogenesis from CO2 and H2 has a significant role as well by keeping a low
hydrogen pressure and thus supporting the growth of bacteria which carry out
anaerobic oxidation of long- and short-chain fatty acids. Methanogenic bacteria
are extremely sensitive to temperature, loading rate and pH fluctuations and
they are inhibited by a number of compounds as has already been reported.
Many studies exist so far on the isolation and kinetic characterization of specif-
ic methanogenic bacteria utilizing acetate and/or hydrogen. For the purposes of
this chapter only representative kinetics of methanogenesis from mixed cultures
are presented in Table 11.
3
Modeling of Anaerobic Digestion
3.1
Models Using Un-Ionized VFA Inhibition as the Primary Key Parameter
The first model that takes into consideration the inhibition of methanogenesis
caused by the volatile fatty acids (VFA) is that of Graef and Andrews [63]. This
study considers only one bacterial population, the acetoclastic methanogens.
Assuming that all VFA can be represented and expressed in acetic acid units,
Table 9. Representative values of kinetic constants concerning the anaerobic oxidation of long chain fatty acids [2]
Temperature KS µmax Y b Reference

(°C) (mgCOD/l) (d–1) (mgVSS/mgCOD) (d–1)
Products coming from 20 4620 0.139 0.04 0.015 [28]

hydrolysis of lipids 25 3720 0.171 0.04 0.015
found in primary sludge 35 2000 0.252 0.04 0.015
Saturated long-chain fatty acids 37 (mean value) [53]

myristic (C14) 105 0.105 0.11 0.01
palmitic (C16) 143 0.110 0.11 0.01
stearic (C18) 417 0.085 0.11 0.01
Unsaturated long-chain fatty acids 37 [53]
oleic (C18) 3180 0.44 0.11 0.01
linoleic (C18) 1816 0.55 0.11 0.01
69
Table 10. Representative values of kinetic constants concerning the bioconversion of propionate and butyrate to acetate
70
Substrate Kinetics for Temperature KS µ max Y b Reference

(°C) (mgCOD/l) (d–1 ) (mgVSS/mgCOD) (d–1)
Propionate propionate 25 1144 0.051 0.04 [54]
Propionate propionate 35 78 0.042 0.01
Butyrate butyrate 35 13 0.047 0.027
Glucose butyrate 37 298 0.86 – – [55]
Propionate propionate 35 17 0.13 – – [56]
(HRT: 14.5d)
Propionate propionate 35 499 1.2 – –
(HRT: 8.2d)
Acetate: propionate: [57]
Butyrate = 2:1:1 mixed acids 35 166 0.414 0.030 0.099
Butyrate butyrate 60 12 0.45 0.019 – [58]
Propionate propionate 33 11 – – – [59]
Table 11. Representative values of kinetic constants concerning methanogenesis from acetate and hydrogen in anaerobic mixed cultures
Culture acclimated in Methanogenesis from Temperature YX/S KS µmax Reference

(°C) (mgVSS/mgCOD) (mgCOD/l) (d–1 )
Municipal wastewater acetate 35 [60]
Municipal wastewater acetate 25 0.050 930 0.25 [54]
Municipal wastewater acetate 36.5 0.28 5 0.49 [48]
Municipal wastewater acetate 33 20 [59]
Glucose acetate 37 198 [61]
Acetate acetate 37 49 [61]
Municipal wastewater hydrogen 30 0.07–0.109 11–69 mgCOD/l/h [62]
H.N. Gavala et al.
Graef and Andrews developed the following stoichiometry [Eq. (13)] for their
conversion to methane.
CH3COOH + 0.032NH3 Æ 0.032C5H7NO2 + 0.92CO2 + 0.92CH4 + 0.096H2O
(13)
where the empirical formula C5H7NO2 corresponds to the composition of metha-
nogenic bacteria.
The growth of biomass and the substrate consumption is assumed to follow
inhibition kinetics according to Eq. (8). Both the substrate and the inhibitor are
the un-ionized VFA (AcH) expressed as acetic acid. The concentration of the un-
ionized form is calculated according to acetate dissociation reaction:
AcH ¤ Ac– + H+ (14)
The model includes a total ion balance and takes into consideration three phas-
es, gas, liquid and biological (solid) phase. Methane is considered to be water
insoluble, whereas the carbon dioxide produced is partly dissolved and partly
escapes to the gas phase. This model has been used to simulate digester start-up
and response to organic overload and was able to predict digester failure due
to a temporary accumulation of VFA, which lowers the pH and subsequently
increases the concentration of un-ionized VFA. The introduction of a first-order
equation describing the rate of microorganisms’ death in the model [Eq. (15)]
gives us the possibility to predict digester failure due to toxic substances.
rK = KT · TX (15)
where rK is the rate of organisms death due to the toxic substance, KT is the tox-
icity rate constant and TX is the concentration of the toxic compound.
Hill and Barth [64] developed a model describing the animal waste digestion
(Fig. 2). Their model considers two microbial groups, the acid-formers and
the acetoclastic methane-formers and it is using un-ionized VFA (expressed as
acetic acid) inhibition of both microbial groups. Furthermore, it considers a
hydrolytic step and ammonia (un-ionized) inhibition of methane-formers.
This double inhibition has been incorporated into the growth kinetics of the
methanogens according to the Eq. (16).
µmax
µ = 0000 (16)
KS AcH NH3
1+8+8+8
AcH KI, 1 KI, 2
Kleinstreuer and Poweigha [65] and Marsili-Libelli and Nardini [66] published
simulation studies of a digester receiving soluble and insoluble organic com-
pounds, respectively. Their models are based on un-ionized VFA inhibition of
methanogenesis and the basic steps considered are presented in Fig. 3.
Moletta et al. [67] developed a model for the anaerobic digestion of glucose
(Fig. 4). It considers two steps: glucose biodegradation to acetate and methano-
genesis from acetate and it is based on un-ionized VFA inhibition of both acido-
genesis and methanogenesis. The model simulated satisfactorily the methane
production during batch experiments with pea bleaching wastewater and with a
synthetic medium consisting of sucrose and acetic acid.
Fig. 2. Block diagram of the Hill and Barth [64] mathematical model
Fig. 3. Block diagram of the Kleinstreuer and Poweigha [65] (a) and the Marsili-Libelli and
Nardini [66] (b) mathematical models
Fig. 4. Flow chart of the Moletta et al. [67] model

Fig. 5. Flow chart of the Smith et al. [68] model
A model developed by Smith et al. [68] considers three steps (Fig. 5) assum-
ing that the insoluble organic material used as feedstock (biomass) consists of
two components, a rapidly and a slowly biodegradable one. In the first step, the
two insoluble biomass components are converted to soluble intermediates that
serve as substrate for volatile fatty acid-producing bacteria during the second
step. Finally in the third step, the methanogenic bacteria convert volatile fatty
acids to methane and carbon dioxide. Smith et al. used two inhibition types: a)
the un-ionized volatile fatty acids inhibition of the methanogenesis step and b)
the total volatile fatty acids inhibition of the acidogenesis step. However, the
model has not been experimentally verified.
Finally, Märkl [69] published a study concerning the quantitative analysis of
the modern biogas reactor systems. This study was based on the model of Graef
and Andrews [63] and especially on its physicochemical assumptions.
3.2
Models Using Total VFA Inhibition as the Primary Key Parameter
The first model considering total VFA inhibition was that of Hill [70]. This mod-
el was developed in order to simulate methanogenesis from animal wastes. The
model considers five bacterial groups and four steps (Fig. 6). During the first
step complex organic material enters the digester and is converted by extracel-
lular enzymes to soluble, biodegradable organic matter. A set of “biodegradabil-
ity constants” from another study [71] was used in this hydrolysis stage. During
the second step (acidogenesis), the soluble organic matter is biodegraded main-
ly to butyrate, propionate and acetate. In the third step (acetogenesis) acetate is
produced from butyrate and propionate whereas the fourth step (methanogen-
esis) refers to the methane production from acetate and hydrogen. All five
bacterial groups catalyzing the three last steps are assumed to be inhibited by
total VFA concentration. This inhibition is expressed both in the growth rate
according to Eq. (8) and microbial decay rate according to Eq. (17), which was
developed by Hill et al. [72].
bmax
b = 04 (17)
Kb
1+8
VFA
where b is the specific decay rate [Eq. (7)], bmax is the maximum specific decay
rate and VFA is the concentration of total VFA.
Fig. 6. Flow chart of the Hill [70] model
Comparison of steady-state predictions with experimental data from six-

teen pilot and full-scale biogas plants digesting animal wastes validated the
model of Hill (1982). The same model was used for the design of param-
eters and operating characteristics of swine and poultry [73], beef cattle
[74] and dairy cattle manure [75] anaerobic digestion systems. Additionally,
Durand et al. [76] used the Hill’s (1982) model for predicting the steady state
and dynamic performance of swine manure digesters and a satisfactory
agreement between the experimental results and the theoretical predictions
was noted.
The model of Kalyuzhnyi [77] is the last one of a series of models [78–82]
dealing with anaerobic digestion of glucose (Fig. 7). It consists of five steps and
considers five bacterial groups: the acidogens that ferment glucose to butyrate,
propionate, acetate and ethanol at an experimentally defined stoichiometry
with the simultaneous production of hydrogen and carbon dioxide, the obligate
proton reducers that convert butyrate and propionate to acetate, the ethanol
degrading acetogens, the acetoclastic methanogens and finally the hydrogeno-
trophic methanogens. All steps are pH-dependent according to the pH function
[Eq. (18)] of Angelidaki et al. [83]. Hydrogen inhibition of the acidogenic and
both acetogenic steps, acetate inhibition of the butyrate-degrading step and
ethanol and butyrate inhibition of both methanogenic steps is taken into con-
Fig. 7. Flow chart of the Kalyuzhnyi [77] model
sideration. Model validation has been made using batch kinetic experiments
with glucose.
1 + 2 · 100.5(pKl–pKh)
F (pH) = 00002 (18)
1 + 10(pH – pK h ) + 10(pKl – pH)
where pKl and pKh denote the lower and upper pH values where the microbial
growth rates are approximately 50% of the uninhibited rate.
3.3
Models Considering the Different Composition of Wastewater
The models described so far do not take the different compositions of waste-
water into account. Specifically, models dealing with the anaerobic digestion of
complex wastewaters, such as animal slurries, considered the organic content as
a whole, which was hydrolyzed and degraded with an experimentally measured
rate. This assumption on the one hand simplifies the models and makes their
use easier, but on the other hand limits their applicability since such models are
useful only for the anaerobic digestion of the specific wastewater they are devel-
oped for.
The first model suggesting that the complex biodegradable particulate part of
a wastewater is hydrolyzed to protein, carbohydrates and lipids and subsequently
to amino acids, simple sugars and fatty acids respectively, is that of Bryers [84].
However, due to the lack of information, the particulate matter, proteins, carbo-
hydrates and lipids are considered as a whole, while the amino acids and simple
Fig. 8. Flow chart of the Bryers [84] model
sugars are lumped together (Fig. 8). Consequently, in the first step hydrolysis
takes place resulting in amino acids, sugars and fatty acids while in the second
and third steps intermediates such as propionate, butyrate and acetic acid are
derived from amino acids/sugars and fatty acids acidogenesis. The fourth step
corresponds to the acetogenesis while the fifth and sixth steps are the methano-
genesis from acetate and hydrogen, respectively. The model predicted fairly well
the observations in two different experimental systems that treated biomass
particulates anaerobically after some parameter optimization concerning initial
bacterial compositions.
In 1996 a mathematical model for the codigestion of piggery, olive-mill and
dairy wastewaters in continuous stirred tank reactors (CSTR) was developed by
Gavala et al. [85]. It was assumed that wastewaters consist mainly of carbohy-
drates and proteins (undissolved and dissolved) and other dissolved organic
matter (fatty acids and lipids are the major constituents of the latter). The mod-
el considers four steps and three bacterial groups, the acidogens, the acetogens
and the acetolytic methanogens (Fig. 9). The model was based on batch kinetic
experiments and is capable of predicting adequately the COD and fatty acids
dependence on the operating conditions when an anaerobic culture acclimated
to a specific wastewater starts being fed a mixture of other agroindustrial
wastewaters [86]. Thus, the model can be useful for predicting the short-term
response of a digester subjected to feed changes and thus avoiding system fail-
Fig. 9. Flow chart of the Gavala et al. [85] model
ure, as well as for maximizing the co-digestion process efficiency. The changes
of anaerobic culture’s biological characteristics during the acclimation process
to each one of the three aforementioned wastewaters were determined as well
[31]. It was found that a dairy acclimated culture was characterized by the
higher total biomass percentage in acidogens, whereas methanogen and aceto-
gens concentrations were higher in the piggery and olive-mill acclimated cul-
tures, respectively. On the other hand, different bacterial species of acidogens
had predominated in each culture. This suggests that the composition of the
wastewater in different organic compounds (carbohydrates, proteins, lipids etc.)
should definitely be taken into account when designing anaerobic digestion
processes.
In the study of Jeyaseelan [87] a model for anaerobic digestion of municipal
wastewater was proposed considering its composition in carbohydrates, pro-
teins, lipids and other organics. However, this study was a theoretical one since
no experimental part is involved and the kinetic parameters used were collect-
ed from the available literature.
Some other models consider the different composition of wastewater as well
[88–92]. However, these models use different key parameters than the ones
already reported and thus they are presented in the next two sections.
3.4
Models Using H2 as the Primary Key Parameter
The first model using H2 as the key parameter that regulates the production of
fatty acids from glucose is that of Mosey [93]. The main topic of this study is the
investigation and the expression of the effect that the dissolved hydrogen con-
centration has on the regulation of the redox potential inside the bacterial cell
and subsequently on the produced mixture of volatile fatty acids.
The model considers four bacterial groups: a) the fast-growing (minimum
doubling times around 30 minutes) acid-forming bacteria that ferment glucose
to a mixture of butyrate, propionate and acetate, b) the slow-growing (minimum
doubling times of 1.5–4 days) acetogenic bacteria that convert the butyrate and
propionate to acetate,c) the slow-growing (minimum doubling times of 2–3 days)
acetoclastic methanogenic bacteria that produce methane from acetate and d)
the relatively fast-growing (minimum doubling times around 6 hours) hydro-

gen-utilizing methanogens that produce methane from carbon dioxide.
The key assumption made by Mosey is that the formation of butyrate, propi-
onate and acetate from glucose is regulated by the availability of hydrogen.
The main hypothesis of his study is that the relative availability of the reduced
(NADH) and oxidized form (NAD+) of the carrier molecule nicotinamide ade-
nine dinucleotide controls both the overall rate of the conversion and the com-
position of the mixture of acids formed. The ratio [NADH]/[NAD] is a function
of the hydrogen partial pressure in the gas phase and it is expressed according
to Eq. (19).
[NADH] [NADH] –3
04 = 1500 · PH2 or 04 = 1.5 · 10 · H (19)
[NAD] [NAD]
where PH2 is the partial pressure of hydrogen in the gas phase and H is the con-
centration of hydrogen (ppm by volume) in the digester gas. Eq. (19) applies
under the following restrictions: a) the bacteria maintain a constant internal pH
value of 7.0 regardless of the variations in the pH value of their growth medium
and b) gaseous hydrogen diffuses both freely and rapidly into and out of the bac-
terial cells. Consequently, the partial pressure of hydrogen inside the bacteria is
the same as the partial pressure of hydrogen in the digester gas and the redox
potential of the bacteria is the same as the potential of the growth medium.
The model considers hydrogen-regulated production of volatile fatty acids
from glucose via the Embden-Meyerhof-Parnas metabolic pathway and also
hydrogen regulated acetogenesis from butyrate and propionate. It includes yield
equations for all the four bacterial groups that are involved in the overall process
of glucose fermentation. The yield equations are based on the production of ATP
at each step and on the assumption that one mole of ATP provides sufficient
energy for the formation of about 10 g of biomass [94].
The metabolic pathways inside acid-forming bacteria are shown in Fig. 10.
Steps and reaction rates are shown in Tables 12, 13 and 14.
Mosey’s model combines in a very fine way biochemical and microbiological
considerations. It is the first model that takes into consideration the varied stoi-
chiometry of the produced fatty acids during the acidogenesis step of glucose.
According to this model and during stress situations that usually result in to
high H2 concentrations, acidogenesis is diverted to the production of propionate
(and butyrate) which then are degraded slowly. This is consistent with the exper-
imentally observed persistency of propionate concentrations during stress situ-
ations. Nevertheless, the Mosey model has not been experimentally verified.
Thereafter, many models were based on the assumption that H2 is the key
parameter that regulates the production of fatty acids from glucose. The study
of Pullammanapallil et al. [95] is mainly based on Mosey’s model and addition-
ally it includes a description of the gas phase and acetoclastic inhibition by
undissociated fatty acids.
Costello et al. [96] developed a model taking into consideration that lactic
acid may also be an important intermediate in the anaerobic degradation of glu-
cose. Their model consists of five bacterial groups (Fig. 11) and the main differ-
ences between this model and the model of Mosey [93] is that the glucose is con-
Fig. 10. Schematic biochemical pathways of the Mosey [93] model. The model of Pullam-
manapallil et al. [95] is based on this scheme as well
verted to a mixture of butyric, acetic and lactic acids with the subsequent degra-
dation of the lactate to propionic and acetic acids. These two steps are inhibited
and regulated by hydrogen through the redox reactions of NAD. The conversion
of butyrate and propionate to acetate is subjected to hydrogen inhibition as
well. pH inhibition functions were applied to each group of bacteria while
product inhibition was taken into consideration for the acid-forming, lactic
acid and acetogenic bacteria by using non-competitive and competitive inhi-
bition terms. In the study of Costello et al. [97] an attempt at experimental veri-
fication of the aforementioned model was made by using independent sets
of experimental data. In some cases the model was able to provide a reasonable
comparison between the theoretical predictions and the experimental results;
however, in most cases an under-estimation of the propionic and butyric acid
concentrations as well as an over-estimation of the total biogas flow rate was
observed.
Keller et al. [98] and Romli et al. [99] used the model of Costello et al. [98] for
the prediction and simulation of experimental results coming from a two-stage
high-rate anaerobic wastewater treatment system fed with diluted molasses.
This system consisted of a continuous stirred tank reactor (CSTR) as the acidi-
Table 12. Stoichiometric reactions, rate and cells yield equations for the acidogenesis step during glucose fermentation
80
Stoichiometric reactions Rate and cells yield equations
d [gluc] RG kG · XG · [gluc]
03 = 046 whereas RG = 004
dt [NADH] (KG + [gluc])
1 + 04 +
[NAD ]
d [but] RG
C12H12O6 Æ CH3CH2CH2COOH + 2CO2 + 2H2 + 2ATP 03 = 0460005
dt [NADH] 2 [NAD+]
1 + 04 · 1 + 04
[NAD+] [NADH]
d [prop] 2RG
C12H12O6 + 2H2 Æ 2CH3CH2COOH + 2H2O + 2ATP 022 = 0460005
dt [NADH] [NAD+]
1 + 04 · 1 + 04
[NAD+]
[NADH]

d [acet] 2RG 2RG
C12H12O6 Æ H2O Æ 2CH3COOH + 2CO2 + 4H2 + 4ATP 021 = 046612 – 0460002
dt [NADH] [NADH] 2 [NAD+]
1 + 04 1 + 04 · 1 + 04
[NAD+] [NAD+]
[NADH]

dXG d [acet] d [prop] [but]
¢ · 76 + Yprop
7 = Yacet ¢ · 77 + Y but
¢ · 72 – XG · b
dt dt dt dt
Where RG : unregulated rate of uptake of glucose (mmoles/L/d). [prop]: concentration of propionic acid (mM).
kG : maximum rate constant (mmoles/mg/d). [but]: concentration of butyric acid (mM).
KG : Michaelis-type constant (mM). ¢ :
Yacet biomass yield coefficient (20 mg/mM acetate).
XG : concentration of glucose fermenters (mg/L). ¢ :
Yprop biomass yield coefficient (10 mg/mM propionate).
[gluc]: concentration of glucose (mM). ¢ :
Y but biomass yield coefficient (20 mg/mM butyrate).
H.N. Gavala et al.
[acet]: concentration of acetic acid (mM). b: decay coefficient (0.2/day).

Table 13. Stoichiometric reactions, rate and cells yield equations for the acetogenesis step during glucose fermentation
d [but] RB kB · XB · [but]
CH3CH2CH2COOH + 2H2O Æ 2CH3COOH + 2H2 + 2ATP 02 = 029 whereas RB = 0228
dt [NADH] (KB + [but])
1 + 041
[NAD+]
dXB d [but]
7 = YB · 02 – XB · b
dt dt
d [prop] RP kP · XP · [prop]
CH3CH2COOH + 2H2O Æ CH3COOH + CO2 + 3H2 + 1ATP 77 = 00 whereas RP = 005

dt [NADH] (KP + [prop])
1 + 761
[NAD+]
dXP d [prop]
7 = YP · 04 – XP · b
dt dt
Where RB , RP : unregulated rate of uptake of butyrate and propionate (mmoles/L/d).

kB , kP : maximum rate constant for butyrate and propionate (mmoles/mg/d).
KB , KP : Michaelis-type constant for butyrate and propionate (mM).
XB , XP : concentration of butyrate and propionate utilizers (mg/L).
YB : biomass yield coefficient (20 mg/mM butyrate).
YP : biomass yield coefficient (10 mg/mM propionate).
b: decay coefficient (0.2/day).
81
Table 14. Stoichiometric reactions, rate and cells yield equations for the methanogenesis step
during glucose fermentation
d [acet] kA · XA · [acet]
CH3COOH Æ CH4 + CO2 03 = 004
dt (KA + [acet])
dXA d [acet]
7 = YA · 76 – XA · b
dt dt
d [H2] kH · XH · [H]
4H2 + CO2 + CH4 + 2H2O 01 = 704
dt KH + [H]
dXH d [H2]
8 = YH · 74 – XH · b
dt dt
Where kA , kH : maximum rate constant for acetate and hydrogen (mmoles/mg/d).

KA : Michaelis-type constant for acetate (mM).
KH: Michaelis-type constant for hydrogen expressed as partial pressure
of hydrogen in the digester gas (atm).
XA , XH : concentration of acetate and hydrogen utilizers (mg/L).
YA: biomass yield coefficient (2.5 mg/mM acetate).
YH: biomass yield coefficient (2.5 mg/mM hydrogen).
b: decay coefficient (0.2/day).
[H2]: concentration of hydrogen gas in the solution (mM).
[H]: partial pressure of hydrogen in the gas (atm).
fication reactor and a fluidized sand-bed reactor (FBR) as the methanogenic

reactor. They stated – although not giving details – that a physico-chemical reac-
tion system was included in the model to calculate the pH at any time given the
concentration of the acidic and basic species in the reactor. The model was pret-
ty well capable to predict the pH, the alkali consumption rate at the acidification
reactor, the gas generation and composition and the effluent concentration in
organic acids when hydraulic step changes at various organic loading rates were
taking place. On the contrary, theoretical predictions were not in good agree-
ment with experimental results when a recycle stream from the second to the
first reactor was introduced [98].Also the system responses in combination with
the model predictions during pH changes and shock loads are described in
Romli et al. [100, 101].
In the study of Ruzicka [102] an extension of Mosey model is proposed tak-
ing into account the fact that in some cases formation of higher acids than
butyrate takes place during saccharide fermentation. Other modifications also
are suggested in Ruzicka [103] and are based on hydrogen transport across the
cell membrane as well as on reaction phenomena in the cell involving hydrogen
and NAD. This interesting approach, mentioned by the author as a “nonequilib-
rium concept”, could explain the contradictory experimental observations how,
on the one hand, small amounts of hydrogen could completely inhibit glucose
cleavage and how glucose uptake could proceed even in a medium fully saturat-
Fig. 11. Schematic biochemical pathways of the Costello et al. [96] model. The studies of Keller
et al. and Romli et al. [98, 99, 101] are based on this scheme as well
ed with hydrogen, on the other. Unfortunately, no experimental validation of this

model exists so far.
The model of Batstone et al. [92] was developed in order to simulate the
anaerobic degradation of complex wastewaters such as the slaughterhouse efflu-
ent. It is a complex model consisting of nine generic biological groups and three
enzymatic groups (Fig. 12). In this study the catabolic reactions are as follows:
the lipids, particulate proteins and carbohydrates are hydrolyzed by exo-enzymes
to long-chain fatty acids and simple sugars, soluble proteins and carbohydrates,
respectively. Acetic acid is coming from the further biodegradation of long-
chain fatty acids via b-oxidation whereas acidogenesis of proteins results in
acetate, propionate, butyrate and valerate. The pathway of protein degradation is
assumed to be via coupled Stickland reactions. The soluble carbohydrate degra-
dation follows H2 regulation of VFA formation as suggested by Mosey [93] and
the pathways are the same as used by Costello et al. [96], thus resulting in acetate,
butyrate and lactate. Subsequently, the lactate is biodegraded to propionic and
acetic acids. All the aforementioned volatile fatty acids are biodegraded to
acetate and finally the acetoclastic and the H2-utilizing methanogens produce
Fig. 12. Schematic biochemical pathways of the Batstone et al. [92] model
methane. The degradation rates of the substrates are subjected to hydrogen and
pH inhibition. The model includes physico-chemical reactions determining the
pH of the liquid phase and the gas-liquid transfer of carbon dioxide. Concen-
trations of saline and free organic components as well as ammonia and carbon
dioxide are calculated using acid-base equilibrium relationships. The model was
validated by performing experiments with a two-stage, high-rate anaerobic
treatment plant that treated wastewater from a pig slaughterhouse [104].
3.5
Models Using NH3 as the Primary Key Parameter
Ammonia is considered as a major inhibitor of the methanogenesis process

especially when animal wastes are being digested. The inhibition is mainly
caused by the un-ionized form of ammonia and thus it is very much dependent
on the pH value. Detailed description of the ammonia chemistry, diffusion and
release from liquid manure can be found in the review of Ni [105]. The first mod-
el using un-ionized ammonia inhibition is that of Hill and Barth [64] already
described earlier in this chapter. During the last decade many models have been
developed including ammonia inhibition as one of the key parameters and most
of them focus on the anaerobic digestion of animal wastes. Kiely et al. [106]
developed a model for anaerobic digestion of piggery slurry, primary sewage
sludge and the organic fraction of municipal solid waste. The model was based
on those of Hill and Barth [64] and Moletta [67] (Fig. 4). It consisted of two
stages (hydrolysis/acidogenesis producing acetate and methanogenesis) and
considers un-ionized VFA inhibition of both steps and un-ionized ammonia
inhibition for the methanogenesis step [Eq. (16)]. The model was validated using
experimental results from lab-scale continuous stirred-tank reactors and
despite its simplicity the theoretical results fitted satisfactorily the experimen-
tal ones.
Digesters fed with substrate with a high ammonia content, such as manure,
exhibit a self-regulation of the pH and self-resistance on un-ionized ammonia
toxicity that can be described as follows: when free ammonia concentration
exceeds the toxicity threshold, inhibition of methanogenesis occurs which leads
to an accumulation of VFA with a subsequent decrease of pH and thus reduction
of the free ammonia concentration. This mechanism tends to stabilize the
process at a certain volatile fatty acids concentration and pH level. The first
model that mentioned this mechanism is that of Angelidaki et al. [83]. It is a
complex model (Fig. 13) consisting of four microbial groups: the glucose fer-
menting acidogens, the propionate degrading acetogens, the butyrate degrading
Fig. 13. Flow chart of the Angelidaki et al. [83] model including pH and ammonia regulation
scheme
acetogens and the acetoclastic methanogens. The primary substrates are soluble
and insoluble carbohydrates whereas a fraction (almost 32%) of the total ammo-
nia is bound to the insoluble fraction and is released during hydrolysis. The
stoichiometry of the microbial reactions is based on the study of Hill [70] with
minor modifications to some coefficients. Equilibrium relationships for ammo-
nia, carbon dioxide and pH as well as gas phase dynamics and temperature
effects are included. Total VFA inhibition of hydrolysis, total acetate inhibition of
acetogenesis and free ammonia inhibition of methanogenesis is assumed in the
model. The type of inhibition used is the non-competitive one [Eq. (9)]. For the
last two steps, acetogenesis and methanogenesis, the effect of the pH on the
microbial growth rate was described by a Michaelis pH function, normalized to
give a value of 1.0 as center value [Eq. (18)]. The model was validated with ther-
mophilic (55 °C) laboratory scale anaerobic digestion experiments with cattle
manure where the feed concentration of ammonia was increased from 2.5 to
6.0 g-N/l. Experimental results and theoretical simulations showed on the one
hand that the process was inhibited but stabilized at a lower level of methane
production on the other.
The above-mentioned model was extended in order to be able to simulate the
anaerobic treatment of cattle manure with olive oil mill effluent [107]. Two more
bacterial groups were added to the model: the lipolytic and the long-chain fatty
acids-degrading bacteria (Fig. 14). Subsequently, the model was extended once
more for simulating the anaerobic bioconversion of complex substrates to bio-
Fig. 14. Extension of the Angelidaki et al. [83] model in order to include lipid and protein
anaerobic biodegradation [91, 107]
gas [91]. Hydrolysis of undissolved proteins and two more bacterial groups (the
amino-acid- and valerate-degrading acidogens and acetogens, respectively) are
also considered (Fig. 14). Specifically, the substrate in this model is defined by its
organic and inorganic composition. The organic part consists of carbohydrates,
proteins, lipids and volatile fatty acids. The inorganic part includes ammonia-N,
phosphate-P, carbonate-C, hydrogen sulfide, anions and cations (i.e., Ca2+, Mg2+,
and K+). The latter plays an important role in determining the pH and buffer
balance of the process. Product inhibition of VFA degradation to acetate was
further included along with inhibition of all the bacterial groups by the long-
chain fatty acids. The model was validated with thermophilic (55 °C) experi-
ments in (a) laboratory-scale CSTRs receiving a mixture of cattle manure and
glycerol trioleate or gelatin and (b) full-scale reactors receiving a mixture of
cattle manure, bentonite-bound oil and a proteinaceous wastewater coming
from bone extraction.
Another model that considers the ammonia inhibition of acetate conversion
to methane is that of Siegrist et al. [89, 90]. This model was developed in order
to describe the dynamic behavior of the mesophilic anaerobic digestion of
sewage sludge according to the reaction scheme shown in Fig. 15. Gujer and
Zehnder [1] first developed this reaction scheme. The model consists of five
microbial groups: the acidogens that ferment the amino acids and sugars to
Fig. 15. Reaction scheme for anaerobic digestion of domestic sewage sludge [1, 89, 90]
Fig. 16. Schematic biochemical pathways of the Vavilin et al. [88] model
butyrate, propionate and acetate, the microorganisms that perform the b-oxida-
tion of fatty acids to hydrogen and acetate, the acetogens that produce acetate
from butyrate and propionate and finally the acetogenic and hydrogen-utilizing
methanogens. The model includes equilibrium relationships for ammonia, car-
bon dioxide and pH and gas phase dynamics as well. The propionate and acetate
degradation and hydrogen consumption are subjected to pH dependence. Addi-
tionally, propionate and fatty acid degradation have non-competitive inhibition
terms for increased hydrogen pressure and acetate concentration whereas for
acetate conversion a non-competitive inhibition term for free ammonia is
included. The model is verified with load variation experiments in laboratory
and full-scale digesters.
Vavilin et al. [88] developed a model named “methane” in order to describe
the anaerobic digestion of complex organic matter. According to Fig. 16, the
model considers hydrolysis of the biodegradable organic matter to dissolved
organic substrate by hydrolytic enzymes released by acidogenic bacteria.
Acetate- and propionate-producing bacteria with the simultaneous release of
hydrogen, ammonia and carbon dioxide consume the dissolved organic sub-
strate, which is a mixture of carbohydrates, proteins and lipids. Propionate is
biodegraded to acetate, which is used for the production of methane along with
the hydrogen. Simultaneously, the sulfate-reducing bacteria, resulting in the

release of hydrogen sulfide, use the propionic and acetic acids. The inhibition
effect of non-ionized molecules of ammonia, sulfide and propionate was taken
into consideration as well.
The innovation brought by this model is the consideration of two more
groups of bacteria than the ones already reported in this chapter: the bacteria
that perform sulfate reduction coupled with the utilization of acetate and propi-
onate, respectively. Also, the aforementioned model takes into account that the
metabolism of the carbohydrates, proteins and lipids is regulated by the hydro-
gen partial pressure and thus the latter controls the relative production of pro-
pionate and acetate. This is accomplished by the consideration of two different
groups of acidogens: the propionate- and the acetate-producers.
The model “methane” was used successfully for the simulation of the results
of batch experiments on anaerobic digestion of food industry wastewater. In
1996, the same scientists developed a theory describing the hydrolysis as a two-
phase process. The model “methane” was used to compare the results of four
types of hydrolytic kinetics during anaerobic digestion of swine waste, sewage
sludge, cattle manure and cellulose. Experimental results from different studies
were used for this purpose [21].
3.6
Recent Developments on Anaerobic Digestion Modeling:
Anaerobic Modeling Task Group Work Presentation
An international anaerobic modeling task group was established in Japan in

1997 in order to formulate a common platform for the establishment of an
anaerobic model (http://www.awmc.uq.edu.au/admtg/tg). This task group has
now formulated an anaerobic model and the key assumptions of this generic
model are presented below. Conversion processes taken into account are biolog-
ical and physico-chemical ones and the carbon flow chart is presented in Fig. 17.
Acidogenesis, acetogenesis and methanogenesis are the three main biological
steps whereas the degradation of complex particulate matter is considered as a
combination of an extracellular, partly non-biological disintegration step and a
extracellular biological hydrolysis step. All extracellular steps follow first-order
kinetics [Eq. (10)] and substrate uptake and biomass growth and decay proceed
according to Eqs. (6) and (7), respectively. The stoichiometric yields of volatile
fatty acids during the acidogenesis step of carbohydrates are considered to be
constant with no hydrogen regulation function suggested so far. Additionally,
Stickland pathways are considered for the estimation of products yield from
amino acids. pH influence on all biological processes is included according to
the empirical function proposed by Angelidaki et al. [Eq. (18)]. Hydrogen inhi-
bition of acetogenesis from long-chain fatty acids (LCFA), propionate, butyrate
and valerate and for hydrogenotrophic methanogenesis is proposed along with
non-competitive free ammonia inhibition of acetolytic and hydrogenotrophic
methanogenesis. Physico-chemical processes such as liquid-liquid transforma-
tions (ion association/dissociation) and gas-liquid transfers of CO2, CH4 and H2
are included in the model since they are considered very important for anaero-
Fig. 17. Flow chart of the generic anaerobic model as suggested by the international anaerobic
modeling task group
bic systems. The IWA Anaerobic Digestion Model No 1 was presented during the
9th World Congress on Anaerobic Digestion [108]. However, discussions about
the final version of the model are still under way.
4
Conclusions
A systematic classification of the mathematical models describing the anaerobic
digestion process in suspended growth systems was made in this chapter. This
effort focused on the overview of the most important models found in the liter-
ature so that the reader can acquire the knowledge of how the mathematical
modeling of the anaerobic digestion developed throughout the years. It is a well-
known fact that anaerobic digestion is a complex process affected and regulated
by many factors. Also, anaerobic digestion is applied to the treatment of a wide
range of wastes/wastewater with significant differences in their composition.

In order to have appropriate mathematical models that combine simplicity
and accuracy and are applicable to almost every type of waste/wastewater, an
integrated approach should focus on the a) formulation of a common platform,
which will take into account all the important parameters and factors in-
fluencing the process, b) classification of the wastes/wastewater based on their
different composition in organic and inorganic compounds and c) formulation
of a model for each category selecting each time the key elements from the
common platform, e.g., NH3 effect should be taken into account in case of waste-
water with high concentration of NH3 or proteins; on the other hand the intro-
duction of the NH3 effect into a mathematical model concerning anaerobic
digestion of dairy wastewater will only add complexity with no practical advan-
tage. In this way, it will be possible to fully exploit the information and knowl-
edge gained through decades of scientific research on the area of anaerobic
digestion process.
5
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Received: April 2002

CHAPTER 6
Molecular Biology of Stress Genes in Methanogens:

Potential for Bioreactor Technology
Everly Conway de Macario · Alberto J. L. Macario
Wadsworth Center, Division of Molecular Medicine, New York State Department of Health;
and Department of Biomedical Sciences, School of Public Health, The University at Albany
(SUNY), Albany, New York 12201-0509, USA. E-mail: everlym@wadsworth.org
Many agents of physical, chemical, or biological nature, have the potential for causing cell
stress. These agents are called stressors and their effects on cells are due to protein denatura-
tion. Cells, microbes, for instance, perform their physiological functions and survive stress
only if they have their proteins in the necessary concentrations and shapes. To be functional a
protein shape must conform to a specific three-dimensional arrangement, named the native
configuration. When a stressor (e.g., temperature elevation or heat shock, decrease in pH,
hypersalinity, heavy metals) hits a microbe, it causes proteins to lose their native configura-
tion, which is to say that stressors cause protein denaturation. The cell mounts an anti-stress
response: house-keeping genes are down-regulated and stress genes are activated. Among the
latter are the genes that produce the Hsp70(DnaK), Hsp60, and small heat protein (sHsp) fam-
ilies of stress proteins. Hsp70(DnaK) is part of the molecular chaperone machine together
with Hsp40(DnaJ) and GrpE, and Hsp60 is a component of the chaperonin complex. Both the
chaperone machine and the chaperonins play a crucial role in assisting microbial proteins to
reach their native, functional configuration and to regain it when it is partially lost due to
stress. Proteins that are denatured beyond repair are degraded by proteases so they
do not accumulate and become a burden to the cell. All Archaea studied to date possess
chaperonins but only some methanogens have the chaperone machine. A recent genome sur-
vey indicates that Archaea do not harbor well conserved equivalents of the co-chaperones
trigger factor, Hip, Hop, BAG-1, and NAC, although the data suggest that Archaea have proteins
related to Hop and to the NAC alpha subunit whose functions remain to be elucidated. Other
anti-stress means involve osmolytes, ion traffic, and formation of multicellular structures. All
cellular anti-stress mechanisms depend on genes whose products are directly involved in
counteracting the effects of stressors, or are regulators. The latter proteins monitor and mod-
ulate gene activity. Biomethanation depends on the concerted action of at least three groups
of microbes, the methanogens being one of them. Their anti-stress mechanisms are briefly
discussed in this Chapter from the standpoint of their role in biomethanation with emphasis
on their potential for optimizing bioreactor performance. Bioreactors usually contain stres-
sors that come with the influent, or are produced during the digestion process. If the stressors
reach levels above those that can be dealt with by the anti-stress mechanisms of the microbes
in the bioreactor, the microbes will die or at least cease to function. The bioreactor will mal-
function and crash. Manipulation of genes involved in the anti-stress response, particularly
those pertinent to the synthesis and regulation of the Hsp70(DnaK) and Hsp60 molecular
machines, is a promising avenue for improving the capacity of microbes to withstand stress,
and thus to continue biomethanation even when the bioreactor is loaded with harsh waste.
The engineering of methanogenic consortia with stress-resistant microbes, made on demand
for efficient bioprocessing of stressor-containing effluents and wastes, is a tangible possibility
for the near future. This promising biotechnological development will soon become a reality
due to the advances in the study of the stress response and anti-stress mechanisms at the
molecular and genetic levels.

96 E. Conway de Macario · A. J. L. Macario
Keywords. Stress, Stress genes, Methanogens, Anti-stress mechanisms, Multicellular struc-

tures
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
1.1 Terminology, Stress, Heat-Shock Proteins, Chaperones,
and Anti-Stress Mechanisms . . . . . . . . . . . . . . . . . . . . . . 98
1.2 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
1.3 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
1.4 Life on Earth, Evolution, and Stressors . . . . . . . . . . . . . . . . 100
1.5 Methanogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
2 Stress Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

2.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.2 Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.3 Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.4 Strategies and Methods for Study and Utilization of Stress Genes . . 103
3 Molecular Chaperones . . . . . . . . . . . . . . . . . . . . . . . . . 103

3.1 Definition and Functions . . . . . . . . . . . . . . . . . . . . . . . 103
3.2 Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.3 Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.4 Mechanisms and Manipulation . . . . . . . . . . . . . . . . . . . . 105
4 Stress Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

4.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.2 Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.3 Stressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5 Stress Genes and Molecular Chaperones in Archaea . . . . . . . . . 106

5.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.2 Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.3 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.4 Expression and Regulation . . . . . . . . . . . . . . . . . . . . . . . 109
6 The Hsp70(DnaK) Chaperone Machine in Methanogens . . . . . . 109

6.1 Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
6.2 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.3 Stressor-Response Relationships . . . . . . . . . . . . . . . . . . . 114
6.4 Other Stressors Pertinent to Methanogenic Bioreactors . . . . . . . 114
6.5 Factors that Modify the Stress Response . . . . . . . . . . . . . . . 116
6.6 Other Methanogens . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.7 Co-Chaperones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Molecular Biology of Stress Genes in Methanogens: Potential for Bioreactor Technology 97
7 The Hsp60 (Chaperonin) System in Methanogens . . . . . . . . . . 123

7.1 Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.2 Structure and Potential for Bioreactor Technology . . . . . . . . . . 124
8 Other Stress or Stress-Related Molecules, Genes and Proteins,

and Anti-Stress Mechanisms in Methanogens . . . . . . . . . . . . 125
8.1 Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
8.2 Osmolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
8.3 TrkA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
8.4 Prefoldin or GimC . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
8.5 Small Heat-Shock Proteins (sHsp) . . . . . . . . . . . . . . . . . . . 128
8.6 PPIase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
8.7 Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
8.8 Putative Stress Genes and Proteins Found in Fully Sequenced
Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
9 Other Manifestations of the Stress Response . . . . . . . . . . . . . 130

9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
9.2 Thermoprotectants . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
9.3 Multicellular Structures . . . . . . . . . . . . . . . . . . . . . . . . 131
10 Perspectives and Applications . . . . . . . . . . . . . . . . . . . . . 138

10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
10.2 Diversity of Methanogens . . . . . . . . . . . . . . . . . . . . . . . 138
10.3 Dynamics of Methanogenic Subpopulations in Bioreactors . . . . . 138
10.4 Diversity of Stressors . . . . . . . . . . . . . . . . . . . . . . . . . . 141
10.5 Diversity of Response . . . . . . . . . . . . . . . . . . . . . . . . . 141
10.6 Diversity of Methanogens: A Source of Useful Microbes? . . . . . . 142
10.7 Cooperation Between Molecules and Between Cells . . . . . . . . . 145
10.8 Proteases as Builders . . . . . . . . . . . . . . . . . . . . . . . . . . 145
10.9 Intrinsic Stress Resistance . . . . . . . . . . . . . . . . . . . . . . . 146
11 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . 146
12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
1
Introduction
1.1
Terminology, Stress, Heat-Shock Proteins, Chaperones, and Anti-Stress Mechanisms
Terms will be defined in the text when pertinent. A few are introduced here to
abate the sense of awe some uninitiated readers might experience when they
encounter specialized words for the first time. This ought to make the Chapter
more “user friendly.”
Stress is a word applied in many fields of intellectual endeavor and does not
need a lengthy explanation. It will be used in this Chapter to indicate an altered
status of the cell caused by an agent (stressor) of a physical, chemical, or biolog-
ical nature [1]. This status is the stress response, which manifests itself by an
increase in the products of a series of genes (stress genes). These produce the
stress proteins, also called for historical reasons, heat-shock proteins, abbreviat-
ed Hsp [2]. While the genes’ names are written in italics, those of their products
are in Roman characters.
A gene, when active, is transcribed to messenger RNA (mRNA), which in turn
is translated in the ribosome into a linear series of amino acids to form a
polypeptide or protein. Thus, when one measures intracellular levels of the
mRNA from a given gene, an estimate of the degree of activity of that gene is
obtained. mRNA levels may increase in a cell also via other mechanisms, but
these will be explained in the text.
A protein is synthesized as a string of amino acids but to become functional-
ly competent it has to fold into a three-dimensional configuration, i.e., it has to
accommodate itself to what is called the native configuration for that particular
protein. There are many occasions in which this functional configuration is par-
tially or totally lost, a process named protein denaturation. Denatured proteins
must be renatured, or eliminated, lest they cause serious problems to the cell.
The major effect of stressors is protein denaturation. Consequently, the stress
response is made of events unchained by protein denaturation, and many of
these events aim at counteracting it.
Some Hsp are molecular chaperones [2]. They assist other polypeptides to
acquire their native configurations, or to regain them if they have been partial-
ly denatured. Proteins that are denatured beyond repair are eliminated by pro-
teases, i.e., enzymes that digest the damaged molecules [3–5].
There are several groups of Hsp, but the two most studied are those that con-
stitute the molecular chaperone machine and the chaperonin system [6–11].
Both will be explained in the text.
NOTE. This review was completed in January 2000. Since then other genomes have been
sequenced and new publications have appeared. Most relevant is a genome survey in search of
archaeal co-chaperones, a discussion of which was added to this article, after completion of
the original manuscript. Other updates may be found in Frontiers of Bioscience, Vol. 5 and 6,
Special Issue, Anti-stress mechanisms in Archaea, at:
http://www.bioscience.org/current/special/macario.htm; Genome Res. 12:532–542, 2002; and
Crit. Rev. Biochem. Mol. Biol., December 2002
Stress genes may be active in the absence of recognizable stress. This physio-
logical activity is called basal or constitutive to distinguish it from that induced
by stressors. The two gene activities, constitutive and stress-induced, are impor-
tant to the cell. They have to be properly regulated, so the cell can perform all its
functions under physiological conditions, and can react speedily and efficiently
in the face of stress, and survive. It follows that the gene-regulatory mechanisms
are as important to the cell as the stress genes themselves. Gene regulation is
mediated by proteins called transcription and regulatory factors that are the
product of other genes. Some of these factors interact with critical DNA
sequences named cis-acting sites or elements, usually located near the genes
they regulate.
There are other components of the stress response beyond stress genes and
chaperones. A few will be discussed in this Chapter, such as simple compounds
that protect the cell against stressors and are called thermoprotectants, and mul-
ticellular structures [12]. The latter structures allow cells to survive adverse con-
ditions as they are shielded inside a large body surrounded by extracellular
material.
It should be easy to envisage from the above general considerations how
important anti-stress mechanisms are for biomethanation. The microbial cells
involved in the bioconversion of wastes in bioreactors are constantly exposed to
stressors. If the cells are not prepared to withstand these attacks by stressors
they will die, or at least cease to function.
There is a very promising future for the use of stress genes and proteins in the
optimization of biomethanation technology.A rational application of these anti-
stress means must be based on adequate knowledge of the mechanisms involved
in transcription and regulation of stress genes. The aim of this Chapter is to pre-
sent in a simplified manner part of what is known about the stress response in a
group of microbial cells that are key to biomethanation.
1.2
Objectives
This Chapter, as stated above, will introduce the topic defined in the title to non-
specialists in molecular biology or stress. Data will be presented in Tables with
only a brief explanation in the text, in which selected references will be quoted
for consultation by those interested in more details. Moreover, recent reviews on
the stress genes and proteins will be quoted extensively because they contain
practically all the information available at the end of 1999 [8, 12, 13]. In addition,
the few pertinent publications that have appeared since then will be discussed.
The information on stress genes and proteins will be discussed mainly to
indicate how it might be pertinent to methanogenesis and, more specifically, to
the designing, monitoring, improving, and controlling of anaerobic methan-
ogenic bioreactors (digestors).
1.3
Scope
An attempt will be made to cover all aspects of the stress response, and the stress
genes and proteins that might be applicable to bioreactor technology, focusing
on the methanogens. However, the emphasis will lie on the systems that are bet-
ter known, namely the molecular chaperone machine and the chaperonins. In
addition, a rather detailed discussion of multicellular structures that play a crit-
ical role in resistance to stressors will be presented.
Likewise, we will focus on two organisms, Methanosarcina mazeii and
Methanosarcina thermophila, whose stress response and molecular chaperone-
machine genes are the best studied among methanogens [12, 14]. They are key
for methanogenesis in bioreactors and in many other ecosystems of biotechno-
logical relevance [15–19]. M. mazeii has an optimal temperature for growth
(OTG) of 37°C and is a key element for mesophilic digestion, while the other,
M. thermophila, has an OTG of 50°C and plays a central role in thermophilic
bioreactors.
This Chapter is based on an extensive search of written and electronic litera-
ture, and on consultations with colleagues, but only a minimal list of essential
references will be quoted and work done in our laboratories will be primarily
surveyed.As stated above, there are comprehensive reviews available that may be
used by the reader to advantage, and that allow us to simplify this Chapter, and
to direct its focus on matters that are, or might be, relevant to methanogenesis
and bioreactor technology.
1.4
Life on Earth, Evolution, and Stressors
Life on Earth today resulted from the evolution of organisms that survived
through the changes in temperature, pH, oxygen levels, etc. that the planet expe-
rienced since the most primitive life form appeared. Since these environmental
changes, and many others that must have occurred during the millennia, are cell
stressors, one is inclined to think that stress genes have played a decisive role in
determining which organisms survived. It would seem reasonable to think that
life forms endowed with the best anti-stressor means (among which stress genes
and proteins are paramount) would be those that withstood the stressful condi-
tions when they appeared more or less suddenly, until the conditions either
changed back to a more benign character or the organism adapted itself to the
new situation. In the latter case, the stressful conditions were no longer such.
They became normal, physiological conditions for the adapted organism. This
mechanism may be one of the reasons why we see today such a variety of habi-
tats that are optimal for their aboriginal organisms [20], but may be deadly to
foreigners. This is a crucial concept while defining stressors. An agent, for
instance a certain level of temperature, may be a potent stressor for a given
organism while it may be optimal for the growth and physiology of another [1].
The same applies to acidity, alkalinity, salinity, etc. It is with these ideas in mind
that one may now look at the list of common cell stressors displayed in Table 1.
Table 1. Cell stressors a
Type Name description b
Physical Heat; several types of irradiation, including ultraviolet light; pressure;

sound
Oxygen H2O2; oxygen-derived free radicals or reactive oxygen species (ROS);
anaerobiosis to aerobiosis shift; hypoxia-anoxia
pH Alkalinity; acidity; pH shift
Osmotic Changes in the concentration of salt, sugars, other osmolytes
(hyper- or hypoosmotic shock)
Nutritional Starvation: multiple; specific (carbon, glucose, nitrogen, phosphate,
nitrate)
Antibiotics Puromycin; tetracycline; nalidix acid
Alcohols Ethanol; methanol; butanol; propanol; octanol
Metals Cadmium; copper; chromium; zinc; tin; aluminum; mercury; lead; nickel
Insecticides, Lindane; diazinon; paraquat; thiram; tributyltin
pesticides
Mechanical Compression; shearing
Other Benzene and derivatives; phenol and derivatives; mutagens; ammonia;
arsenite; arsenate; amino acid analogues; nicotine; anesthetics;
desiccation
a Reproduced modified from reference [1] with permission from the copyright owner.
b These agents cause stress in cells from the three phylogenetic domains, Bacteria, Archaea
(both prokaryotes), and Eucarya (eukaryotes).
1.5
Methanogens
All living cells have been classified into three main lines of evolutionary descent
based mostly on comparative analyses of the sequences of the small subunit of
ribosomal RNA (rRNA) [21–24]. The lines or phylogenetic domains are: Archaea
(formerly archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes).
Methanogens belong to the Archaea, and consequently they are prokaryotes
with many characteristics that distinguish them from the other prokaryotes, the
bacteria.
Methanogens are very important organisms for a number of reasons. The
most pertinent to this Chapter is that they are key elements in the bioconversion
of organic matter with the generation of methane gas, i.e., biomethanation [17,
19, 25]. They are widespread, diverse, and capable of metabolizing a range of dif-
ferent substrates. Therefore, they are useful in waste treatment under a variety
of conditions, and have the potential for application in the bioconversion of a
range of materials in many different locations in our planet.A major objective of
this Chapter is to indicate possibilities for improving methanogens, and to sug-
gest means to do it, so they become very resistant to stressors and thus able to
function efficiently even in very inhospitable environments.
2
Stress Genes
2.1
Definition
In a broad sense, stress genes are those that work during and after a stressor hits
a cell differently as compared with the pre-stress situation [1]. Stress genes begin
to be transcribed, or increase their transcriptional activity, in response to the
stressor’s impact on the cell. In contrast to other cellular genes, the stress genes
are activated upon stress. Non-stress genes are down-regulated or shut off by
stress.
In agreement with a broad definition, stress genes form a large group encom-
passing those that produce the molecular chaperones and chaperonins (the
most representative of the group) [2] and many others [12, 26]. Among the lat-
ter are those that participate in the formation of multicellular structures, of
which we know very little despite their obvious importance in cell survival and
function.
2.2
Classification
The stress genes and their proteins (Hsp) are classified according to the molec-
ular mass of the latter as shown in Table 2. The Hsp70 and Hsp60 families are the
best known in Archaea, including methanogens [8, 12, 27, 31, 32]. There is also
information on the components of the molecular chaperone machine other than
Hsp70(DnaK), namely the Hsp40(DnaJ) and GrpE proteins, particularly in M.
mazeii and M. thermophila, as we shall discuss later in this Chapter. While these
gene/protein families are very important and their study should continue if one
wants to develop means to improve methanogens for biotechnologic purposes,
another important group shown in Table 2 is “Other”. Under this heading, there
Table 2. Classification of Hsp into families according to their molecular mass a
Family Name Mass (kDa) Found in methanogens

(synonyms within parentheses) b
Heavy (High M.W.; Hsp100) 100 or higher No c

Hsp90 81–99 No
Hsp70 (Chaperones) 65–80 Yes
Hsp60 (Chaperonins) 55–64 Yes
Hsp40 (DnaJ) 35–54 Yes
Small Hsp (sHsp) <35 Yes
Other (proteases, etc.) Various Yes
a Reproduced from reference [2] with permission from the copyright owner.
b For additional information on the various families see references [7, 10, 11, 27–36].
c Not yet investigated, or investigated but not yet found, or found but incompletely character-
ized (see also references [8, 37]).
are several gene/proteins that are surely crucial for cell survival in the face of
stress but that are not yet well understood [12]. This group offers a great poten-
tial for research and as a source of genes and molecules to engineer more resis-
tant methanogens as we shall see in other sections of this Chapter.
2.3
Evolution
The evolutionary history of stress genes and proteins is extremely interesting

from both the theoretical and practical standpoints. The topic has been reviewed
in detail [8, 12, 32, 38–40], and we shall not deal with it here again. We encour-
age the reader to consult the references quoted, and others cited in them, to
become acquainted with the evolution of these important genes, and to under-
stand why they are useful for constructing evolutionary trees, and how they can
be used for such a purpose.
What are the practical dividends of understanding stress gene/protein evolu-
tion? It provides a comprehensive picture of their presence and variations in
many organisms, and thus their functions and adaptations in a variety of cir-
cumstances. Thus, it helps us to understand the functions of the stress proteins
as a whole, and those of their specialized domains, which in turn is instrumen-
tal to designing strategies for engineering more efficient genes and molecules
that will “fortify” methanogens pertinent to bioreactors.
2.4
Strategies and Methods for the Study and Utilization of Stress Genes
Because of the diversity of methanogens mentioned above, including the capac-

ity to grow in a wide variety of ecosystems, the methods for studying them are
very varied [21, 25, 41, 42]. It follows that the methods for manipulating the
stress genes and proteins from methanogens are also diverse and may be quite
complex.
A wide array of species possibly also exists, with many families, but this type
of diversity has not yet been fully assessed because only a limited number of iso-
lates have been thoroughly characterized.We shall come back to this topic in the
course of the Chapter to show the array of possibilities that are available for dis-
covering useful methanogens and for obtaining useful genes and proteins.
3
Molecular Chaperones
3.1
Definition and Functions
A molecular chaperone is a protein that assists others to fold correctly, refold if

partially denatured (unfolded) by stressors, and translocate to the cell’s locale
(e.g., the periplasm in bacteria, or the mitochondria and the endoplasmic retic-
ulum in eukaryotes) where they reside and function [6, 9, 43]. They are also
implicated in the degradation of intracellular proteins that are damaged or

abnormal to the extent that they might aggregate and precipitate, and thus cause
serious problems to the cell [3–5].
In addition to the functions listed above, molecular chaperones have other
activities, the list of which grows rapidly as more research is done on these
important molecules. For example, they participate in the regulation of their
own synthesis [44–46] and in the disaggregation and refolding of partially dena-
tured polypeptides [47–49], and some of them interact with nucleic acids [50].
Furthermore, it has been suggested that molecules other than proteins (e.g.,
lipids) can play a role as chaperones in protein folding [51].
In this Chapter, the molecular chaperones that are the product of stress genes
will be treated in more detail. However, others that are less well studied will also
be mentioned to stimulate the curiosity of the reader and, hopefully, to promote
research aiming at clarifying their functional role. This should provide a wider
basis for biotechnologic developments than that furnished by the analysis of the
classical molecular chaperones exclusively.
3.2
Interactions
Molecular chaperones usually exercise their functions by interacting with other

molecules that are chaperones themselves, or that act as co-chaperones or
co-factors, forming complex multimolecular assemblies [6, 8, 9, 35, 43, 52].
Examples are the molecular chaperone machine formed by the chaperones
Hsp70(DnaK), Hsp40(DnaJ), and GrpE; and the thermosome or chaperonin
complex, as we shall see later in this Chapter. Also, the molecular chaperone
machine may interact with chaperonins in the process of de novo polypeptide
folding. Thus, in the chaperoning process and in the regulation of their own syn-
thesis, chaperones do not act alone but in association with teammates. It follows
that if one aims at, for example, controlling the levels of stress proteins in a cell
to make it more resistant to stressors over periods of time longer than usual, one
must understand their self-regulatory circuits and the array of other molecules
involved in this mechanism.
3.3
Families
As mentioned above, the Hsp, many of which are molecular chaperones, are clas-
sified into families according to their molecular mass, see Table 2. It is important
to bear in mind that while many molecular chaperones are stress proteins in the
sense that their levels are increased by stress, others are not. Also, the reverse is
true, namely, many stress proteins are not molecular chaperones. Their levels
increase in response to stress, or they are more active as a consequence of stress,
but they do not assist other proteins to fold, re-fold, or mobilize; they have
other functions.
3.4
Mechanisms and Manipulation
It is well known that stress proteins, including those that are molecular chaper-
ones, increase in quantity inside the cell in response to stressors. This increase is
one of the landmarks of the stress response [1]. There are several mechanisms
by which the concentration of a given protein may be increased in a cell, for
example, increases in the rate of transcription of the gene that encodes the pro-
tein, life-time of the mRNA, efficiency of mRNA translation, and life-time of the
protein itself. Hence, there are at least four points to consider when designing
strategies to increase the cell’s resistance to stressors. One may focus on tran-
scription initiation and elongation and try to improve these processes to make
them faster and more efficient. Or one may try to do the same with the process
of translation at the level of the ribosome. In addition, one may aim at decreas-
ing the degradation rate of the mRNA, or the protein, and thus lengthen their life
spans. Obviously, a combination of these approaches seems the most promising
strategy, however complicated it might be.
4
Stress Response
4.1
Definition
Now that we have introduced the stressors, stress genes and proteins, and the
molecular chaperones, it is timely to define the stress response. This is a complex
series of events unchained by a stressor upon hitting a cell [1]. The stress genes,
proteins, and molecular chaperones are the main players in these events. They
increase in concentration and/or activity to counteract the effects of the stres-
sor.
The central, most damaging effect of stressors is on cellular proteins that
become denatured, i.e., they lose their native configuration [1, 12]. Hence, the
stress response aims at avoiding protein denaturation, renaturing (refolding)
partially damaged molecules, and degrading those damaged beyond repair.
4.2
Characteristics
A stress response manifests itself in several ways [1, 12]:

(i) Most, if not all, house-keeping genes are down-regulated or shut off;
(ii)The stress genes are activated;
(iii)
Many cellular proteins become denatured in various degrees;
(iv)The cell may change its motility and move more or less than before
stress;
(v) The cell membrane and wall may change to become more resistant to the
stressor, more or less permeable to certain compounds, ions, etc.;
(vi) The cell may aggregate with others via synthesis of an intercellular con-
nective material and build multicellular structures of various degrees of
complexity; and
(vii) Other phenomena may occur, ranging from alterations in the intracellular
osmolytes through changes in the electrolytes and other components.
All the above events, or some of them, may occur in a cell in response to a given
stressor, but down-regulation of housekeeping genes and activation of stress
genes are by definition essential landmarks. These two main components are
evidenced by a change in the levels of the proteins that these genes encode: the
products of the housekeeping genes diminish, even become undetectable,
whereas the products of the stress genes increase. Thus, in monitoring microbes
in a bioreactor to check their functional status and potential for recovery if
stressed, one may measure the levels of representatives of these two groups of
proteins, or their respective mRNAs. This in fact ought to be a straightforward,
relatively simple approach to the monitoring and controlling of bioreactors to
prevent, or at least anticipate, failure with disruption of a waste-treatment oper-
ation.
4.3
Stressors
Anything endowed with the capacity to elicit a stress response is a stressor [1].
The most common or best studied are listed in Table 1. It is clear from the list
that stressors are ubiquitous. They can be found in air, soil, water, all kinds of
foods and nutrients, and in a great variety of natural or manufactured materials.
Many of them reach the microbes in methanogenic bioreactors with the influent
or are produced in the bioreactor itself [17, 53–56]. It is therefore not surprising
that bioreactor malfunction is a rather frequent occurrence, even when the
mechanical and most chemical conditions seem to be adequate. It follows that
monitoring bioreactors ought to include testing the degree of stress of the
microbes by measuring manifestations of the stress response.
5
Stress Genes and Molecular Chaperones in Archaea
5.1
Overview
This topic has been reviewed recently [12]. It will, therefore, not be necessary
to treat it again here in any detail, except in what pertains directly to biometha-
nation.
The study of stress genes in Archaea began very recently by comparison with
their counterparts in organisms of the other two domains, Bacteria and Eucarya.
The earliest studies on the stress response in Archaea may be traced back to the
late 1980s, but it was only in 1991 that an archaeal stress gene, hsp70(dnaK), was
cloned and sequenced for the first time [57]. The same year, a chaperonin gene
was also cloned and sequenced [7]. Since then, a few others have been se-
quenced, some as part of genome sequencing projects [12]. In parallel, func-
tional studies in vitro have been carried out to elucidate how and when these
genes are expressed and how they respond to stress. Nevertheless, very little is
known about these genes in comparison with those from bacteria and eukary-
otes. A lot of work remains to be done before one can think of using archaeal
genes, or their products, for improving methanogens pertinent to bioreactor
technology.
5.2
Evolution
The evolution of archaeal stress genes, hsp70(dnaK) in particular, has attracted

the attention of a number of investigators. Considerable information exists on
the evolution of hsp70(dnaK), which has revealed very interesting features [8, 32,
39, 40]. For example, the gene is absent from several archaeal species, including
some methanogens, Table 3, seemingly in contradiction of the generally ack-
nowledged fact that this gene is very important, if not essential, for life, espe-
cially for surviving stress. In fact, the discovery that the gene has a discontinu-
ous distribution among Archaea may be considered one of the major contribu-
tions of researchers working with Archaea to the fields of evolution, stress
Table 3. Occurrence, or lack thereof, of the hsp70(dnaK) gene among Archaea and represen-
tatives of thermophilic and hyperthermophilic bacteria a
Organism OTG b hsp70 Genome size Demonstrated

(°C) (dnaK) (Mb) by:
ARCHAEA
Methanosarcina
mazei S-6 37 Yes 2.8 S, N, W, seq.c
mazei JC3 37 Yes n.d.d N
mazei LYC 37 Yes n.d. N
sp. JVC 37 Yes n.d. N
acetivorans C2A 37 Yes 2.7 N
barkeri 37 Yes 2.7 S
thermophila TM-1 50 Yes 2.7 S, N, seq.
Methanospirillum hungateii 37 No n.d. S
Methanobacterium 65 Yes 1.7 seq.
thermoautotrophicum DH
Methanococcus
voltae 37 No n.d. S, W
vannielii 37 No n.d. S, P
jannaschii 85 No 1.7 S, seq.
Methanothermus fervidus 85 No n.d. S, P
Methanopyrus kandleri 100 No n.d. S, P
Haloarcula marismortui 45 Yes n.d. seq.
Table 3 (continued)
Organism OTG b hsp70 Genome size Demonstrated

(°C) (dnaK) (Mb) by:
Halobacterium
cutirubrum 45 Yes n.d. seq.
halobium 45 Yes n.d. S, P
Thermoplasma acidophilum 55 Yes 1.7 seq., P
Sulfolobus solfataricus 70 No 3.1 S, P
Sulfolobus sp. 70 No n.d. S
Archaeoglobus fulgidus 83 No 2.2 seq., P
Desulfurococcus mobilis 85 No n.d. S, P
Thermococcus tenax 88 No n.d. S, P
Pyrococcus
furiosus 100 No 2.0 seq.
horikoshii 100 No 1.7 seq.
woesei 100 No n.d. S, P
abyssi 100 No 1.8 seq.
Pyrobaculum aerophilum 100 No 2.2 seq.
Aeropyrum pernix K1 100 No 1.7 seq.
BACTERIA
Thermus thermophilus 70 Yes n.d. seq.
Thermomicrobium roseum 70 Yes n.d. seq.
Thermotoga maritima 80 Yes n.d. seq.
Aquifex
aeolicus 83 Yes n.d. seq.
pyrophilus 83 Yes n.d. seq.
a Reproduced from reference 12 with permission from the copyright owner.
b OTG, optimal temperature for growth.
c S, N, and W, Southern, Northern, and Western blotting, respectively; P, PCR; seq., sequenc-
ing of gene or genome.
d n.d., not determined.
e R. Weiss, personal communication.
f S.A. Fitz-Gibbon, personal communication.
response, and molecular chaperones in general. Several of the critical questions

posed by this finding have been discussed recently [8, 12]. The finding has had a
significant impact on our views regarding the evolutionary conservation of the
gene, its role in cell physiology and survival, and its substitute in those species
that do not have it (but still need to maintain an optimal set of functional pro-
teins via folding and refolding under “normal” conditions and in the face of
stress).
Also very interesting is the evolution of the gene encoding the Hsp60 archaeal
chaperonin, of which one, two, or three representatives may be found depending
on the species [31, 38]. The proteins encoded in these chaperonin genes have
received different names: chaperonin subunit 1 and 2, a and b (and g in case
there were three in the same organism), or TF55 and TF56. Comparative analy-
ses of amino acid sequences using computer programs that reveal phylogenetic
relationships suggested that multiple subunit species arose several times, inde-
pendently, during archaeal evolution.
5.3
Structure
The analyses of sequences mentioned above have revealed other features of

interest. For example, the variation in the number of chaperonin subunits
depending on the species is intriguing. It may have important consequences on
the structural details of their assembly when they build up the chaperonin com-
plex (for details on this complex, including three-dimensional reconstructions,
see [12]). These structural details may in turn play a decisive role on the way the
complex functions as a chaperoning machine. A complex formed by a single
type of subunit may work differently from complexes formed by two, or three,
types of subunits. This remains to be determined and points to another area in
which research with archaeal chaperonins will most likely have reverberations
on biomethanation technology. No doubt, the engineering of chaperonin com-
plexes with the proper structure will help cells to make functionally competent
proteins (such as the enzymes needed for methanogenesis) even under stress.
5.4
Expression and Regulation
Very little is known about the expression of the genes that produce the compo-
nents of the chaperone machine, the chaperonin complex, and other stress pro-
teins and molecular chaperones in Archaea. The topic has been recently
reviewed [12], and the reader is encouraged to consult this article in order to
understand its relevance to the art and science of fortifying cells, to make them
resistant to stress. Some detailed information is available for the methanogens
M. mazeii and M. thermophila, which will be discussed in subsequent Sections
of this Chapter.
6
The Hsp70(DnaK) Chaperone Machine in Methanogens
6.1
Components
As mentioned above, some methanogens lack the hsp70(dnaK) gene, and the
genes for the other components of the machine, Hsp40(DnaJ), and GrpE, Table 3.
The latter two genes seem to always accompany hsp70(dnaK) [8]. If hsp70(dnaK)
is missing, hsp40(dnaJ) and grpE are also absent in the genome. This parallelism
has been demonstrated whenever enough sequence data have been available.
Fig. 1. hsp70(dnaK)-locus genes of the methanogens for which sequences are available that
include genes up- and downstream of hsp70(dnaK). The genes are represented by rectangular
boxes from the 5¢ to the 3¢ end (left to right) with their names above their respective boxes in
the locus on top (dnaK and dnaJ are used instead of hsp70(dnaK) and hsp40(dnaJ) for sim-
plicity). The figures within the boxes indicate the number of amino acids encoded. The lines
joining the boxes represent the intergenic regions with their lengths, in base pairs, shown
underneath. The sequences of M. thermophila TM-1 grpE and trkA are still incomplete (what
is available would encode 53 and 401 amino acids, respectively). Accession numbers and
other details are provided in Tables 3–6. Reproduced from references [12] and [14] with per-
mission from the copyright owners
Thus, one may conclude that the product of hsp70(dnaK) cannot operate with-
out the products of the other two. Needless to say, this conclusion from sequenc-
ing data alone must translate into functional inferences, and impact on the way
one plans to engineer microbes to make them stronger in the face of stress.
Manipulation of only hsp70(dnaK) would not suffice. The other two genes
should also be included, so the three of them would be expressed in a coordi-
nated fashion for their products to act in unison, namely, to form a balanced
chaperone machine, functionally efficient.
The hsp70(dnaK), hsp40(dnaJ), and GrpE genes from methanogens that have
been cloned and sequenced are listed in Tables 4, 5, and 6, respectively. The first
hsp70(dnaK) loci to be fully sequenced in methanogens (and in the whole
domain Archaea) are depicted in Fig. 1.
6.2
Expression
Expression of the Hsp70(DnaK) molecular chaperone machine genes in

response to the stressor heat has been studied in M. mazeii S-6, Fig. 2, and to a
lesser extent in M. thermophila TM-1 [14, 59–64].
Data in Fig. 2 demonstrate that the genes in M. mazeii S-6 respond to heat
shock and, thus, that they are in this regard similar to counterparts in the organ-
Table 4. hsp70(dnaK) genes in methanogens a
Organism Accession Base pairs/amino Promoter/terminator/ Other structures Expression

number acids encoded RBS b
Methanobacterium AE000894 1791/596 n.r./n.r./gaggtg (-8) c Downstream repeats; n.r.

thermoautotrophicum DH stemloops
Methanosarcina mazei S-6 X60265 1860/619 aaactttaattaa (-79)/ Up- and downstream Heat-shock
inverted repeats/ repeats; stemloops inducible
aggatataa (-5)
Methanosarcina Y17862 1833/610 aacttttatcta (-60)/ Palindrome; Heat-shock
thermophila TM-1 tctttttt (+38)/ distinctive features inducible
agtgaggataaa (-7) upstream
a Data extracted from reference [12] with permission from the copyright owner.
b RBS, ribosome-binding site.
c n.r., not reported; (–) and (+) refer to position of center of sequence upstream from the translation start codon or downstream from the translation
stop codon, respectively.
Molecular Biology of Stress Genes in Methanogens: Potential for Bioreactor Technology
111
Table 5. hsp40(dnaJ) genes in methanogens a
112

Methanobacterium AE000894 1131/376 acatttttttatt (-63) Up- and downstream n.r.

thermoautotrophicucm DH /n.r./ aggtg (-9) c repeats; stemloops
Methanosarcina X60265 1170/389 aaacctgttcaca Up- and downstream Heat-shock
mazei S-6 (-100)/t-rich region/ repeats; stemloops inducible
aacagggaatctg (-8)
Methanosarcina AJ010152 1167/388 aaacctgcact (-55)/tcttttt Inverted repeat n.r.
thermophila TM-1 (+30), t-rich region/ upstream; t-rich
atgacagggaa (-11) region downstream
a Data extracted from reference 12 with permission from the copyright owner.
b RBS, ribosome binding site.
Table 6. grpE genes in methanogens a

Methanobacterium thermo- AE000894 525/174 aaafftttatata (-87)/ Upstream repeats, n.r.

autotrophicum DH n.r/aggtg (-7) c stemloops
Methanosarcina mazei S-6 X74353 630/209 aactatttataga (-69)/ Up- and downstream Heat-shock
inverted repeat repeats; stemloops inducible
(+76)/atggg (-11)
a Data extracted from reference 12 with permission from the copyright owner.
E. Conway de Macario · A. J. L. Macario

Fig. 2. Northern blots with M. mazeii S-6 total RNA (10 µg/lane) showing an increase in the
transcripts of hsp70(dnaK) (A), hsp40 (dnaJ) (B), and grpE (C), and a decrease in the tran-
script of orf16 (D), in response to heat shock. (E) dot blot showing a decrease in the transcript
of orf11-trkA in response to heat shock. Hybridizations were done in all cases with radiola-
beled probes specific for the respective genes. In A, B, and D, I the gel is stained with ethidium
bromide showing the ribosomal RNAs, 23S and 16S, while II is the corresponding Northern
blot. Lanes A: total RNA from M. mazeii S-6 cells maintained at the optimal growth tempera-
ture of 37°C, i.e., non-heat-shocked cells. Lanes B–C, or B–D: total RNA from cells heat-
shocked at 45°C for increasing time periods, from 15 to 60 min. The sizes of the transcripts in
A–D are indicated in kilobases (kb). Transcripts were detected for all the genes in non-heat-
shocked cells. Heat shock caused an increase in the transcripts of hsp70(dnaK), hsp40(dnaJ),
and grpE. The reverse occurred for orf16, and orf11-trkA. The latter two genes overlap, and are
cotranscribed, whereas the other genes are transcribed monocystronically. Reproduced from
references [59, 60, 62, 64] with permission from the copyright owners
isms of the other two phylogenetic domains. However, the M. mazeii genes
resemble eucaryal homologues in as much as the message is monocistronic, in
contrast to the bacterial counterparts. The latter are transcribed polycistroni-
cally into an mRNA molecule at least as long as the sum of the lengths of the
three genes [12, 65]. In contrast, the mRNAs from the M. mazeii genes are dis-
tinct from one another, and their individual lengths are about the same as those
of the respective genes.
The other two genes in the M. mazeii S-6 hsp70(dnaK0 locus, orf16 and orf11-
trkA, Fig. 1, respond differently [62, 64, 66]. Their transcripts decrease instead of
increasing, after heat shock, Fig. 2.
6.3
Stressor-Response Relationships
In order to be able to develop methanogens with improved stress resistance –

that can be used for bioconversion of wastes in harsh, changing environments –
one must first understand the range of conditions within which the stress genes
are able to respond, and also, one must learn how the cell responds at the
extremes of that range.
Data in Fig. 3 demonstrate that the three genes of the chaperone machine
triad in M. mazeii S-6 (OTG 37°C) respond very well at temperatures between
45 and 60°C, even though cell viability is considerably diminished already at
55°C [63].
The conclusion from the data is that while an increase in temperature above
certain limits kills many M. mazeii S-6 cells, the heat-shock response of the
chaperone machine triad is still in operation, at least with respect to increasing
the genes’ transcripts. This observation suggests that in a seriously damaged
microbial population, with many of its members dying, there are still many cells
capable of mounting a sizable stress response. The data also suggest that by
improving the cells’ ability to mount a stress response, using genetic engineer-
ing procedures for example, one will increase the size of the surviving, func-
tional subpopulation, and thus insure continuity in the bioconversion process
and avoid bioreactor failure.
It must be mentioned, however, that it remains to be established whether
damaged cells still capable of mounting a stress response as measured by an
increase in the stress genes’ transcripts, are also capable of proceeding with
the entire pathway of protein biogenesis and produce functional stress pro-
teins. This is a very promising area of research pertinent to biomethanation
technology.
6.4
Other Stressors Pertinent to Methanogenic Bioreactors
The list of cell stressors that can affect methanogens is long, as one may infer
from the sample listed in Table 1. Examples of stressors pertinent to industrial
and other effluents and environments are heavy metals and sound.
Cadmium (Cd++) and sound do elicit a stress response in M. mazeii S-6, as
shown by the data in Figs. 4 and 5, respectively [58]. It is likely that other
methanogens, and other microbes pertinent to methanogenesis in bioreactors,
are also susceptible to be stressed by Cd++ and sound. It follows that
methanogens and the other components of methanogenic consortia ought to be
able to withstand stress caused by heavy metals and sound to a degree above and
beyond that of cells in other, less stressful environments.
Fig. 3. Response of the M mazeii S-6 genes grpE, hsp70(dnaK), and hsp40(dnaJ) to heat shock
at various temperatures demonstrated by slot-blotting. The levels of mRNA for grpE,
hsp70(dnaK), and hsp40(dnaJ) (top three panels) are represented by vertical bars expressed in
the OD. X mm units given by the densitometer. The respective slot blots (10 µg/slot of total
RNA) are shown at the foot of the bars, while the heat-shock temperatures are indicated in the
horizontal axis at the bottom of the figure (°C). Hybridization was done with the respective
labeled probes. The culture density is shown in the bottom panel. The OD660 was determined
at time 0 (open bars) and at 30 min (hatched bars), in cultures maintained at 37°C or heat-
shocked during this 30-min period at the temperatures indicated at the foot of the bars. Repro-
duced from reference [63] with permission from the copyright owner
grpE
dnaJ dnaK
% area of peak
a b c d
Fig. 4. Response of the M. mazeii S-6 genes grpE, hsp40(dnaJ), and hsp70(dnaK) to the stres-
sors cadmium (Cd++) and heat. The bars represent levels of mRNA determined by slot-blot-
ting with probes for the grpE, hsp40(dnaJ), and hsp70(dnaK) genes. The total RNAs were from
cells grown at 37°C (i.e., the optimal temperature for growth for M. mazeii S-6) in medium
without Cd++ (a), and in medium with 5 or 27 mM CdCl2 (b and c, respectively); and from cells
grown in medium without Cd++ but heat-shocked at 45°C for 30 min (d). Note that the mRNAs
from the three genes increased after heat shock by comparison with the levels before heat
shock (constitutive or basal levels; compare a vs. d). Likewise, the presence of Cd++ in the
medium also induced an increase in the three mRNAs. This effect was more marked with 27
than with 5 mM CdCl2; compare a vs. b and c; and b vs. c. Reproduced from reference [58] with
permission from the copyright owner
Another compound very pertinent to methanogenic bioreactors is ammonia.

Bioconversion of wastes from humans and other animals with feces and urine is
inhibited when the ammonia present in these wastes reaches certain levels [53,
54]. Data in Fig. 6 demonstrate that ammonia induces a stress response in
M. mazeii S-6 as evidenced by the increase in the levels of the mRNAs from the
molecular chaperone machine genes [67].
Interestingly, ammonia also induces a response by the adjacent gene trkA. The
response of this gene seems to be tightly regulated in view of its strict dose
dependency. Overall, the results show that concentrations of ammonia over
20-fold higher than that which is adequate for physiological growth of M. mazeii
S-6 cause stress in this methanogen (see also Sects. 6.6 and 8.3).
6.5
Factors that Modify the Stress Response
It is well established that a number of cellular activities and physiological func-

tions are associated with, or are dependent on, the cell cycle and the growth
Fig. 5. Response of the M. mazeii S-6 hsp70(dnaK) gene to the stressors heat and sound. The
levels of hsp70(dnaK) mRNA were determined by slot blotting (A) and were measured by den-
sitometry (B). RNAs were from cells cultured at the optimal temperature for growth, i.e., 37°C
(slot-blots 1 and 3, counting from the top down); or from cells heat-shocked at 45°C for 30 min
(slot blot 2); or from cells maintained at 37°C and exposed to sound (90 decibels) for 15, 30,
60, and 120 min (slot blots 4, 5, 6, and 7, respectively). Notice the increase of hsp70(dnaK)
mRNA after heat shock (compare peaks 1 vs. 2), and after sound stress (compare peaks 3 vs.
4–7). Reproduced from reference [58] with permission from the copyright owner
phase. Thus, growth phase affects many cellular properties. Among these is the
stress response. In M. mazeii S-6, both the basal (constitutive) and heat-shock
induced levels of the mRNAs from the molecular chaperone machine genes are
affected by growth phase, as illustrated by the data in Fig. 7, which pertain to the
hsp70(dnaK) gene [63]. The highest levels of this gene’s mRNA were induced by
heat stress in cells in early stationary phase, as compared to the levels induced in
cells in the exponential and late-stationary phases. The highest basal levels of
mRNA were observed in cells in late stationary phase. This, together with the
diminished response to heat stress, suggests that cells in late stationary phase are
stressed. The degree of stress gene activity in late-stationary phase in the
absence of an added stressor is higher than in the other two phases. Concomi-
tantly, in late stationary phase, the capacity to respond to an added stressor is
impaired.
Fig. 6. Effect of ammonia on grpE, hsp70(dnaK), hsp40(dnaJ) and orf11-trkA mRNA levels in
M. mazeii S-6. Total RNA was extracted from single cells cultivated in medium with the stan-
dard concentration of NH4Cl (i.e., 1 g/L; lanes A), or from cells incubated for 30, 60, or 180 min
in medium containing either 10 (lanes B, C, and D, respectively) or 25 (lanes E, F, and G, respec-
tively) g/L of NH4Cl, and electrophoresed (10 µg/lane) in a denaturing gel. The upper portion
of each panel represents the gel stained with ethidium bromide showing the 23S and 16S
rRNAs, whereas the bottom portion displays the respective Northern blot hybridized with
probes for grpE (top left panel), hsp70(dnaK) (top right), hsp40(dnaJ) (bottom left) and orf11-
trkA (bottom right). The sizes of the rRNAs, and those of the hybridization bands in kilobases
(kb) are indicated to the right. Reproduced from [63] with permission from the copyright
owner
dnaK
37°C
45°C
1 2 3
Fig. 7. Response of hsp70(dnaK) to heat shock in M. mazeii S-6 cells at different growth phas-
es. Top panel. Slot blots of total RNA (10 µg/slot) from cells in exponential (1), and early (2)
and late (3) stationary phases, before (37°C) and after (45°C) a heat shock at 45°C for 30 min,
hybridized with a probe for hsp70(dnaK). Bottom panel. Densitometric readings (OD. X mm)
of the slot blots shown in the top panel, before and after heat shock (open and hatched bars,
respectively). Reproduced from reference [63] with permission from the copyright owner
The observations describe above are critical for developing strategies to mon-
itor the performance of microbes in bioreactors, and to control and improve the
microbial populations so that they are always capable of responding to stressors,
and to proceed with methanogenesis, despite changes in growth rates and envi-
ronmental factors.
6.6
Other Methanogens
The studies referred to above were done using M. mazeii S-6, which is a key
organism in mesophilic methanogenic ecosystems, including bioreactors [19,
68–73]. There are, in addition, data pertaining to M. thermophila TM-1 (OTG
50°C), which is important for methanogenesis in many thermophilic ecologic
niches and bioreactors [15, 17, 18, 71, 74, 75].
The effect of heat-shock of various durations on the chaperone machine
genes of M. thermophila TM-1 was determined. Illustrative data for
hsp70(dnaK) and hsp40(dnaJ) are shown in Fig. 8, top panel [14]. Cells were
heat-shocked at 60 or 65°C for 15, 30, or 60 min. Gene transcripts were minimal
before stress, but after a 60-min heat shock at 60°C they increased. A similar
trend was already evident after a 15-min heat shock at 65°C, and the levels of
transcripts were still higher after longer heat shocks by comparison with cell
heat-shocked at 60°C. The mRNA from hsp40(dnaJ) increased little under all the
conditions tested, suggesting that this gene is regulated differently as compared
with hsp70(dnaK).
It is clear from the results in Fig. 8 that 65°C is a critical temperature for
M. thermophila TM-1. The cells suffer a stress and mount a stress response after
only 15 min of exposure at 65°C. It may be concluded that assessing the levels of
hsp70(dnaK) transcripts will provide a sensitive indicator of TM-1-cell stress in
a bioreactor with temperatures above 60°C. By the same token, the data also
indicate that the bioreactor temperature must be maintained below 60°C to
insure the well being of M. thermophila TM-1 (and the same is probably true for
other methanogens that have OTGs similar to TM-1).
The response of M. thermophila TM-1 to ammonia stress was also studied
[14]. An increase in the mRNA from hsp70(dnaK) was induced by all three
ammonia doses tested: 5, 10, and 25 g/L in the series of experiments shown in
Fig. 8, bottom panel. Interestingly, the effect of 5 g/L correlated with exposure
length: no mRNA increase after a 10-min exposure, clear increase after 30 and
60 min, and clear but slightly lower increase after 120 min.
A dose-dependent response to ammonia stress was also observed for the trkA
gene, Fig. 8, bottom panel, lower section.A clear increase in this gene’s transcript
was produced by the 5 g/L-10 min dose. Longer exposure times caused tran-
script increases that were less and less marked as the times increased. A differ-
ent pattern was observed for the 10 g/L dose. The trkA mRNA augmented pro-
gressively as the incubation time with ammonia increased from 10 to 60 min, but
a 120-min exposure caused about the same effect as 60 min. The highest dose
tested, 25 g/L, caused the same increase in the trkA mRNA levels at all the incu-
bation times tested with ammonia, except for the 30-min exposure, which pro-
duced a slight but evident higher increase.
Fig. 8. Top panel. Response of M. thermophila TM-1 to heat shock. Northern blotting of total
RNA (10 µg/lane) from TM-1 hybridized with a probe for hsp70(dnaK), top and middle sec-
tions, or for hsp40(dnaJ), bottom section. Lanes 1 and 5 contained RNA from cells maintained
at 50°C. The other lanes contained RNA from cells heat-shocked for 15 min (lanes 2 and 6),
30 min (lanes 3 and 7), or 60 min (lanes 4 and 8), at 60°C (top section) or at 65°C (middle and
bottom sections). The left half of each section displays the gel stained with ethidium bromide
to show the 16 and 23 rRNAs, while the right half shows the respective Northern blot with the
size of the hybridization bands in kilobases (kb). Bottom panel. Response of M. thermophila
TM-1 to increasing concentrations of ammonia assessed by slot blotting to determine levels
of hsp70(dnaK) and trkA transcripts after incubation with the stressor for various time peri-
ods. Total RNA was extracted from exponentially-growing cells and 10 µg of RNA/slot was
used. The blots were hybridized with biotin-labeled probes for hsp70(dnaK) and trkA (top and
bottom sections, respectively). In each section the slot blots are at the bottom, and the vertical
bars above the slot blots represent the respective densitometric readings. Reproduced from
reference [14] with permission from the copyright owner
These data, together with those obtained with M. mazeii S-6 and described in
the previous Sect. 6.4, indicate that the stress response to the stressor ammonia
is dose dependent, and that it must be tightly regulated. The levels of ammonia
in the medium in which the cells grow, and the length of time during which the
cells are exposed to this compound, play critical roles in determining the degree
of the stress response to ammonia (and probably have distinctive effects on the
response to other stressors that may act simultaneously in real-life situations).
The implications for bioreactor management and technology of the data dis-
cussed above are manifold. For example, the level of stress caused by ammonia
can be monitored by assessing the levels of mRNAs from one or more stress
genes, including trkA. Also, it is clear from the data that ammonia can be a pow-
erful cell stressor, and that its effects are more pronounced as the dose and expo-
sure times increase. Lastly, a time of exposure to elevated levels of ammonia as
short as 2 h will cause severe cell stress.
The effects of relatively long heat shocks on both Methanosarcina species are
illustrated by data in Fig. 9 [63]. The results show that, contrary to bacteria [12],
M. mazeii S-6 and M. thermophila TM-1 (and most likely many other
methanogens) can withstand heat shocks longer than 15–30 min without a
decrease in the stress response as measured by the levels of the hsp70(dnaK)-
locus gene products. Even after a 3-h heat shock, the M. mazeii S-6 hsp70(dnaK)
mRNA is quite increased.
Fig. 9. Response of the hsp70(dnaK) gene from M. mazeii S-6 and M. thermophila TM-1 to
heat shocks of various durations. Left panel. Northern blots of total RNA (10 µg/lane) extract-
ed from S-6 cells before heat shock (lane 0, in both sections), or after a heat shock at 45°C for
the length of time indicated in the horizontal axis, in minutes (min) or hours (h). Hybridiza-
tion was done with a probe for hsp70(dnaK). The size of the hybridization bands in kilobases
(kb) is indicated to the right. Right panel. Total RNA (20 µm/lane) from M. thermophila TM-1
was run in a denaturing gel and stained with ethidium bromide to show the 23S and 16S
rRNAs (top section). The respective Northern blot obtained with a probe for hsp70(dnaK) is
shown below (bottom section). Lanes from left to right are: RNA from cells maintained at 50°C
(lane 0, non-heat-shocked) and RNAs from cells heat-shocked at 60°C for 1, 2, or 3 h (lanes 1,
2, and 3, respectively). The size of the hybridization bands in kilobases (kb) is shown to the
right. Reproduced from reference [63] with permission from the copyright owner
The capacity of methanogens to respond efficiently to temperature elevations

above their optima, even if they are exposed to these temperatures for hours, is a
useful and encouraging feature. It shows that the need to correct the temperature
of a bioreactor before it causes irreversible cell damage is not of extreme urgency.
Likewise, it also shows that any measure that would improve the cell’s stress
response, even slightly, will add time to the period during which corrections to
the bioreactor conditions can be made. This time gain should greatly facilitate
bioreactor operation, and reduce the frequency of total, irreversible failures.
6.7
Co-Chaperones
In view of the discontinuous distribution of the chaperone machine among

Archaea and the occurrence of chaperonins in these organisms (see follow-
ing Section), it is pertinent to ask whether Archaea have the co-chaperones –
also named chaperone co-factors – known to coexist, and interact with the
machine and the chaperonins in bacteria and eukaryotes. Examples of co-
chaperones are the bacterial trigger factor (TF), and the eucaryal Hop, Hip,
BAG-1, and NAC.
A recent survey of five fully sequenced archaeal genomes, including one with
the machine and four lacking it, but all containing chaperonins, showed absence
of conservation of the genes encoding the co-chaperones [75a]. There were no
genes readily identifiable by common genome-searching methods as being the
homologues of the five co-chaperones listed above. However, two families of
molecules were identified that might be related to Hop and to one of the sub-
units of NAC. These results, which open the road to a more detailed analysis of
the chaperoning mechanisms in Archaea as compared to those of bacteria and
eukaryotes, are available in the Internet at:
http://www.bioscience.org/2001/v6/d/macario/fulltext.htm
7
The Hsp60 (Chaperonin) System in Methanogens
7.1
Examples
Extensive descriptions and discussions of the archaeal chaperonin system are

available in the literature [7, 10, 12, 76, 77]. Therefore, only aspects directly or
indirectly pertinent to methanogens will be touched in this chapter.
Examples of chaperonins in methanogens are listed in Table 7. The functions
of these genes’ products have not been elucidated in any detail either in vivo or
in vitro. However, extrapolating from what is known from studies in other
archaeal, non-methanogenic species, and in eukaryotes and bacteria (references
in [12]), it may be said that the chaperonins of methanogens are likely to play a
critical role in de novo protein biogenesis. They may also play a role during the
stress response, and in the cell recovery after stress, but these roles although
probable have not yet been demonstrated.
Table 7. A sample of hsp60 (chaperonin) genes in methanogens a
Organism Chaperonin Base Promoter Terminator RBS b

(accession number) pairs
Methanobacterium Chaperonin 1617 n.r. c n.r n.r

thermo- (a-subunit)
autotrophicum (mt0794)
DH Chaperonin (mt0218) 1659 n.r. n.r. n.r.
Methanococcus Chaperonin 1626 n.r. n.r. n.r..
jannaschii (U67542)
Methanococcus MTTS (AB015435) 1632 tttatata t-rich region n.r.
thermo- (–75) d (+20)
lithotrophicus
Methanopyrus Thermosome 1635 tttaaata c-rich region aggtgat
kandleri (Z50745) (–60), (+18)
atgc (–42)
a Data extracted from reference 12, with permission from the copyright owner.
c n.r., not reported.
d (–) and (+) refer to position of center of sequence upstream from the translation start codon
or downstream from the translation stop codon, respectively.
7.2
Structure and Potential for Bioreactor Technology
The chaperonins form multimeric complexes of comparatively very large size

(thousands of kDa) with a spheroidal or cylindrical shape, and with a central
cavity that serves as a protected chamber inside which polypeptide folding is
thought to occur [12].
The implications for biotechnology and bioreactors are significant. The phys-
iological performance of methanogens is tied to a protein balance within the
normal range. Protein balance here is understood as the entire set of proteins in
a cell, which is composed of many functionally distinct subsets. Each subset
must be maintained within the normal range of number of molecules (concen-
tration), and each molecule must be kept with its structural and conformational
integrity (native configuration). Most of these properties are maintained by the
concerted action of the molecular chaperone machine, the chaperonins, and
other molecules including proteases. It follows that research ought to be direct-
ed towards the elucidation of how the chaperonin system in methanogens with
different OTGs, and pertinent to anaerobic digestion of wastes (e.g., M. mazeii
S-6 and M. thermophila TM-1, and others) assembles itself and functions under
normal circumstances, and under stress. Regulation of the chaperonin genes
ought to be clarified in order to manipulate them in a way that will maintain pro-
tein biogenesis during stress to ensure continuous biomethanation.
8
Other Stress or Stress-Related Molecules, Genes and Proteins,
and Anti-Stress Mechanisms in Methanogens
8.1
Examples
A number of molecules different from those discussed in the preceding Sections

appear, or increase in concentration, in response to stressors [12]. Illustrative
examples of molecules that have been studied are listed in Table 8. These mole-
cules are integral parts of the stress response as the molecular chaperones and
chaperonins. Similar genes, and other pertinent examples have been identified
in the genomes of methanogenic species that have been fully sequenced, as
shown in Table 9. Also, some methanogenic species have a cell envelope with
extraordinary stability [42, 78].
8.2
Osmolytes
Some of the molecules listed in Table 8 (e.g., inositol compounds) are not pro-
teins and participate in maintaining the internal osmotic pressure. Compounds
of this sort are called osmolytes and they come into action when the osmolarity
of the medium surrounding the cell increases or decreases (osmotic shock) [13,
79–81]. These compounds are of paramount importance for cells that inhabit
hypersaline environments, or that suddenly encounter such environments, for
example when the influent of a bioreactor contains unusual concentration of
salts. In addition, the enzymes that participate in the synthesis import of osmo-
lytes are also stress proteins in as much as they must be active during stress, and
must produce the osmolytes to protect the cells from osmotic stress.
8.3
TrkA
TrkA has been identified in M. mazeii S-6, M. thermophila TM-1, and in other
methanogens whose genomes have been fully sequenced [14, 62, 66]. It is a pro-
tein member of the Trk K+ transport system in Escherichia coli and other bacte-
ria [82, 83]. By analogy, the archaeal homologue is assumed to be also involved
in the transport of this cation.
TrkA is involved in maintaining the K+ balance of the cell [82, 83]. The inter-
nal K+ balance of methanogens in anaerobic bioreactors may be affected by fac-
tors in the immediate environment. Ammonia is one of these factors, known to
inhibit methanogenesis [55, 56]. Ammonia may reach inhibitory levels when a
bioreactor is fed with protein-rich wastes or swine manure, for example [53].
The unionized ammonia causes a pH increase. An intracellular pH increase will
result in a K+ efflux coupled to a H+ influx in order to counteract the pH increase
[55, 84]. TrkA is involved in this counter transport. As described in a previous
Section, the trkA of both M. mazeii S-6 and M. thermophila TM-1 respond to
Table 8. Other examples of stress, or stress-related, genes and proteins that have been found
and studied in methanogens a
Gene/protein Protein mass Organism Presumed Inducer

(kDa) function
Crx protein trio 40.8, 42.3, Methanobacterium Copper or Copper

and 42.9 bryantii general resistance
Betaine transporter n.r. b Methanosarcina Maintains internal Osmotic
thermophila TM-1 ionic balance stress
Inositol n.r. Methanococcus Maintains internal Osmotic
compounds igneus ionic balance stress
TrkA 44.1 Methanosarcina Maintains internal Ammonia
mazei S-6 K + balance
Prefoldin or 14–23 c Methanococcus Protein folding n.r
jannaschii;
GimC Methanobacterium n.r. n.r.
thermo-
autotrophicum
Small heat-shock n.r. Methanococcus RNA stabilization, n.r.
protein (sHsp) jannaschii thermotolerance
ClpB n.r. Methanosarcina Affects growth and n.r.
acetivorans survival at hightem-
peratures, involved
in proteolysis
PPIase (peptidyl 19.4–31 d Methanococcus Accelerates rate n.r.
prolyl cis-trans 16 or 42 d thermo- limiting step in
isomerase) lithotrophicus, protein folding
Proteasome 24 and 22 e Methanosarcina Protein n.r.
hermophila TM-1, degradation
25.8 and Thermoplasma
22.3 e acidophilum
a Data extracted from reference 12 with permission from the copyright owner. See also
Table 9.
b n.r., not reported.
c Six subunits within the indicated size range in eukaryotes but only two subunits in Archaea.
d Depends on the method used.
e a- and b -subunit, respectively.
ammonia stress as shown by an increase of its mRNA. Since it may be assumed

that this gene’s product, TrkA, is involved in maintaining a physiological level of
intracellular K+ also in methanogens, and since this cation is essential for the
molecular chaperone machine to function [refs. in 62, 66, 67], one may hypo-
thesize that trkA is a stress gene. It probably plays a major role in cell physiology
and survival in bioreactors. More research on this interesting topic with many
Table 9. Stress related gene/protein-homologues identified in sequenced genomes from

methanogens a
Organism Gene/protein ID b
Methanococcus Heat-shock protein X MJ1682

jannaschii Heat-shock protein 31 MJ0285
DNA repair protein 45 MJ0869
DNA repair protein RAD51 MJ0254
DNA repair protein RAD2 MJ1444
PPIase MJ0278
PPIase MJ0825
Proteasome a-subunit MJ0591
Proteasome b-subunit MJ1237
Survival protein MJ0559
Methanobacterium Heat-shock protein X MTH569
thermoautotrophicum Heat-shock related protein X MTH1817
DH Heat-shock protein class I MTH859
DNA repair protein rad2 MTH1633
DNA repair protein radA MTH541
PPIase MTH1125
PPIase B MTH1338
Proteasome, a-subunit MTH686
Proteasome, b-subunit MTH1202
Survival protein (SurE) MTH1435
a Excluding the Hsp70(DnaK) chaperone machine and the Hsp60 (chaperonin) family.
Reproduced from reference 12 with permission from the copyright owner.
b ID, identification number in genome project.
potential applications for the monitoring and control of bioreactors should be

done. This research will lay the foundations for using the trkA gene to fortify
cells and make them more resistant to the ammonia and other stressors, which
may also provoke imbalances of intracellular electrolytes.
8.4
Prefoldin or GimC
Another multimeric complex named prefoldin or GimC seems to be involved in

protein folding in eukaryotes and in Archaea, including methanogens [85–87].
Six subunits have been identified in eukaryotes, but only two have been found in
Archaea.
The role of this complex in the stress response is unclear. The genes coding
for the subunits do not seem to be activated by stressors. Nonetheless, we will
discuss prefoldin in this Chapter because of its probable participation in protein
folding in vivo. Very little is known in this regard but current research will soon
add to our knowledge of this “chaperone machine” and may unveil functions
that are essential for survival during stress, and/or for recovery after stress. Both
these functions are important for methanogens in bioreactors.
The representative from Methanobacterium thermoautotrophicum named
MtGimC has been studied in some detail [86]. It is a complex of 87 kDa made of
two different subunits, a and b. Preliminary studies have indicated that the com-
plex is a hexamer consisting of two a and four b subunits. The a subunit is the
equivalent of the eukaryotic subunits Gim2 and Gim5, while b is the homologue
of the eukaryotic Gim 1, 3, 4, and 6 subunits.
A preliminary in vitro search for possible chaperone functions of MtGimC
showed that it:
(i) Formed a complex with unfolded actin, and bound this substrate with rel-
atively low affinity;
(ii) Suppressed aggregation of unfolded hen lysozyme (14 kDa);
(iii) Prevented aggregation of chemically unfolded bovine mitochondrial rho-
danese (30 kDa) and glucose dehydrogenase (39 kDa);
(iv) Formed complexes with non-native dihydrofolate reductase (DHFR;
23 kDa) and firefly luciferase (62 kDa);
(v) Stabilized non-native actin for at least 15 min, which allowed transfer of
actin to TRiC (the eukaryotic chaperonin complex) for folding in the pres-
ence of ATP; and
(vi) Prevented aggregation of unfolded rhodanese (as mentioned above) and
allowed its folding by the bacterial chaperonin GroEL.
While the above series of observations demonstrate a certain degree of partici-
pation of MtGimC in preventing the aggregation of partially denatured poly-
peptides, and in assisting folding by way of interaction with TRiC in the case
of actin or GroEL for rhodanese, how much the results reflect in vivo, physio-
logically meaningful situations remains to be seen. Further analysis in vitro, and
new studies in vivo should be done to elucidate the functions of GimC in
methanogens, its mechanism of action, preferred substrates, and activity (or
lack thereof) during stress. One may anticipate that important information is
going to emerge from these analyses, which will be very useful in understanding
the intracellular situation of stressed methanogens and in thinking of ways for
coping with it so the cell will be able not only to survive, but also to continue the
bioconversion pathway unabated.
8.5
Small Heat-Shock Proteins (sHsp)
The sHsp are currently the focus of active investigation in organisms of the
three phylogenetic domains, including the methanogens [12]. An sHsp from
Methanococcus jannnaschii has been purified and crystallized [33]. Like other
stress proteins, it forms a large multimeric complex. It protects other proteins
from heat denaturation and prevents aggregation of partially denatured
polypeptides in vitro [34]. More research is necessary to determine the role of
this, and other, sHsp in vivo. Such research should provide the basis for design-
ing strategies to use sHsp and/or their genes for improving the mechanisms
against protein denaturation in methanogens, and thus their resistance to stres-

sors. In this regard, it is noteworthy that sHsp form large complexes, as the chap-
eronins for example do. These complexes are seemingly essential for the chap-
erones in general to exercise their function of assisting other proteins to fold and
refold. One of the aims of research in this area, with direct implications for bio-
methanation technology, should be the elucidation of how the multimeric struc-
tures form, what stressors do tend to damage these structures, and what keeps
them from being disrupted by stressors. Obviously, information on these areas
will help in identifying the most damaging stressors, and in developing means
to avoid their accumulation in a bioreactor, and tools, genetic and otherwise, to
strengthen the stability of the multimeric complexes.
8.6
PPIase
PPIase (for peptidyl prolyl cis-trans isomerase) is an enzyme that is found in
many organisms of the three phylogenetic domains [12]. It mediates peptidyl-
prolyl isomerization, an important step in protein folding. There are various
forms of the enzyme, similar to each other, that have been grouped into three
families: cyclophilins (Cyp), FK506-binding proteins (FKBPs), and parvulins
[29, 30, 88–90]. A PPIase from Methanococcus thermolithotrophicum that
belongs to the FKBP family has been characterized in some detail [88]. The
genes encoding other PPIases have been found in the genomes of other
methanogens, as seen in Table 9.
The observations described above demonstrate that methanogens possess a
complex battery of tools, including PPIases, to generate and maintain a balanced
set of proteins within the ranges of concentrations and configurations required
for growth and survival. Study of PPIases will help in understanding protein
folding in methanogens pertinent to bioreactors, and will pave the way to devis-
ing means for protecting the folding machinery from damage due to stressors.
8.7
Proteases
Proteases constitute a large group of enzymes, some of which should be consid-
ered under the umbrella of stress.We will not discuss them here in any detail but
refer to reviews available in the literature [3–5, 91, 92]. Suffice it to say that pro-
teases are involved in the degradation of abnormal proteins lest they interfered,
or might interfere, with the trafficking of normal proteins and other functions
inside the cell. Abnormal protein in this context means molecules that are par-
tially or completely unfolded due to stress or to some structural alteration
(mutation, or post-synthetic modification that went wrong). These abnormal
molecules tend to misfold, aggregate, and build up precipitates. If these are too
large, they will be an obstacle to the physiological movement of molecules inside
the cells, and cause a disturbance in many functions. Hence, abnormal molecules
must be refolded into a correct configuration or, if this is impossible, they must
be eliminated. Molecular chaperones participate in folding, refolding, dissolving
aggregates, and degradation. For the latter purpose, some molecular chaperones
present the abnormal polypeptide to the protease for digestion. When all the
preventive and corrective measures aiming at keeping the proteins in the correct
concentrations and configurations fail, or are overwhelmed owing to an excess
of substrate for the chaperoning systems, proteases are called into action. These
enzymes degrade the abnormal polypeptides and, in doing so, they not only rid
the cell of aggregates, but they also generate building blocks (i.e., amino acids)
for the synthesis of new protein molecules.
Proteases are surely also involved in the construction of multicellular struc-
tures (see Section below), a process that requires the action of many molecules
in addition to proteases. The formation of multicellular structures requires also
the migration of these molecules towards the cell’s outside, as we shall discuss in
a subsequent Section of the Chapter.
Interestingly, proteases tend to form multimeric complexes. One example is
the proteasome, which is a large multimolecular machine similar to the chaper-
onin complex [91–95].
8.8
Putative Stress Genes and Proteins Found in Fully Sequenced Genomes
The availability of full genome sequences has opened the doors to computer-
assisted searches for stress genes, or candidate (putative) stress genes, which
encode proteins likely to play a role in the stress response but which have not yet
been isolated and tested in the laboratory. A sample of these genes/proteins
found in two genomes from methanogens (Methanococcus jannaschii and
Methanobacterium thermoautotrophicum) that have been sequenced is dis-
played in Table 9. Excluded from the list are the genes for the members of the
Hsp70(DnaK) chaperone machine and those for the Hsp60 (chaperonin) sys-
tem, both groups already discussed in previous Sections (see Tables 4–7). It is
important to re-emphasize that the chaperone machine genes are not present in
the genome of M. jannaschii, as discussed previously (see Table 3), whereas the
chaperonin genes do occur in this methanogen and in M. thermoautotrophicum.
The functions of the genes/proteins listed in Table 9 remain to be determined.
This is a challenging task for the near future made attractive because of the
availability of the clones that contain the genes, and the promise of information
useful to devise strategies and tools for improving methanogens so that they will
develop increased resistance to stressors.
9
Other Manifestations of the Stress Response
9.1
Introduction
In addition to the components of the stress response described in the preceding

Sections of this Chapter, which have been identified in methanogens and other
Archaea, there are other pertinent molecules, anatomical structures, and events
that must be discussed [12]. These are either induced or are associated in some
meaningful way with the stress response, the molecular chaperoning process,
the development of stress resistance also called thermotolerance or stress toler-
ance [26], or the recovery of cells after stress. What follows is a brief account of
several of these stress-related molecules, structures, and phenomena that are
important for the survival and functioning of methanogens, and that have
potential for the devising of means to improve bioreactor performance despite
changing environmental conditions.
9.2
Thermoprotectants
Cells produce compounds that somehow improve their thermotolerance. Some

of these are sugars and simple molecules such as di-myo-inositol phosphate
(DIP) and cyclic diphosphoglycerate (cDPG). They have been demonstrated, for
instance, in the hyperthermophilic methanogens Methanopyrus kandleri (OTG
100°C) and Methanothermus fervidus (OTG 85°C). A more detailed discussion
on the functions and possible mechanism of action may be found in recent arti-
cles with pertinent bibliography [13, 81, 96]. See also Table 8.
9.3
Multicellular Structures
A few methanogenic species have the ability to build multicellular structures,

either by themselves (single-species structure) or in association with one or
more different species (multispecies structure) [17, 25, 42, 97, 98]. These struc-
tures may be formed in response to stressors and confer more resistance to them
by comparison with the isolated cells growing as independent, free units.
Morphologically, the multicellular structures appear as flat sheets of one or
very few cell-diameters in thickness, or as globular masses vaguely spheroidal in
shape with diameters equivalent to many (e.g., 10–20) cell diameters. The cells
are kept together by an intercellular connective material, whose components are
not yet fully elucidated, and that ought to be considered elements of the stress
response as a working hypothesis for future research (see below).
Examples of single-species multicelluar structures are produced by M. mazeii
S-6, which can be flat (named lamina) or globular (named packet), as illustrated
in Fig. 10 [97, 98]. The packet morphotype is considerably more resistant to
mechanical, chemical, and physical stressors, and to antibiotics, than the single-
cell morphotype (AJLM and ECdeM, unpublished data). For instance, induction
of a heat-shock response measurable by an increase in the mRNAs from the mol-
ecular chaperone machine genes (as shown in Figs. 2 and 3, for example)
requires higher temperatures and longer exposure times in packets than in sin-
gle cells. Illustrative data for the grpE gene are presented in Fig. 11 [60]. The sin-
gle-cell morphotype showed a more pronounced response than that of the pack-
ets to a heat-shock at 45°C for 30 or 60 min. In fact, the packets showed a
response only after a heat shock of 60 min.
Other multicellular structures directly pertinent to anaerobic methanogenic
bioreactors are the globular multispecies consortium termed granule [17], and
Fig. 10. Multicellular structures formed by M. mazeii S-6. Packets (A) and lamina (B) are dis-
played along with the single-cell morphotype (C), for comparison (see references [97, 98]).
The diameter of the single cells is 1–3 µm, and the magnification factor is the same for the
three panels. The photographs were taken with phase contrast optics of wet samples from live
cultures between glass slide and cover slip. Reproduced from reference [12] with permission
from the copyright owner
the biofilm [72, 99, 100]. They are usually composed of a variety of methanogens
and bacteria interlaced in a food web. Histological thin sections of a granule
from a thermophilic bioreactor are shown in Fig. 12, where the spheroidal
shape may be inferred from the visible segment of the outer profile [101].
Methanosarcinal packets can be seen in panel A, while panel B shows laminar
structures formed by M. thermophila TM-1 as demonstrated by the antigenic
fingerprinting method. The methanogens form colonies of various shapes and
sizes that are lodged in the supporting scaffolding provided by the intercellular
connective material as represented in the model shown in Fig. 13 [101].
Little is known about the mechanism of granule formation (granulogenesis)
at the molecular and genetic levels, or about the biochemistry and synthesis of
the components of the intercellular connective material. Also, the functions of
this material, beyond the obvious mechanical support for cells, are largely
unknown. These functions are surely more complex than just providing a scaf-
fold for the growth of cellular colonies. They must also include insulation, trans-
port of nutrients and catabolites in opposite directions, concentration of
micronutrients, passive barrier or active defense against agents of various kinds
(chemical, physical, and biological such as antibiotics), and others that future
research ought to discover. Granules have an inner communication network
made of a small tubes [101], as shown in Fig. 14. These tubes can conceivably be
the route for nutrients to reach the cells inside the granule, and the way of escape
for catabolites and other cellular products away from the cells.
There is some information about the composition of the intercellular con-
nective material in Methanosarcina packets [102, 103], but beyond that there is
not much that would allow the developing of means to manipulate this material
Fig. 11. Heat resistance of a multicellular structure formed by a methanogen as compared

with its own single-cell phenotype. Primer-extension mapping of the transcription initiation
site for M. mazeii S-6 grpE. A radiolabeled oligonucleotide primer complementary to bases 57
through 77 within the grpE coding region was used with 10 µg of total RNA from single cells
(lanes 1 to 3) or packets (lanes 4 to 6) per test. Single cells and packets were grown at 37°C
(lanes 1 and 4) or heat shocked at 45°C for 30 (lanes 2 and 5) or 60 (lanes 3 and 6) min. The
primer-extended products were electrophoresed in a 6% acrylamide sequencing gel in paral-
lel with the products of a sequencing reaction that was done with the same primer and the
dideoxychain-termination method (lanes G, A, T, and C). These lanes show the complemen-
tary (anti-sense) strand sequence. The coding (sense) strand sequence and the initiation site
(asterisk) are shown on the left. Reproduced from reference [60] with permission from the
copyright owner
at the molecular and genetic levels for biotechnologic purposes. This is a very
important area for investigation since efficient methanogenesis in bioreactors
depends on the presence of a stable population of microbes retained in position
within the granule, and inside the bioreactor, in the appropriate spatial relation-
ship with one another. This three-dimensional distribution of different species
is key to the metabolic interactions between them, as required by the food web
leading to waste bioconversion with generation of methane.
Fig. 12. Spheroidal multicellular structure (granular consortium, or granule) formed by

methanogens associated with bacteria in a thermophilic (50°C), anaerobic, methanogenic
bioreactor, as seen in a thin histological section. (A) Cross section of the granule showing the
cortex and medulla (see reference [101]) and a large island of methanosarcina packets
(arrows). Hematoxylin-eosin (magnification ¥800). (B) Another section of the same granule
in which the presence of Methanosarcina thermophila TM-1 (optimal temperature for growth,
50°C) is demonstrated with a antibody probe for TM-1 by immunofluorescence. The
methanosarcina cells are arranged mostly in laminae (see Fig. 10; magnification ¥4000).
Reproduced from reference [12] with permission from the copyright owner
Most likely, granules are among other things a mechanism to protect the cells
from stressors, as may be inferred from the data mentioned above, obtained
with methanosarcinal packets. Also, because of their large size and weight as
compared with individual cells, the granules will not be washed away by the cir-
culating bioreactor contents, and thus they will maintain a steady functional
profile.
Fig. 13. Computer-assisted three-dimensional representation of a granular consortium like

the one shown in the preceding figure, depicting the aggregates or bundles formed by
methanogens. Methanobacterium thermoautotrophicum, surface (SC) and inner (IC) colonies;
Methanosarcina thermophila packets (P) and laminae (L); Methanosaeta (Methanothrix) rods
in bundles of more or less intertwined filaments (Mx); Methanobrevibacter arboriphilus (Ma),
clouds that appear in cross-section as lawns of variable density; and Methanobrevibacter
smithii (Ms), thin clouds that look like sparse lawns in cross-section. The filaments formed by
the Methanosaeta rods are shown for the sake of clarity in only two areas but they are more
generalized. Reproduced from reference [101] with permission from the copyright owner
The structure of the granule is complex (see Figs. 12–14) [75, 101, 104],
including well defined zones, such as the cortex and the medulla, and subzones
that probably represent functionally specialized areas. In addition, there are
the small tubes (Fig. 14), which provide still another proof that a granule is a
complex anatomic structure with a complicated physiology, seemingly well
equipped to withstand stressors. It is then important to realize that understand-
ing how a granule forms and maintains its integrity as a functional unit in an
environment as full of stressors as the bioreactors influents is essential for the
developing of means to monitor and control biomethanation, and to correct it
when the bioreactors malfunction. The same type of considerations apply to the
biofilm [70, 72, 73, 99, 100, 105], an example of which is presented in Fig. 15.
Fortification of cells to withstand stressors should, therefore, also include
improvements in their granule- or biofilm-formation ability. In this regard, all
the molecules that form the intercellular connective material and the enzymes
that synthesize as well as those that translocate them to the cell’s outside should
be considered components of the stress response.As such, they should be targets
for investigations aiming at developing means to improve granulogenesis, and
biofilm formation, and, thereby methanogenesis.
Fig. 14. Superficial, histological thin section of a granule like the one shown in Fig. 12, pass-
ing through the cortex. Visible are circular openings that are cross-sections of the tubes that
crisscross the granule (possibly communicating different zones of it between themselves and
with the immediate surroundings of the granule [101]). Hematoxylin-eosin (magnification
¥800). Reproduced from reference [12] with permission from the copyright owner
Fig. 15. Example of a microbial consortium in the form of biofilm made of methanogens and
associated, syntrophic bacteria visualized by scanning electron microscopy (SEM). The
biofilm was attached to the substratum (curler-type polypropylene) in a fixed-bed anaerobic
methanogenic bioreactor processing synthetic waste water containing acetate, propionate, and
butyrate at 35 °C. The samples were collected from the top (A and B), middle (C and D) and
bottom (E and F) of the bioreactor 57 days after its inoculation with sludge from another
digestor treating municipal sewage. Discernible are cells that were identified as related to M.
mazeii S-6 (single cells, 1), Methanosaeta (Methanothrix) soehngenii (2), Methanospirillum
hungatei (3), and Desulfovibrio sp. (4). M. mazeii occurred as single cells (best visible in B)
and as laminae (see Fig. 10). The exopolymer of the intercellular connective material in the
laminae appeared as filaments, as illustrated in D. Scale bars (in µm) are shown at the right-
bottom corner of each panel. Reproduced from reference [73] with permission from the copy-
right owner
10
Perspectives and Applications
10.1
Introduction
The study of the stress response, stress genes and proteins, other components of
the stress response, and molecules and phenomena pertinent to resistance
against stressors and recovery after stress is essential to deal with stressors,
counteract them, and avoid or abate their effects. Stressors of many kinds reach
bioreactors with the influent, or are produced inside the bioreactors. It is there-
fore of paramount importance to develop means to deal with the problems
caused by stressors. As repeatedly stated in this Chapter, before preventive and
corrective means can be developed, information from basic and applied
research is needed. This research should focus on several topics, some of which
will be dealt with in the following portions of this Chapter.
10.2
Diversity of Methanogens
In the preceding Sections we have referred to methanogens in bioreactors and

focused chiefly on M. mazeii S-6 and M. thermophila TM-1. These two
Methanosarcina species are key for methanogenic bioconversion in meso- and
thermophilic environments, respectively. However, other methanogens also
occur in bioreactors, Fig. 13. In fact, there is considerable diversity of
methanogenic species, strains, and immunotypes in bioreactors as demonstrat-
ed as early as 1988, Fig. 16 [69]. An important conclusion drawn from these and
subsequent findings is that the study of the stress response, stress genes and pro-
teins, and anti-stress mechanisms should be extended to other methanogens, in
addition to methanosarcinas (see also Sect. 10.6).
10.3
Dynamics of Methanogenic Subpopulations in Bioreactors
Qualitative and quantitative analyses using immunologic and other comple-

mentary methods have revealed that the population of methanogenic organisms
in bioreactors (and several other ecosystems) is composed of subpopulations,
each of these representing a different species [15, 18, 69–71, 74, 75, 100]. Sub-
populations have also been identified within a single species. Time-course stud-
ies have demonstrated that methanogenic subpopulations change in distribu-
tion and in size (number of organisms in each subpopulation), during bioreac-
tor operation [70, 75]. An illustrative study is displayed in Fig. 17. A few species
of methanogens identified in a bioreactor fed with sulfite evaporator condensate
were followed over a period of 14 months [70]. Some stressful manipulations
were done to the bioreactor during this period. Methanogenic subpopulations
were assessed by qualitative and quantitative methods at different time points.
The results showed that:
Fig. 16. Diversity of methanogens in bioreactors. Methanogens identified in a series of 14 dif-

ferent bioreactors (DIGESTOR A-N) with antibody probes and the antigenic fingerprinting
method using indirect immunofluorescence and the quantitative slide immunoenzymatic
assay, SIA. The variety of methanogens occurring in these bioreactors as a group and in each
one of them is evident from the total number (14) of species identified and the range of species
found in the individual bioreactors (from one, bioreactor K, up to 8, bioreactor B). In most cas-
es the methanogens found were not identical to the reference species, neither were they of the
same immunotype within each species identified.Abbreviations are: Mx., Methanothrix; Msp.,
Methanospirillum; Mbr., Methanobrevibacter; Mc., Methanococcus; Mb., Methanobacterium;
Ms., Methanosarcina. Reproduced from reference [69] with permission from the copyright
owner
(i) The subpopulations differed in size at the beginning, thus adding an extra
dimension (quantitative) to the diversity already evident from the variety
of species present;
(ii) The subpopulations closely related to Methanobrevibacter smithii ALI and
M. mazeii S-6 were the most abundant at the beginning, while the subpop-
ulations closely related to Methanobacterium formicicum MF, Methanobre-
vibacter arboriphilus AZ, and M. arboriphilus DC, were the smallest;
Fig. 17. Diversity and dynamics over time (months) of methanogenic subpopulations in
bioreactors subjected to manipulations known to cause cell stress (e.g., change in pH, nutri-
ents’ availability, and configuration of functional space). Samples A and D (abscissa) were tak-
en from different levels inside the chamber at the beginning, when the bioreactor reached sta-
ble conditions, i.e., steady flow of substrate and yield of biogas. Seven months later, sample F
was obtained at a time in which modifications in pH and substrate chemical oxygen demand
(COD) were introduced. Immediately thereafter, configuration changes were also made, and
seven months later, samples H and J were collected, from different levels. Methanogens were
identified and quantified in each sample as follows: Methanobacterium formicicum MF (a);
Methanobacterium arboriphilus AZ (b) and DC (c); Methanobrevibacter smithii ALI (d) and
PS (e); a rod related to Methanosarcina thermophila TM-1 (f); Methanosarcina barkeri W (g);
and Methanosarcina mazeii S-6 (h). Each bar represents the number (arithmetic mean ±
range; n = 2) of organisms per species identified – that in most cases were not identical to the
reference organism. In g and h, open and closed bars represent the packets and single-cell
morphotype, respectively, of M. mazeii S-6 (see Fig. 10). The wavy lines at the top of the bars
for samples H and J in panel f indicate abundance beyond the quantifiable by the method
used. Reproduced from reference [70] with permission from the copyright owner
(iii) Some subpopulations increased after seven months (e.g., MF, DC, ALI, and
Methanobrevibacter smithii PS), while others remained the same (AZ) or
decreased (Methanosarcina barkeri W, and S-6);
(iv) Seven months after stressful manipulations of the bioreactor performed
within a short interval, which had caused a decrease in all methanogens
(data not shown), practically all the subpopulations had recovered;
(v) The two Methanosarcina species recovered to some extent but only in the
form of packets, namely the phenotype most resistant to stressors;
(vi) The presence of Methanosarcina packets at the end of the observation peri-
od was even more striking if one considers that at the beginning there were
virtually only single cells, and suggests that stressors along the way select-
ed against single cells and perhaps induced them to form multicellular
structures.
Efforts to improve bioreactor operation and yield ought to take into account the
diversity of methanogens involved and their time-course dynamics in terms of
quantity, described above. Those species more productive of methane will have
to be targeted first for improving their anti-stress machinery using genetic engi-
neering procedures, or stress-gene inducers that are not harmful, such as drugs
that mimic physiologic stress-gene inducers. These drugs could be added to the
influent at doses predetermined to induce stress genes without secondary,
unwanted effects on the cells.
10.4
Diversity of Stressors
A major goal of future research aiming at improving bioreactor technology

should be the identification of stressors that might affect methanogens and oth-
er pertinent microbes. A list of representative stressors for all kinds of cells is
displayed in Table 1, but only a minority of them have actually been tested with
methanogens, as shown in Table 10. These stressors are relevant to bioreactor
technology because they are found in relatively high levels in the effluents from
many factories, homes, farms, and other man-made sources that require anaer-
obic bioconversion in bioreactors.
10.5
Diversity of Response
Another important task for the near future will be that of characterizing in detail
the response to the different stressors that are relevant to methanogenic biotech-
nology. It is well established that a series of components of the stress response
are the same for any stressor. These are the basic or common components, which
are those discussed in this Chapter for the most part. It is very likely, however,
that the stress response has, in addition to the basic components, other elements
Table 10. Examples of stressors, other than heat, tested with methanogens a
Stressor Organism
Hyperosmolarity Methanococcus igneus, Methanococcus thermolitholitrophicus,

Methanosarcina thermophila TM-1, Methanosarcina mazei S-6
Pressure Methanococcus thermolithotrophicus, Methanococcus jannaschii
Ethanol Methanococcus voltae
Copper Methanobacterium bryantii
Cadmium Methanosarcina mazei S-6
H2O2 Methanococcus voltae
Ammonia Methanosarcina mazei S-6, Methanosarcina thermophila TM-1
Sound Methanosarcina mazei S-6
a Data extracted from reference [12] with permission from the copyright owner.
that are specific for each stressor or family of similar stressors. It will be
extremely interesting and useful to identify at least some of the stress-response
components that are specific for each of the stressors most relevant to bio-
methanation technology. They could be genes/proteins, signal transducers,
membrane sensors or receptors, gene-activating and gene-repressing factors,
molecules for signaling the formation of multicellular structures, etc. Also, the
mechanism of action of some of these molecules may differ depending on the
stressor. A case in point would be a gene activator that would induce a stress
gene by one mechanism (e.g., using a heat-shock cis-acting element) if the stres-
sor is heat, and by another if the stressor is a heavy metal (e.g., by interacting
with a metal element in the DNA instead of binding to a heat-shock element).
One can also hypothesize that the response to stress by ammonia implicates
DNA elements and transcription and regulatory factors that are different from
those used in the response to a heat shock, at least for the activation of the trkA
gene.
10.6
Diversity of Methanogenes: A Source of Useful Microbes?
A rational approach to the improvement of bioreactor technology includes the

manipulation of relevant genes to construct better methanogens, more resistant
to stressors and also more efficient bioconverters. This must be based on knowl-
edge provided by basic and applied research on the molecular biology and bio-
chemistry of the various components of the stress response, as outlined
throughout this Chapter up to this point. A second avenue towards assembling a
very efficient and resistant microbial population inside a bioreactor is the search
for “good” microbes in natural ecosystems. If one or more are found with the
characteristics required, they could be used for bioconversion in bioreactors, or
as a source of useful genes.
The diversity of methanogens we have mentioned several times before prob-
ably reflects their universality [106]. They can be found in a wide variety of eco-
logic niches. An idea of the ubiquity of methanogens is provided by data in
Table 11 [AJLM and ECdeM, unpublished data]. In it, we have listed the sources
of methanogenic isolates recorded as tested and identified immunologically in
our laboratories between 1981 and 1986, and the number of isolates from each
source. The variety of sources is evident and encompasses ecologic niches with
very different characteristics. We have also identified methanogens in other
ecosystems, different from those mentioned in Table 11, such as the Antarctic
continent [107], deep subterranean aquifers [108], and temperate marine waters
[109], just to mention a few. In each ecosystem explored, whenever it was possi-
ble to study at least a few isolates, species diversity was evident, and within
species a diversity of immunotypes was usually discovered. For example,
46 methanogens isolated from human feces were all identified as Methanobre-
vibacter smithii, but they were distributed into at least seven groups with dis-
tinctive antigenic mosaics demonstrable with a panel of six monoclonal anti-
bodies [110]. These findings show that the diversity of methanogens, even with-
in a single species, is quite remarkable, and that with the appropriate tools
Table 11. Methanogenic isolates from various ecosystems identified by antigenic fingerprint-
ing during the period 1981–1986
Isolate Ecosystem Total Antigenically identifiable

Source studied
Country Yes No
USA human feces 67 67 0

USA dental plaque 14 12 2
USA animal feces 16 11 5
USA rumen herbivores 11 9 2
USA cockroach digestive tract 2 2 0
The Netherlands marine ciliate 1 0 1
Germany; USA marine sediments 14 12 2
Germany hot spring 1 1 0
Germany swamp 1 1 0
USA peat lands 5 1 4
Germany; France soil 4 3 1
USA fresh-water sediments 5 5 0
United Kingdom landfills 12 11 1
Japan; USA; France waste-water sludge 5 5 0
Germany; USA; bioreactors (digestors) 39 33 6
France
Canada; Germany; undetermined 21 17 4
Japan; New Zealand;
USA
TOTAL 218 190 (87%) 28 (13%)
(e.g., a panel of calibrated antibody probes) this diversity can be demonstrated

fairly easily.
Another source of methanogens for possible use in biotechnology is the sea.
In one study of the water column of the Chesapeake Bay, we demonstrated sev-
eral species, Fig. 18. Some were related more or less closely to the reference
organisms available in culture collections but others were not [109]. The deeper
the water layer, the more abundant were the methanogens less similar to the
known species.
The diversity of methanogens demonstrated by the antigenic fingerprinting
method is phenotypic. It might not reflect to the last detail structural diversity
at the genome level. Nevertheless, phenotypic diversity does suggest genomic
differences, particularly functional ones. It shows that even if the gene contents
of different genomes are very similar, their functional patterns are not. Genes
active in one phenotype may be inactive in another. Thus, there is diversity in the
pattern of regulatory mechanisms. What are the regulatory genes involved in
determining the phenotypes? Which are the most useful phenotypes? An impor-
tant endeavor in the near future should be the identification of useful pheno-
types, and of the genes involved in producing them. It will then be possible to
search for the “good” microbes, stress resistant and efficient for biomethanation,
or to make them by means of genetic engineering procedures.
It is evident from the data described above, more recently confirmed by oth-
ers with different methods [111], that microbial diversity is probably enormous,
Fig. 18. Diversity of methanogens in an aquatic ecosystem. Methanogens were isolated from
the water column of Chesapeake Bay (USA) and characterized by antigenic fingerprinting and
other methods. Samples for isolating the microbes were collected from three different layers
of the water column (abscissa). Twelve, eight, and thirteen isolates from the upper, middle
(pycnocline), and lower layer, respectively, were characterized and identified. Each isolate is
represented by a square with the number inside indicating the species-strain most closely
related, as follows: 3, Methanosarcina barkeri MS; 6, Methanosarcina barkeri R1M3; 18,
Methanosarcina mazeii S-6; 19, Methanosarcina barkeri W; 20, Methanosarcina thermophila
TM-1; and 0 (zero), unrelated to the known reference methanogens. The striped bars indicate
the percentage of methanogenic isolates that were unrelated to the reference organisms,
namely they were novel methanogens, not yet available in the pure-culture collections. The
relative abundance of isolates related to the Methanosarcinae is noteworthy, as is the increased
abundance of novel methanogens with increasing water-column depth. Data extracted from
reference [109] with permission from the copyright owner
and that what we have so far uncovered is only a very minimal portion of it. We
have seen only the tip of the iceberg, as it were. Hence, there is hope that a search
for methanogens in nature will yield abundant dividends in terms of species
useful for methanogenic biotechnology, endowed with the necessary resistance
to the stressors that usually threaten bioreactor stability and efficiency. This
search for naturally “good” organisms can be complemented with genetic engi-
neering to make them optimal not only to withstand stress but also to proceed
through the methanogenic pathway with speed and efficiency.
10.7
Cooperation Between Molecules and Between Cells
Stress proteins like the molecular chaperones function as members of a team or

molecular machine with several interconnected and interacting parts. Members
of a machine, for example the Hsp70(DnaK)-Hsp40(DnaJ)-GrpE molecular
chaperone machine, interact with each other, and also with other molecules and
machines [6, 9, 43, 49, 52]. Chaperonins and sHsp also assemble into large mul-
timeric complexes [7, 11, 33, 76, 77]. It is obvious that natural selection has
favored these complexes, and one must infer that they are functionally better
than the sum of the separate activities of their single components. Alternatively,
one might think that multimerism is a requirement for the functioning of cer-
tain types of molecules as multicellular communities (tissues, organs, and their
primitive prokaryotic counterparts) would be for cells.
A parallelism to molecular multimerism seems to occur with the tendency to
form multicomponent (communities, tissues, organs) structures by cells. These
forms of association for function seem to be far more effective under physio-
logic circumstances and in the face of stress than solitary molecules or cells.
Molecular machines, tissues and organs, and microbial consortia, all appear to
be landmarks of evolutionary success. The main conclusion one may draw from
these observations is that strategies for optimizing bioreactor technology ought
to include the development of means that enhance the formation of multicom-
ponent machines at the molecular and cellular levels.
10.8
Proteases as Builders
Proteases are essentially destructive in as much as they degrade molecules into

smaller parts [4, 5, 91]. However, this process may be essential in some instances
for building complex multicellular structures. Enzymatic digestion of molecules
by proteases frees the space occupied by the initial, larger whole, when the small-
er parts have been used up or removed (e.g., washed away with fluids in circu-
lation, or engulfed by cells). The freed space can then be occupied by another
component of the complex, this time a more appropriate one for that particular
location. Alternatively, the voided space may remain empty of solids and thus
become a vesicle or tube for storage or circulation, respectively. It is likely that
proteases take an active role in the formation of the tubes that crisscross the
granular microbial consortia discussed earlier in this Chapter, and shown in Fig.
14. There can be little doubt that this internal circulatory system is essential for
the survival of cells inside the structure, or at least for distribution of nutrients
within it. It also is a convenient way for removal of catabolites and for delivery of
products from one cell (or colony) to another (see Fig. 13), or to the outside
(e.g., methane).
The observations discussed above suggest that proteases should also be the
target of basic and applied research that would provide the basis for engineer-
ing more efficient microbial consortia. For example, a consortium should have a
circulation network commensurate with its size, and the needs and metabolic
activities of all its cellular constituents, regardless of the location of these con-
stituents in the whole structure.
10.9
Intrinsic Stress Resistance
The molecules of organisms that have high or very high OTG, or that grow under
high hydrostatic pressure, are able to function under these extreme conditions
(as compared to those that are optimal for humans, for instance). The molecules
are endowed with intrinsic stress resistance; the mechanisms implicated in
this resistance are only now beginning to be examined [13, 112, 113]. This is an
interesting point for investigation, potentially useful for methanogenic bio-
technology.
11
Conclusion and Perspectives
Stress proteins, molecular chaperones, formation of functional multimeric
structures by molecules and cells, thermoprotectants, ion transporters, and oth-
er anti-stress mechanisms ultimately depend on the presence of genes properly
regulated, capable of responding to the attack of stressors. Hence, elucidation
of the gene regulatory mechanisms is an important step towards optimizing
biomethanation. Tools to study gene regulation and to manipulate genes in
methanogens are being developed [114–117]. Likewise, means to manipulate
microbial cells, including methanogens and syntrophs, using antibodies and
related techniques are available [118]. The perspectives for rapid progress are,
therefore, promising. If the study of stress genes, proteins, and other anti-stress
mechanisms, and the identification of novel microbial species continue, it will be
possible in the near future to optimize the biological component of bioreactors.
Furthermore, it will be possible to monitor bioreactor function to anticipate fail-
ure, and to repair it in case of malfunction, by removing unwanted microbes
and/or introducing those pre-selected or pre-engineered (genetically) to meet
the requirements for stress resistance and optimal biomethanation. It has been
demonstrated that it is possible to introduce a microbial species in a granular
consortium to add to it a lacking metabolic ability [119]. The consortium was
thus endowed with the capacity to bioconvert a substrate that could not be
metabolized prior to the microbial graft. This procedure is easy to perform and
has a promising future as a means to build consortia with stress-resistant
microbes tailored to bioconvert specific substrates. Biofilms and granules con-
structed on demand with stress-resistant microbes, which are also efficient for
biomethanation of specific substrates (e.g., a certain type of waste), should be a
reality in the first decade of the third millennium.
Acknowledgement. Work in the authors’ laboratories has been supported over the years by
grants from NYSERDA, DOE, and NSF. We thank our collaborators of yesterday and today, too
numerous to mention by name (many appear in the list of references), for their help. We also
thank the members of the Photo-Art unit of this Center for their excellent assistance with
graphs and photographs.
12
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Received: December 2001

CHAPTER 6
Molecular Ecology of Anaerobic Reactor Systems

J. Hofman-Bang 1 · D. Zheng 2 · P. Westermann 1 · B. K. Ahring 1 · L. Raskin 3
1 Environmental Microbiology and Biotechnology, Biocentrum DTU, The Technical
University of Denmark, Building 227, 2800 Lyngby, Denmark.
E-mail: jhb@ssi.dk; E-mail: peter.westermann@biocentrum.dtu.dk;
E-mail: bka@biocentrum.dtu.dk
2 Alpha Therapeutic Corporation, Los Angeles, CA 90032, USA.
E-mail: dandan.zheng@alphather.com
3 Department of Civil and Environmental Engineering, University of Illinois
at Urbana-Champaign, Urbana, Illinois 61801, USA. E-mail: lraskin@uiuc.edu
Anaerobic reactor systems are essential for the treatment of solid and liquid wastes and con-
stitute a core facility in many waste treatment plants. Although much is known about the basic
metabolism in different types of anaerobic reactors, little is known about the microbes respon-
sible for these processes. Only a few percent of Bacteria and Archaea have so far been isolated,
and almost nothing is known about the dynamics and interactions between these and other
microorganisms. This lack of knowledge is most clearly exemplified by the sometimes un-
predictable and unexplainable failures and malfunctions of anaerobic digesters occasionally
experienced, leading to sub-optimal methane production and wastewater treatment.
Using a variety of molecular techniques, we are able to determine which microorganisms
are active, where they are active, and when they are active, but we still need to determine why
and what they are doing. As genetic manipulations of anaerobes have been shown in only a
few species permitting in-situ gene expression studies, the only way to elucidate the function
of different microbes is to correlate the metabolic capabilities of isolated microbes in pure cul-
ture to the abundance of each microbe in anaerobic reactor systems by rRNA probing.
This chapter focuses on various molecular techniques employed and problems encoun-
tered when elucidating the microbial ecology of anaerobic reactor systems. Methods such as
quantitative dot blot/fluorescence in-situ probing using various specific nucleic acid probes
are discussed and exemplified by studies of anaerobic granular sludge, biofilm and digester
systems.
Keywords. rRNA, rDNA, PCR, Biofilm, UASB, Granular sludge
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
2 Nucleic Acid-Based Analysis of Anaerobic Bioreactors . . . . . . 154

2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
2.2 Retrieving Nucleic Acid Sequences . . . . . . . . . . . . . . . . 156
2.2.1 Nucleic Acid Isolation . . . . . . . . . . . . . . . . . . . . . . . 157
2.2.2 PCR Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2.2.3 Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
2.2.4 rDNA Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . 159
2.2.5 Community Fingerprints . . . . . . . . . . . . . . . . . . . . . . 160
2.2.6 Quantification Based on Sequence Retrieval . . . . . . . . . . . 161
2.2.7 Quantitative PCR . . . . . . . . . . . . . . . . . . . . . . . . . . 162
2.2.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

152 J. Hofman-Bang et al.
2.3 Oligonucleotide Probes . . . . . . . . . . . . . . . . . . . . . . . 163

2.3.1 Probe Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2.3.1.1 Probe Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2.3.1.2 Target Accessibility . . . . . . . . . . . . . . . . . . . . . . . . . 165
2.3.2 Quantitative Slot (Dot) Blot Hybridization . . . . . . . . . . . . 165
2.3.2.1 Hybridization Stringency . . . . . . . . . . . . . . . . . . . . . 166
2.3.2.2 Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
2.3.2.2.1 Interpreting the Quantification Results . . . . . . . . . . . . . . 166
2.3.2.2.2 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
2.3.2.2.3 Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
2.3.2.3 Factors that May Interfere with Quantification . . . . . . . . . . 167
2.3.2.3.1 Membrane Saturation . . . . . . . . . . . . . . . . . . . . . . . 167
2.3.2.3.2 Target Accessibility . . . . . . . . . . . . . . . . . . . . . . . . . 167
2.3.2.3.3 Co-Extracted Substances . . . . . . . . . . . . . . . . . . . . . . 168
2.3.2.3.4 In Vitro Transcribed rRNA . . . . . . . . . . . . . . . . . . . . . 168
2.3.3 Reverse Genome Sample Probing . . . . . . . . . . . . . . . . . 169
2.3.4 Whole Cell or in Situ Hybridization . . . . . . . . . . . . . . . . 169
2.3.4.1 Hybridization Stringency . . . . . . . . . . . . . . . . . . . . . 170
2.3.4.2 Cell Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
2.3.4.3 Signal Enhancement . . . . . . . . . . . . . . . . . . . . . . . . 171
2.3.4.3.1 Indirect Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
2.3.4.3.2 Enzyme-Labeled Oligonucleotides . . . . . . . . . . . . . . . . . 172
2.3.4.3.3 Multi-Probe and Multi-Labeling . . . . . . . . . . . . . . . . . . 173
2.3.4.3.4 Amplification of the Target Sequence . . . . . . . . . . . . . . . 173
2.3.5 Solution-Based Hybridizations (Molecular Beacons) . . . . . . . 173
2.4 FISH and Reporter Systems . . . . . . . . . . . . . . . . . . . . 175
2.5 FISH and Antibody Probes . . . . . . . . . . . . . . . . . . . . . 176
2.6 FISH and Microautoradiography . . . . . . . . . . . . . . . . . 178
2.7 Peptide Nucleic Acid Probes . . . . . . . . . . . . . . . . . . . . 179
3 rRNA-Based Analyses of Anaerobic Reactors . . . . . . . . . . . 180

3.1 Biofilm Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . 180
3.1.1 Biofilm Formation . . . . . . . . . . . . . . . . . . . . . . . . . 180
3.1.2 Biofilm Composition and Dynamics . . . . . . . . . . . . . . . . 189
3.2 Granular Sludge Reactors . . . . . . . . . . . . . . . . . . . . . 190
3.2.1 Granular Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.2.2 Microbial Composition of Granules . . . . . . . . . . . . . . . . 191
3.2.3 Structure of Granular Sludge . . . . . . . . . . . . . . . . . . . . 193
3.2.4 The Granulation Process . . . . . . . . . . . . . . . . . . . . . . 194
3.3 Continuously Stirred Tank Reactors (CSTR) . . . . . . . . . . . 194
3.3.1 Microbial Composition in CSTRs . . . . . . . . . . . . . . . . . 194
3.3.2 Microbial Dynamics in CSTRs . . . . . . . . . . . . . . . . . . . 196
4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 197
5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Molecular Ecology of Anaerobic Reactor Systems 153
1
Introduction
Most anaerobic microbial processes are characterized by close association of
numerous functional groups of microorganisms. The understanding of anaero-
bic processes has improved greatly during recent decades with advances made
in microbial physiology, biochemistry, ecology, kinetics, and mathematical
modeling. These contributions have led to an expansion of anaerobic processes
by introducing better designs and operational controls. However, the under-
standing of anaerobic processes is far from complete. Understanding the micro-
bial ecology in anaerobic reactor systems requires
(1) identification and classification of microorganisms,
(2) quantification of microbial abundance, and
(3) quantification and identification of activity.
Morphology and other microbial traits have previously been used for identifi-
cation and quantification of microbial populations. Grotenhuis et al. [1] micro-
scopically counted cell numbers of methanogens and identified aceticlastic
methanogens based on morphology, and hydrogenotrophic methanogens by
visualizing autofluorescence at 420 nm. Morphology and ultrastructure have
also been used extensively in scanning or transmission electron microscopy
studies to show the location of certain microorganisms in anaerobic granules
[2, 3]. Information gained from morphology-based techniques is, however,
ambiguous and limited since most microorganisms are small in size, and simple
in morphology and ultrastructure.
In the absence of special morphological features or autofluorescence, physio-
logical and biochemical traits have been used for identification. Furthermore,
enrichments on defined substrates have been helpful to identify prevalent
species in anaerobic granules [4], and Most Probable Number (MPN) estimates
have been used frequently for quantification of different trophic groups of
anaerobic microorganisms [1, 4]. These methods are, however, cultivation-
dependent and therefore limited by the ability of microorganisms to grow under
laboratory conditions. It is well known that only a very small fraction of the
microorganisms in nature is culturable by present cultivation techniques,
because of unrecognized nutrient and growth conditions, or the interruption of
intrinsic interdependencies such as syntrophic interactions [5]. Amann et al.
estimated that the culturability ranges from 0.001% in seawater to 15% in acti-
vated sludge [6]. Culturing may be especially difficult for anaerobes due to their
low growth rates and fastidious nutritional and environmental requirements.
Recently, more direct methods have been developed for identification, quan-
tification, and localization of microorganisms in environmental samples.
Immunology techniques utilize monoclonal or polyclonal species-specific anti-
bodies to detect and even quantify the abundance of cultivable microorganisms
in environmental samples [7–9]. In combination with electron microscopy, anti-
bodies have been used to localize microorganisms in sections of anaerobic gran-
ules [1]. The major disadvantages of immunotechnology are the need for axenic
cultures or defined co-cultures to produce the specific antibodies, and the high
specificity that limits the detection to the species or subspecies level [10–12]. In
addition, cross-reactions often might cause a problem [13, 14]. Adsorption to
cross-reacting cells can broaden the specificity of an antibody [15], but this
approach is limited by the number of species that are used for the specificity test.
Molecular phylogeny, which employs nucleic acid sequences to document the
history of evolution, has provided a new basis for the direct identification and
quantification of microorganisms [16]. Nucleic acid-based methods allow
microbial community characterization without cultivation. So far, ribosomal
RNA (rRNA) and ribosomal DNA (rDNA) have been the most commonly used
target nucleic acids in microbial ecology studies. This chapter focuses mainly
on the use of rRNA- and rDNA-based methods for the study of anaerobic
reactor systems. In addition, some other molecular approaches are discussed
briefly.
We first present the fundamentals and principles of different nucleic acid-
based techniques to study anaerobic reactor systems. The second part of the
chapter reviews literature in which rRNA- and rDNA-based techniques have
been applied to studies of anaerobic bioreactors.
2
Nucleic Acid-Based Analysis of Anaerobic Bioreactors
This section provides an overview of the most widely used or potentially appli-
cable rRNA- and rDNA-based methods, but also presents studies in which func-
tional diversity has been investigated by analyses of expressed messenger RNA.
When available, we have used examples from anaerobic bioreactor work.
2.1
Background
Studying microbial ecology requires identification of microorganisms, based

upon a comprehensive classification system that ideally should reflect the evo-
lutionary relatedness of organisms [5].As pointed out by numerous authors, tra-
ditional classification systems based on phenotypic characteristics (morpholo-
gy, physiology, and structure of cell components) offer little information on evo-
lutionary relatedness and require cultivation for identification [5, 17].
In the mid-1960s, Zuckerkandl and Pauling pointed out that molecular
sequences could document evolutionary history [18]. Due to the pioneering
work of especially Carl Woese, the rRNAs have become the most commonly used
molecules for phylogenetic analyses. rRNA or the corresponding rDNA are par-
ticularly suitable as evolutionary chronometers [19–21] since
(1) they are key elements of the cells and are functionally and evolutionarily
homologous for all organisms;
(2) they are very conserved in overall structure;
(3) their regions of different conservation levels allow phylogenetic analysis
and design of probes and primers;
(4) they are very abundant in most cells (103 to 105 copies) [6], and are easily
recovered and detected;
(5) the small subunit (SSU) rRNA (16S and 16S-like rRNA) and the large rRNA
of the large subunit (LSU) of the ribosome (23S rRNA and 23S-like rRNA)
are sufficiently long for statistically significant comparisons; and
(6) their genes have so far not been shown to be transferable among organisms.
Using 16S rRNA comparative sequence analysis, Woese and colleagues devel-
oped the first universal phylogenetic tree, which reflects the evolutionary relat-
edness of all organisms, grouping them into three domains: Eucarya, Archaea,
and Bacteria [22–24].
16S rRNA sequences are most commonly used for molecular ecology investi-
gations since a huge number is available through the Ribosomal Database Pro-
ject II (16,277 aligned and 30,322 unaligned 16S rRNA sequences in June, 2000)
[25]. However, 23S rRNA-based analyses should become more common when
more sequence data become available, since 16S rRNA comparisons sometimes
fail to resolve very closely related species [20, 21, 26]. Internal transcribed spac-
er (ITS) regions that separate rRNA genes may provide additional information
for resolving very close phylogenetic relationships [27]. A complication related
to the use of rRNA as a target for the quantification of population abundance is
the limited information currently available on the number of rRNA operons pre-
sent on microbial genomes. Information on the level of gene redundancy present
in the rRNA operons has recently been catalogued in the Ribosomal RNA Oper-
on Copy Number Database (rrndb) [28]. Besides the ribosomal RNA genes, oth-
er gene sequences have been used for phylogenetic analyses, including genes for
the elongation factor Tu, and F1F0ATPase b-subunit [29].
A phylogenetic analysis allows the identification of a microorganism based
only on a molecular sequence, eliminating the need for cultivation. In other
words, a sequence can be retrieved from an environmental sample, sequenced,
and compared to known sequences for identification of the corresponding
organism [19]. If the retrieved sequence is new, characteristics associated with
housekeeping functions of the cells (e.g., characteristics of ribosomes, DNA
replication machinery, biosynthetic pathways and their regulation mechanisms)
can be inferred from closely related species [5, 17]. The metabolic diversity of
cells, however, is more variable than reflected by the housekeeping functions,
due to events such as lateral transfer of metabolic genes and symbiotic fusions
[17]. Thus, caution has to be taken when metabolic characteristics of a newly
identified microorganism are inferred from characteristics of close phylogenet-
ic relatives.
Based on “signature” sequences of specific groups of microorganisms, probes
can be designed and used to identify and quantify these microorganisms in
complex microbial ecosystems. The strategies based on rRNA sequences analy-
sis for characterizing a microbial community are summarized in Fig. 1. It should
be noted that many of these strategies are also applicable to other genes.
Fig. 1. Strategies based on rRNA sequence analysis for characterization of microbial commu-
nities without cultivation (arrows indicate the interconnected use of methods, experimental
materials, and information in the study of microbial ecosystems. RT-PCR: reverse transcrip-
tion to produce DNA from RNA, followed by PCR. DGGE: denaturing gradient gel elec-
trophoresis. RFLP: restriction fragment length polymorphism. Modified from [5, 6]
2.2
Retrieving Nucleic Acid Sequences
Methods for retrieving nucleic acid sequences from environmental samples are
mainly used to detect and identify microorganisms, although some quantitative
methods are being developed, especially for populations present in low num-
bers. The sequences obtained should ideally represent the diversity present in
the sample, but all methods introduce at least some bias and may not identify all
populations present as discussed below.
The process starts with the extraction of nucleic acid (DNA or RNA) from a
sample. Extracted DNA can be randomly digested and cloned (shotgun cloning).
Subsequently, the clones are screened for rRNA genes using dot/colony blot
hybridization. More commonly, however, the rRNA genes in DNA extracts are
specifically amplified by PCR, or rRNA genes are produced by reverse tran-
scription from rRNA in RNA extracts followed by PCR (i.e., RT-PCR). Next, PCR
or RT-PCR products are cloned or separated by gel electrophoresis (denaturing
gradient gel electrophoresis [DGGE], temperature gradient gel electrophoresis
[TGGE], terminal restriction fragment length polymorphism [T-RFLP]). The
rDNA clone library or the DNA bands from the electrophoresis gel can be
sequenced and the obtained sequences are deposited in sequence databases.
Subsequently, phylogenetic trees showing the diversity of the corresponding

environmental sample can be constructed by comparative sequence analysis.
Details of these methods and their applications can be found in a number of
reviews [5, 6, 17, 19, 20, 26, 27, 30, 31]. Brief descriptions of the methods and of
some factors that affect the overall results are discussed below.
2.2.1
Nucleic Acid Isolation
Ideally, a sample should be representative and free from bias, especially when
quantification is the objective. It should also contain sufficient biomass for sub-
sequent analyses. In addition, the presence of materials that interfere with nucle-
ic acid recovery or manipulation (e.g., humic substances) should be avoided or
such compounds should be removed from the sample if possible. If the sample
is collected for RNA extraction, nuclease activity should be reduced as much as
possible [32]. Measures such as quick freezing, storing at –80°C, avoiding thaw-
ing the sample before extraction, and preventing the introduction of foreign
nucleases should be practiced.
Several methods have been published in the recent years for extraction of
intact RNA from environmental samples, such as manure [33], sediment, soil,
and water samples [34], sediment and microbial mat samples [35], and rumen
fluid [39]. Recovering RNA or DNA quantitatively from all cells in a complex
community without bias can be difficult. In general, mechanical lysis methods
have shown less bias than enzymatic lysis methods, leading to the recovery of
intact high molecular weight nucleic acids [36].
Ibrahinm et al. reported on a rapid method for extracting high purity rRNA
from manure [33]. The bead-beating-based method involves citrate buffered,
low-pH phenol and chloroform extractions. This method effectively disrupts the
cell wall of cells that are difficult to break such as Gram-positive bacteria and
methanogens. Citrate has been shown to strongly inhibit RNases [37]. Humic
acids were removed from the samples by repeated washes with low-pH citrate
prior to cell disruption.
Moran and coworkers used a low-pH, hot-phenol extraction method and sub-
sequent gel filtration with Sephadex G-75 spin columns for sediment, soil, and
water samples [34]. Since they used lysozyme to open the cells, this method may
introduce a bias, since lysozyme is not equally effective for all types of cells.
Alm and Stahl compared different lysis solutions and subsequent vortexing in
low-pH phenol and chloroform to extract RNA from sediments and microbial
mats [38]. Use of a guanidine thiocyanate/b-mercaptoethanol lysis solution
resulted in an efficient recovery of intact, high purity rRNA. When they in-
creased the ratio of lysis buffer to sample volume from a 1:1 to 5:1, an order of
magnitude more rRNA was extracted. Adding the sample to the lysis buffer
while mixing, instead of the opposite also proved to be important. This extrac-
tion method left the final extract with considerable amounts of organic conta-
mination.
Raskin and coworkers used a low-pH, hot phenol, bead-beating extrac-
tion method to isolate RNA from rumen fluid [39]. They demonstrated that
the total amount of RNA recovered per gram of rumen fluid neither changed
significantly with the duration of the beating, nor with the amount of beads
used. However, the amount of RNA recovered from Gram-positive Rumino-
coccus cells increased significantly when the beating period was extended. In
the same study, the total amount of RNA recovered in replicate extractions
showed very high variability. The efficiency of RNA recovery was also investi-
gated [39]. The study demonstrated proportional recovery of RNA for a specific
target population. The study also demonstrated loss of RNA in the phenol
phase and during precipitation/rinse/resuspension steps of the extraction
process.
Yu and Mohr developed a fast method (one hour) to simultaneously extract
DNA and RNA [36]. Bead beating combined with 2 M ammonium acetate pre-
cipitation of proteins in the presence of DEPC (diethyl pyrocarbonate) resulted
in intact rRNA and non-sheared DNA. No phenol or chloroform was used. By
adding RNase or 200 mM NaOH, either DNA or RNA was extracted. The nucleic
acids were of a sufficient quality to perform PCR and RT-PCR. This method has
been adapted to extract intact DNA or rRNA from municipal solid waste and
cow manure without any washing prior to extraction in our laboratory. The
resuspended nucleic acids obtained after extraction are colorless, indicating that
humic substances and other impurities are removed effectively during the
extraction procedure.
Humic substances coextracted with nucleic acids may interfere with subse-
quent enzymatic reactions (such as PCR) [27]. They can also interfere with
membrane hybridizations (see below). A number of methods has been devel-
oped for removing the humic substances. These include polyvinylpolypyrroli-
done (PVPP) adsorption, gel purification, and dilution [27, 34, 35]. Furthermore,
DNA is often present in RNA extracted from environmental samples [38, 40].
The influence of DNA on membrane hybridization is discussed below. When
necessary, DNase can be used to remove DNA, although concerns of partial
degradation of RNA due to impurities in commercial DNase should be consid-
ered [40].
2.2.2
PCR Reaction
The polymerase chain reaction (PCR) can be used to amplify DNA sequences
from environmental samples. The PCR products can be analyzed by techniques
such as DGGE (denaturation gradient gel electrophoresis), TGGE (temperature
gradient gel electrophoresis), T-RFLP (terminal restriction fragment length
polymorphism), or SSCP (single stranded conformation polymorphism), which
have the potential to separate the PCR products originating from different DNA
sequences representing populations in the original samples. The PCR products
can also be cloned and subsequently sequenced to allow identification of popu-
lations. For details about PCR, the reader is referred to a review by Steffan and
Atlas and “The PCR application manual, 2nd ed. [41, 42]. Since the amount of
DNA produced by PCR ideally increases exponentially during the amplification,
errors occurring early in the process will result in biased results [6].
Factors that cause bias of PCR are:

– “Universal” primers or other specific primers are designed based on
sequence information available in databases (obtained from cultured organ-
isms and clones) [5]. However, primers targeting multiple groups of organ-
isms may not amplify all target genes since the primer sites are not complete-
ly conserved.
– Bias can be caused by an inappropriate annealing stringency, which results in
amplification of genes that are not intended to be amplified [27].
– There is some evidence that PCR does not amplify all rRNA sequences in the
sample to the same extent (preferential amplification) [6, 27].
– Contaminating sequences from chemicals and enzymes can be erroneously
included in the analysis.
– Chimeric sequences are often produced [27] due to the presence of partial
fragments of rDNA in DNA extracts, partially reverse transcribed DNA when
performing RT-PCR, or premature PCR products acting as primers in a sub-
sequent PCR cycle [6].
2.2.3
Cloning
Cloning can produce large amounts of DNA segments originally isolated from
environmental samples. The DNA fragments can be produced after digestion
with restriction enzymes of the DNA extracted from a sample (i.e., shotgun
cloning), or after PCR or RT-PCR (if RNA is the template). Compared to cloning
after PCR, shotgun cloning introduces less bias and produces clones of multiple
genes at the same time [5]. Cloning after PCR is rapid and convenient, but can
be biased [5, 27]. The bias can be introduced during the PCR step as discussed
above or during cloning. For instance, the use of rare-cutting restriction
enzymes during cloning might also cut amplified rDNA [6]. In addition, it is pos-
sible that different rRNA gene fragments are cloned with different efficiencies.
2.2.4
rDNA Sequences
Cloned DNA fragments can be sequenced to study the phylogenetic diversity of

the microbial community from which the sample was originally obtained. The
resulting sequences can be compared to sequences in databases to identify the
closest phylogenetic relatives.
There are a number of problems that need to be taken into consideration
when using this technique. First, when the retrieved sequences exhibit high
similarity to sequences available in databases (98% to 99% identity), it is
difficult to rule out PCR errors [27]. Secondly, it is difficult to convert the rRNA
similarity to the nomenclature level of species or genus [6]. In general, more
than 97% 16S rRNA identity indicates that two sequences belong to the same
species, which typically corresponds to DNA:DNA hybridization values above
70%. The definition of a similar threshold for genera is not as clear, but 16S
rRNA differences greater than 5–7% may be used to support a new genus [43].
Finally, the heterogeneity of 16S rRNA genes increases the complexity. It has
been observed that some species express different 16S rRNA genes at different
growth stages, or multiple 16S rRNA genes are expressed at the same time [27].
For example, sequence heterogeneity was found in Paenibacillus polymyxa [45],
strains of the Mycoplasma mycoides cluster [44], and in Clostridium para-
doxum [45a].
2.2.5
Community Fingerprints
Several fingerprinting techniques, such as DGGE, TGGE, RFLP, T-RFLP, and

SSCP, have been developed to screen clone libraries, to estimate the level of
diversity in environmental samples, to follow changes in community structure
(e.g., trace one or more populations over time) and to compare diversity and
community characteristics in various samples. These techniques usually involve
gel electrophoresis that can separate different DNA segments of a community
rDNA library.
DGGE (denaturing gradient gel electrophoresis) separates DNA fragments of
equal length (obtained after PCR of DNA extracted from an environmental sam-
ple) into distinct bands on a chemical denaturing gradient polyacrylamide gel.
PCR amplification of the 16S rRNA gene utilizing conserved primers targeting
either the V3 or the V8 +V9 variable regions is normally used to produce a
300–500 bp fragment. Larger fragments are typically not used as the DGGE tech-
nique cannot resolve these into distinct bands [46]. One of the primers used has
a GC-clamp consisting of a 30 nucleotide GC-rich 5¢ end, which maintains the
two denaturated single stranded DNA fragments together in the denaturing gel.
As the double stranded DNA migrates through the gel experiencing increasing-
ly higher denaturant concentrations, the double stranded DNA separates into
two single strands at a specific point and the migration stops due to the larger
volume of the denaturated molecule kept together by the GC clamp. The DGGE
technique has been used to characterize the microbial diversity in different envi-
ronments such as activated sludge [47], sediments [46], lake water [8], hot
springs [48], soils [49, 50], biofilm [51]. DGGE has been used to monitor changes
in complex communities [48, 52–55] and to identify microorganisms present in
wall painting [56].
The banding pattern reveals the community components at best semi-quan-
titatively due to the possible bias caused by PCR and difficulties to quantify the
amount of DNA associated with a band. An advantage of the technique is that it
can resolve the microbial diversity of up to 15 different species by optimizing the
denaturing gradient concentration in the gel. By using narrow gradients, rDNAs
that differ in only one bp can be separated in DGGE [46]. A drawback of the
technique is that the reproducibility is not optimal; one DNA fragment may
generate more than one band on the gel and a DNA sample analyzed on two
different gels may not generate the same band pattern [57]. In addition, it is
possible that a band in a DGGE gel may contain different sequences with
similar denaturation characteristics.
Alternatively, the obtained rDNA can be digested with restriction enzymes

and analyzed on an agarose gel. This technique is called restriction fragment
length polymorphism (RFLP) [58] or amplified ribosomal DNA restriction
analysis (ARDRA) [59]. The banding pattern obtained has been used to identi-
fy different genotypes of microorganisms [60] and to monitor population
changes in environmental samples [61, 62]. The RFLP patterns have also been
used to deduce the phylogeny of axenic cultures of microorganisms [63–67].
Compared to cloning, DGGE and RFLP are faster and less laborious, and bands
of interest can be cut out, extracted from the gels, and cloned and sequenced
directly.
SSCP (single-strand conformation polymorphism) is based on the separation
of the double stranded DNA PCR product by NaOH prior to non-denaturing
polyacrylamide gel electrophoresis. The single stranded DNA forms secondary
structures (analogous to the cloverleaf structure of tRNAs). Scheinert and
coworkers employed this technique to differentiate between 15 Mycoplasma
species and to analyze a mixed sample of six different species based upon analy-
sis of the spacer region between the 16S rRNA and the 23S rRNA gene [68]. The
advantage of the technique is that even point mutations can be detected as a
change of conformation in the secondary structure of the single stranded DNA.
In the study of the Mycoplasma species, the variable size of the PCR product of
the spacer region (280–1300 bp) further discriminated the populations while the
16S rDNA DGGE technique might not have been able to do so since the gel band
pattern would have been too compressed. A critical parameter in the SSCP tech-
nique is to control the temperature in the gel tank to reduce smearing. As the
available sequence data of the rRNA spacer region is limited compared to the
16S rRNA sequence databases, the SSCP technique is mainly used to differenti-
ate between different cloned PCR products.
2.2.6
Quantification Based on Sequence Retrieval
Theoretically, the abundance of a population can be inferred from the frequen-

cy of a particular sequence appearing in the sequence collection obtained from
an environmental sample. For instance, the microbial community structure of
an anaerobic fluidized-bed reactor (treating wine distillation wastewater) was
characterized by PCR and sequencing [69]. The PCR was conducted using three
pairs of primers specific for the three domains. The authors obtained 460 and
96 clones from Bacteria and Archaea, respectively. Of these 556 clones, 76% were
Bacteria, 10% corresponded to Methanobacterium formicicum, 4% represented
Methanosarcina frisius, 8% were Methanosarcina barkeri, and 2% represented
other Archaea. Within the bacterial domain, there were 6% high G + C Gram-
positives, 4 % Planctomyces, 33% low G + C Gram-positives, 4% Spirochaetes,
12% delta Proteobacteria, 2% gamma Proteobacteria, 1% beta Proteobacteria,
2% alpha Proteobacteria, 26% Cytophaga-Flexibacter-Bacteroides, and 7%
green non-sulfur bacteria. As discussed by the authors, this method has many
shortcomings when used as a quantitative tool. First, the primers were designed
based on previously isolated cultures, thus it is not free from the well-known cul-
tivation-derived limitations. Secondly, bias can be introduced during PCR and

nucleic acid extraction as previously discussed.
2.2.7
Quantitative PCR
Recently, a number of quantitative PCR methods have been developed that have
the potential of detecting low-level populations in environmental samples.
One of these methods is competitive PCR, in which an internal standard is
added to the sample. The sample and the internal standard are amplified using
the same pair of primers. The corresponding assumptions are: The gene of inter-
est and the internal standard are equally accessible to primers after denatura-
tion; both templates have the same efficiency to hybridize to the primer and to
be extended by the polymerase; substrate exhaustion affects the extension of
both templates equally [70]. However, in a competitive PCR experiment [70], the
authors observed a bias that was strongly dependent on the number of replica-
tion cycles. They demonstrated that reannealing of genes progressively inhibit-
ed the formation of template-primer hybrids.
Taqman PCR is another quantitative method exploited for detection of low-
level populations. This method takes advantage of the 5¢ to 3¢ exonuclease activ-
ity of the Taq DNA polymerase [71]. A probe targeting one strand of the PCR
template is labeled at the 5¢ end, and its 3¢ end is phosphorylated to prevent
extension. During PCR, the Taq polymerase extends the ordinary primer along
the template strand. When it meets the probe that binds to the template strand,
it cleaves the 5¢ terminal nucleotide and produces mono- or oligonucleotides,
which are shorter than the original probe. In the first Taqman study [71], the
probe was labeled with 32P. Autoradiography after TLC (thin layer chromatogra-
phy) was needed to detect the hydrolyzed probes. The original Taqman method
was modified [72] to allow rapid analysis by labeling the probe with a fluores-
cent dye at the 5¢ end and with a quencher at the seventh nucleotide from the 5¢
end. The dye fluoresces when the probe is cleaved between the dye and the
quencher (Fig. 2). Therefore, there is no need for post-amplification separation.
Subsequently, Taqman PCR was demonstrated to be quantitative since the
intensity of fluorescence was proportional to the amount of PCR product, and
under appropriate conditions, to the initial number of the templates [73]. How-
ever, it is necessary to assume that the efficiency of the PCR to amplify DNA
from an environmental sample is similar to the efficiency of the PCR used for
constructing the standard curve, before this method can be used to quantify
populations in environmental samples. Since this assumption may not always be
valid, bias can occur.
2.2.8
Summary
Despite the potential biases of the various methods discussed above, retrieving
16S rRNA sequences directly from environmental samples allows the investiga-
tion of microbial communities without cultivation. The use of these techniques
Fig. 2. Taqman PCR. Modified from [72]
has revealed that the microbial diversity is much greater than was anticipated
based on cultivation studies.
2.3
Oligonucleotide Probes
The first step of oligonucleotide probe hybridizations consists of probe design

as illustrated in Fig. 1. The probes are used in various types of hybridizations to
detect, quantify, and localize the target sequences or cells in a sample, in a nucle-
ic acid extract, or in a clone library. For more information on environmental
application of nucleic acid hybridization, several reviews are available [10, 39,
74, 75].
This section focuses on probes rationally designed, using the phylogenetic
framework provided through comparative analysis of sequences available in
databases [76]. In particular, the large collection of 16S rRNA sequences makes
it possible to design a nested set of 16S rRNA-targeted oligonucleotide probes
with different levels of specificity. By comparing aligned 16S rRNA sequences,
unique regions can be found that are shared only by the target population(s).
Empirically designed probes have traditionally been generated from a
genomic recombinant library or simply are the total genomic DNA obtained
from a target organism [76]. These probes have not been used much in micro-
bial ecology research in the last decade because of limitations with nesting and
quantification. An obvious advantage of oligonucleotide probes is that targets
differing in a single nucleotide can be discriminated under appropriate experi-
mental conditions. A few applications of empirically designed probes have been
published. DeLong et al. [12] studied the correlation between growth rates of
Escherichia coli, the average ribosome contents, and the fluorescence conferred
by hybridization probes. They observed that with decreasing growth rates the
hybridization signal quickly approached the limit of detection of epifluores-

cence microscopy or flow cytometry. Using oligonucleotides carrying multiple
labels both in the hybridizing probe and in a non-complementary tail did not
significantly increase the sensitivity [77]. A possible way to identify cells with
low metabolic activity, i.e., low amount of ribosomes, is by applying polynu-
cleotide probes carrying multiple fluorescent reporter molecules [78]. In this
study a polynucleotide probe (ca. 200 to 300 nucleotides in length) was generat-
ed by transcription of a cloned probe sequence from the 23S rRNA gene from
Pseudomonas stutzeri. The probe was selected to target the variable domain III
region in the 23S rRNA molecule. Whole-cell hybridization proved that the
polynucleotide probe was superior to the oligonucleotide probes for in situ
detection of cells with low cellular rRNA contents. Also, the larger probes could
differentiate between two closely related organisms Pseudomonas stutzeri and
Pseudomonas diminuta.
Heuer and coworkers utilized digoxigenin-labeled probes targeting the 16S
rRNA molecule to fingerprint the microbial community in rhizosphere samples
taken from potato plants [79]. Briefly, PCR amplified 16S rDNA genes (the V6
region) were separated by temperature gradient gel electrophoresis (TGGE),
cloned, sequenced and used to probe dot blotted 16S rDNA amplified from bac-
terial isolates. To optimize the specificity of the probes, flanking conserved
regions were removed. One truncated probe hybridized to three different bacte-
rial isolates all with different electrophoretic mobility in the TGGE. Subsequent
sequencing of these isolates revealed an identical V6 region but different V7 and
V8 regions explaining the above observations. Also, the polynucleotide probes
were shown to discriminate between targets differing only by two nucleotides.
The advantage of polynucleotide probes compared to oligonucleotide probes
is their higher specificity. Even though the specificity of oligonucleotide probes
is sufficient for many applications, more specific probes are sometimes needed
to discriminate between two closely related organisms. Several groups of organ-
isms have been identified which share almost identical 16S rRNA sequences but
among which DNA:DNA hybridization values are lower than 70% [80].
2.3.1
Probe Design
2.3.1.1
Probe Specificity
The specificity of a newly designed probe has to be tested before it can be used
with confidence. The specificity can be checked using rRNA databases such as
the CHECK_PROBE software provided in the Ribosomal Database project
(http://www.cme.mwu.edu/RDP/html/analyses.html) [25] and the Oligonu-
cleotide Probe Database (OPD) [81]. Alternatively, the BLAST network service
available from the National Center for Biotechnology Information at
http://www.ncbi.nlm.nih.gov can be used. Due to the limited collection of rRNA
sequences compared to the total estimated prokaryotic diversity, there is a pos-
sibility that some yet undiscovered sequences are targeted by probes designed
for other organisms [27]. As pointed out by Ward et al., “probes should be
regarded as tools subject to refinement” [27]. Probe nesting is another way to
check the specificity of a probe [39]. Theoretically, the sum of quantification
results from an environmental sample at one taxonomic level (e.g., family)
should equal the quantification result of the taxa at one higher level (e.g., order).
If this is observed, all the probes used are probably specific. If the population size
obtained for, e.g., an order, however, is larger than the sum of populations rec-
ognized at the family level, an unknown family not targeted by the family probes
might exist [39].
2.3.1.2
Target Accessibility
This needs to be considered when probes are used for in situ or whole cell
hybridization, since the higher order structure of the target rRNAs in this type
of hybridization is intact and since rRNAs are associated with ribosomal pro-
teins [6]. Fixation can denature the higher order structure. However, since the
influence of fixation is hardly predictable, there is no easy estimation of accessi-
bility to the target [6]. Some of the 16S rRNA and 23S rRNA sites that have been
successfully targeted were summarized by Amann and coworkers [6, 82].A more
systematic study of target accessibility was reported by Frischer and co-workers
[83]. Five probes each consisting of 12 nucleotides were designed to target the
515, 786, 1063, 1341, and 1369 sites of Escherichia coli 16S rRNA. Hybridization
signals of all the five probes were equal in hybridization to cell blotted mem-
brane, but different in whole-cell hybridization. Only probe 1341 gave a good
signal in whole-cell hybridization. Probe 515, which targeted a ribosomal pro-
tein-binding site, showed moderate signals, but the inhibition by the proteins
seemed to be outcompeted by a longer probe. The study showed that probe 786,
which targeted a loop site, gave a moderate signal for unknown reasons. Target-
ing the self-complementary sites did not seem to be a problem since probe 1369,
which targeted a less self-complementary site, gave a lower signal than
probe 1341, which targeted a higher self-complementary site. Another study
also showed that targeting highly structured sites with probes consisting of
30 nucleotides resulted in good signals [84]. More recently, Fuchs et al. con-
vincingly conducted a systematic study on the accessibility of 16S rRNA target
sites in E. coli by probing with more than 200 probes along the 16S rRNA mole-
cule showing regions with high accessibility and other regions with low accessi-
bility [85]. Fuchs et al. also showed improved accessibility to otherwise low
accessibility regions by applying unlabeled helper oligonucleotides binding next
to the labeled probe’s target site [86].
2.3.2
Quantitative Slot (Dot) Blot Hybridization
In general, the quantitative slot (dot) blot hybridization involves the application
of rRNAs extracted from environmental samples on membranes together with a
dilution series of RNA from an axenic culture (reference RNA). The membranes
are then prehybridized, hybridized with the probes, and washed. Usually mem-
brane hybridizations are conducted at low stringency (high salt concentrations
and low temperatures). The washing step is then used to remove all excess non-
specific binding probe. The signals from the environmental samples are quanti-
fied by comparison with the reference rRNA. The abundance of a specific popu-
lation is expressed as a percentage of the total rRNA determined by a universal
probe targeting all rRNA. Alternatively, results can be reported in terms of µg of
rRNA per sample volume or weight.
2.3.2.1
Hybridization Stringency
The washing conditions are critical in order to distinguish between target

sequences and sequences with one or more mismatches. The specificity is usu-
ally controlled by temperature. The optimum washing temperature (Tw) is rec-
ommended to be equal to, or slightly higher than, the dissociation temperature
(Td) which is the temperature at which 50% of the duplexes remain intact dur-
ing a specified washing period [87]. Another closely related parameter is the
melting temperature (Tm) which is defined as the equilibrium temperature
where half of the duplexes are dissociated [76, 87]. Thus, Tm is defined for equi-
librium conditions and is time-independent, whereas the Td value is time-
dependent. There are a number of empirical equations that can be used to esti-
mate the Tm and Td of a duplex [76, 87]. These equations provide guidelines for
probe design, especially when certain Td values are needed (e.g., the design of a
number of probes for simultaneous in situ hybridization experiments) [39].
However, since the Td is a function of numerous factors [76, 87] such as duplex
structure and length, sequence, nucleotide content, number and type of mis-
matches, terminal unpaired bases, as well as the hybridization and washing con-
ditions, the experimental determination of Td is highly recommended.
2.3.2.2
Quantification
2.3.2.2.1
Interpreting the Quantification Results
When interpreting the quantification results obtained from membrane hybrid-

ization experiments, it is important to consider that rRNA abundance does not
equal cell abundance, since the number of rRNA molecules in each cell lies with-
in a very broad range (103 to 105) [6]. However, since the cellular rRNA content
of a cell often is correlated to its growth rate [12], the abundance of the rRNA is
an indication of the metabolic activity. Since one genotype can be related to sev-
eral phenotypes, derivation of specific physiological activities from rRNA abun-
dance should be carried out with caution. Although the absolute abundance of a
population in terms of mass of rRNA per weight or volume of a sample is desir-
able, this type of quantification result should also be carried out with caution
due the high variability of RNA recovery in the extraction process as previously
discussed.
2.3.2.2.2
Sensitivity
The sensitivity of membrane hybridizations can be as low as 0.1% when an iso-

tope-labeled probe is used [6, 27]. Based on observations in our laboratory, this
sensitivity can be obtained by proper loading (highest loading without saturat-
ing the membrane, see [40]) and high radioactivity of the probes. This has the
consequence that populations having a rRNA abundance below 0.1% of the total
rRNA in a sample cannot be detected by membrane hybridization. However,
low-abundance populations can be detected with the aid of PCR.
2.3.2.2.3
Variation
By comparing different types of commercially available membranes, [32] it was

shown that the detectability and local variation differed significantly from one
type of membrane to another. The local variability of a membrane (Type I)
ranged from 10 to 50%, and was believed to be the primary cause for variation
in quantification results. Several other types of variability during membrane
hybridizations were tested [39]. It was demonstrated that variability introduced
by denaturation/dilution (Type II) and prehybridization/hybridization (Type
III) were not statistically significant. The washing step (Type IV), however, could
introduce significant variation. Therefore, it was recommended to apply each
sample in triplicate to compensate for the Type I variation. Samples and refer-
ence rRNA series should also be washed in the same tube or beaker to avoid
Type IV variation.
2.3.2.3
Factors that May Interfere with Quantification
2.3.2.3.1
Membrane Saturation
Membrane saturation can be one of the many reasons that a non-linear

hybridization response is observed [39, 40]. The saturation of Marga Charge
membranes (MSI, Westborough, MA) was determined to be around 150 ng
nucleic acid/slot using 32P-labeled E. coli rRNA [40].
2.3.2.3.2
Target Accessibility
Accessibility of probes to the 16S rRNA sequence can be different from site to
site, even in membrane hybridizations [32]. This study showed that the
hybridization signal increased as the denaturation conditions increased (in
terms of temperature, time, and the concentration of glutaraldehyde) for the
target site 628 ( E. coli 16S rRNA numbering). However, for site 1392, higher lev-
els of denaturation resulted in lower signals. It was hypothesized that a higher
level of denaturation might result in loss of signal because site 1392 has rela-
tively low levels of secondary structure [32]. In another study, McMahon et al.
[88] also showed that the effect of denaturation conditions on membrane
hybridization signals was target site-dependent. The difference in accessibility
along the 16S rRNA could therefore cause bias in quantification if different
probes are used.
2.3.2.3.3
Co-Extracted Substances
The presence of as little as 5.6 µg humic substances in 10 ng RNA can lower

hybridization signals [38].Although DNA does not contribute much to non-spe-
cific binding during membrane hybridization [38], high concentrations of DNA
(higher than 10 ng in 10 ng RNA) can reduce hybridization signals [40]. Since a
total of 20 ng nucleic acids (DNA and RNA) is much lower than the saturation
limit of the membrane [40], the inhibition is caused by mechanisms other than
saturation. However, since hybridization signals are reduced for both specific
and universal probes, results expressed in terms of percentage should not
change significantly due to the presence of inhibitory compounds, but detect-
ability is reduced.
2.3.2.3.4
In Vitro Transcribed rRNA
An advantage of the oligonucleotide probe method to previously used antibody

methods is that a probe can be designed and synthesized without the avail-
ability of an axenic culture. However, there is still a requirement for pure
culture rRNA for probe characterizations, for specificity studies, and for
standards during quantitative membrane hybridizations. In vitro transcribed
RNA or rcDNA (obtained by reverse transcription) was suggested as a substi-
tute for native RNA by Ward et al. [27] and this method has been used in some
studies [89–92]. However, the behavior of the in vitro transcribed rRNA and
rcDNA was not compared to native rRNA in those studies. Polz and Cavanaugh
[93] and McMahon et al. [88] both agreed that in vitro transcribed rRNA can
be used to determine the Td values of the native rRNA, although there is
some disagreement between the two studies. Polz and Cavanaugh found that
transcribed rRNA resulted in 2 to 3 °C higher Td values compared to native
rRNA using probes S-D-Bact-0338-a-A-18 for Bacteria and S-S-V.ang-0219-a-
A-20 for Vibrio anguillarum. McMahon et al. found, however, that transcribed
rRNAs have the same Td values as the native rRNA for probes S-*-Synb-
0222-a-A-19, S-S-S.fum-0464-a-A-19, S-F-Synm-0700-a-A-23, and S-*-Univ-
1390-A-a-18. Both studies demonstrated bias when the transcribed rRNAs were
used for quantification by membrane hybridization, even though Polz and
Cavanaugh claimed that the bias was not statistically significant due to the
high variability of the hybridization signals. This will restrict the usage of in
vitro transcribed RNA for absolute quantification of microbial population
abundance.
2.3.3
Reverse Sample Genome Probing
The reverse sample genome probing technique was developed by Voordouw and
coworkers in the mid-1990s [95]. Instead of using a probe targeting a genus or a
family, genomic DNAs from reference strains are immobilized on a membrane.
DNA extracted from an environmental sample is then isotope-labeled by nick
translation and used as probes. This method is well suited to study diversity of
microbial groups like sulfate-reducing bacteria, syntrophs or methanogens.
rRNA probing detects how many known and unknown species containing the
probe sequence are present in a sample while the reverse sample genome prob-
ing detects specific species or strains. To carry out the same task by rRNA prob-
ing, many hybridizations would need to be performed using different probes.
The DNA probe is much more specific because it contains thousands of genes. A
prerequisite, however, is a proper selection of the reference strains to minimize
cross-hybridization to closely related strains. Each DNA reference spot only
detects other genomes present if they share enough homology. This technique
has been shown to discriminate down to the species level [96].
Voordouw and coworkers have used the technique to study diversity in oil-
containing environments [96, 97]. Twenty-six sulfate-reducing bacteria (16
belonging to the genus Desulfovibrio) plus other reference species were used to
probe oil field samples to detect environmental nitrate- and sulfate-reducing
bacteria. Hybridization signals were shown to specific Desulfovibrio species but
not to others. Thereby the authors were able to identify and to quantify the rel-
ative amount of the different species present in a single hybridization event. A
problem with this technique is that the DNA extracted from the samples may
originate from dead cells. Also, the generation time of specific populations has
to be considered to ensure that environmental changes are reflected in the bac-
terial populations. rRNA probing may detect changes in population sizes and
activities that occur within days, while the reverse sample genome probing can
do the same on a week scale, but in higher detail.
2.3.4
Whole Cell or in Situ Hybridization
Amann et al. define whole cell hybridization as hybridization performed with

morphologically intact cells, and in situ hybridization as whole cell hybridiza-
tion targeting cells in their natural habit [6, 82]. The term “fluorescence in situ
hybridization (FISH)” is used for both whole cell and in situ hybridizations. The
perspectives of whole cell and in situ hybridizations are discussed below.
1) FISH can show the three-dimensional spatial distribution and morphology of
uncultured cells. For instance, Harmsen et al. [90, 98] used fluorescently
labeled probes to reveal the internal structure of anaerobic granules. This
technology was also used to show the dense aggregation of Paracocci in a den-
itrifying biofilm [99], as well as locations of Nitrosomonas and Nitrobacter in
a nitrifying biofilm [100]. With careful design of probes and their fluorescent
labeling, the distribution of at least seven different types of microorganisms

can be shown on one slide at the same time [101].
2) Following FISH labeling, target cells can be counted and concentrations
can be expressed in terms of cell numbers. Flow cytometry in combination
with FISH can also be used to count a large number of cells in a short time
[10].
3) Since the number of ribosomes in each cell is assumed proportional to its
growth rate, quantification of the signal from each cell may be used to infer
its growth rate. This approach was used to detect the in situ growth rate of sul-
fate-reducing bacteria in a biofilm [102]. The results should, however, be
interpreted with caution, since it is not known if the relation between the
ribosome number and the growth rate is similar for cells under different con-
ditions [6]. The sensitivity of FISH can be very high, 1 in 106 cells (compared
to 0.1% in membrane hybridization and 1 in 103 for cloning).
4) Probe specificity can be controlled even for very complicated communities by
using multiple probes with different color labels that target the same organ-
ism at different sites in their 16S or 23S rRNA sequences.
2.3.4.1
Hybridization Stringency
In contrast to membrane hybridizations, FISH is usually conducted at a relative-

ly high stringency, i.e., a stringency that can differentiate target from non-target
cells. The wash step is merely used to remove excess probes. Although tempera-
ture could be used as the parameter for controlling stringency [103], salt or for-
mamide concentrations are more often used [99, 104]. This is more convenient
since only one temperature and hence, one oven is needed for hybridization
reactions at different stringencies.
The optimal stringency for FISH is determined in a similar way as a Td study.
Target cells (perfect match) as well as non-target cells with a few mismatches
should be used for stringency tests. Hybridizations are conducted at a number
of stringencies. The average hybridization intensities obtained for a large num-
ber of cells (e.g., 100 to 200 cells) are plotted versus the stringencies in terms of
formamide or salt concentration. The optimal hybridization stringency is deter-
mined as the point where the signals from non-target cells are low while those
from target cells are still strong (see [104] and [99] for examples). Using the
same hybridization and wash solutions employed for membrane hybridization,
Amann and coworkers carried out a Td study with whole cells using a 32P-labeled
probe [105]. It was shown that there was no significant difference in Td values
between the two methods. Empirical equations are available for the conversion
of stringencies expressed in terms of temperature, salt concentration, and for-
mamide concentration [76]. These, again, can be used to predict the necessary
stringency for whole cell hybridization, but are not a substitute for experimen-
tal examinations.
2.3.4.2
Cell Fixation
The first step of FISH is fixation, which permeates the cell wall and cell mem-
brane to allow the penetration of probes. Insufficient fixation can be a cause of
low signal [6]. It is recommended to check the permeability of the cell walls
before further studies involving hybridization with well-labeled universal or
domain-specific probes are carried out [6].
Fixatives can be ethanol or methanol, aldehydes, enzymes, or heat [6]. In gen-
eral, paraformaldehyde (PFA) and 50% ethanol offer good results for both
Gram-positive and Gram-negative cells for fluorescently labeled oligonucleotide
probes [106]. PFA is often preferred for the fixation of Gram-negative cells, since
PFA might cross-link the thick Gram-positive cell wall to such an extent that the
probe cannot pass through the wall. The 50% ethanol fixation is less used for
Gram-negative cells, since the stability of ethanol-fixed Gram-negative cells is
low. Heating or ethanol-formalin (v/v = 9:1) fixation also have proven to be suit-
able for Gram-positive cells [107]. Some of the Gram-positive cells may need lyt-
ic enzymes, hydrophobic solvents (toluene or diethyl ether), or acids for proper
fixation [82]. PFA solution (4%) has proven to be a good fixative for most of the
Archaea tested in a study by Burggraf et al. [108]. However, 4% PFA was too mild
in some cases due to the rigid cell wall of Methanopyrus kandleri, Methanother-
mus fervidus, Methanobacterium thermoautotrophicum, and Halococcus mor-
rhuae [108]. Sørensen et al. [109] found that 4% PFA fixation of Methanosarcina
mazeii resulted in disruption of the cells. They demonstrated satisfying fixation
results by washing the M. mazeii cells in saline-formaldehyde (1.6% formalde-
hyde and 0.85% NaCl).
Fixation time also plays an important role. In a study by de los Reyes et al.
[104], one minute fixation in 4% PFA is optimal for mycolic-acid-containing
Gordona amarae, Rhodococcus rhodochrous, and Mycobacterium semegmatis.
Longer fixation caused excessive cross-linking of the proteins in the cell wall
preventing the access of probes.
A number of fixation methods, including PFA fixation, ethanol/formaldehyde
fixation, solvent extraction using chloroform/methanol, acid methanolysis, and
acid hydrolysis, were evaluated for mycolic-acid-containing actinomycetes and
some other Gram-positive and Gram-negative cells [110]. It was demonstrated
that the optimum fixation was species-dependent. The wide variety of cell wall
types complicated a proper fixation of all cells in a complex community by a
single treatment method. It was suggested that different fixation methods
should be used corresponding to the cell wall properties of the populations to
be detected [106].
2.3.4.3
Signal Enhancement
Besides poor fixation, low signals can be the result of non-complementarity be-
tween the probe and the target, ineffective probe labeling, non-optimal hybridiza-
tion conditions, low ribosome numbers, or low accessibility of the target site.
Several methods can be used to enhance the signal from cells having a
low ribosome content, and thus increase the sensitivity of detection [see below].
It should be noted that none of these methods can improve the signal quanti-
tatively.
The sensitivity of detection can alternatively be improved by using instru-
ments such as CCCD (cooled charged-coupled device) cameras, which can
detect very low levels of emitted light, and confocal laser scanning microscopy
(CLSM), which can exclude out-of-focus fluorescence.
2.3.4.3.1
Indirect Assays
In this technique the oligonucleotide probe is labeled with digoxigenin. After

hybridization, the digoxigenin is detected by a binding protein labeled with a
fluorescent dye or an enzyme. The signal increase by fluorescently labeled bind-
ing proteins is limited, since the molar fluorescent dye/protein ratio cannot
exceed 3. Enzyme-labeled binding protein has the advantage that hybridization
is detected by the precipitation of suitable substrates, eliminating interference
from background fluorescence or autofluorescence. The major problem with
indirect assays is the limited permeability of the cell wall to relatively large flu-
orescent dyes or enzyme-labeled binding proteins. Usually, enzymatic or more
rigid fixation methods are needed, even for Gram-negative cells [111, 112].
2.3.4.3.2
Enzyme-Labeled Oligonucleotides
Enzyme-labeled oligonucleotides are formed by covalent linking of the oligonu-

cleotide probe to an enzyme. After hybridization, the probe is detected by the
precipitate formed from suitable substrates. Similar to the indirect assay, auto-
fluorescence and background fluorescence do not interfere with this method.An
improvement compared to indirect assays is the lack of problems with non-spe-
cific binding of the binding proteins. A study by Urdea et al. (1988) showed that
enzyme-labeled oligonucleotide probes had lower detection limits than fluores-
cently labeled probes and that their sensitivity was comparable to 32P-labeled
probes [113]. Since the probe is smaller compared to binding proteins of indi-
rect assays, this method can be used for most of the Gram-negative cells and
some Gram-positive, ethanol-fixed cells. Lysozyme treatment of Gram-negative
cells is also suitable for this method [114], while SDS might be used for treat-
ment of Archaea [114]. In a study of the bacterial community in the gut of an
earthworm Fischer et al. (1995) used fluorescence-, peroxidase- (enzyme-
labeled), and digoxigenin- (indirect assay) labeled probes [115]. The authors
showed that the peroxidase- and digoxigenin-labeled probes were limited by the
requirement of enzymatic fixation, diffuse images of stained cells, and interfer-
ence with DAPI.
2.3.4.3.3
Multi-Probe and Multi-Labeling
In general, the multi-probe method using several monolabeled fluorescent

probes that target the same cell can increase the hybridization signal [103, 105].
However, limitation of specific target sites in a cell restrict the signal increase of
this method. Alternatively, a probe can be labeled with several fluorescent dyes.
But for unknown reasons, this method does not result in a significant improve-
ment of signal intensity [6].
2.3.4.3.4
Amplification of the Target Sequence
Amplification of the target sequence before detecting has been shown effective
in detection of weak hybridization signals [27]. In situ PCR with a fluorescent
primer was used to localize the single prokaryotic cells in a complex communi-
ty [116]. Non-specific amplification, however, might be a potential problem
associated with this method.
2.3.5
Solution-Based Hybridizations (Molecular Beacons)
When performing membrane hybridization or FISH, it is necessary to remove

excess probe from the hybridization mixture before the detection step. This
requires immobilization of cells or nucleic acids on solid surfaces (slides or
membranes), which may lower the sensitivity of hybridization due to non-spe-
cific binding of probes to the surface [117]. It also prohibits real-time monitor-
ing of nucleic acid synthesis and location of specific nucleic acids in living cells
[117]. Furthermore, these hybridization methods are labor intensive and cannot
be automated. The use of solution hybridization techniques, in contrast, offers
advantages such as fast kinetics and the suitability for automatic analysis [74].
Molecular beacon techniques eliminate the need of removing excess probe after
hybridization, and thus provide the feasibility for quick and automated
hybridization.
A molecular beacon is a probe that contains a stem-and-loop structure
(Fig. 3) [117]. The loop part consists of a sequence that is complementary to the
target sequence, while the stem part consists of two short sequences (arms)
located at each end of the probe and complementary to each other. One of the
arms is end-labeled with a fluorescent dye, while the end of the other arm con-
tains a quencher. The quencher is selected so that its absorption spectrum over-
laps the emission band of the fluorophore.When the molecular beacon is closed,
the fluorescent dye and the quencher are held closely together by the stem. As a
result, no fluorescence is emitted. When the molecular beacon hybridizes to a
target, or when the temperature is higher than the Tm of the stem, the fluorescent
dye is spatially removed from the quencher and the fluorophore emits light
upon exitation (Fig. 3).
Fig. 3. Concept of the molecular beacon technique. Modified from [117, 118]
Tyagi and Kramer [117] showed that the hybridization was very fast and that
the reaction rate increased as a function of the beacon concentration, the target
concentration, the salt concentration, and the temperature. The molecular bea-
con technique was found to be very specific when oligonucleotides were used as
a target. Almost no hybridization was observed to targets with one mismatch or
one deletion. Molecular beacons, therefore, can be used for detection of living
cells or for real-time monitoring of PCR reactions. In addition, it is possible to
detect a number of different populations in the same reaction tube if molecular
beacons are labeled with different types of fluorescent dyes. In a comparative
study of membrane hybridizations with oligonucleotide probes and solution
hybridizations with molecular beacons, Schonfield et al. [118] obtained similar
results for the detection of 16S rRNA. However, several problems were encoun-
tered by the authors. Firstly, the specificity of molecular beacons was not as high
as reported by Tyagi and Kramer [117]. Only an 80% signal decrease was
observed when one mismatch occurred in the target sequence. Secondly, denat-
uration had to be carried out carefully. Chemical denaturants may not be
applicable, since they denature the molecular beacons if they are not removed
from the solution before hybridization. In addition, denaturants cause high
background fluorescence interfering with the detection of molecular beacons.
Schonfield et al. denatured the RNA by heating (95°C for 5 min) followed by
overnight hybridization at 39°C. Thirdly, the post-hybridization mixture need-
ed to be centrifuged to remove particles that otherwise interfered with the
detection of the molecular beacons. Nevertheless, this study showed that the
molecular beacon technique reduced the total time needed for analysis of a
sample from 3–4 days to 12 hours, and it was possible to use the technique for
detecting 16S rRNA in environmental samples.
2.4
FISH and Reporter Systems
Direct hybridization methods (e.g., FISH) are only possible when the abundance
of target nucleic acids is sufficiently high. Detecting genomic DNA or mRNA
using in situ hybridizations is therefore difficult or impossible since the amount
of target is too low. The amount of target can be increased by in situ RT-PCR
applied to whole, permeabilized cells. By this technique, it is possible to address
the questions regarding gene expression under different growth conditions and
the influence of metabolites and chemicals on the different pathways at the single
cell level. This approach is very laborious when compared to direct studies with
well known model systems like Echerichia coli, Saccharomyces cerevisiae, and
Bacillus subtilis, but at the current state of technology it is the only possibility.
To our knowledge the only report describing gene expression in anaerobic reac-
tor systems on the mRNA level is by Lange et al. [119]. This paper describes the
expression of the heat shock gene dnaK in Methanosarcina mazei S-6 under dif-
ferent stress situations in situ. Paraformaldehyde-fixed cells were permeabilized
by lysozyme and heat treatment, and the cellular DNA was removed by DNAse
treatment.By means of a semi-nested RT-PCR protocol,DIG-labeled primers were
used to amplify the dnaK gene product. Detection of the dnaK reporter molecule
was based on the HPPN/Fast Red detection system and the binding of anti-
digoxigenin-AP conjugate. Key parameters in this technique are the permeabi-
lization of the cells and the number of cycles in the RT-PCR step. Analyses of dif-
ferent ecosystems by this technique, therefore, have to be carefully and individual-
ly optimized as especially Gram-positive bacteria and many archaeal species have
a cell wall that is difficult to permeabilize.As opposed to traditional mRNA analy-
sis techniques, e.g., Northern blotting, the in situ RT-PCR technique may reveal
heterogeneous gene expression in microbial populations [120], providing a more
detailed picture of the physiological state of populations.
A few reporter systems have been developed in organisms present in anaero-
bic reactor systems. In the methanogenic archaeon Methanococcus maripaludis,
genetic manipulations are now possible, as a naturally occurring plasmid in
Methanococcus has been modified to include the genes encoding the puromycin
resistance marker and the reporter gene lacZ encoding a b-galactosidase [105,
121, 122]. A uidA b-glucuronidase reporter gene has been used in Methanococ-
cus voltae [123]. By fusing the nifH promotor region to the lacZ coding region,
mutational analysis showed the presence of a regulatory palindromic sequence
repressing the nifH gene expression.
In the genus Methanosarcina, a plasmid from Methanosarcina acetivorans
was reported to be able to replicate in 9 of 11 Methanosarcina strains with high
transformation efficiency [124]. Lange and Ahring have utilized the plasmid
found by Metcalf to construct a reporter system based on the heat shock pro-
motors of dnaK and grpE in Methanosarcina mazei S-6 and the lacZ marker
gene. The resistance marker was the puromycin cassette. This system showed a
nice correlation between the amount of stress and the activity of b-galactosi-
dase. However, after several transfers of the transformants, the presence of the
promotor-lacZ cassette could no longer be confirmed due to the size of the plas-
mid (12 kb), but the strains retained their resistance towards the original level of
puromycin [125]. These findings should encourage new investigations to con-
struct smaller plasmids to allow routine genetic manipulations in the genus
Methanosarcina. Also, other resistance markers are needed as the puromycin
marker is one of a few shown to work in methanogens [126–128].
In the extremely halophilic archaeon Haloferax alicantei, a b-galactosidase
protein was purified and the sequence of the N-terminal part of the protein was
determined which facilitated the cloning of the gene [108]. b-Galactosidases
from other organisms do not function in the halophiles due to salt concentra-
tions above 4 M NaCl. Since plasmids already have been found in the halophilic
Archaea, in vivo studies of gene expression in this group of organisms is now
possible [127].
Future gene candidates to be used as reporter genes may be the luxA and the
luxB gene system; these genes have been expressed in the anaerobe Clostridium
perfringens under the transcriptional control of the a-toxin promotor region
under anoxic conditions [129]. However, this gene system has only limited rele-
vance in anaerobic prokaryotes, as the lux protein complex needs oxygen to pro-
duce light. The green fluorescent protein (GFP) also requires oxygen to mature
into a functional fluorescent protein [130]. Errampalli et al. [131] have recently
reviewed the applications of the GFP protein as a molecular marker in environ-
mental studies, so we refer to this paper for further reading. The GFP may be
valuable as a reporter gene in anaerobic laboratory systems if organisms trans-
formed with the GFP gene can be detected by fluorescence when subsequently
exposed to oxygen [130]. It will, therefore, not be possible to detect the GFP
reporter system in situ, but ex situ as a tool to monitor gene expression and to
characterize promotor regions. Further work has to be done on the applicabili-
ty of the GFP in anoxic and anaerobic systems.
An unusual blue protein Ambineela, has been isolated from the archaeon
Acidianus ambivalens [132]. Spectrum analyses of this protein show two emis-
sion bands around 395 nm and 625 nm. The protein does not require any tran-
sition metals in order to display this blue color. Ambineela might be a possible
reporter gene candidate in archaeal species for flow cytometric measurements
or absorbance measurements in axenic cultures.
2.5
FISH and Antibody Probes
Molecular studies of anaerobic reactor systems started using antibodies raised

against different microbial groups and labeled with fluorochromes [133–139].
The technique was used in phylogenetic studies to construct 2D pictures of the
spatial distribution of different groups of microorganisms in granular sludge
and flocs. Several drawbacks are, however, associated with the production and
use of antibodies in microbial ecology studies [10]. In order to raise the desired
antibodies, axenic cultures are required to induce antibody production by the
animals implying that only known organisms can be targeted. The antibodies
are less specific than oligonucleotide probes and antibodies raised against one
organism may target different epitopes on the cell leading to putative cross-reac-
tivity to other non-target cells. Furthermore, probing with antibodies does not
discriminate between dead and living cells.
An advantage of antibody probing is that cells having different metabolic
activities are equally visualized. The difference in metabolic activity, on the oth-
er hand, is one of the problems of oligonucleotide probing when targeting the
16S rRNA molecule. The cells of a population do not contain an equal amount of
ribosomes [27, 119, 120] and as the oligonucleotides normally are quite small,
only a certain amount of fluorochrome can be targeted to each cell. If the ribo-
some content in the cell, therefore, is low, the fluorescent signal cannot be dis-
tinguished from the background fluorescence.
To circumvent this problem, antibody probing can be combined with fluores-
cent oligonucleotide probes targeting the 16S rRNA molecule. The antibody
probe and the oligonucleotide probe can be labeled with different colors and by
applying different filters when examining the samples in the microscope, only
cells labeled by both probes are visible.
Raskin and coworkers have successfully applied both antibody and oligonu-
cleotide probes in situ for the characterization of Gordona species in activated
sludge and anaerobic digesters [140, 141]. Gordona species are slow growing fil-
amentous bacteria commonly found in activated sludge foam causing opera-
tional problems in wastewater treatment plants. Quantification of Gordona
species in activated sludge samples and in anaerobic digester samples by fluo-
rescent antibodies showed that Gordona species biomass accounted for 10–28%
of the volatile suspended solids in a full-scale activated sludge system and for
8–19% in anaerobic digester sludge. Quantification using traditional Gram
staining and filament counting for the same activated sludge sample resulted in
significantly lower values of 2–10% [142].
Analyzing seven full-scale wastewater treatment plants with antibody probes
or FISH targeting Gordona species showed that the antibody probing technique
led to estimates of a higher number of Gordona species than when FISH was
used [141]. Simultaneous staining with antibody probes and FISH showed that
some branched filaments were clearly stained by the antibodies, but gave a low
signal when using the FISH probe. The ribosome content of the cells, therefore,
indicated that individual cells differed in metabolic activity. These findings
point to the difficulties when estimating the number of species and the meta-
bolic state of the species in an environmental sample. Quantifying the number
of Gordona species by membrane hybridization targeting 16S rRNA and FISH
targeting 16S rRNA, expressed as biomass by measuring cell length and calcu-
lating the cell volume, gave different results. As observed in other studies [143],
relative cell numbers, therefore, cannot be reliably extrapolated from rRNA
abundance levels.
Although both immunostaining and FISH might yield non-specific signals
from non-target microorganisms, the possibility of misidentifying the same
non-target microorganisms by two independent methods should be low. The
simultaneous application of a polyclonal antibody serum and an oligonu-
cleotide probe targeting 16S rRNA also offers the advantage of detecting slow
growing or metabolically less active microorganisms while maintaining high
phylogenetic specificity in complex environments [140].
2.6
FISH and Microautoradiography
To identify and to evaluate the metabolic activity of a single cell simultaneously,

microautoradiography (MAR) has successfully been used in combination with
FISH. MAR is used to study the microscale distribution of radiolabeled com-
pounds. The radiolabeled compound is taken up by either active or passive
transport across the cell membrane. After appropriate treatment, the radiola-
beled cell sample is placed in contact with a layer of radiosensitive emulsion. The
emulsion is then developed after days or weeks of exposure to the radioisotope
by means of standard photographic procedures. Subsequent microscopy of the
cell sample will show cells covered with silver grains if the radiolabeled com-
pound has been taken up or metabolized. Cells on the developed slide can then
be investigated further by FISH to correlate the phylogenetic affiliation and
metabolic activity.
Nielsen et al. and Ouverney et al. have combined FISH and MAR protocols
to correlate cell identity and function in activated sludge reactors [144–147].
The uptake of 14C- or 3H-labeled substrates (acetate, glucose, ethanol, glycine,
leucine, and oleic acid) in bulking sludge was investigated in seven industrial or
municipal activated sludge treatment plants. The authors concluded that the
taxonomic variability assessed using FISH with probes specific for type 021 N,
Thiothrix and Leucothrix is high since only some of the filaments morphologi-
cally identified as type 021 N hybridized with the 021 N probe. Moreover, no fil-
aments took up all the tested substrates, and type 021 N from the various treat-
ment plants varied in their uptake capabilities. The study demonstrates that
strain differences with regard to substrate utilization are likely to occur among
bacteria within the same genus.
Some parameters to obtain good results with the MAR technique are:
– The choice of radioactive tracer: image resolution will decrease if high ener-
gy isotopes are used.
– The selection of incubation conditions: amount of biomass and isotope, ratio
of “hot” to “cold” substrate, presence of electron acceptors and incubation
time.
– The sample handling and fixation: the sample must be washed thoroughly to
remove excess isotopes.
– Other staining procedures: various cell stains such as Gram and Neisser stain-
ing for identification of filamentous microorganisms and fluorescent non-
specific stains such as Acridine orange or DAPI targeting DNA may be com-
bined with MAR. The dye CTC may be used to discriminate between viable
and dead cells.
The limitation of the MAR technique is low resolution unless cryosectioning of
the sample is conducted and 3D distribution of non-fluorescent silver grains in
the emulsion is accurately analyzed by confocal laser scanning microscopy.
2.7
Peptide Nucleic Acid Probes
Peptide nucleic acids (PNA) are artificial oligoamides reported for the first time
by Nielsen et al. [148]. PNA are capable of forming not only double-stranded
duplexes, but also triple-stranded complexes with poly- or oligonucleotides.
Due to their neutral backbone, PNA can hybridize to nucleic acids in the absence
of the counterions needed to stabilize nucleic acid duplexes. Thus, PNA probes
exhibit superior hybridization characteristics compared to DNA probes under
conditions where double-stranded DNA is electrostatically unstable, e.g., at low
salt conditions. PNA probes have been used for a wide range of applications and
the advantages of PNA over DNA are numerous: rapid hybridization, hybridiza-
tion independent of the salt concentration, resistant to nuclease and protease
attack, more specific and shorter probes can be used for higher sensitivity [149].
Prescott and Fricker [149] have used PNA oligonucleotide probes for in situ
detection of Eccherichia coli in water. They targeted the PNA biotinylated probes
towards the V1 region of the E. coli 16S rRNA molecule and the specific detec-
tion was carried out in less than 3 hours. The specificity of the PNA probe
against E. coli was confirmed by comparative dot-blot analyses using the genera
Klebsiella, Enterobacter, and Citrobacter.
Work done by Perry-O’Keefe et al. [150] has shown the superior hybridization
conditions under which PNA probes can detect DNA target sequences compared
to DNA probes. Investigations of parameters like hybridization rate and salt
concentrations showed that PNA probes hybridized much faster under a wide
range of salt concentrations. Because the PNA molecule is not charged, pre-gel
hybridization can be performed with subsequent transfer to nylon membrane
and fast detection.
Another new method of detection of nucleic acids is the application of PNA
probes together with the BIAcore biosensor system. The principle behind this
system is the detection of PNA hybridizing to DNA through signaling to a sur-
face plasmon resonance unit. Specific hybridization occurs in 10 min within a
flow stream at room temperature [151].
This fast and reproducible technique is promising for the detection and quan-
tification of rRNA extracted from environmental samples, as it is much faster
than the traditional techniques employed today. The problem with the sec-
ondary structure of the rRNA may be overcome by an addition of formamide
which will inhibit double strand and hydrogen bond formation, thereby reduc-
ing the secondary structure of the rRNA but still allowing the PNA probe to bind
to its target sequence.
3
rRNA-Based Analyses of Anaerobic Reactors
3.1
Biofilm Reactors
3.1.1
Biofilm Formation
Anaerobic biofilm development was monitored by Araujo et al. [152]. They

investigated biofilm formation with respect to the methanogens and the prop-
erties of biofilm formation on hydrophilic and hydrophobic surfaces. FISH and
confocal laser microscopy were used to quantify the microbial composition and
to elucidate the spatial distribution of microbes in the biofilm. Table 1 outlines
the probes used in this study.
In this experiment, axenic cultures of Methanobacterium formicicum, Me-
thanosaeta concilli and Methanosarcina barkeri were grown together for a
month before introduction into a sterile, anaerobic chemostat connected to a
modified Robbins device (MRD) [153]. The system was operated at 30°C ± 3°C
and the biofilm was grown on either polypropylene or glass discs inside the
MRD. The carbon sources were methanol, formate and acetate. Samples ware
taken on days 0, 2, 7, and 9. Even though the setup was supposed only to analyze
for methanogens, contaminants were observed when samples were taken. Dur-
ing the experiment, the chemostat microbial community changed from 100%
Archaea at day 0 to contain 40% Bacteria within 2 days.After 7 days the Bacteria
constituted 80% of the total microbial community. The number of Methano-
sarcina barkeri and Methanosaeta concilii decreased from about 15% day 0 to
close to 0% day 9. The number of Methanobacterium formicicum cells was
reduced from 80% to 10% within 9 days. The findings were not reflected in
the composition of the biofilm growing on the discs. After 7 days, the bacterial
population had only increased to about 15%. Methanobacterium formicicum
predominated in all the biofilms ranging from 40% to 80% after 9 days.
Methanosaeta concilii cells were found at low numbers up to 2.4% while
Methanosarcina barkeri was not detected. No difference in biofilm colonization
was observed between the polypropylene and the glass discs.
In a second experiment crushed granular sludge was used as seed in the
chemostat and the biofilm in the MRD was grown on polypropylene discs at
33°C ± 2°C. Initially, the inoculum in the chemostat consisted of 13% sulfate-
reducing bacteria (SRB) and 93% Archaea mostly Methanobacterium species.
Methanosarcina species accounted for 5%.After eleven days the SRB population
had decreased to 1.7% although the bacterial population still constituted 13%.
Since no sulfate was present, the SRB population probably functioned as proton
reducers in syntrophic association with hydrogen and formate-consuming
methanogens. From day zero to day eleven Methanobacteriaceae increased from
74% to 85% while Methanosarcinales decreased from 11% to 4%. Microscopic
observations of the biofilm by confocal laser microscopy showed areas where
no growth occurred, and areas up to 9 µm where growth occurred. This was
Table 1. Various 16S rRNA probes used in environmental studies
Reactor type T Carbon sources Probe technique Probes used Target organisms Reference
Fixed-bed M Glucose FISH S-D-Bact-0338-a-R-18 Virtually all bacteria Amann et al. 1999
anaerobic S-*-Srb-0385-a-R-18 All sulfate-reducing bacteria
bioreactor S-Ss-Pt1–647-a-R-19 Unknown Desulfuromonas sp.
S-Ss-Pt2–647-a-R-19 Unknown Desulfovibrio vulgaris sp.
Fixed-bed M Lactate FISH S-Ss-Pt2–647-a-R-19 Unknown Desulfovibrio vulgaris sp. Kane et al. 1993
anaerobic Dot blot
bioreactor
Modified M Methanol, FISH, CLSM S-D-Bact-0338-a-R-18 Virtually all bacteria Araujo et al. 2000
Robbins formate, acetate

device reactor
S-D-Arch-0915-a-R-20 Virtually all Archaea
Acetate, butyrate, FISH, CLSM S-*-Srb-0385-a-R-18 All sulfate-reducing bacteria
formate +
Ethanol and S-F-Mcoc-1109-a-R-20 Methanococaceae
methanol
S-F-Mbac-1174-a-R-22 Methanobacteriaceae
S-O-Mmic-1200-a-R-21 Methanomicrobiales
S-O-Msarc-860-a-R-21 Methanosarcinales
Fixed-bed M Glucose FISH S-*-Univ-1392-a-R-15 Virtually all organisms Raskin et al. 1995
anaerobic Dot blot S-D-Bact-0338-a-R-18 Virtually all Bacteria Raskin et al. 1996
bioreactor S-D-Arch-0915-a-R-20 Virtually all Archaea
S-D-Euca-0502-a-R-16 Virtually all Eucarya
S-F-Mcoc-1109-a-R-20 Methanococaceae
181
182
Table 1 (continued)
S-F-Msar-0821-a-R-24 Methanosarcina
S-F-Msae-0825-a-R-23 Methanosaetaceae
S-F-Dsv-0687-a-R-16 Desulfovibrionaceae
S-G-Dsbm-0221-a-R-20 Desulfobacterium
S-G-Dsb-0129-a-R-18 Desulfobacter
S-*-Dscoc-0814-a-R-18 Desulfococcus+Desulfosarcina +
Desulfobotulus
S-G-Dsbb-0660-a-R-20 Desulfobulbus
Anaerobic M Glucose Dot blot S-U-Univ-1392-a-R-15 Virtually all organisms Raskin et al. 1994
chemostat
Solid waste M, T Municipal waste FISH S-D-Bact-0338-a-R-18 Virtually all bacteria

digester
Sewage sludge M Sewage sludge S-D-Arch-0915-a-R-20 Virtually all Archaea

digester S-D-Euca-0516-a-R-16 Virtually all Eucarya
S-F-Mcoc-1109-a-R-20 Methanococales
S-F-Mbac-0310-a-R-22 Methanobacteriales
S-F-Msar-821-a-R-24 Methanosarcinaceae
S-G-Msar-0821-a-R-21 Methanosarcina sp.
S-F-Msae-0825-a-R-23 Methanosaeta
One-phase, M, T Sewage sludge Dot blot S-*-Univ-1390-a-R-18 Virtually all organisms Raskin et al. 1995
two-phase
J. Hofman-Bang et al.
Sewage sludge S-D-Bact-0338-a-R-18 Virtually all bacteria
digesters S-D-Arch-0915-a-R-20 Virtually all Archaea
S-D-Euca-0502-a-R-16 Virtually all Eucarya
S-F-Msar1414-a-R-21 Methanosarcinaceae
S-G-Dsb-0804-a-R-18 Desulfobacter group
S-G-Dsbm-0221-a-R-20 Desulfobacterium spp.
S-G-Dsb-0129-a-R-18 Desulfobacter spp.
S-*-Dscoc-0814-a-R-18 Desulfococcus multivorans

Desulfosarcina variabilis
Desulfobotulus sapovorans
S-G-Dsbb-0660-a-R-20 Desulfobulbus spp.
CSTR digester: M Cow manure Dot blot S-F-Synm-0700-a-R-23 Strain FSS7 Hansen et al. 1999
Biogas plant + lipids Strain FMS2
Syntrophospora bryantii
Syntrophomonas sapovorans
Syntrophomonas wolfei subsp. wolfei
Syntrophomonas wolfei subsp. LYB
S-G-Synm-0126-a-R-19 Syntrophomonas sapovorans
Syntrophomonas wolfei subsp. wolfei
S-S-S-wol-0180-a-R-21 Syntrophomonas wolfei subsp. wolfei
S-S-S.sap-0181-a-R-20 Syntrophomonas sapovorans
S-S-S.bry-0181-a-R-21 Syntrophospora bryantii
183
Table 1 (continued)
184
CSTR reactor M Glucose Dot blot S-G-Msar-0821-a-R-24 Methanosarcina Fernandez et al.

S-G-Msae-0381-a-R-22 Methanosaeta 2000
Temp. phase T Sewage sludge Dot blot S-G-Dtm-0229-a-R-18 Desulfotomaculum Hristova et al.
digester 2000
Municipal solid
waste
CSTR digester T Municipal S-*-Dtm(a)-0229a-R-18 Selected Desulfotomaculum sp.

wastewater
Paper mill S-*-Dtm(be)-0152-a-R-20 Selected Desulfotomaculum sp.
wastewater
S-*-Dtm(c)-0428-a-R-19 Selected Desulfotomaculum sp.
S-*-Dtm(cd)-0216-a-R-19 Selected Desulfotomaculum sp.
S-*-Dtm(bcd)-230-a-R-18 Selected Desulfotomaculum sp.
Lab-scale M, T Municipal Dot blot S-U-Univ-1390-a-R-18 Virtually all organisms Zheng and Raskin,
digester waste 2000
Temp. phased T Activated sludge S-D-Arch-0915-a-R-20 Virtually all Archaea

digester S-F-Mcoc-1109-a-R-20 Methanococaceae
S-F-Msar1414-a-R-21 Methanosarcinaceae
S-G-Msae-0733-a-R-22 Methanosaeta
S-G-Msae-0322-p-R-22 Methanosaeta competitive probe
S-S-M.con-0381-a-R-22 Methanosaeta concilii
UASB M Ethanol, Dot blot S-D-Arch-0915-a-R-20 Virtually all Archaea Zheng et al. 2000
waste water
Glucose FISH S-O-Mmic-1200-a-R-21 Methanomicrobiales
Glucose+ S-F-Mbac-0310-a-R-22 Methanobacteriaceae
propionate S-F-Mcoc-1109-a-R-20 Methanococaceae
S-G-Msar-0821-a-R-24 Methanosarcina
S-S-M.con-0381-a-R-22 Methanosaeta concilii
S-S-M.the-0396-a-R-22 Methanosaeta thermophila
UASB M Sulfate-rich mill Dot blot S-D-Bact-0338-a-R-18 Virtually all bacteria Elferink et al. 1998
paper waste water S-D-Arch-0915-a-R-20 Virtually all Archaea
S-*-Srb-0385-a-R-18 Gram-negative sulfate-reducing bacteria
S-G-Dsbm-0221-a-R-20 Desulfobacterium
S-G-Dsb-0804-a-R-18 Desulfobacter
S-S-D.amn-0454-a-R-20 Desulfohabdus amnigenus
S-*-Dscoc-0804-a-R-18 Desulfococcus, Desulfobacterium
Desulfosarcina, Desulfobacter,
Desulfobotulus
185
Table 1 (continued)
186
S-*-Sbac-0222-a-R-19 Syntrophobacter fumaroxidans

Syntrophobacter pfennigii
Desulforhabdus amnigenus
S-S-S.pfe-0460-a-R-21 Syntrophobacter fpennigii
S-S-S.wol-0223-a-R-19 Syntrophobacter wolinii
S-Ss-SYN7–0177-a-R-23 Syntrophic propionate-oxidizer SYN7
S-F-Mcoc-1109-a-R-20 Methanococales
UASB M Propionate Dot blot S-*-Univ-1390-a-R-18 Virtually all organisms Harmsen et al.
+ sulfate 1996 a
FISH S-D-Bact-0338-a-R-18 Virtually all bacteria
S-S-SYN7–0177-a-R-23 Syntrophic propionate-oxidizer SYN7
S-S-S.wol-0223-a-R-19 Syntrophobacter wolinii
S-S-MPOB-0222-a-R-19 MPOB
S-S-S.pfe-0460-a-R-21 Syntrophobacter fpennigii
S-F-Dsv-0687-a-R-16 Desulfovibrio
S-O-Mmic-1200-a-RA-21 Methanomicrobiales
UASB M Sucrose+sulfate FISH S-D-Bact-0338-a-R-18 Virtually all bacteria Harmsen et al.
VFAs+sulfate S-D-Arch-0915-a-R-20 Virtually all Archaea 1996 b
S-S-MPOB-0222-a-R-19 MPOB
S-S-Spfe-0460-a-R-21 Syntrophobacter fpennigii alias
KOPROP
UABS M Paper mill Dot blot S-D-Bact-0338-a-R-18 Virtually all bacteria Elferink et al. 1997
wastewater S-*-Sbac-0222-a-R-19 Syntrophobacter fumaroxidans
Syntrophobacter pfennigii
Desulforhabdus amnigenus
S-S-D.amn-0454-a-R-20 Desulfohabdus amnigenus
UASB M, T Alcohol distillery FISH S-D-Arch-0915-a-R-20 Virtually all Archaea Tagawa et al. 2000
Sucrose+VFAs S-G-Msae-0757-a-R-18 Methanosaeta
Dairy milk S-F-Mbac-1174-a-R-22 Methanobacteriaceae
Food brewery
Bean jam
Potato waste
Municipal sewage
UASB T Sucrose+VFAs FISH S-D-Bact-0338-a-R-18 Virtually all bacteria Imachi et al. 2000
+Yeast extract
Dot blot S-D-Arch-0915-a-R-20 Virtually all Archaea
S-Ss-TGP-0690-a-R-20 Strain SI
UASB M, T Sucrose, VFAs PCR Sekiguchi et al.

1998
187
188
Table 1 (continued)
UASB M, T Sucrose, VFAs CLSM S-D-Bact-0338-a-R-18 Virtually all bacteria Sekiguchi et al.
FISH S-D-Arch-0915-a-R-20 Virtually all Archaea 1999
S-F-Msar-1414-a-R-21 Methanosarcinaceae
S-*-MUG28–0701-a-R-20 rDNA clone MUG28
S-*-GNSB-0633-a-R-20 rDNA clones in green non-sulfur
bacteria
UASB M Propionate, Dot blot S-D-Bact-0338-a-R-18 Virtually all bacteria Hofman-Bang et al.
butyrate S-D-Arch-0915-a-R-20 Virtually all Archaea 2001
S-*-Srb-0385-a-R-18 Gram-negative sulfate-reducing
bacteria
S-*-R6B-7–0980-a-R-18 Clostridium-like bacteria
S-*-R1B-16–0980-a-R-18 Clostridium-like bacteria
explained by the syntrophic growth of the SRB population in conjunction with

the methanogens. The high number of Methanobacteriales found may be
explained by the competitive advantage over Methanococcales and Methanosar-
cinales, when hydrogen and formate kinetics are compared.
Amann et al. [155] investigated the role of sulfate-reducing bacteria (SRB) in
the establishment and development of a biofilm. This was done by comparative
sequence analysis and FISH in biofilm targeting selected SRB populations.
Biofilms were grown on coverslips and reached a thickness of 10 µm. Two clones
were identified as delta-Proteobacteria. One was closely related to Desul-
furomonas acetoxidans. The other was closely related to Desulfovibrio vulgaris.
Specific probes constructed to target these two SRB did not hybridize to any cells
in the biofilm. If specific probes constructed on the basis of the two cloned 16S
rDNA genes were used, single cells could be identified. If the PCR primer
S-*-Srb-0385-a-R-18, used to construct the clone library, however, was used as a
fluorescent probe, only one of the populations could not be identified. The gen-
eral observation was that the biofilm formation was patchy and that the identi-
fied cells were gathered in colonies. The authors also concluded that the differ-
ent results obtained with the FISH probes demonstrate that a better insight in
the SRB diversity is needed to construct more specific probes.
3.1.2
Biofilm Composition and Dynamics
Raskin et al. [154] studied the competition and coexistence of sulfate-reducing

bacteria (SRB) and methanogens in anaerobic biofilms. Four biofilm reactors,
MA, MB, SA, SB, were operated on glucose as sole carbon source with (SA, SB) or
without sulfate (MA, MB). After eleven months of operation, sulfate was added
to MB and omitted from SB. MA and SA were maintained as control reactors.
Probing of samples taken throughout the experiment using the probes shown in
Table 1 was carried out.
Reactor MA contained 25% methanogens. Specific probing showed that
Methanobacteriales accounted for 12% and Methanosarcinales, Methanomicro-
biales, Methanococcales each occurred in the range of 4 –5 %. Prior to sulfate
addition, SRB comprised a significant fraction of the community in the
methanogenic reactors. Desulfovibrio and Desulfobacterium genera were pre-
sent in high amounts (16% and 2.8%, respectively). As previously mentioned,
these findings may be explained by the capability of several SRB to grow syn-
trophically on lactate, ethanol, propionate, and pyruvate.
After the addition of sulfate to the MB reactor, sulfate reduction started after
6 hours, reaching steady state levels within a few days. Desulfovibrio and Desul-
fobacterium sharply increased to 26% and 7.7%, respectively. These levels de-
creased subsequently to 20% and 3.5%, followed by a slow increase to 35% and
4.5%, respectively. Desulfobulbus and Desulfobacterium spp. increased shortly
after the addition of sulfate, but later fell to the levels observed before sulfate
addition. Desulfosarcina, Desulfococcus, and Desulfobotulus spp. all fell below the
levels observed before the sulfate addition (from 2% to 0.5%). 100 days after the
sulfate addition, the acetate levels increased in the reactor for about 100 days.
This change in acetate was not accompanied by detectable population changes,

thus emphasizing the need for a careful evaluation of the concept “steady-state”
in anaerobic reactors. Even though the methane production decreased to unde-
tectable levels after the addition of sulfate, the methanogenic populations still
constituted about 8%. Obviously, the ribosome content was maintained for
months in the methanogens despite the inactivity of their primary metabolic
pathways.
After the sulfate was removed from the inflow of reactor SB, methanogenic
populations slowly increased to steady-state levels dominated by Methanobacte-
riales (18%). The relative abundance of other methanogens remained fairly con-
stant. Following sulfate removal, Desulfovibrio and Desulfobacterium popula-
tions decreased to levels comparable to the control reactor (MA). These obser-
vations show that hydrogen is the key substrate when the metabolism shifts from
sulfidogenesis to methanogenesis.After several hundred days of operation with-
out sulfate, the microbial community structure and function of the SB reactor
was similar to that of the methanogenic control reactor MA.
Similar to observations from other sulfate-rich environments, the SRB were
shown to outcompete methanogens in high-sulfate biofilms. Also, the presence
of Desulfovibrio and Desulfobacterium spp. only varied a factor 2 as a function
of the presence or absence of sulfate. Moreover, Desulfosarcina, Desulfococcus,
and Desulfobotulus spp. turned out to be better adapted to the biofilms without
sulfate. The content of ribosomes in methanogens only slowly decreased upon
sulfate addition although methane could not be detected. Wagner et al. [143]
have made similar observations when probing denitrifying bacteria in waste-
water treatment plants. It therefore seems reasonable to correlate microbial
responses to environmental perturbations to increased ribosome content, while
it is more problematic to correlate decreasing microbial activity to decreasing
ribosome content.
3.2
Granular Sludge Reactors
3.2.1
Granular Sludge
Granular sludge consists of conglomerates of anaerobic microorganisms, which

are still visible as granules after settling and is considered a major form for
immobilization of microorganisms in anaerobic wastewater treatment systems
[156]. Similar to biofilms, granular sludge provides minimized mass transfer
limits, optimal micro-environment, and protection for microorganisms such as
methanogens and syntrophic bacteria. Granules typically form in upflow anaer-
obic sludge blanket (UASB) reactors, although they also might be found in oth-
er anaerobic systems, such as expanded granular sludge blanket (ESGB) reactors
[157, 158], upflow sludge bed filters (UBF) [2, 159], compartmentalized UASB
reactors [160, 161], anaerobic migrating blanket reactors (AMBR) [162], anaer-
obic baffled reactors (ABR) [163], and anaerobic sequencing batch reactors
(ASBR) [164]. Granular sludge has been used successfully to treat wastewaters
having varying COD contents. The loading can be as high as 20 g COD/L [165],
or as low as 1 g COD/L, such as for domestic wastewater [166]. Anaerobic gran-
ular sludge reactors can be operated at mesophilic, thermophilic, and even psy-
chrophilic conditions. After acclimation, granular sludge can treat wastewater
containing refractory, toxic, or xenobiotic compounds [167–174]. Even though
UASB reactors are usually operated at neutral pH, granules can adapt to high pH
(8.1–8.5) [175] or low pH (6.0) [176]. UASB reactors have also been used to treat
sulfate rich wastewater (SO42–/COD >1) [177].
To further optimize the process, it is of great interest to understand the micro-
bial species composition, and structure of granular sludge, as well as the granu-
lation processes.
3.2.2
Microbial Composition of Granules
Zheng and Raskin [178] used membrane hybridization with radioactively

labeled oligonucleotide probes to quantify methanogens in granules from two
lab-scale UASB reactors. The two reactors were fed synthetic wastewater
(COD = 4 g/L) containing glucose or glucose/propionate as the only energy
source. In both reactors, the granular sludge contained around 40% archaeal 16S
rRNA of which aceticlastic methanogens, Methanosarcinales were dominating.
Quantification by a species-specific probe revealed that Methanosaeta concilii
was the predominant species among the Methanosarcinales. This finding was
consistent with the observation that Methanosaeta-like filaments often are dom-
inating in granules. Among the hydrogenotrophic methanogens, Methanobacte-
riaceae were dominant in the glucose-fed reactor (about 5%). In the glucose/
propionate-fed reactor, Methanobacteriaceae and Methanomicrobiales each
made up around 5% of the microbial populations.
Zheng and Raskin also analyzed granules sampled at different heights from a
full-scale UASB reactor treating wastewater from a corn wet milling plant [178].
The archaeal 16S rRNA constituted between 30 to 50% of total 16S rRNA. The
highest numbers of Archaea were found in granules sampled from the top of the
reactor. Methanosarcinales and Methanobacteriaceae were the two dominant
methanogenic groups. Methanomicrobiales constituted less than 5% while
Methanococcaceae were almost absent. Within the Methanosarcinales, which
constituted between 7.4 and 27.9%, only 2.2 to 2.8% were Methanosaeta concilii,
and less than 1% were Methanosaeta thermophila and Methanosarcina species.
The authors concluded that genera such as Methanolobus, Methanococcoides,
Methanohalophilus, Methanohalobium, and Methanosalsus within Methano-
sarcinales may be present in high numbers in these granules.
The population changes of propionate-oxidizing bacteria after granule for-
mation in potato-processing industry wastewater adapted to different substrates
was investigated by Harmsen et al. [90]. Very low amounts of propionate-utiliz-
ing, sulfate-reducing bacteria Desulfobulbus spp. (around 2% of total 16S rRNA)
and the syntrophic propionate degrader strain SYN7 (less than 1% of total 16S
rRNA) were found in the inoculum. After adaptation, the Desulfobulbus spp.
increased up to 35% in the granules fed propionate and sulfate, while SYN7
increased to around 10% in the granules fed only propionate. The universal
probe used in this study underestimated the amounts of bacterial 16S rRNA,
which may have introduced significant biases [179]. Sekiguchi et al. [180] ana-
lyzed the microbial diversity of two types of methanogenic granular sludge,
sampled from a mesophilic (35°C) and a thermophilic (55°C) UASB reactor
treating synthetic wastewater containing sucrose, propionate, and acetate. Clone
libraries of 16S rDNA were constructed using a prokaryote-specific primer set,
followed by partial sequencing of the cloned rDNAs. It was found that 19% of the
clones from the mesophilic granules and 22% from the thermophilic granules
were closely related to methanogens, while the rest were Bacteria.A major group
of bacterial clones from the mesophilic granules showed homology to the delta
subclass of the Proteobacteria (27%) harboring syntrophic bacteria and sulfate-
reducing bacteria. The bacterial clones from the thermophilic granules, how-
ever, were mainly Thermodesulfovibrio spp. (19%), green non-sulfur bacteria
(18%) and low G+C Gram-positive bacteria (18%). The authors also found that
the microbial diversity of the thermophilic granules was lower compared to the
mesophilic granules.
The population changes in granular sludge in a sucrose-fed five-compart-
ment AMBR system were monitored when staging was established [181]. Using
probes for methanogens and Archaea, the authors demonstrated that the high-
est amounts of methanogenic 16S rRNA was found in the middle compartment,
where 42% of the total 16S rRNA belonged to Archaea. Methanosaeta account-
ed for 32% of the 16S rRNA, Methanobacteriaceae for 8%, and Methanomicro-
biales for 2%. Throughout the process, Methanosaeta spp. were always the pre-
dominant aceticlastic methanogens. Even though acetate concentrations in the
side compartments were as high as 600 mg/L, Methanosarcina spp. always
amounted to less than 1%. With respect to hydrogenotrophic methanogens,
Methanobacteriaceae occurred in highest amounts followed by Methanomicro-
biales. Methanococcaceae were almost absent. Interestingly, syntrophic bacteria
such as Syntrophomonas and Syntrophobacter, and sulfate-reducing bacteria
Desulfobulbus occurred in similar amounts in the different compartments even
after the staging was established.
Microbial diversity of syntrophic bacteria in granular sludge in UASB reac-
tors was studied by Hofman-Bang et al. [182]. Microbial enrichment was con-
ducted in a UASB reactor at 32°C fed with butyrate and propionate. After three
months of stable operation, the granular sludge was divided between three new
UASB reactors fed with butyrate and propionate. One reactor was kept as a con-
trol reactor, a second reactor was fed 10 mM sulfate, and in a third reactor the
temperature was increased by 10°C. After three months of operation, the granu-
lar sludge from each reactor was again divided into two new UASB reactors fed
with either butyrate or propionate and operated for six weeks. Community fin-
gerprinting (DGGE) from the six reactors showed a low number of bands indi-
cating a bacterial enrichment consisting of 3–5 different species. Bacterial clone
libraries were constructed from each reactor and 15–30 clones from each library
were fully sequenced. Phylogenetic analysis indicated that bacterial clones were
85.9%–99.7% homologous to members of the Gram-negative d-Proteobacteria
and the Gram-positive Syntrophomonas cluster.
3.2.3
Structure of Granular Sludge
The microbial structure of granular sludge has been studied using fluorescence in
situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. Granules
collected from a full-size UASB reactor treating potato-processing wastewater
were shown to have a layered structure [90]. The thick outer layer of the gran-
ules was dominated by Bacteria and contained very few methanogens. Desulfob-
ulbus spp. were present in this layer. The inner layer of the granules had two
types of microcolonies arranged in concentric circles. One type only contained
methanogens and the other type consisted of Bacteria tangled with filamentous
methanogens. Two lab-scale reactors were inoculated with the granules, one was
fed propionate (methanogenic) and the other was supplied with propionate and
sulfate (sulfidogenic). After 8–12 weeks of adaptation, the granules in the
methanogenic reactor had lost the thick outer bacterial layer. Hybridization
with bacterial probes revealed two types of microcolonies yielding strong and
weak signals, respectively. Microcolonies yielding strong signals were found to
contain syntrophic propionate oxidizers and methanogens. Granules that adapt-
ed in the sulfidogenic reactor had established a new outer layer dominated by
Desulfobulbus.
Harmsen et al. observed three layers in granules that were originally obtained
from a system treating sugar beet wastewater and then adapted to sucrose for six
months [90]. The external layer contained mainly Bacteria. The middle layer
consisted of syntrophic microcolonies containing propionate-degraders and
Methanobrevibacter spp. (detected by the Methanobacteriaceae-specific probe
and morphologically similar to Methanobrevibacter). In this layer, transmission
electron microscopy and FISH revealed microcolonies of Methanosaeta spp.
located next to the syntrophic microcolonies. The core contained inorganic
material with large cavities and some methanogens were detected by an
Archaea-specific probe. In the same study, the authors showed that granules
from a system fed a sugar beet wastewater that were adapted to a mixture of VFA
(butyrate:propionate:acetate = 42:32:24), created similar structures as the
sucrose-adapted granules, except for an additional thick layer between the
external and the middle layer rich in Methanosaeta spp. and Methanosarcina
spp. microcolonies. The authors explained this extra layer by the high acetate
concentration in the feed. High concentrations of acetate are inhibitory to syn-
trophic propionate degradation due to the unfavorable thermodynamic condi-
tions. Methanosaeta spp. and Methanosarcina spp. present in the thick layer
could remove the acetate before it reached the syntrophic microcolonies. Since
the feed contained only butyrate, propionate, and acetate, the authors concluded
that bacteria in the external layer were mainly butyrate degraders.
Granules from a mesophilic and a thermophilic lab-scale UASB reactor were
analyzed by Sekiguchi et al. [183]. Granules from both reactors had an outer lay-
er dominated by Bacteria and an inner layer dominated by Archaea and an
unstable center. Methanosaeta spp. were the predominant Archaea in both types
of granules. In granules from the mesophilic UASB reactor, some Methanobac-
teriaceae cells were observed together with Bacteria. Some Methanomicrobiales
cells were also detected that were spread in the granules. In granules from the
thermophilic reactor, some Methanobacteriaceae and Methanosarcina cells
were detected. The authors also tried to locate Bacteria that were dominating in
the clone libraries from their previously mentioned study [180]. In mesophilic
granules, Desulfobulbus cells were found in the outer layer of the granules. Cells
closely related to Syntrophobacter were shown to form microcolonies together
with Methanobacteriaceae cells in the mesophilic granules. A probe targeting
green non-sulfur bacteria revealed filamentous cells on the surface of the ther-
mophilic granules.
3.2.4
The Granulation Process
The potentials for Methanosaeta concilii and propionate-degrading syntrophic

consortia to serve as nuclei for granulation were tested by monitoring the gran-
ulation processes from non-granular sludge in two laboratory-scale upflow
anaerobic sludge blanket reactors treating synthetic wastewater [184]. The influ-
ent of one reactor contained glucose as the only energy sources, while the influ-
ent of the other reactor contained glucose and propionate. The two reactors were
started following the recommended procedures and the effluent acetate concen-
trations were maintained below 200 mg/L. Quantitative membrane hybridiza-
tions and FISH were used to monitor the changes of microbial communities and
to investigate the cell aggregate structures during the granulation processes.
Methanosaeta concilii demonstrated good settling ability and a large population
developed in the microbial community. The increase in population size was cor-
related with the significant increase in cell aggregate sizes at the early stage of
granulation. Methanosaeta concilii cells were found to serve as backbones in the
small cell aggregates having other archaeal and bacterial cells attached to them.
They remained dominant in larger cell aggregates and in the mature granules.
These findings support the hypothesis that Methanosaeta serves as nuclei for
granulation. Syntrophic propionate-oxidizing bacteria, on the other hand,
exhibited poor settling capability and were easily washed out from the system.
Their contribution to granulation, therefore, is probably minimal.
3.3
Continuously Stirred Tank Reactors (CSTR)
3.3.1
Microbial Composition in CSTRs
Zheng and Raskin reevaluated the probes available for detection of Methano-
saeta species in mesophilic and thermophilic anaerobic digesters [178]. Gen-
erally Methanosaeta species are detected with the S-F-Msae-0825-a-A-23 probe.
However, some of the Methanosaeta spp. 16S rRNA sequences have a deletion
in the target site of this probe at position 838 (based on E. coli numbering). To
circumvent the problems with this probe, new probes targeting the genus
Methanosaeta according to Table 1 were constructed and tested on anaerobic
digesters. Also, specific probes for Methanosaeta concilii and Methanosaeta

thermophila were constructed. Three probes targeting the genus Methanosaeta
were tested against reference strains with or without mismatches. The probes S-
G-Msae-0733-a-A-22 and S-G-Msae-0781-a-A-22 were not able to discriminate
between target and non-target sites since the optimal wash temperatures were
too close to each other. A third probe, S-G-Msae-0332-a-A-22 was designed and
shown to be specific to the genus Methanosaeta.
To evaluate the probes, environmental samples from various mesophilic and
thermophilic digesters were analyzed for the presence of Methanosaeta spp.
In general, the two new probes detected a higher level of Methanosaeta than
the old S-F-Msae-0825-a-A-23 probe. This could indicate putative deletions
in target sites of some Methanosaeta species. Moreover, the amount of Me-
thanosaeta spp. in the digesters decreased when acetate concentrations in-
creased, and increased when acetate concentrations decreased. Methanosarcina
increased with increasing acetate concentrations which is in agreement with
previous findings[185].
Hansen et al. [186] quantified the syntrophic fatty acid-b-oxidizing bacteria
in a mesophilic biogas digester treating cow and swine manure. Syntrophic fat-
ty acid b-oxidizing bacteria were found in the Gram-positive Syntrophomon-
adaceae cluster. This family currently contains three genera, Syntrophomonas,
Syntrophospora, and Thermosyntropha as well as two lost strains FSM2 and FSS7
[187–190]. Probing of samples from the digester showed that members of
Methanomicrobiales were the most abundant hydrogenotrophic methanogens
constituting 10% of the prokaryotes. Syntrophomonadaceae were estimated to
comprise 0.2–1% and probing for the different syntrophic genera showed that
only members of the genus Syntrophomonas degrading butyrate were present.
Methanosarcina spp. were the only methanogens present apart from the
Methanomicrobiales. This is consistent with previous studies demonstrating
that Methanosarcina spp. are the main acetate utilizers in Danish biogas plants
[191]. The relatively low numbers of butyrate degrading bacteria imply that
these have high metabolic rates corresponding to the low energy yield of VFA
oxidation.
Raskin et al. [192] quantified the abundance of sulfate reducing bacteria
(SRB) and methanogens in twenty-one single-phase and two-phase full-scale
anaerobic sewage sludge digesters by oligonucleotide probing of 16S rRNA. It
was determined that methanogens in well-functioning mesophilic, single-phase
digesters accounted for 8–12% of which Methanosarcinales and Methanomicro-
biales constituted the majority. Methanobacteriales and Methanococcales only
played a minor role.
The SRB were present in significant amounts. Desulfovibrio and Desulfobul-
bus spp. dominated the community while Desulfobacter and Desulfobacterium
were less abundant. Desulfosarcina, Desulfococcus and Desulfobotulus spp. were
not detected. Even though sulfate was present in small amounts in the sludge, the
relative high level of SRB found in most of the digesters indicates that SRB still
can compete with the methanogens for available electrons and/or with proton-
reducing syntrophs for fermentation products such as propionate, butyrate, lac-
tate, or ethanol [192].
Hristova et al. [194] constructed genus- and subgenus-specific probes for

Gram-positive SRB. Thermophilic anaerobic digesters were probed to evaluate
the probes and to quantify the abundance of Desulfotomaculum spp. Desulfo-
tomaculum spp. accounted for about 2% of the total bacterial population at the
start up of a thermophilic CSTR digester inoculated with mesophilic digester
sludge and cow manure. After 31 days of operation the amount had decreased to
0.3%. Probing against Desulfovibrio, however, indicated that this genus account-
ed for 1.7%. This may be explained by the Desulfovibrio being more competitive
then Desulfotomaculum under the low sulfate concentrations found in the
reactor.
Probing another thermophilic digester operated for a longer period showed
Desulfotomaculum at a stable level of 2%. However, the sulfate concentration
was two times higher than in the start-up reactor, and the long operation time
may have stabilized the digester allowing Desulfotomaculum to gain foothold.
Godon et al. [195] have determined the microbial diversity in an anaerobic
digester treating wine distillation waste by extensive sequencing of clone
libraries of 16S rDNA. 579 clones were partially sequenced and analyzed by
operational taxonomic unit (OTU) phylogenetic analysis; 146 OTUs were found
of which 133 belonged to Bacteria, 6 to Archaea and 7 to Eucarya. The bacterial
clones were distributed among at least eight of the major groups of the Bacteria
domain. Despite the large bacterial diversity, the 20 most frequent bacterial
OTUs represented 50% of the total clones.
3.3.2
Microbial Dynamics in CSTRs
Ahring and coworkers investigated the archaeal population dynamics in a

digester treating cow manure during a temperature shift from 55°C to 65°C
[196]. Thermophilic anaerobic digestion of complex organic wastes is usually
carried out at 50–55°C. Previous studies demonstrated that an increase in the
operational temperature to 65°C caused a disturbance of the biogas process
reflected in an increase in VFA concentrations [191]. This indicates that the pop-
ulations participating in the terminal part of the biogas process are much more
sensitive to a temperature increase than the fermentative populations responsi-
ble for VFA production. Probing of samples from the digester showed that the
archaeal population increased significantly as a consequence of the temperature
increase and that the bacterial population decreased correspondingly. Analyses
of the archaeal population showed that the Crenarchaeota increased dramati-
cally at the cost of the Euryarchaeota after the temperature increase. Meth-
anosaetaceae were never detected, Methanosarcinaceae disappeared and Meth-
anococcus increased to a relatively high amount after the temperature change.
These findings indicate that our knowledge about the diversity in thermophilic
reactors is limited and that much more work is needed to elucidate the popula-
tion dynamics at elevated temperatures.
Fernandez et al. [67] conducted glucose perturbation studies on CSTR
digesters under mesophilic conditions to evaluate the relationship between sta-
bility and community structure. Two digesters were studied; one (HS) was inoc-
ulated with fluid from a bioreactor operating for 200 days on glucose and the
other (LS) was inoculated with fluid from a bioreactor operating for 60 days on
glucose. The effect of shock loading the digesters with glucose was monitored by
morphological analysis, 16S rRNA probing, restriction analysis of amplified
rDNA, and partial 16S rDNA sequencing. The HS reactor was characterized by
good replicability, a high proportion of spirochete-like and short thin rod mor-
photypes, a dominance of spirochete-related 16S rDNA genes, and a high per-
centage of Methanosarcina-related 16S rRNA. The LS reactor was characterized
by higher morphotype diversity dominated by cocci, a predominance of Strep-
tococcus-related and deeply branching spirochete-related 16S rDNA genes, and
a high percentage of Methanosaeta-related 16S rRNA.
In the HS reactor, the glucose shock caused a dramatic shift in the relative
abundance of fermentative bacteria, resulting in a temporary displacement of
spirochete-related ribotypes by Eubacterium-related ribotypes followed by a
return to the pre-shock community structure. The LS reactor was less affected,
and the Streptococcus-related organisms still dominated after the glucose shock,
although changes in the relative abundance of some of the members were
detected by morphotype analysis.
4
Concluding Remarks
During the last decade, environmental microbiology has changed markedly as a
consequence of the exploitation of molecular biology methods for answering
questions such as which organisms are active and where and when do they show
activity. The number of isolated microbes has increased markedly and based
upon molecular analyses, researchers are able to improve experimental designs
and to isolate new bacteria. Also, as our knowledge of microbial metabolism is
increasing, we are coming closer to be able to answer questions related to what
microbes are doing in situ.
Molecular microbiology has increased our field of vision and our resolution
capability. Now, traces of DNA from one gene are often sufficient to assign the
bacterium from which the DNA originated in a phylogenetic context. Whole
genomes are being sequenced using the same effort needed for sequencing of a
single gene a few decades ago. Sequence information can now be used to link
metabolism, phylogeny, and ecology. Sequence databases are growing exponen-
tially in size and bioinformatics, therefore, will inevitably play an increasing role
in microbial ecology. From an evolutionary point of view, massive amounts of
DNA sequences and powerful computers have made it possible to conduct phy-
logenetic analysis on these data and to broaden our view of how life on Earth
evolved. In the foreseeable future, genome sequence analysis will to a higher
degree link 16S rRNA/23S rRNA gene sequences to metabolic functions of whole
bacterial families.
To a large degree, the different techniques developed for microbial diversity
screening discussed in this chapter answer the questions of who is present.
To a limited extent, we are able answer questions on who is where since
homogeneous cell accessibility and probe specificity still are two key problems
in in situ probing. Furthermore, we still need to develop methods to deter-

mine activity levels in situ (i.e., who is doing what in which location at
which point in time?). For instance, interactions and interdependence among
different microbial populations are probably much more pronounced than
generally assumed. The metabolic expression pattern of a single microbial
cell probably differs from the neighboring cell yielding a multi dimensional
patchwork. Several major constraints, therefore, have to be overcome, and
several new concepts have to be adapted and resolved to fully exploit the use
of molecular methods in understanding complex ecosystems such as anaerobic
reactors.
5
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Received: March 2002

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Preface

In November 1776, Alessandro Volta performed his classic experiment disturb-

Lyngby, January 2003 Birgitte K. Ahring

Perspectives for Anaerobic Digestion

Keywords. Anaerobic digestion, Carbon-flow, Microbiology, Antimization, Gas yild, Effluent

2 Microbiology of Anaerobic Digestion . . . . . . . . . . . . . . . . 3

3 Anaerobic Digestion Plants . . . . . . . . . . . . . . . . . . . . . 12

4 Anaerobic Digestion as a Way to Add Extra Value . . . . . . . . . 14

5 Optimization of Anaerobic Digestion . . . . . . . . . . . . . . . . 15

Advances in Biochemical Engineering/

6 Optimization of Effluent Quality . . . . . . . . . . . . . . . . . . 23

A major value of anaerobic digestion is linked to the production of biogas

Fig. 1. The anaerobic degradation process

Fig. 2. Carbon flow in anaerobic environments with active methanogens

Fig. 3. Carbon flow in anaerobic environments without active methanogens

2. The obligate hydrogen-producing acetogenic bacteria convert propionate

Fig. 4. Interspecies hydrogen transfer

Syntrophic relationships have also been found to be of importance for conver-

Fig. 5. Modified anaerobic degradation process with syntrophic acetate conversion

Fig. 6. Start-up using thermophilic digested seed material

Fig. 7. Unbalanced growth of methanogens (dots) and fermentative bacteria (rods)

possessing an optimum growth rate at thermophilic temperatures. Probing of

methanogens, the first reaction is often an increase in the concentration of VFA

Fig. 9. Addition of 5 % fish oil will double the biogas production

Fig. 10. Simultaneous production of bioethanol and biogas

Several methods have been discovered to increase the digestibility of manure or

Fig. 11. Major goals for anaerobic digestion of today

A handful of wastewater plants in the world have further implemented the

The AD process can be conducted in a single-step or multi-step process [47].

Fig. 12. Different reactor configurations for anaerobic digestion of waste

Fig. 13. Operation of today’s biogas plant

Fig. 14. Future operation Operator

Improving bioprocesses by implementation of microbial populations with

show that better performance can be obtained when lipid-containing waste is

pathogens. The anaerobic environment seems to have an additional effect,

bic digestion including the possibility to remove a resistant virus by pretreat-

Agricultural waste can contain persistent organic contaminants such as pes-

Acknowledgement. I would like to thank Zuzana Mladenovska, Hinnerk Hartmann, Thomas

27. Uemura S, Tseng I-C, Harada H (1995) Environ Technol 16:987

Received: March 2002

Metabolic Interactions Between Methanogenic

Keywords. Competition, Sulfate reduction, Denitrification, Acetogenesis, Inhibition

2 Metabolic Interactions in Methanogenic Bioreactors . . . . . . . . 32

Advances in Biochemical Engineering/

3.4.4 Competition for Organic Acids and Ethanol . . . . . . . . . . . . . 46

Competition between two or more populations of microorganisms is a negative

The competitive interactions among anaerobic microorganisms can be roughly

Table 1. The respiration hierarchy

Acceptor Product E0¢ (V)

Oxygen O2 Water H2O +0.82

In contrast to aerobic conditions where most heterotrophic microorganisms

In natural environments, the substrate concentration for most organisms is

Several compounds, which serve as electron donors to respiring bacteria, might

From an immediate evaluation of redox potentials of methanogenesis and nitrate

pure culture of an aceticlastic methanogen (Methanosarcina mazei) and mixed

In environments where sulfate is present, sulfate-reducing bacteria will compete

Table 2. Acetogenic and methanogenic reactions, and sulfate-reducing reactions involved in

Syntrophic Acetogenic reactions

biomass concentration, Michaelis-Menten kinetics may be used to predict which

Table 3. Selected growth kinetic data of hydrogenotrophic sulfate-reducing bacteria and

Microorganism Ks µmax Yield a Km Vmax

Microorganism Ks µmax Yield a Km Vmax

The aceticlastic sulfate-reducers that prefer freshwater conditions, such as