Christian Andersson Thesis-Succinic PDF

DOC TOR A L T H E S I S
ISSN: 1402-1544 ISBN 978-91-86233-17-4

Lule University of Technology 2009
Christian Andersson Biobased Production of Succinic Acid by Escherichia coli Fermentation
Department of Chemical Engineering and Geosciences

Division of Chemical Engineering
Biobased Production of Succinic Acid

by Escherichia coli Fermentation
Christian Andersson
Lule University of Technology
Biobased Production of Succinic Acid

by Escherichia coli Fermentation
Christian Andersson
Division of Chemical Engineering

Department of Chemical Engineering and Geosciences
Lule University of Technology
SE-971 87 Lule
Sweden
February 2009
Tryck: Universitetstryckeriet, Lule

ISSN: 1402-1544
ISBN 978-91-86233-17-4
Lule
www.ltu.se
Abstract
The prospects of peak oil, climate change and the dependency of fossil carbon have urged
research and development of production methods for the manufacture of fuels and chemicals
from renewable resources (biomass). The present thesis illustrates different aspects of
biobased succinic acid production by a metabolically engineered E. coli strain. The main
areas of the thesis are sugar utilisation and feedstock flexibility, and fermentation inhibition,
both due to toxic compound derived from the raw material and the fermentation products
themselves.
The first part of this thesis aimed to investigate the fermentation characteristics of AFP184 in
a medium consisting of corn steep liquor, inorganic salts and different sugar sources without
supplementation with high-cost nutrients such as yeast extract and peptone. The effects of
different sugars, sucrose, glucose, fructose, xylose, equal mixtures of glucose-fructose and
glucose-xylose, on succinic acid production kinetics and yields in an industrially relevant
medium were investigated. AFP184 was able to utilise all sugars and sugar combinations
except sucrose for biomass generation and succinate production. Using glucose resulted in
the highest yield, 0.83 (g succinic acid per g sugar consumed anaerobically). Using a high
initial sugar concentration resulted in volumetric productivities of almost 3 g L-1 h-1, which is
above estimated values for economically feasible production. However, succinic acid
production ceased at final concentrations greater than 40 g L-1.
To further increase succinic acid concentrations, fermentations using NH4OH, NaOH, KOH,
K2CO3, and Na2CO3 as neutralising agents were performed and compared. It was shown that
substantial improvements could be made by using alkali bases to neutralise the
fermentations. The highest concentrations and productivities were achieved when Na2CO3
was used, 77 g L-1 and 3 g L-1 h-1 respectively. A gradual decrease in succinate productivity was
observed during the fermentations, which was shown to be due to succinate accumulation in
the broth and not as a result of the addition of neutralising agent or the subsequent increase
in osmolarity.
To maintain high succinate productivity by keeping a low extracellular succinic acid
concentration fermentations were interrupted and cells recovered and resuspended in fresh
media. By removing the succinate it was possible to maintain high succinic acid productivity
for a prolonged time. Cells subjected to high concentrations of succinate were also able to
regain high productivity once transferred into a succinate-free medium.
In the last part of the thesis succinic acid production from softwood dilute acid hydrolysates
was demonstrated. This study involved establishing the degree of detoxification necessary for
growth and fermentation using industrial hydrolysates. Detoxification by treatment with lime
and/or activated carbon was investigated and the results show that it was possible to produce
succinate from softwood hydrolysates in yields comparable to those for synthetic sugars.
The work done in this thesis increases the understanding of succinic acid production with
AFP184, illustrate its limitations, and suggests improvements in the current technology with
the long term aim of increasing the economical feasibility of biochemical succinic acid
production.
Keywords: Succinic acid, Escherichia coli, Flexibility, Productivity, Inhibition, Softwood
ii
Acknowledgements
First of all I would like to thank my supervisor Professor Kris A. Berglund. Thank you for
including me in your team. Your positive nature and endless array of ideas are a constant
source of inspiration.
I would like to extend my deepest gratitude to my assistant supervisor Dr. Ulrika Rova. Your
hard work and undying attention to detail are the primary reasons for the realisation of this
thesis. Thank you for all the work you have put in.
I am also grateful to my colleagues Josefine Enman, Magnus Sjblom, Jonas Helmerius, and
David Hodge. Working with you has been a privilege.
My master thesis workers over the years, Andreas Lennartsson, Ksenia Bolotova, Mario
Winkler, and Ekaterina Petrova are all acknowledged. The efforts and work you put in has
been of great assistance to me.
I also thank all my students. Teaching you has been a journey I would not trade for anything.
I just hope that you have been able to learn as much from me as I have from you.
To all my friends and colleges at the Department of Chemical Engineering and Geosciences,
thank you for all the good times.
Till min bste vn Ivan Kaic, min bror Fredrik Andersson, samt mina frldrar Margaretha
och Jarl Andersson, ni finns alltid dr och ert std har fr mig varit ovrdeligt.
Finally, my beautiful fianc, Antonina Lobanova, thank you for all your love and patience. I
will never forget the support and encouragement you have given me.
Perstorp 2009,
Christian Andersson
iii
iv
List of Papers
This thesis is based on the work contained in the following papers, referred to in the text by
Roman numerals.
Effects of Different Carbon Sources on the Production of Succinic Acid

Using Metabolically Engineered Escherichia coli
Christian Andersson, David Hodge, Kris A. Berglund, and Ulrika Rova
Biotechnology progress, 23(2), 381 -388, 2007
II
Inhibition of Succinic Acid Production in Metabolically Engineered

Escherichia coli by Neutralising Agent, Organic Acids, and Osmolarity
Christian Andersson, Jonas Helmerius, David Hodge, Kris A. Berglund, and Ulrika
Rova, Biotechnology Progress, 25(1), 116-123, 2009
III
Maintaining High Anaerobic Succinic Acid Productivity by Cell

Resuspensions
Christian Andersson, Ekaterina Petrova, Kris A. Berglund, and Ulrika Rova
Submitted to Journal of Industrial Microbiology and Biotechnology
IV
Detoxification Requirements for Bioconverision of Softwood Dilute

Acid Hydrolyzates to Succinic Acid
David Hodge, Christian Andersson, Kris A. Berglund, and Ulrika Rova
Enzyme and Microbial Technology, in press (online December 11, 2008)
Other Publications by the Author
Process for Producing Succinic Acid from Sucrose

Christian Andersson, Ulrika Rova, and Kris A. Berglund,
U.S. Patent Application (2007), 2007/0122892
Process for the Production of Succinic Acid
Kris A. Berglund, Christian Andersson, and Ulrika Rova,
International Patent Cooperation Treaty (2007), WO 2007/046767
vi
Contents
INTRODUCTION .................................................................................................1
SCOPE OF THE PRESENT WORK ..................................................................................................................... 2
PRODUCTION OF BIOBASED FUELS AND CHEMICALS ................................... 3

THE INTEGRATED BIOREFINERY................................................................................................................... 4
FERMENTATIVE PRODUCTION OF ORGANIC ACIDS .................................................................................... 7
BIOBASED SUCCINIC ACID PRODUCTION ................................................................................................... 10
ESCHERICHIA COLI AS A PLATFORM FOR INDUSTRIAL PRODUCTION OF

SUCCINIC ACID ................................................................................................15
ESCHERICHIA COLI SUGAR METABOLISM.................................................................................................. 15
Glucose ..............................................................................................................................15
Fructose............................................................................................................................ 19
Xylose ............................................................................................................................... 19
Other Sugars .................................................................................................................... 20
EFFECTS OF PH AND ORGANIC ACIDS ON ESCHERICHIA COLI METABOLISM ....................................... 20
Energy Generation and pH Homeostasis ....................................................................... 20

Organic Acids Metabolic Uncoupling and Anion Accumulation ................................ 22
Acid Resistance Systems.................................................................................................. 24
Membrane Transport Systems........................................................................................ 26
Membrane Transport during Organic Acid Production ................................................ 27
OSMOTIC STRESS IN ESCHERICHIA COLI ................................................................................................... 30
Osmotic Pressure, Osmolarity, and Turgor pressure.....................................................30

The Role of Compatible Solutes in the Response of Escherichia coli to Osmotic Stress..31
INDUSTRIAL RAW MATERIALS ..................................................................................................................... 34
Pretreatment and Hydrolysis.......................................................................................... 34

Detoxification................................................................................................................... 36
RESULTS AND DISCUSSION............................................................................ 39
SUGAR UTILISATION (PAPER I AND IV) ...................................................................................................... 40
INHIBITION OF SUCCINATE PRODUCTION (PAPER II) ............................................................................. 43
EFFECTS OF REMOVING SUCCINIC ACID (PAPER III) ............................................................................... 47
INDUSTRIAL RAW MATERIALS AND DETOXIFICATION (PAPER IV) ........................................................ 51
CONCLUSIONS ................................................................................................ 55
FUTURE WORK ................................................................................................57
REFERENCES .................................................................................................. 59
PAPERS I IV
vii
viii
Introduction
The prospects of peak oil, climate change and the dependency of fossil carbon have
moticvated research and development of production methods for the manufacture of fuels
and chemicals from renewable resources (biomass). The main focus so far has been
replacement of oil for transportation fuels, but the utilisation of petroleum for production of
chemicals and materials could represent up to 15% of the petroleum usage [1].
White biotechnology, also called industrial biotechnology, is a fast evolving technology with
the potential to substantially impact the industrial production of fuels and chemicals.
Conventional, non-biological processes can be replaced by biochemical conversion of
biomass resulting in reduction of greenhouse gas emissions and energy usage for the
production of fuels and chemicals. By definition, the technology uses the properties of living
organisms, such as yeast, moulds, bacteria, and plants, to make products from renewable
resources. When utilising living systems the reactions involved generally occurs under mild
conditions
with
high
product
specificity,
hence
reducing
the
formation
of
undesirable/harmful by-products. One well-known example of white biotechnology is glucose

fermentation by the yeast Saccharomyces cerevisiae for the production of fuel ethanol.
In order for biobased production of chemicals to be cost-competitive with fossil based
alternatives it is essential to develop fermentations that produce building blocks, i.e.
molecules that can be converted into a number of high-value chemicals or materials.
Examples of building block or platform molecules are organic acids like lactic and acetic acid.
Of special interest are molecules containing multiple functional groups, e.g. dicarboxylic
acids. The presence of more than one functional group offers a greater range of possible
reaction paths and thus an extended product portfolio. One such example is succinic acid,
which is considered as one of twelve top chemical building blocks manufactured from
biomass [2]. In addition to its own use as a food ingredient and chemical, succinic acid can be
used to derive a wide range of products including: diesel fuel oxygenates for particulate
emission reduction; biodegradable, glycol free, low corrosion deicing chemicals for airport
runways; glycol free engine coolants; polybutylene succinate (PBS), a biodegradable polymer
that can replace polyethylene and polypropylene; and non-toxic, environmentally safe
solvents that can replace chlorinated and other VOC emitting solvents [3]. Therefore
biobased production of succinic acid offers the opportunity for a widespread replacement of
fossil fuel based chemicals.
Introduction
Scope of the Present Work

The present work addresses different aspects of succinic acid production by fermentation
using metabolically engineered Escherichia coli. The objective was to further the
understanding of the process and bring large-scale biobased production of succinic acid
closer to realisation. In the current work, the fermentative capability of an E. coli mutant,
AFP184, towards selected sugars was studied. The metabolic products from the
fermentations were quantified and productivity and yield on each sugar was determined. The
influence of osmotic stress and concentration of the main end-product, succinic acid, on
productivity and cell viability was also established. Finally, fermentation of softwood dilute
acid hydrolysates was demonstrated and the degree of detoxification necessary to generate a
fermentable hydrolysate is determined.
Production of Biobased Fuels and Chemicals

Changing the raw material for transportation fuels and everyday chemicals from nonrenewable to renewable requires, apart from research in production technologies,
sustainability of the raw material supply. The yearly biomass production must be shared
between many interests, including human food, forage crops, and raw materials for the
industry. The annual global production of plant biomass has been estimated to roughly
1701012 kg, out of which approximately 75% are carbohydrates, 20% lignin, and 5% lipids,
proteins, and other compounds [1]. Currently biomass as a production resource is
underutilised, but increasing interest in biomass for production of fuels and chemicals will
raise the demand on available biomass [4]. For this purpose it is important to certify that the
supply of biomass is enough to sustain the present biomass consumers as well as the growing
biobased fuel and chemical sectors. In the U.S. an estimated annual potential of 1.31012 kg
biomass is available for conversion without interfering with current biomass uses [5], and
hence offers a large potential for expanding the present production of biobased fuels and
chemicals. By converting the carbohydrate fraction of these 130 billion tons to chemicals with
an overall process yield of 0.5 kg chemicals per kg carbohydrate processed generates a net 50
billion tons worth of chemicals. Although current prices for biomass of interest for industrial
conversion to chemicals (e.g. agricultural residues and forest biomass) are lower than the
price of crude oil [4, 6], biologically produced fuels and chemicals have problems competing
with their petroleum based counterparts. Industrial use of renewable feedstocks suffers from
low raw material usage as only part of the biomass is converted into products. This is mainly
due to lack of process or plant integration; the biocatalyst used can only achieve high yields
on a selected part of the substrate while the rest is left unconverted as the production sites
utilising the other parts of the feedstock often are not located within close proximity to each
other. To increase raw material utilisation investments in new plants must be made.
Fermentation products are also produced in dilute water solutions and the cost of
downstream processing is high. Many types of biomass, especially lignocellulosics, are
inherently resistant to processing and together with poor raw material utilisation and high
capital costs it translates into high production costs and hence reduced competitiveness.
The situation in the petrochemical industry is the reverse. Even if the feedstock (crude oil) is
traded at a higher price than agricultural residues or forest materials, the processing costs are
generally lower due to well implemented and mature processes and technologies. Utilisation
of the raw material is very high and different processes refining various parts of the crude oil
into high-value products are linked together in a petrochemical refinery [4]. The

petrochemical industry converts a set of initial building-blocks derived from crude oil in wide
spectrum of products, literally thousands, and if the biotechnical process industries are to be
competitive they need to develop in the same direction. The solution at present is an
integrated production facility analogous to the petrochemical refinery called a biorefinery [7].
The Integrated Biorefinery

The main concept of an integrated biorefinery is efficient conversion of all feedstock
components into value-added products by combinations of thermochemical, biochemical,
chemical and physical processes (Fig. 1). By process integration, the use of all feedstock
components will be maximised including the use of by-products and waste streams. Today
there exists a number of biorefineries or potential biorefineries such as corn dry and wet
milling plants and pulp and paper mills [4, 7].
Grains
Agricultural residues
Forest biomass
Municipal solid waste
Feedstocks
Biochemical
Thermochemical
Processing
Physical
Chemical
Products
Fuels
Chemicals
Materials
Foods
Figure 1. Principal sketch of the biorefinery concept.

Biorefineries are further classified as phase I, II or III biorefineries with phase III being the
most sophisticated and versatile. A phase I biorefinery is typically limited to one type of
feedstock (e.g. grain), producing one main product and one or two by-products/wastes,

hence having very little operational flexibility. Phase II biorefineries also use one type of
feedstock, but can tailor production to fit current demands. Wet-milling plants are an
example of a phase II biorefinery producing fermentations products, starch, corn syrup, corn
oil, gluten, and meal. The phase III biorefineries are able to process varying feedstocks into a
great number of products offering high flexibility of processing and adaptability to both
product demand and raw material supply. Biorefineries typically use regionally available
biomass; in the US biorefineries are constructed around corn wet, and dry mills and in Brazil
around sugar cane processing plants. In Sweden the main source of biomass is the forest, in
particular softwood, and it is suitable to develop biorefineries based on a lignocellulosic
feedstock, the LCF biorefinery [7]. The LCF biorefinery is a phase III biorefinery with the
potential to convert lignocellulosic biomass into energy, cellulose/hemicellulose derivatives,
microbial fermentation products, lignin, and extractives. Plant biomass offers almost endless
processing alternatives and product compositions. Carbohydrates in biomass, like cellulose,
can be fermented to biofuels or chemicals. Lignin can be used as a natural binder, adhesive,
in the production of carbon fibres or as a solid fuel. Plant biomass also contain proteins, fats,
dyes, vitamins, flavouring agents, minerals and extractives and biorefinery concepts can
except for supplying the chemical industry, generate products for food, feed, personal
hygiene, and medicine.
An example of an LCF biorefinery is hydrolysis and fermentation of softwood for production
of succinic acid and ethanol (Fig. 2). The monomeric sugars derived from the softwood
hydrolysate are distributed to different bioreactors. Part of the sugar is fermented to ethanol
by S. cerevisiae and part is fermented to succinic acid by E. coli AFP184 (used in this work).
The sugar not utilised by the yeast is recycled to the E. coli reactor. Spent yeast cells can be
lysed and used as an in-house source of nitrogen and vitamins. The CO2 produced during
ethanol fermentation is fed to the E. coli bioreactor since AFP184 utilise CO2 in producing
succinate. Benefits from combining these two fermentations will be seen in a higher sugar
utilisation and recovery of the carbon lost as CO2 in a conventional ethanol plant. The ethanol
is distilled and used as transportation fuel, or reacted with succinate to form
diethylsuccinate. The fatty acids and oils contained in the biomass can be utilised for
biodiesel production. The succinate and diethylsuccinate make up useful building blocks for
the chemical industry.
Bacterial
Fermentation
Hydrolysis
Separation
Residual sugar
Sugar
Succinic Acid
Diethyl Succinate
CO2
Energy
Biomass
Yeast
Fermentation
Separation
Ethanol
Vegetable Oils
Chemical
Reactor
Separation
Biodiesel
Glycerol
Figure 2. An LCF biorefinery based on ethanol and succinic acid fermentation.

Biorefineries could also be constructed around currently existing industry. A pulp and paper
mill could for example be modified into a lignocellulosic biorefinery, benefiting from already
having the infrastructure and labour force to handle and process forest biomass [8]. In Kraft
pulping wood chips are boiled together with sodium hydroxide and sodium sulphide in large
digesters. The aim is to separate lignin and hemicellulose from the cellulose fibres without
degrading the fibres. After separating the fibres from the spent pulping liquor (black liquor)
the fibres are processed into paper pulp and the liquor is concentrated by multi-effect
evaporation and processed to recover the cooking chemicals. Converting a Kraft mill into an
integrated biorefinery involves recovering the hemicellulose and lignin fractions and turning
them into high-value products. The hemicellulose is most easily attained by extracting it
before the pulping process using acid, alkali or hot water as solvent [8, 9]. Lignin can be
recovered from the black liquor either by precipitation by acidification or ultrafiltration [10].
An example of an LCF biorefinery based on a Kraft pulp mill with the hemicellulose extracted
prior to pulping [8] and the lignin precipitated before the chemical recovery processes is
shown in Fig. 3. The mill in Fig. 3 has also been equipped with a gasifier, in which the black
liquor after evaporation is gasified, generating green liquor containing the pulping chemicals
and hot synthesis gas [11]. The produced synthesis gas is cooled in a gas cooler generating low
and medium pressure steam. The syngas can after cooling be further processed into
chemicals or liquid fuels. The concept presented in Fig. 3 aims to use the extracted
hemicellulose for chemicals production by fermentation and to produce pulp from the

cellulose. Converting the biomass into high-value products and materials provides a great
opportunity for a Kraft pulp mill to expand its business and increase its revenue [8, 12].
Evaporation
Alkaline
solution
Water Syngas
Wood
chips
Black liquor
Extractor
Steam
Extracted
hemicellulose
Gas
cooler
Acidifier
Digester
Fermentation
Pulping
chemicals
Lignin
precipitation
MP Steam
Gasifier
Black
liquor
Lignin
Pulp
Cooling water
Chemicals
Recovered
pulping
chemicals
Chemicals
recovery
Green liquor
Figure 3. An LCF biorefinery based on a Kraft pulp mill.
Fermentative Production of Organic Acids

Organic acids make up a highly important group of building-block chemicals that can be
produced from renewable resources by microbial fermentation. They occur as end-products
and/or intermediates in metabolic pathways of yeast, filamentous fungi, and bacteria (Fig. 4).
Presently large-scale production facilities for biomass derived organic acids exist for citric,
acetic, and lactic acid [13]. Citric acid has by far the largest production volumes with more
than 1.6 million tonnes being produced per year. It is mainly used as an acidulant in food
products. The primary organisms used are the fungi Aspergillus niger, which produce final
citrate titres of close to 200 g L-1, and the yeast Yarrowia liplytica reported to generate citric
acid concentrations of 140 g L-1 [14, 15]. General production conditions include high substrate
loads, low manganese and high dissolved oxygen concentration, constant agitation, and low
pH [14].

Glucose
Glycolysis
Pyruvate
Lactate
CO2
Acetate
Reductive
Arm
Oxaloacetate
Malate
TCA Cycle
Citrate
Oxidative
Arm
Fumarate
Succinate
Figure 4. Organic acids as intermediates/end-products of microbial carbohydrate

metabolism.
Lactic acid is a three-carbon monocarboxylic acid. It has been used by humans for a long
time, primarily by fermentation of foodstuff as a means of preservation. Recently the interest
in optically pure lactic acid for production of polylactic acid (PLA), a biodegradable plastic,
has stimulated industrial-scale production of lactic acid. The most widely used biocatalysts
are different species of lactic acid bacteria, but also fungi like Rhizopus oryzae are known to
be good lactic acid producers [16]. Other bacteria such as E. coli have been engineered for
homofermentative lactic acid production and have in mineral salt media been shown to
produce lactic acid in excess of 100 g L-1 at volumetric productivities of > 2 g L-1 h-1 and yields
around 95% of the theoretical [17]. Other acids with potentially large markets include malic,
fumaric, and succinic acid [13].
Malic and fumaric acid are both four-carbon dicarboxylic acids which precede succinic acid in
the reductive arm of the tricarboxylic acid cycle (TCA cycle or Krebs cycle, Fig. 4). These TCA
intermediates can accumulate in filamentous fungi species when cultivated under nitrogen
limited conditions; which arrest cell growth and hence carbon can be redirected towards the
reductive arm of the TCA cycle [14]. Presence of carbon dioxide is also necessary since the
pathways utilise CO2 fixation during conversion of glucose to malic and fumaric acid. Both

acids are polymer precursors currently produced from maleic anhydride. Apart from
polymers, malic acid is also used in beverages, candy, and food, in metal cleaning, textile
finishing, pharmaceuticals, and paints [14]. The best malic acid producing organism is
Aspergillus flavus which in CaCO3 neutralised fermentations can accumulate extracellular
malic acid to concentrations of 113 g L-1 at glucose yields of 0.94 g g-1 [18]. However, the
volumetric productivity is very low, 0.59 g L-1 h-1. E. coli is a well-known organism that has
been successfully engineered for organic acid production, benefiting from good
productivities, easy handling, and the possibility of using low-cost media [19, 20]. Attempts
have been made to engineer E. coli for malic acid production [21]. Obtained volumetric
productivities were in the range of 0.75 g L-1 h-1 and further work is needed to develop strong
malic acid producing mutants.
Fumaric acid was produced by fermentation during the 1940s using Rhizopus arrhizus [14],
but production was abandoned with the development of more efficient petrochemical routes.
Production of fumaric acid with Rhizopus strains is done under aerobic conditions and good
oxygen transfer is therefore necessary. However, Rhizopus species often grow on both the
reactor impellers and walls resulting in reduced transfer rates. Insufficient oxygenation in R.
oryzae leads to ethanol being produced instead of fumarate yield [22]. In batch
fermentations it is necessary to control the pH by addition of a neutralising agent. The best
results have been obtained when CaCO3 was used [23]. Except maintaining the fermentation
pH utilisation of CaCO3 serves two purposes. It binds to fumarate creating calcium fumarate.
The low aqueous soluibility of calcium fumarate leads it to precipitate during fermentation,
hence lowering the extracellular organic acid concentration [24]. Keeping the extracellular
acid concentration low benefits transport of produced organic acids out from the cells and
reduce the effects of product inhibition. The carbonate in CaCO3 also serves as a source of
CO2 for the enzyme pyruvate carboxylase that is active in the carboxylation of pyruvate to
oxaloacetate (Fig. 4) [25]. Oxaloacetate is then further reduced to malate and finally fumarate
in the reductive arm of the TCA cycle. Using CaCO3 in glucose batch fermentations with R.
arrhizus has produced fumaric acid in concentrations around 100 g L-1 with mass yields and
productivities of 0.7-0.8 and 1-2 g L-1 h-1 respectively [26, 27]. The downside of using CaCO3
is that it increases the viscosity of the broth. The cells can also interact with the calcium
precipitates further increasing viscosity, lowering oxygen transfer rates, and complicating cell
recycling. Another disadvantage of the use of CaCO3 the expensive and tedious downstream
processing, which involves heating and acidification with sulphuric acid and the connected
generation of large amounts of gypsum [24, 27]. In order to improve productivity
fermentation should be combined with simultaneous product recovery. This was done with R.
oryzae grown on rotating discs partly submerged in a fermentation medium [22]. The head

space of the rotary biofilm reactor was filled with air and as the discs revolved the cells were
subjected both to high oxygen transfer in the air and to carbon dioxide in the medium. The
produced fumarate was continuously removed and recovered by passing the fermentation
broth over ion-exchange/adsorption columns. Since the cells were grown on the rotating
plastic discs they remained in the reactor when the broth was circulated over the adsorption
columns. The study demonstrated a yield of 0.85 g fumarate per g glucose and a volumetric
productivity of 4.25 g L-1 h-1. The productivity could be maintained for close to two weeks. An
issue with the rotary biofilm reactor that might limit its usefulness in industrial installations
is its scale-up potential. Fumaric acid fermentations with Rhizopus strains have chiefly been
demonstrated on glucose and although fumaric acid production by R. arrhizzus from xylose
has been shown, productivities were low [28]. More research on conversion of other sugars
than glucose, and industrial raw materials, such as lignocellulosic hydrolysates should be
performed. For this means and in effort to increase yields and productivities metabolic
engineering of either Rhizopus species or bacteria that already grows on and ferments five
carbon sugars well constitute a largely untapped possibility [23].
Biobased Succinic Acid Production

Succinic acid, a dicarboxylic acid with the molecular formula C4H6O4, was first discovered in
1546 by Georgius Agricola during dry distillation of amber. Currently, succinic acid is
manufactured from petrochemical resources through oxidation of n-butane or benzene
followed hydrolysis and finally dehydrogenation. Succinic acid is currently as a surfactant,
detergent extender, foaming agent, in the food industry for pH reduction, as a flavour and
antimicrobial agent, and in the manufacture of health products such as vitamins and
pharmaceuticals [3]. Succinic acid can also be used to synthesise other chemical starting
blocks e.g. tetrahydrofuran and butanediol. Both being large-volume chemicals currently
used as solvents and for polymer production [13]. Potential new markets for biobased
production of succinic acid are expected to come from the synthesis of biodegradable
polymers; polybutylene succinate (PBS) and polyamides, and various green solvents [2].
Since succinic acid is a common intermediate in the energy metabolism it can also be
produced by microbial conversion of biomass utilising fungal or bacterial fermentation [29].
Different conversion pathways for succinic acid applications from fermentation are outlined
in Fig. 5.
10

New applications
Cleaners
Food and feed
additives markets
Deicing chemicals
Runway
Commercial,
residential, industrial
Fermentation
Succinic Salts
Separations
Acidulants
Food and beverage
Agicultural
chemicals
Herbicides
Polymerisation
Succinic Acid
Esterification
Diesel fuel additives
Solvents/Surfactants
Processing
Cleaning
Paint industry
Cosmetics
Dyes/pigments
Chelating agents
Detergent additives
Water treatment
Corrosion
inhibitors
Thermoset resins
Dehydration
Succinate Esters
Succinic Anhydride
Condensation
Specialty Chemicals
Itaconic Acid
Polymers
Figure 5. Value-added products that can be derived from succinic acid.

Biochemical production of succinic acid has been demonstrated using a number of organisms
including Bacteroides ruminicola and Bacteroides amylophilus, Anaerobiospirillum
succiniciproducens,
Actinobacillus
succinogenes,
Mannheimia
succiniciproducens,
Corynebacterium glutamicum, and Escherichia coli [30-36]. One of the most thoroughly
investigated organisms is A. succiniciproducens. It is a strict anaerobe and has been
characterised in a number of studies both with regards to preferred medium composition
[37-41] and processing conditions [42, 43]. Achievements with A. succiniciproducens include
conversion of hardwood hydrolysates into succinate with a mass yield of 0.88 gram succinate
per gram glucose [44] and recently continuous production in an intergrated membrane
bioreactor-electrodialysis system with an impressive volumetric productivity around 10 g L-1
h-1 [45]. The main drawback with the organism is that it does not seem to tolerate succinic
acid concentrations in excess of 30-35 g L-1.
A facultative anaerobe, M. succiniciproducens, isolated from bovine rumen has been shown
to produce succinate as its main fermentation product with yields in the order of 0.7 gram
succinate per gram sugar consumed [32] and with succinate concentration of approximately
50 g L-1 [46]. The volumetric productivities demonstrated with M. succiniciproducens has
been high, up to 3.9 g L-1 h-1 [47], but final titres and yields should still be improved. The
11

organism has been also been demonstrated to ferment industrially derived glucose and xylose
from hardwood hydrolysates [48].
A. succinogenes is also a facultative anaerobe inhabiting the bovine rumen. The organism has
been shown to produce succinic acid in impressive concentrations (105.8 g L-1) with good
yields (approximately 0.85 gram per gram glucose) [49-51]. The fermentations that have
reached succinate concentrations of more than 80 g L-1 have been neutralised with MgCO3.
When using NH4OH or sodium alkali final concentrations were in the range of 60 g L-1, which
is lower than concentrations achieved by a number of E. coli strains [19, 52, 53]. The
fermentations were conducted in either vial flasks or 1 L reactors and the results have not
been repeated in pilot or production-scale bioreactors.
Under anaerobic conditions E. coli is known to produce a mixture of organic acids and
ethanol [54]. Typical yields from such fermentations are 0.8 moles ethanol, 1.2 moles formic
acid, 0.1-0.2 moles lactic acid, and 0.3-0.4 moles succinic acid per mole glucose consumed. If
the objective is to produce succinic acid, the yield of succinic acid relative the other acids
must be increased. In the 1990s USDOE initiated the Alternative Feedstock Program (AFP)
with the aim to develop a number of metabolically engineered E. coli strains with increased
succinic acid production. The two most promising mutants developed by the program were
AFP111 and AFP184 [31, 55, 56]. AFP111 is a spontaneous mutant with mutations in the
glucose specific phosphotransferase system (ptsG), the pyruvate formate lyase system (pfl)
and in the fermentative lactate dehydrogenase system (ldh) [31]. The mutations resulted in
increased succinic acid yields (1 mole succinic acid per mole glucose) [56]. AFP184 is a
metabolically engineered strain where the three mutations described above were deliberately
inserted into the E. coli strain C600 (ATCC 23724), which can ferment both five and six
carbon sugars and possess strong growth characteristics [55]. AFP184 is has been used
throughout the current work. Other interesting succinic acid producing E. coli biocatalysts
include strains engineered to make succinate during aerobic metabolism [34], AFP111
overexpressing heterologous pyruvate carboxylase [33, 52], and strains developed from
combinations of metabolic engineering and natural selection able to produce high succinic
acid titres in mineral media [19, 57].
Technologies for biological production of succinic acid from renewable resources have seen
massive improvements in the past five to ten years. Nevertheless, further improvement is still
needed if the biochemical conversion methods should be cost-competitive in a long term
perspective. Regardless of the chosen biocatalyst the manufacturing cost of succinic acid is
affected by productivity and yield, raw material costs and utilisation, and recovery methods.
12

From a downstreaming perspective, succinic acid concentrations should be high and the
quantity of other fermentation products (carboxylic acids and ethanol) low. High levels of byproducts reduce the succinate yield and increase the cost of separation. The presence of
colouring substances, often from added complex nutrients, and other impurities will also
interfere with product separation, requiring additional purification steps, for example
treatment with activated carbon and filtration. Therefore a potential biobased succinic acid
production process should promote both efficient fermentation and product recovery. To this
end a biocatalyst able to generate high succinate titres without by-product formation should
be used. The organism should also utilise a wide range of sugar feedstocks in a medium
supplemented minimum amounts of complex nutrients and to be economically feasible
preferably achieve volumetric productivity above 2.5 g L-1 h-1 [2].
13
14
Escherichia coli as a Platform for Industrial Production of

Succinic Acid
Escherichia coli Sugar Metabolism

In E. coli sugars are metabolised in different ways depending on structural similarities
between the sugars and the properties of the enzyme systems responsible for uptake and
degradation. The present work deals with succinic acid production from monosaccharides
available in Sweden. Raw materials are likely to come from wood hydrolysates, but
agricultural residues and products e.g. wheat straw and sugar beets, are also possible sources
of carbohydrates. Focus in this thesis is on the metabolism of glucose, fructose, and xylose;
glucose and xylose being the most abundant sugars in lignocellulosic biomass, while fructose
and glucose are the components of sucrose, a widely available disaccharide. Metabolism of
other hemicellulose sugars; arabinose, galactose, and mannose, was demonstrated during
fermentation of softwood hydrolysates and will also be discussed (paper IV). In the following
sections the metabolism of each of the sugars will be described and the effects of the
mutations in AFP184 discussed.
Glucose
The first step in the glucose metabolism of E. coli is glycolysis (also called EmbdernMeyerhof- Parnas (EMP) pathway). In glycolysis one mole of glucose is taken up by the cell,
phosphorylated and converted into 2 moles of pyruvate generating small amounts of
metabolic energy. In the energy metabolism of E. coli the fate of pyruvate depends on the
environmental conditions. When oxygen is available, pyruvate molecules enter the TCA cycle
and are oxidised to CO2. Under anaerobic conditions E. coli will instead undergo mixed acid
fermentation and produce a mixture of organic acids and ethanol [54]. However, when
metabolising glucose under anaerobic conditions the mutations in the pfl and ldh genes
significantly influence the ratio of the products formed. The mutations in AFP184 direct the
metabolic fluxes so that the only end-products that accumulate in any significant amounts
are succinic acid and acetic acid. The mutations in the ldh and pfl genes limit the generation
of the by-products lactic acid, formic acid, ethanol and acetic acid, although a small amount
of pyruvic acid also accumulates. The sugar metabolism of AFP184 for glucose, fructose, and
xylose is outlined in Fig. 6.
15
Escherichia coli as a Platform for Industrial Production of Succinic Acid

Anaerobic
succinic
acid
production
by
AFP184
requires
CO2.
The
enzyme
phosphoenolpyruvate carboxylase directs the carbon flow from phosphoenolpyruvate (PEP)

to oxaloacetic acid by fixing CO2. Oxaloacetate is then reduced to malate, fumarate, and
finally succinate via the reductive arm of the TCA-cycle. From a carbon balance a yield of 2
moles of succinic acid for every mole of glucose consumed is obtained. Carrying out a redox
balance shows that ~15 % of the carbon must be committed to the glyoxylate shunt to
generate the necessary amount of reducing equivalents (NADH). The maximum theoretical
yield of succinate from one mole of glucose is thus 1.71 (1.12 gram per gram glucose). The
anaerobic activity of the glyoxylate shunt has been demonstrated for AFP111 after aerobically
inducing isocitrate lyase [33]. Since AFP184 carry the same mutations as AFP111 it is
assumed in the present study that the glyoxylate shunt also is active in AFP184.
16

Xylose
Glucose
Xylulose
PEP
ATP
Pyruvate
ADP
ATP
ADP
Glucose 6-P
PEP
Xylulose 5-P
Ribose 5-P
Glyceraldehyde 3-P
Sedoheptulose 7-P
Pyruvate
Fructose 6-P
Fructose
ATP
ADP
Glyceraldehyde 3-P
2H, ATP
CO2
PEP
ATP
Erythrose 4-P
Fructose 6-P
2H, CO2
ATP
Pyruvate
2H
Acetate
Lactate
Oxaloacetate
ADP
2H, CO2
LP
Acetyl-CoA
Glyceraldehyde 3-P
4H
2H
Malate
Glyoxylate
Ethanol
Isocitrate
Fumarate
2H
Citrate
Succinate
Figure 6. Anaerobic glucose, fructose and xylose metabolism of AFP184. Note that some
metabolites are excluded. The reactions blocked by the mutations in AFP184 are shown by
crosses.
E. coli has two hydrophobic membranes surrounding its cytoplasm, the cytoplasmic (or
inner) membrane and the outer membrane. The outer membrane contains special proteins,
porins, which forms pores across the membrane that allow glucose and other small solutes to
diffuse through the membrane into the periplasm (the volume between the inner and outer
membrane). The diffusion of glucose across the outer membrane into the cytoplasm is a
passive process depending on the concentration gradient across the membrane. The two
main porins for glucose uptake during concentrations above 0.2 mM are OmpC and OmpF
17

[58], as described under the section Osmotic stress in Escherichia coli. Normally in E. coli,
glucose is taken up and phosphorylated by the phosphotransferase system (PTS), which is a
system of transport proteins [59, 60] that internalise glucose (Fig. 7).
Periplasm
Cytoplasm
Glucose 6-P
IICDMan
OmpC
IIABMan
P
PEP
EI
HPr
Pyruvate
EI
HPr
IIBGlc
IICGlc
IIAGlc
Glucose 6-P
Glucose
Glucose
P
OmpF
Glucose 6-P
Glucokinase
Glucose + H+
Outside
cell
GalP
H+
ADP ATP
ADP ATP
Glucose 6-P
Glucokinase
Glucose
MglA
MglC
MglB
ADP ATP
Figure 7. Glucose uptake systems in E. coli.

The glucose uptake process starts with Enzyme I (EI) taking a phosphate group from PEP,
generating phosphorylated EI and pyruvate. EI is said to be non sugar-specific, meaning that
its activity is not linked to a specific sugar substrate; instead it will assist in the uptake of
many sugars for example fructose, glucose, and mannose [58]. The phosphate group is then
transported to a sugar specific enzyme II complex via the phosphohistidine carrier protein
(HPr). In E. coli there are 21 different enyme II complexes [59]. The ones responsible for
glucose uptake and phosphorylation are IIGlc and IIMan where the former is more efficient. The
IIGlc complex consists of the glucose-specific enzymes, IIAGlc and IIBCGlc . IIAGlc is a soluble
enzyme and IIBCGlc is an integral membrane protein permease.
The gene, ptsG, encoding the glucose-specific permease IIBCGlc normally represses the genes
encoding the enzymes in the IIMan complex. AFP184 was metabolically engineered to have the
glucose-specific permease knocked out, resulting in an organism where the genes for the IIMan
complex is no longer repressed and glucose uptake can commence through the complexes
IIABMan and IICDMan. The affinity and capacity of the mannose permease system is somewhat
lower for glucose than the glucose specific uptake system and lower growth rates has been
reported in mutants relying on the IIMan complex [61].
18

Two other systems that are important for glucose internalisation in E. coli, which lacks a
functional glucose-specific permease, are the GalP and Mgl systems [58]. GalP is a
galactose:H+ symport that has the ability to transfer both galactose and glucose into the
cytoplasm. The Mgl system consists of a galactose/glucose periplasmic binding protein
(MglB), an integral membrane transporter protein (MglC), and an ATP-binding protein
(MglA). Glucose imported via GalP or Mgl is not phosphorylated. The phosphorylation
necessary before entering glycolysis is performed by the cytoplasmic enzyme glucokinase.
Instead of using PEP as a phosphate donor, this phosphorylation is ATP dependent. Uptake
and phosphorylation by glucokinase has been reported as slower than when conducted by the
PTS [62].
Fructose
The fructose metabolism of AFP184 (Fig. 6) is similar to glucose, but with the main difference
that the PTS for fructose is intact and fructose is thus internalised and phosphorylated by the
fructose specific PTS and not by the mannose PTS or by glucokinase [63-65]. A redox balance
for succinate production by fructose metabolism gives a maximum theoretical molar yield of
1.20 (mass yield 0.79). The lower succinate yield is due to more PEP being committed to
pyruvate during uptake and phosphorylation of fructose reducing the availability of PEP for
conversion to oxaloacetate.
Xylose
Xylose metabolism differs from the two other sugars since xylose is not a PTS substrate.
Uptake of xylose is instead governed by chemiosmotic effects [66, 67]. An initial increase of
external pH has been reported as xylose is consumed by E. coli [67]. This increase was
interpreted as an influx of protons (or efflux of hydroxyl groups) accompanying a
protonmotive force driven transport of xylose into the cells. Once inside the cell, xylose is
isomerised by xylose isomerase to xylulose which is phosphorylated by xylulose kinase to
xylulose 5-phosphate [66, 68]. Xylulose 5-phosphate then enters the pentose phosphate
pathway and after conversion to either fructose 6-phosphate or glyceraldehyde 3-phosphate
it enters the glycolytic pathway (Fig. 6). The maximum theoretical yield of succinate from
xylose calculated by a redox balance is 1.43 moles per mole xylose (mass yield 1.12).
19
Other Sugars
Arabinose, galactose, and mannose are all sugars present in softwood hemicellulose and
hence interesting for industrial production of succinic acid from lignocellulosic biomass. Due
to the mutation in the PTS of AFP184 the metabolism of these sugars are not repressed by the
presence of glucose (Paper III). Mannose, a PTS sugar, is metabolised in manner similar to
fructose since the PTS for mannose is not subjected to any mutations. Galactose enters the
cell via either the GalP-proton symport or the Mgl system, it is phosphorylated by
galactokinase and through a series of reactions it enters glycolysis as glucose 1-phosphate
[69, 70]. Arabinose catabolism starts by conversion to D-ribulose, which subsequently is
phosphorylated by the enzyme L-fuculokinase [71]. Arabinose enters glycolysis after further
conversion as hydroxyacetone phosphaste (in equilibrium with glyceraldehyde 3-phosphate).
Effects of pH and Organic Acids on Escherichia coli Metabolism

Organic acids and acid stress affect microbial cells by influencing energy generation,
intracellular pH homeostasis, and the integrity of cellular proteins and DNA [72]. For the
present aim of producing large quantities of organic acid by fermentation to be successful,
the impact of organic acids on cell physiology, functionality, and viability needs to be
considered.
Energy Generation and pH Homeostasis

The chemiosmotic theory outlines that a proton concentration gradient across the
cytoplasmic membrane is generated and used as a pool of energy for ATP synthesis [73]. An
essential component for generating the proton gradient is the relative impermeability of the
cytoplasmic membrane to protons. Protons are channeled into the cell by active transport via
the membrane bound enzyme ATP synthase. This enzyme concomitantly catalyses the
phosphorylation of ADP from the energy obtained by the movement of protons along the
proton gradient.
The energy stored in the proton concentration gradient is called the protonmotive force
(PMF) and is formed by two contributors; the difference in proton concentration (pH) and
the difference in charge, called membrane potential (), between the cytoplasm and
surrounding medium. The PMF is written as the sum of the membrane potential and the pH
gradient and is given in V or mV.
20

PMF = ZpH
(1)
where Z, which converts the proton concentration difference to electric potential, is given by:
Z = ln 10
RT
F
(2)
where F is Faradays constant and R the ideal gas constant and T is the absolute temperature.
The PMF in E. coli actively undergoing cell division, is in the range of -140 to -200 mV [74].
If pH increases the membrane potential is reduced to keep a stable PMF. A very large PMF
would create a massive pull on extracellular protons and result in increased influx and
acidification of the cytoplasm.
There are two main factors that affect the pH homeostasis system in E. coli; the pH of the
medium and the concentration of organic acids produced during fermentation. In the advent
of a reduction of the extracellular pH, bacteria respond by changing the activity of the ion
transport systems responsible for bringing protons into the cytoplasm [75]. E. coli is a
neutrophile and thus grows best at near neutral pH. The cytoplasm usually has a pH
somewhat above that of the surrounding medium. For growth of E. coli in a near neutral
medium the internal pH is in the range of 7.4 to 7.9 [76]. When E. coli is subjected to extreme
acid stress the pH gradient across the membrane increases and creates a strong influx force
for protons. The cytoplasmic membrane of E. coli has very low proton permeability, but at
large pH the net influx of protons increases nevertheless. It is hypothesised that protons
travel across the membranes by using protein channels or damaged parts of the lipid bilayers
[74]. It has been shown that E. coli cannot maintain a pH of more than 2 pH units [74]
resulting in acidification of the cytoplasm. E. coli has been demonstrated to survive acid
stress down to pH 2-3 for several hours [77]. However, as the cytoplasmic pH decrease due to
improper enzyme function, protein denaturation and damage on the purine bases of DNA,
the low pH environment will eventually kill the cells (for a description of how E. coli survive
acid stress see Acid Resistance Systems).
The second source of cytoplasmic pH perturbations, weak acids, works differently. Organic
acids, like acetate, lactate, and succinate are all produced during anaerobic fermentation of
various carbon sources e.g. glucose. Those acids have pKa values in the range of 4 and in a
cultivation medium at pH 7, which is a common cultivation pH for E. coli, the acids dissociate
and lower the pH. In an industrial fermentation the pH is kept constant at optimal levels by
addition of acid and base. If the pH of the medium is lowered to values below the pKa of the
21

organic acids, the acids remain undissociated and hence uncharged. An uncharged organic
acid can travel across the cell membrane by passive diffusion [75]. Once inside the slightly
alkaline cytoplasm the acid dissociates increasing the proton concentration inside the cell,
lowering the cytoplasmic pH and dissipating the trans-membrane proton gradient and the
protonmotive force [78]. Studying the effects on growth of different organic acids derived
from hemicellulose hydrolysates in cultures of the ethanologenic E. coli LY01 it was shown
that the acids reduced growth more at lower pH than at neutral or slightly alkaline [79]. The
effects of a lower pH and the presence of organic acids are thus more severe when present
together.
Organic Acids Metabolic Uncoupling and Anion Accumulation

The effect of organic acids on growth is not only due to the lowering of the cytoplasmic pH,
but also includes anion specific effects. The growth inhibition caused by acetate anions is
proposed to be linked to methionine biosynthesis [80]. It was suggested that the inhibition
caused by acetate is an effect of the anion affecting an enzyme in the methionine biosynthesis
pathway leading to accumulation of homocysteine, a metabolite that has been shown to
inhibit growth in E. coli.
As explained above, the protons generated by dissociation of organic acids in the cytoplasm
affects cells both by reducing the cytoplasmic pH thus damaging protein structures and DNA,
and by reducing pH thus disrupting the protonmotive force. The reduction of pH can be
mediated by what is called an uncoupler. Synthetic uncouplers are lipophilic compounds that
travel across cell membranes in both undissociated as well as in dissociated form (Fig. 8).
The protonated form of the species crosses the membrane and releases its proton upon entry
into the alkaline cytoplasm. The deprotonated form is then pushed to the external side of the
membrane by the electrical gradient. The anion is again protonated, diffuses into the
cytoplasm and releases another proton. Protons however are not able to cross the membranes
and remain in the cytoplasm. The cycle continues until the protonmotive force is completely
disrupted [81].
22

Inside cell
Outside cell
HX
HX
H
-
Figure 8. Mechanism of uncoupling. HX represents the undissociated acid and X- the acid
anion.
Whether organic acids are able to act as true uncouplers is a matter of debate. It has been
argued against organic acids being true uncouplers due to their inability to diffuse through
the cell membranes, instead they will accumulate in the cytoplasm like protons [81]. This was
contradicted in a study proposing that both acetate and lactate permeates through the
membranes at comparable rates in both undissociated and dissociated form and in this way
they catalytically dissipate the protonmotive force [82]. This model also purports that since
acetate could freely traverse the cell membrane it would not accumulate in the cytoplasm. In
the study it was observed that the intracellular acetate concentrations did not reach levels
predicted by pH. In contrast, another investigation showed that acetate accumulation
increased with increasing external acetate concentration and an equilibrium between the
intracellular acetate and pH occurs when the external acetate concentrations was 60 mM or
more [83]. It was found that acetate accumulated as long as the intracellular pH and thus the
pH gradient was kept high [83, 84]. However, as the intracellular pH decreased acetate
accumulation ceased. The effects of organic acids can thus be two-fold. Even though it cannot
be said that organic acids act as uncouplers per se, the growth inhibiting effects seems to
originate both from acidification of the cytoplasm and of anion accumulation. In E. coli K-12
even low (<50 mM) extracellular acetate concentrations will result in decreased levels of
intracellular ATP [84]. The effect can be attributed to the ATP requirements for the reversible
activity of ATP synthases to pump protons out of the cytoplasm against the proton gradient in
order to keep a slightly alkaline intracellular pH. Acidification of the cytoplasm and the
following reduction in intracellular ATP levels as an effect of proton extrusion is a typically
observed in the presence of an uncoupler [84].
23
Acid Resistance Systems

At low extracellular pH E. coli have a number of ways to control the influx of protons and
thus maintain the cytoplasmic pH. The pH homeostasis of E. coli can be divided into a
passive and an active homeostatic system [85]. The main contributions to the passive system
are the relative impermeability of the lipid bilayer to protons and ions, and the cytoplasmic
buffering capacity. The buffering system uses phosphate groups and carboxylated metabolic
substances to obtain a buffering capacity in the range of 50-100 nmol H+ per pH unit and mg
cell protein [75]. The cell membranes are an essential part of the passive pH homeostasis. E.
coli exposed to acidic conditions have been shown to increase the amount of cyclopropane
fatty acids in the cytoplasmic membrane. The higher concentration of cyclopropane fatty
acids is supposed to increase the survival rate of the cells [86, 87].
The active homeostasic system controls the flow of ions (mainly sodium, potassium, and
protons) across the cell membranes. It has been demonstrated that the role for Na+/H+
antiporters is central in the sodium regulation, but not for cytoplasmic pH control [88, 89].
Potassium uptake on the other hand has a strong correlation with the regulation and
maintenance of an alkaline cytoplasmic pH. It has been demonstrated that E. coli cells
suspended in potassium-free medium quickly reduce their intracellular pH. After
introduction of potassium in the medium, alkalinity of the cytoplasmic pH was restored [90,
91].
The uptake and accumulation of K+ is an important part of pH homeostasis at close to neutral
pH, under extreme acid stress E. coli also use other systems to ensure survival. Today four
well characterised acid resistance systems (AR, Fig. 9) are known [74, 92-95]. AR1 is
activated when cells are grown aerobically in Luria Bertani medium at pH 5.5. This system is
dependent on the alternative sigma factor (rpoS, part of the RNA polymerase holoenzyme
necessary for DNA transcription), and the cAMP receptor protein [92, 96]. The AR1 system is
repressed by glucose. The system provides acid resistance down to pH 2.5 during the
stationary growth phase [94].
24

H+
GABA
rpoS (AR1)
GABA
Glutamate
H+
Glutamate
Glutamate decarboxylase (AR2)
Agmatine
Agmatine
Arginine
H+
Arginine
Arginine decarboxylase (AR3)
H+
Cadaverine
Cadaverine
Lysine
H+
Lysine
Lysine decarboxylase (AR4)
Figure 9. Acid resistance system in E. coli.

Two other stationary phase acid resistance systems were discovered when E. coli was
cultivated in glucose containing media [97]. These systems depend on extracellular glutamate
(for AR2) and arginine (for AR3) [97-100]. Both systems confer acid resistance down to pH 2
and comprise an amino acid decarboxylase and an antiporter. The decarboxylases (GadA or
GadB for glutamate and AdiA for arginine) substitute a carboxyl group on the amino acid
with a proton from the cytoplasm. The reaction products are the decarboxylated amino acid;
-amino butyric acid (GABA) from glutamate and agmatine from arginine and CO2. GABA or
agmatine is excreted from the cells through their corresponding antiport (GadC for glutamate
and AdiC for arginine) and new amino acids are taken up. AR2 is induced in aerobically
grown cultures in the presence of glucose and extracellular glutamate. AR3 also requires
glucose, but differs from AR2 in that it is activated under anaerobic cultivation. Both systems
generate internal pH values in close proximity with the pH optima of the decarboxylases (pH
4 for GadA and GadB and pH 5 for AdiA). If the pH increases above the optimum for the
respective carboxylases, activity will decrease and pH will drop. A fourth acid resistance
system (AR4) was recently discovered [99]. It is similar to AR2 and AR3, but is dependent on
lysine and its efficiency is low. The low efficiency could be explained by a higher pH optimum
for lysine decarboxylase enzyme activity. During extreme acid stress conditions, the
extracellular pH initially lowers the intracellular pH too much and as a consequence the
decarboxylase looses activity and is not able to meet the proton charge and raise the internal
pH [74].
25

When E. coli are grown at near neutral pH, is maintained at roughly -90 mV. In the
stationary phase the membrane potential decreases to approximately -50 mV. Transferring
the culture to acidic pH (pH < 3) reduces to 0. The effect is expected to be caused by a
loss of membrane integrity. Without a functioning membrane it is no longer possible to
generate and maintain ion gradients and extracellular and cytoplasmic concentrations of ions
are evened out, hence reducing the membrane potential. In an investigation of AR2 and AR3
it was found that if glutamate or arginine was present in the medium, E. coli revert its
membrane potential in the same way as some acidophilic organisms do. With glutamate a
membrane potential of +30 mV was achieved and with arginine +80 mV [101]. The proposed
mechanism of protection would be that the positive membrane potential should repel protons
and thus relieve some of the acid stress.
Finally E. coli possess systems for the protection of vital protein. In order to function
properly, cells need to maintain the activity of proteins, including those linked to cellular
membranes and the cell wall. HdeAB, a heterodimer expressed in the periplasm, has been
shown to dissociate into monomers at acid pH. The dissociated subunits bind to unfolded
proteins in the periplasm and prevents unwanted protein aggregation [102]. It is not known
what happens to the proteins once the acid stress is relieved, but mutants lacking hdeAB
genes showed a dramatic loss of viability at acid pH [103]. The protection of periplasmic
proteins thus seems crucial for cell survival at low pH.
Membrane Transport Systems

Organic acid producing organisms utilise different excretion systems in order to transport the
acids from the interior of the cell to its surroundings. All types of transport in and out of the
cell are connected to the properties of the cell membranes and the type of molecule being
exported. The nature of the lipid bilayer membranes allows hydrophobic as well as small
uncharged molecules like water to diffuse across it. The rate of transfer is then controlled by
the difference in concentration of the species in question over the membrane and the
diffusivity of each species in the lipid membrane core. Charged or larger polar molecules are
transported across the membrane using transport proteins; classified as pores, passive
transporters, and active transporters. Active transporters are further classified as primary or
secondary [104].
Pores are transmembrane proteins that resemble a filter by allowing molecules of the right
size and charge to move down a concentration gradient. The process does not require energy
and is similar to diffusion across the membrane in that it is not highly selective and the rate
26

of transfer is limited by the rate of diffusion. Examples of pores in the outer membrane of E.
coli are OmpC and OmpF for glucose uptake; they belong to a special group of pore proteins
called porins. Passive transport proteins, like pores, channel molecules down a concentration
gradient and hence do not require any input of energy. A distinguishing difference is that
passive transporters bind to specific molecules for which they facilitate the transfer across the
cell membrane. The process of passive transport is also known as facilitated diffusion. Active
transporters on the other hand require energy since the transfer takes place against a
concentration gradient. During primary active transport the energy is supplied directly, often
in the form of ATP [105]. Examples of primary transporters are membrane-bound ATPases
and the ATP-Binding-Cassette (ABC) family of transport proteins [106]. The energy can also
be provided indirectly by an ion gradient (often protons or sodium) in which case it is called
secondary active transport [105]. The mechanism of transfer for secondary active transport
and passive transport is similar and can be classified into three categories: uniports
transporting only one solute, symports transporting two solutes simultaneously in the same
direction, and antiports which also transports two solutes, but in opposite directions (Fig.
10).
HA
Outside cell
Cytoplasm
ATP
ADP
Primary transport
Uniport
Symport
Antiport
Figure 10. Transport of protons and acid anions by primary active transport and secondary
active and passive transport through a uniport, symport, and antiport.
Membrane Transport during Organic Acid Production

The driving force for organic acid export from the cell varies with respect to not only the
concentration gradient of the acid, but also with the membrane potential, pH, extracellular
pH, and ATP availability (for primary active transport) [106]. Excreting the produced acids is
a necessity both for maintaining a stable internal pH, a cytoplasmic osmotic environment in
which cellular functions are not compromised, and to avoid end-product inhibition. In E. coli
the intracellular pH is kept constant approximately around 7.5 (see pH Homeostasis and
27

Energy Generation). At this pH organic acids produced in the cellular metabolism will be
deprotonated and hence have very low solubility in the cell membrane, which limits export by
simple diffusion. Specific transport proteins are then necessary to keep transfer rates high
[106]. The system used is dependent on thermodynamic requirements.
For citric acid produced by Aspergillus niger at an extracellular pH of 3, thermodynamic
modelling indicate that passive transport would be sufficient for transporting citrate out of
the cell up to extracellular concentrations above 190 g L-1 assuming an intracellular pH of 7.6
[107]. At an extracellular pH of 3 the excreted citrate will take up protons changing the
charge of the molecule and hence not significantly reduce the concentration gradient of the
anionic species being excreted. The diffusion of citrate from the cytoplasm would thus only be
marginally affected. An issue producing organic acids by fermentation at low pH is that the
acids in the fermentation broth mainly occur in their undissociated form. Undissociated acids
can diffuse back across the cell membrane lowering the cytoplasmic pH, accumulate in the
cytoplasm affecting metabolism or upon excretion back to the broth dissipate the PMF (see
Organic Acids Metabolic Uncoupling and Anion Accumulation).
At an extracellular pH above the pKa of the acid, passive transport can only generate acid
concentrations in the fermentation broth up to a concentration in equilibrium with the
cytoplasmic acid concentration. At higher concentrations the driving force for acid export will
be too low. In order for the cell to still produce and excrete acid active transport must be
utilised. The driving force during primary and secondary active transport of organic acids out
of a cell is the sum of the contributions of the difference in membrane potential, pH, and acid
concentration across the membrane. If ATP is hydrolysed during the translocation process,
the free energy of the reaction also contributed to the total driving force [106]. For secondary
active transport uniports and symports are interesting for industrial organic acid production
from sugar. A substrate/product antiport would only be of interest in e.g. malolactic
fermentation. Thermodynamic driving forces for some selected export mechanisms are
summarised in Table 1 [106]. The calculated driving force must be negative for the transfer
to occur.
Depending on the how the acids are exported the process can either require energy or help
produce metabolic energy by generating an electrochemical proton gradient [105].
Electrogenic export of the produced acid (export of a surplus of charge, e.g. undissociated
acid and an extra proton) will contribute to the generation of electrochemical gradients
between the cytoplasm and the fermentation broth. Homofermentative lactic acid producing
organisms generate one mole of ATP per mole lactate produced from substrate-level
28

phosphorylation [106]. Utilising an ATP dependent export system would thus reduce the
metabolic energy available for maintenance and cell growth to zero. It has been shown that
S. cerevisiae engineered for lactic acid production do not generate enough ATP to sustain
anaerobic growth [108]. The study points to the energy expenditure connected with export of
the produced lactic acid as the reason for the halted growth. When designing biocatalysts for
industrial production of organic acids the export systems available to the organism and not
only the metabolic route to the desired acid should be taken into account.
Table 1. Thermodynamic driving forces for selected export mechanisms
Transport Type
Transport
Transported
Protein
Speciesa
Primary active
ATPase
H+
Primary active
ABC
HA
Sec. active/passive
Uniport
HA
Sec. active/passive
Uniport
A-
Sec. active/passive
Symport
A- and nH+
an
Driving Forceb
n + nZpH +
G ATP
F
[HA]Cyt G ATP
+
[HA]Out
F
[HA]Cyt
Z log
[HA]Out
[HA]Cyt
Z log
+
[HA]Out
[HA]Cyt
Z log
(n 1) + nZpH
[HA]Out
Z log
is the number of protons exported. b GATP is the free energy of ATP hydrolysis (J mol-1), [HA] is the
concentration of undissociated acid in the cytoplasm (Cyt) and broth (Out), is the membrane
potential (V)
29
Osmotic Stress in Escherichia coli
Osmotic Pressure, Osmolarity, and Turgor pressure

In the same way as the lipid membranes of E. coli are vital to pH homeostasis they also
restrict the transport of other ionic compounds, thus generating concentration gradients
between the cytoplasm and the medium.
The phenomenon of osmotic pressure is well described by a vessel divided into two
compartments by a semipermeable membrane. The membrane restricts the transport of
solutes between the two compartments, but permits the flow of water. If compartment 1 is
filled with pure water and compartment 2 with a solution having a certain concentration of
for example an ionic compound the water activity (or concentration of water) in
compartment 2 is lower than in compartment 1. As a result, water will diffuse from
compartment 1 across the semipermeable membrane into compartment 2 and raise the liquid
level. Water will continue to diffuse into compartment 2, lowering the liquid level in
compartment 1 and further raising it in compartment 2 creating a hydrostatic pressure
difference. The influx of water to compartment 2 will continue until the hydrostatic pressure
on compartment 2 can balance the tendency of water to diffuse into the compartment. The
equilibrium pressure achieved is termed the osmotic pressure.
The osmotic pressure is directly proportional to the osmolarity (OsM) of the solution.
Osmolarity in turn is measured as osmoles per litre solvent, where an osmole is the number
of moles of a substance that contributes to the osmotic pressure of a solution. E. coli has been
shown to be able to grow in osmolarities ranging from 0.015 OsM to approximately 1.9 OsM
(in minimal media) or 3.0 OsM (in rich media) [109-111]. An E. coli cell can also be described
in terms of the two compartment model. The interior of the cell responds to increasing or
decreasing concentration in the medium by adjusting the water or solute content in the
cytoplasm. In most cases the solute concentration of the cytoplasm is higher than that of the
surrounding medium and water has a tendency to diffuse into the cell, a state where the cells
are said to be in positive water balance. The hydrostatic pressure difference between the
cytoplasm and the cells environment is called turgor pressure.
The turgor pressure has a major impact on the functionality of the cell. Proteins, enzymes
and other macromolecules have a range of water activity and ionic concentrations in which
they retain proper function. In media that force osmolarities of the cytoplasm outside of this
30

range E. coli cells could lose important cellular functions and mechanisms [112]. When cells
are subjected to hyper- or hypoosmotic shock they will respond by fast influx or efflux of
cytoplasmic water. Hypoosmotic shock means that bacteria are subjected to very low
osmolarities. The result is an influx of water and simultaneous swelling of the cell. Gramnegative cell walls can withstand pressures of up to 100 atm and hypoosmotic treatment has
limited effect on cells [113]. Hyperosmotic shock is the opposite. Cells are subjected to
solutions with very high salt concentrations. This treatment causes water efflux and
shrinkage of the cytoplasmic volume. Severe hyperosmotic shock will lead to plasmolysis; the
cytoplasmic membrane being retracted from the cell wall leaving a gap between cell wall and
cytoplasm. Hyperosmotic environments will negatively affect cell functions causing
inhibition of DNA replication, decreased uptake of nutrients, reduced synthesis of
macromolecules and subsequent ATP accumulation [114-116].
The Role of Compatible Solutes in the Response of Escherichia coli to

Osmotic Stress
Cells exposed to high medium osmolarity export intracellular water resulting in a reduction
in turgor pressure and cytoplasmic volume. The concentrations of intracellular metabolites
increase with the decrease of the cytoplasmic volume. This passive adaptation to the
surrounding osmolarity is often not adequate and could result in inhibitory or toxic
concentrations of the intracellular metabolites. Instead E. coli exposed to hyper- or
hypoosmotic stress adapt by accumulating or releasing certain solutes like potassium ions,
amino acids (glutamate, proline), quaternary amines (e.g. glycine betaine), and sugars (e.g.
sucrose, trehalose). Theses solutes are called compatible solutes because they can be
accumulated to high levels through uptake from the surrounding environment or by de novo
synthesis without negatively affecting most cytoplasmic enzymes [112, 117]. Common for
most compatible solutes is that they cannot traverse the cellular membranes easily, but has to
be transported mainly by active transport mechanisms. Most compatible solutes are
uncharged, with the exception of the most important, K+ and glutamate-, and will thus
minimally interfere with cellular macromolecules. Some compatible solutes do not only help
the cell to survive osmotic stress, but will also positively affect growth rate. These are called
osmoprotectants [117].
Potassium is the major intracellular cation and is accumulated as the first response of E. coli
to osmotic upshock [118]. Potassium is taken up via the transport systems Kdp and Trk. Trk
is a constitutive low affinity, high capacity uptake system and Kdp is a high affinity, low
capacity system that is induced by low turgor pressure when Trk cannot maintain the turgor
31

pressure [119]. Closely coupled with potassium accumulation is the increased de novo
synthesis of glutamate. Glutamate synthesis is strongly dependent on K+ and if a medium
deprived of K+ is used, glutamate synthesis is reduced to approximately 10% of the synthesis
rate obtained when K+ is present [118]. A second response after initial K+ accumulation is the
displacement of K+ and glutamate by neutral compatible solutes that can accumulate to
higher concentrations without inhibiting vital cellular functions. The uptake of neutral
species may occur simultaneously with K+ uptake, but uptake via Trk is more rapid and will
be most important for the osmotolerance in the early stages of an osmotic upshock [117].
Glycine betaine and proline are accumulated following initial uptake of K+. These compatible
solutes interfere less with the cytoplasmic constituents than K+ and glutamate and during
uptake of glycine betaine and proline K+ is excreted from the cells [117]. Glycine betaine
uptake is mediated by the transport systems ProP and ProU [120]. The same systems also
transport proline, but the affinity is higher for glycine betaine [120, 121]. The ProP system is
energised by the protonmotive force while ProU uses energy derived from hydrolysis of ATP
[117]. The first system to respond to an osmotic upshock is the ProP, which is a low affinity
transport system. This transport system is activated within seconds after the cells are
subjected to a high osmolarity environment. At high osmolarities ProP is also the
physiologically more important system, since cells without this system under high osmolarity
conditions are unable to take up proline and glycine betaine from the medium. ProU, on the
other hand, is first activated after several minutes [117]. It is a periplasmic high affinity
transport system that because of its high affinity for its substrates plays an important part in
osmotic adaptation when glycine betaine and proline are present in low concentrations. Both
systems however only accumulate glycine betaine under osmotic stress [121]. Compatible
solutes play an important role in assisting osmotically stressed cells as has been
demonstrated when cells grown in a high osmolarity medium lacking compatible solutes lost
their colony-forming activity, regaining it first after glycine betaine had been added [122].
Most bacteria are unable to synthesise glycine betaine de novo and to use glycine betaine as
an osmoprotectants it must be present in the media [112]. Even though glycine betaine
cannot be synthesised by E. coli from glucose it has been shown that choline is converted to
glycine betaine under osmotic stress [123]. The conversion of choline to glycine betaine is
dependent on O2 or some other terminal electron acceptor. Choline can therefore only be
used as an osmoprotectant during aerobic conditions [123].
Like glycine betaine, proline cannot be synthesised in E. coli and other gram-negative
bacteria. High intracellular concentrations of proline during osmotic stress can only be
32

obtained by an increased uptake. Ectoine, a cyclic amino acid identified as a compatible
solute in halophiles, was shown to improve E. coli growth in high osmolarity media [124]. It
is not metabolised in E. coli and has similar osmoprotective properties as glycine betaine.
Both the ProP and ProU systems are responsible for ecotine uptake, where ProP is the main
uptake system [124].
Trehalose is accumulated when other compatible solutes are absent from the cultivation
medium. It is only accumulated via synthesis since uptake of the sugar from the medium will
result in the phosphorylated form of the sugar, which is not a compatible solute and thus
does little to relieve the bacterium during osmotic stress [125]. Trehalose can accumulate to
intracellular concentrations equal to approximately 20% of the osmolar concentration of
solutes in the cultivation medium [112].
A last part of the osmoregulation of E. coli is the outer membrane porins. For osmotic
regulation the two most important porins are OmpF and OmpC. Their production is
regulated by a number of environmental conditions such as osmolarity, carbons source and
temperature. The total cellular concentration of the porins is more or less constant, but the
ratio between them varies. High osmolarity and temperature increases the levels of OmpC
and concomitantly reduce the levels of OmpF and vice versa. The pore size of OmpF is larger
than of OmpC, which is an explanation to why OmpC is preferentially synthesised in high
salinity media where the nutrients are plentiful and the temperature high; i.e. conditions
which makes diffusion into the cell easy. OmpF on the other hand is needed when nutrients
are scarce and it is important for the bacterium to scavenge the medium for nutrients [112].
Finally it should be noted that the rate of the hyperosmotic shock also is important for
viability and growth. Results from studies with osmotic upshock have shown that if cells are
subjected to moderate osmotic stress their growth at higher osmolarities is not equally
impaired. Osmotolerance can thus be induced by treatment prior to osmotic shock [126].
Furthermore the survival of E. coli in increased osmolarity media also depends on time. A
sudden osmolarity increase by NaCl reduced the viability of the cultures more than if the
increase is conducted during 20 minutes [127]. The results imply that cells are able to
respond to the increase in osmolarity if the increase is slow.
33
Industrial Raw Materials
Pretreatment and Hydrolysis

Implementing fermentative organic acid production on an industrial scale requires the use of
inexpensive, abundant carbohydrates instead of highly purified sugars. The most widely
available feedstock for renewable carbohydrates is lignocellulosic biomass. The composition
of lignocellulosic biomass varies with its origin, but the basic components are the same;
cellulose, hemicellulose, and lignin. Cellulose is a straight, highly crystalline glucose polymer,
which in lignocellulose is hydrogen-bonded with the branched hemicellulose, a
heteropolymer of different monomeric sugars (xylose, mannose, arabinose, and galactose),
glucuronic acid, and acetylated side-chains [128]. The network is further strengthened by
lignin, a polymer of various phenolic compounds. Before being utilised in fermentation
processes the cellulose and hemicellulose must be hydrolysed to fermentable sugars followed
by detoxification to remove antibacterial and inhibitory compounds created during
hydrolysis.
Biomass is hydrolysed using either concentrated acid and moderate temperature, dilute acid
and high temperature or enzymes [129, 130]. The use of enzymatic hydrolysis is usually
preceded by another pretreatment step, such as steam explosion with our without an acid
catalyst, liquid hot water extraction or an organosolv process [130]. The objective is to open
up the physical structure of the biomass making it available for the enzymes by liberating the
hemicellulose and lignin from the cellulose. Acid hydrolysis can be conducted with a number
of mineral acids including sulphuric, hydrochloric, phosphoric, and nitric acid. Hydrolysis of
cellulose using organic acids has also been demonstrated [131, 132]. Indeed acetate released
from hemicellulose can cause autohydrolysis of cellulose and hemicellulose at elevated
temperatures [8]. In concentrated acid hydrolysis acid concentrations in the range of 40-50
wt-% are used. Glucose yields of approximately 90% of the theoretical can be achieved, but
processing of large quantities of acid puts high demands on equipment corrosion resistance,
acid recovery processes and disposal of waste products (gypsum from sulphuric acid usage)
making the technology less attractive [129].
Dilute acid hydrolysis is carried of at 150-200 C with 0.5-5 wt-% acid [129, 133]. Utilising
dilute acid hydrolysis limit problems with acid recovery, but the high temperatures used
degrade the monomeric six-carbon sugars to 5-hydroymethylfurfural (HMF) and the fivecarbon sugars to furfural [134, 135]. Both HMF and furfural have been shown to be potent
34

inhibitors of anaerobic growth in ethanologenic E. coli with an IC50 for growth (concentration
inhibiting 50% of growth) of around 2.5-3 g L-1 [136]. The toxicity was directly related to the
hydrophobicity of the furan in question. Further degradation of HMF results in formation of
levulinic and formic acid (Fig. 11). Formic acid can also be formed from furfural under the
conditions present during dilute acid hydrolysis [137]. Hemicellulose contains acetyl groups
which upon hydrolysis are released as acetate to the hydrolysed liquor. Acetate is also an
inhibitor of growth and product formation in microorganisms [79]. Formic acid (IC50 2.5 g L1)
is more inhibitory than levulinic (IC50 7.5 g L-1) and acetic acid (IC50 9.0 g L-1) for
anaerobically grown E. coli [79]. Similar results were also demonstrated in ethanol
fermentations with S. cerevisiae [134]. During dilute acid hydrolysis of softwood a fraction of
the lignin is also degraded resulting in phenolic compounds being present in the
fermentation medium. Phenolics derived from softwood hydrolysis are highly toxic to
bacterial growth [138]. They dissolve in cell membranes affecting its integrity and causing
unselective transfer across the membrane [139]. Additive or synergistic interactions between
inhibitory compounds can also be a factor contributing to the total inhibitory effect resulting
in an inhibitory outcome that can be either equal to or greater than the sum of the individual
contributions [138]. Apart from organic compounds present in softwood hydrolysates the
concentration of inorganic ions could also affect cellular metabolism [140]. Toxic effects can
be specific for a certain ion e.g. sulphate or trace metal cations accumulated from the
lignocellulosic biomass or pretreatment equipment, or related to the total ionic strength and
osmolarity of the medium (Paper IV). In dilute acid hydrolysis only about 50% of the
theoretical glucose yield is achieved [129].
Figure 11. Degradation products from glucose.

Hemicellulose, a branched polymer, is more susceptible to hydrolysis than cellulose and
during dilute acid hydrolysis the hemicellulose sugars are liberated before the cellulose is
hydrolysed. The monomeric hemicellulose sugars are then degraded during the severe
reaction conditions necessary to obtain high cellulose hydrolysis rates. In order too minimise
35

the formation of hemicellulose degradation products a two-stage dilute acid process has been
developed [141]. In the first stage milder conditions sufficient for hemicellulose hydrolysis
are utilised and in the second stage after removal of the hemicellulose sugars harsher
conditions are used for the cellulose degradation. Yields in excess of 90% of theoretical for
monomeric hemicellulose sugars have been achieved. Glucose yields were however still in the
range of 50% [141]. In the present thesis a two-stage dilute acid hydrolysis of softwood has
been used (Fig. 12). The process is used by SEKAB in the ethanol pilot plant in rnskldsvik,
Sweden. The liquid exiting the first stage is a hydrolysate of hemicellulose, primarily
containing mannose and xylose. The solids from the first stage, consisting of mainly glucose,
are separated and hydrolysed in the second stage, resulting in a glucose rich liquor. After
hydrolysis equal volumes of the liquors from the first and second stage are combined and
evaporated to increase the sugar concentration by a factor 2 (Paper IV).
Softwood Chips
nd
st
1 Stage Acid
Hydrolysis
Solids
2 Stage
2 Stage Acid Hydrolysis Liquor (H2)
Hydrolysis
nd
1st Stage Hydrolysis Liquor (H1)
Evaporation
Combined
Hydrolysis Liquor (H3)
Figure 12. Two-stage dilute acid hydrolysis.
Detoxification
When converting lignocellulosic materials to fuels and chemicals via microbial processing,
one should consider feedstock, hydrolysis method, detoxification, microbial conversion, and
product recovery together. For a dilute sulphuric acid pretreatment process, the hydrolysis
conditions can be considered as a balance between high hemicellulose and cellulose
conversions into fermentable sugars and generation of sugar degradation products inhibitory
to microbial conversion of the sugars. High conversion to monomeric sugars often leads to
increased concentrations of degradation products; hence detoxification operations adding to
the total cost of processing are required.
Detoxification prior to fermentation is often necessary to reduce the effect or quantity of toxic
and inhibitory compounds present in lignocellulosic hydrolysates to a level where the
36

hydrolysate is amendable for bioconversion. It is desirable to keep the detoxification to a
minimum since it adds additional processing steps and thus increase the total cost of the
process. Detoxification methods can be physical, chemical or biological [142]. Physical
detoxification methods remove the inhibitor from the hydrolysate. Methods include phaseequilibrium separations such as liquid extraction, evaporation, ion-exchange, and adsorption
unto e.g. activated carbon [142-144]. Other possible unit operations that could be used are
different types of membrane separations, such as dialysis, pervaporation, reversed osmosis
and other molecular sieve membranes. No single detoxification method of the above
mentioned could remove all inhibitors. Evaporation for example would only be applicable to
volatile inhibitors, while ion-exchange and liquid extraction are limited by the selectivity of
the resin/solvent. Activated carbon is commonly used to remove furans and phenols in
aqueous solutions [144]. Drawbacks with activated carbon are the need for regeneration or
replacement of used activated carbon due to strong/irreversible adsorption of phenolics
[144]. Chemical detoxification methods involve modifications of the inhibitors to less or nontoxic forms. The most important examples are treatment with Ca(OH)2 (overliming) or
NaOH. The detoxification mechanism is both precipitation of the inhibitors due to reduced
solubility at increased pH or formation of low solubility calcium salts and instability of some
inhibitors at high pH [142]. Problems associated with overliming are mainly related to scaling
of equipment, slurrying of the lime at high concentration, controlling lime addition, and
disposal of the filter cake. Overliming has also been shown to degrade sugars to formic acid
[145]. Biological detoxification can be conducted by treating the hydrolysate with an enzyme
or organism different from the organism used in the main fermentation. The enzyme laccase
has for example been shown to remove phenolics from hydrolysates of willow [146]. The
fermenting organism can also by itself detoxify the medium either by metabolising the
inhibitor, e.g. acetate metabolism during aerobic growth, or by physical adsorption of
inhibitors onto dead cells (Paper IV).
37
38
Results and Discussion

In this section a brief overview of the experiments performed in Papers I IV will be given
together with a discussion of the main results. For full descriptions of experimental
procedures and results, please refer to Papers I IV.
The fermentations in this study were conducted in dual-phase mode; each fermentation
consisting of an aerobic phase and an anaerobic phase. This fermentations procedure takes
advantage of E. coli being a facultative anaerobe; i.e. can grow both in the presence or
absence of oxygen. Aerobic cultivation of E. coli results in high growth rates and a fast
increase in cell concentration. When the desired cell concentration is reached, the
fermentation is shifted to anaerobic conditions by shutting of the air supply to the reactor
and instead sparging the medium with CO2. The cells grown aerobically now consume sugar
anaerobically and produce a mixture of organic acids, with succinic acid as the main product
for AFP184.
Economically viable technologies for production of biobased succinic acid require, as was
outlined before (see Introduction), high volumetric productivities and utilisation of a wide
array of feedstock materials in order to produce high yields of succinic acid. Apart from the
biocatalyst and the cultivation conditions, the medium needs to be considered. The medium
should be free of toxic compounds that inhibit fermentation, support growth and production
by being a source of nutrients, and contain a minimum of components that might interfere
with downstream processing of the fermentation broth. Even though the requirements on
detoxification and nutrient quantities vary between biocatalysts the resulting medium is
usually a trade-off between productivity and downstream processing. The type and sensitivity
to impurities of the downstream operations used in a given process also puts different
demands on medium composition.
In the present work fermentations in 1-10 L laboratory bioreactors were performed using a
medium based on corn steep liquor (50% solids) supplemented with inorganic salts and a
sugar solution. The medium was developed to be cost effective and relevant for use in
industrial production of succinic acid. The initial sugar concentration in the fermentations
was 100 g L-1 (except in the hydrolysate fermentations) and the sugars used were glucose,
fructose, xylose, mixtures of glucose/fructose or glucose/xylose, or a softwood hydrolysate
(containing apart from glucose and xylose also arabinose, mannose, and galactose).
39
Sugar utilisation (Papers I and IV)

The biocatalyst used for sustainable production of succinic acid from renewable resources
needs to be able to efficiently utilise a diverse set of plant biomass carbohydrates. In a
biorefinery application the feedstock quality and composition could vary with season, local or
regional availability, and current pricing. The catalyst must be versatile and able to handle
feedstock variations without upsetting production rates and yields. In Paper I the effect of
different sugars on succinic acid kinetics and yields during fermentations with AFP184 were
determined in order to establish the impact of possible feedstock variations. Glucose and
xylose are the most abundant sugars in lignocellulosic biomass, while fructose and glucose
are the components of sucrose, a widely available disaccharide. In this study sucrose, glucose,
fructose, xylose and equal mixtures of glucose:fructose and glucose:xylose were used as
feedstocks in dual-phase fermentations. AFP184 did not grow on or ferment sucrose and
sucrose is thus excluded from the following discussion. In Paper IV fermentations with
mannose, a common monosaccharide in softwood hemicellulose, as the sole carbon source
were also performed (Fig. 13).
In wild-type E. coli high catabolic loads generated by glycolysis at high sugar concentrations
leads to carbon being redirected to acetate instead of entering the TCA cycle, a phenomenon
called overflow metabolism [147]. An important result in the current work was that even at
the high sugar concentrations used (100 g L-1) acetate was not overproduced (Fig. 13). The
result is of significant importance since it shows that by using the present technology cells
can be cultivated to high cell densities in a batch reactor with high initial sugar
concentrations without the need for rigorously controlled fed-batch conditions to keep sugar
concentrations low. It also makes it possible to carry out both aerobic cell cultivation and
anaerobic fermentation with the same media in batch mode.
The fermentation profiles (Fig. 13) show that all sugars used were co-metabolised with
glucose without catabolite repression. The mutation in the PTS interferes with the regulation
of catabolite repression, so that simultaneous utilisation of multiple sugar substrates is
possible [148, 149]. Indeed from the sugar consumption (Fig. 13) it can be seen that when
fermenting the sugar mixtures, fructose was metabolised slightly faster during the aerobic
phase. When a mixture of glucose and xylose was used, the sugars were also fermented
simultaneously, but xylose was consumed at a substantially higher rate.
40
Glucose
100
C o n c e n tra tio n
-1
-1
12
(g L ; C e lls L x 1 0 )
C o n cen tratio n
(g L -1 ; C ells L -1 x 10 1 2 )
80
60
40
20
0
Fructose
100
80
60
40
20
0
10
15
20
25
30
10
Xylose
100
15
20
25
30
Time (hours)
80
c
C o n cen tratio n
(g L -1 )
C o n cen tratio n
(g L -1 ; C ells L -1 x 10 1 2 )
Time (hours)
80
60
40
20
0
Mannose
60
40
20
0
10
15
20
25
30
10
Time (hours)
15
20
25
30
Time (hours)
60
60
Glucose:Xylose
50
C o n cen tratio n
(g L -1 ; C ells L -1 x 10 1 2 )
C o n cen tratio n
(g L -1 ; C ells L -1 x 10 1 2 )
40
30
20
10
0
Glucose:Fructose
50
40
30
20
10
0
10
15
20
25
30
35
10
15
20
25
30
Time (hours)
Time (hours)
Figure 13. Sugar, product and cell concentration profiles for (a) glucose, (b) fructose, (c)
xylose, (d) mannose, (e) glucose:xylose, and (f) glucose:fructose fermentations. Product and
sugar concentrations are given in g L-1 and viable cells in (cells L-1)1012. Symbols used:
glucose (), fructose (), xylose (), mannose (), succinic acid (), acetic acid (), pyruvic
acid (), viable cells (*). Staggered line indicates time of switch to the anaerobic phase.
The fermentation results are summarised in Table 2. Glucose is the preferred sugar, resulting
in the highest productivities, final titres, and yields. The higher yields obtained when glucose
was used as the carbon source are due to the mutation in the PTS (described under Sugar
Metabolism in AFP184). Fermentation of fructose and mannose uses PEP for
phosphorylation committing a larger fraction of the carbon to pyruvate (Fig. 7) and thus
41

reducing the succinate yield. Xylose metabolism uses the pentose phosphate pathway (Fig. 6)
and the theoretical yield on xylose is somewhat lower than on glucose, but higher than on
fructose. However, xylose fermentation resulted in the lowest yield achieved. A carbon
balance can only account for approximately 60% of the carbon. To improve xylose
fermentation, further work is required to identify the metabolic fate of remaining carbon.
Table 2. Fermentation results for AFP184a.
Sugar
(h-1)
YP/S (g g-1)
Final titre
anaerobic
(g L-1)
QP (g L-1h-1)
QP (g L-1h-1)
anaerobic, T22d anaerobic, T32d
Glucose
0.42
0.83
40.6
2.89
1.76
Fructose
0.50
0.66
32.3
1.79
1.26
Xylose
0.57
0.50
24.9
1.49
1.08
Mannose
0.35
0.71
36.1
1.84
1.70
Glu:Frub
0.58
25.2
1.67
1.05
Glu:Xylc
0.60
27.6
1.48
1.08
aSpecific
growth rates were obtained from fermentations conducted in 1 L bioreactors. YP/S is
the mass yield of succinic acid based on the glucose consumed in the anaerobic phase and QP
is the volymetric productivity of succinic acid during the anaerobic phase.
Glu:Fru is an
abbreviation for glucose:fructose. c Glu:Xyl is an abbreviation for glucose:xylose. d anaerobic

productivity at 22 and 32 hours total fermentation time respectively
AFP184 grew on all sugars and sugar combinations. However specific growth rate on glucose
was slower than for growth on xylose or fructose. This result is most likely an effect of the
mutation in the PTS of AFP184. There is also a phosphotransferase permease for fructose
[59, 63], but in strain AFP184 this permease is not affected by any mutations. Apart from the
PTS, E. coli can also transport glucose by galactose permease and phosphorylate it by the
enzyme glucokinase [150]. Glucokinase is not subjected to any mutations in AFP184.
According to literature glucose uptake and phosphorylation by glucokinase is slower than by
the PTS [62], which explains why the growth rate on fructose is higher than the growth rate
on glucose. Unlike fructose, xylose is not transported into the cells via the PTS. Instead xylose
uptake is facilitated by chemiosmotic effects [66, 67]. Interestingly growth on xylose was
initially very slow, while the use of glucose and fructose rapidly initiated exponential growth.
42

Since the seed cultures were grown in a glucose medium, glucose and fructose fermentations
may already have the enzymes required for utilisation of the respective sugars, while in the
case of xylose fermentation, expression of these enzymes might need to be induced before
exponential growth can start.
Organic acid concentrations (including both succinic and other acids) above approximately
30 g L-1 resulted in drastically decreased productivities per viable cell. The general inhibiting
effects of organic acids is well known and described in the section Effects of pH and Organic
Acids on Escherichia coli. The fermentations in this study were conducted with ammonium
hydroxide as base for neutralisation of end products and it has been reported that ammonia
in concentrations above 3 g L-1 inhibits growth [151, 152]. In the present work, ammonium
concentrations of approximately 10 g L-1 were reached at the end of the fermentations with
high succinate production. In other studies investigating changes in growth characteristics of
E. coli under different ammonium sulphate concentrations, it was shown that ammonium
sulphate concentrations of 5% had to be used before any severe inhibition was observed
[153]. When the fermentations conducted in the present work showed a productivity decrease
they were in the anaerobic phase and only very little growth would take place even without
any inhibition. Therefore it is not clear if productivity would be affected by the achieved
ammonium concentrations. The effects of organic acids and ammonia on the fermentation
characteristics of AFP184 were further investigated in Paper II.
Inhibition of Succinate Production (Paper II)

As shown in Paper I, AFP184 produces succinic acid to final concentrations of 25-40 g L-1
with productivities in the range of 1.5-3 g L-1 h-1. The productivities, being initially high
decreased as fermentation products accumulated in the medium. Limited final titres and
productivity reductions can be due to the toxicity of organic acid [79, 154], high medium
osmolaritiy due to unfavourable salts and sugar concentrations [155], and toxicity of the
neutralising chemical used. Toxicity of succinic acid has not been reported before, and
therefore the objective of this work was to uncouple the effect of succinic acid versus base and
osmolarity to demonstrate quantitatively how increased production of succinate effects
productivity. Differences in succinic acid productivity, titre, yield, and cell viability when
using ammonia, alkali hydroxides and alkali carbonates as neutralisers, were studied by
conducting fermentations with NH4OH, KOH, NaOH, K2CO3, and Na2CO3. Effects of organic
acid toxicity and osmotic stress were studied in fermentations with externally added succinic
43

acid, sodium buffer, or by supplementing the growth medium with the osmoprotectant
glycine betaine.
Fermentations profiles for fermentations with different neutralising agents are summarised
in Table 3. The use of alkali carbonates for neutralisation gave higher volumetric
productivities than the alkali hydroxides. Using Na2CO3 and K2CO3 resulted in an increased
volumetric productivity compared to NaOH and KOH. The increased productivity is likely
caused by an increased availability of hydrogen carbonate ( HCO 3 ) from the addition of the
base chemical. The enzyme PEP carboxylase catalysing the carboxylation of PEP to
oxaloacetic acid uses HCO 3 as a substrate for the reaction [156]. The higher productivity
during neutralisation with CO3-bases indicates that the medium is not saturated by the
sparged CO2.
The highest final succinic acid concentration, 77 g L-1, was achieved when Na2CO3 was used
as base. Using NaOH resulted in 69 g L-1, K2CO3 in 64 g L-1 and KOH in 61 g L-1. The only byproduct formed was acetic acid and the lowest concentration, 4.6 g L-1, was obtained when
Na2CO3 was used
Table 3. Summary of fermentation results for different basesa
Final titre
YP/S (g g-1)
QP (g L-1 h-1)
QP (g L-1 h-1)
(g L-1)
anaerobic
anaerobic, T20
anaerobic, (max g L-1)b
NH4OH
43
0.75
2.91
1.85*
KOH
62
0.84
2.62
0.67
K2CO3
64
0.82
3.02
0.72
NaOH
69
0.78
2.47
0.76
Na2CO3
77
0.75
2.95
1.05
Base
YP/S is the mass yield of succinic acid based on the glucose consumed in the anaerobic phase,
and QP is the volumetric productivity of succinic acid during the anaerobic phase after 20 h
(T20) of total fermentation time.
Anaerobic productivity calculated either when
fermentations are terminated or maximum succinic acid concentration is achieved.

*maximum concentration was achieved after 32 hours
44

Productivities per viable cell contain different information than volumetric productivity.
Whereas volumetric productivity gives the total amount of succinate produced per volume
and time unit there is no information regarding the state of the cells. If the volumetric
productivity decreases it is not known if it is due to inhibition or viable cell loss. The
productivity per viable on the other hand cell can reveals the producing capacity of the each
viable cell and thus indicate if the cells are inhibited or not. However, it does not provide any
insight in the total productivity of the batch. In general, productivities per viable cell were
initially high, but decreased after approximately 20 hours of total fermentation time (Fig.
14a). When using NH4OH the succinate productivity completely stopped after 32 hours,
whereas the other fermentations showed gradually decreasing productivities during the
remaining anaerobic phase (Fig. 14a). Viable cell concentrations for fermentations using
NaOH or KOH as the pH regulator showed similar trends. During the first 20 hours of
anaerobic conditions the viability of the cultures decreased significantly, but for the
remainder of the fermentations only small losses in viability occurred (Fig. 14b). Using K2CO3
and Na2CO3 resulted in improved cell viability during the anaerobic phase relative to KOH
and NaOH (Fig. 14b).
60
50
V ia b le ce lls
(c ells L -1 ) x 10 12
Productivity per viable cell

-1 -1
14
(g cell h ) x 10
10
40
30
20
10
0
0
0
20
40
60
80
100
20
40
60
80
100
Time (hours)
Time (hours)
Figure 14. Productivity per viable cell (g cell-1 h-1) x 1014 (a) and viable cell concentration
(cells L-1 x 1012) (b) as functions of time. Productivities are calculated for the anaerobic phase.
Symbols used: KOH, NaOH, K2CO3, and Na2CO3, NH4OH. Fermentations were
done in triplicate and the values are averages of the data range (error bars).
Fermentations were conducted in which 150 mL of either a 140 g L-1 succinic acid solution or
a sodium phosphate buffer (pH 6.6) were added gradually during the anaerobic phase. The
amount of succinic acid produced when the buffer was added was significantly higher than
when the succinic acid solution was added (Fig. 15a and b). In neither of the fermentations
were the viable cell concentration negatively affected (Fig. 15c). Externally added succinic
acid resulted in a decreased anaerobic productivity per viable cell as the total succinic acid
45

concentration increased (Fig 15d). From the experiments conducted with addition of either
succinic acid or sodium buffer it was clear that the main reason for the decrease in
productivity per viable cell was the increase in succinic acid concentration during the
anaerobic production phase (Fig. 15). The osmolarity of the medium appears to have only
marginal effects on succinate productivity. This conclusion was further substantiated by the
results from the fermentations supplemented with glycine betaine (see Paper II). Addition of
osmoprotectants like glycine betaine should improve succinate production if the reduced
productivity was caused by increased osmolarity [122]. It has been shown that increased
intracellular concentrations of the osmolytes trehalose in ethanologenic E. coli did not
improve growth in the presence of formate, lactate, or acetate suggesting that another
mechanism than osmotic stress is responsible for growth inhibition in cultivations with
organic acids [157]. The productivity reduction was thus attributed to organic acid toxicity. In
order to further improve productivity the product acids must be separated from the cells.
Removal of succinic acid and its effects on productivity and cell viability is further discussed
in Paper III.
46

80
80
60
C o n c en tra tio n
(g L -1 )
C o n c en tra tio n
(g L -1 )
40
20
60
40
20
0
0
10
15
20
25
30
10
Time (hours)
20
25
30
10
P ro d u ctivity p e r viab le ce ll
(g ce ll-1 h -1 ) x 10 14
80
c
V iab le ce lls
(c ells L -1 ) x 10 12
15
Time (hours)
60
40
20
d
8
0
0
10
15
20
25
30
10
15
20
25
30
Time (hours)
Time (hours)
Figure 15. Fermentations with succinic acid or salt-buffer addition; (a) total (), produced
(), and added () succinic acid concentration when 140 g L-1 succinic acid was added, (b)
succinic acid concentration equivalent to the amount of added sodium ions () and produced
succinic acid (), (c) viable cells per litre x 1012 for fermentation with succinic acid () and
Na+-buffer () addition, (d) productivity per viable cell (g cell-1 h-1) x 1014 for fermentation
with succinic acid () and Na+-buffer () addition. Viable cell counts were done in duplicate
and the values are averages of the data range (error bars). The broken lines indicate the
transition to the anaerobic phase. In all figures () represents a control using 2 M Na2CO3.
Effects of Removing Succinic Acid (Paper III)

As outlined in Paper II, the decreased succinic acid productivity is due to organic acid
inhibition and maintaining high productivity is most likely connected to the removal of
succinic acid from the cells. The actual separation of succinic acid from the fermentation
broth can be accomplished by various separation operations e.g. crystallisation [158] or
electrodialysis [45]. Since the inhibitory effects of succinic acid observed for AFP184 becomes
evident first at concentrations of approximately 40 g L-1 processing options include carrying
out continuous fermentations, removing the succinate continuously by leading off a stream
47

from the bioreactor to the chosen separation equipment and then returning it to the reactor
or recovering the succinate once it reaches certain predetermined threshold concentrations.
The objective of this study was to investigate the effects of succinic acid on the fermentation
kinetics, yield, and cell viability with the aim of restoring the initially high productivities in
cultures exposed to high concentrations of succinic acid. Succinic acid fermentations were
interrupted at predetermined times, correlating to different succinic acid concentrations and
varying physiological states of the cells. The cells were harvested and resuspended in fresh
succinate free media for continued fermentation. Resuspending the cells in fresh media also
offers the possibility to modify the media the cells are being resuspended in. Removing all
nutrients except glucose and the potassium buffer was attempted to limit the amount of
complex nutrients and colouring substances in the fermentation as these add to the cost and
effort needed to separate and purify the succinate [13]. In this study, fermentations with cell
resuspensions after 23, 40, or 60 hours of total fermentation time were conducted. Multiple
resuspensions were also investigated by harvesting and resuspending the same cells three
times; after 23, 45, and 70 hours of total fermentation time.
Fig. 16 shows the variation of volumetric and cellular succinic acid productivity for
fermentations with cell resuspension. Resuspending the cells after 23 hours of total
fermentation time resulted in both maintained cellular and volumetric productivities (Fig.
16a and b). For fermentations with cell resuspension after 40 or 60 hours, the cellular
productivity was decreased as a result of the accumulated succinic acid (Fig. 16a and c). The
decrease in cellular productivity follows very closely that of the control fermentation (without
resuspension). After resuspending the cells both the 40 and 60 hour resuspensions regained
full cellular productivity. Hence the cells as such can recover from the inhibition, as long as
the produced succinic acid is removed. This result shows that succinic acid does not
permanently damage the cells, and that the inhibitory effect of the organic acid is reversible.
In contrast, the volumetric productivity did not recover to the same extent as the cellular.
Resuspending the cells after 40 hours nearly reached the volumetric productivities achieved
in the beginning of the anaerobic phase, whereas resuspension after 60 hours only recovered
to approximately half of the initial volumetric productivity. The reason is that volumetric
productivity, as opposed to cellular productivity, is dependent on the concentration of viable
cells. Cellular productivity is only affected by the succinate concentration and once that is
reduced the productivity increases. Since the volumetric productivity is the sum of what all
cells in the culture produce and it does not recover to the initial values it means that the
concentration of viable cells has decreased.
48
10
3.5
P ro d u ctivity
(g L -1 h -1 )
P ro d u ctivity
(g cell -1 h -1 ) x 10 1 4
12
8
6
4
2
3
2.5
2
1.5
1
0.5
20
40
60
80
20
Time (hours)
60
80
12
10
3.5
P ro d u ctivity
(g L -1 h -1 )
P ro d u ctivity
(g cell -1 h -1 ) x 10 1 4
40
Time (hours)
8
6
4
2
3
2.5
2
1.5
1
0.5
20
40
60
80
100
Time (hours)
20
40
60
80
100
Time (hours)
Figure 16. Productivities as functions of time; a) and c) cellular productivity (g cell-1 h-1 x
1014); b) and d) volumetric productivity (g L-1 h-1). Symbols used in the figures represent when
resuspensions were done: - 23 hours, - 40 hours, - 23 hours (glucose media), - 60
hours, - 23/45/70 hours, - control without harvesting/resuspension.
The concentration of viable cells, as shown in Fig. 17, decreases with fermentation time
irrespective of whether the cells are resuspended or not. It appears that the cell viability is not
connected to succinic acid concentration, being dependent only on the time the cells spend in
the anaerobic environment. During anaerobic metabolism for succinate production AFP184
generates zero ATP. The implication is that during succinate production there will be no
energy available for replication or maintenance and hence the anaerobic viability of AFP184
is clearly limited. The absence of a net ATP generation also puts restrictions on the acid
excretion systems. With no available metabolic energy AFP184 is limited to non-energy
requiring export systems during succinate production. Through electrogenic export of an acid
(acid to proton stochiometry greater than 1) it is possible to generate an electrochemical
gradient which can translate into metabolic energy [159]. Achieving redox balanced
anaerobic glucose metabolism in AFP184 also commits carbon from PEP to pyruvate and the
oxidative arm of the TCA cycle. The PEP to pyruvate conversion results in the formation of 1
mole of ATP for every mole of PEP that is dephosporylated (Fig. 6). The availability of
49

complex nutrients can also enter and affect the cellular energy metabolism supplying more
metabolic energy and driving acid excretion, cell maintenance or possibly replication.
C on cen tratio n
(C ells L -1 ) x 10 1 2
60
50
40
30
20
10
0
0
20
40
60
80
100
Time (hours)
Figure 17. Viable cells per litre x 1012 for fermentations with cell resuspensions. Symbols
used in the figure represent when resuspensions were done: - 23 hours, - 40 hours, - 23
hours (glucose media), - 60 hours, - 23/45/70 hours, - control without
harvesting/resuspension.
Resuspending the cells in a medium consisting of only glucose and a potassium phosphate
buffer resulted in cellular productivities (Fig. 16a) and succinate yields of the same order as
fermentations with CSL, however the cell viability was reduced faster (Fig. 17) and although
the complex nutrients are not necessary for efficient succinate productivity they might be
significant for prolonging the anaerobic viability. The decrease in cell viability compared to
fermentations with CSL resulted in a relatively lower volumetric productivity.
The results from this study are encouraging, providing clear indications that substantial
increases in succinic acid production can be achieved by shielding the cells from the
produced end-product. Further research should be aimed at integrating low-cost, efficient
separation processes with fermentation and at genetic engineering of the anaerobic
metabolism of AFP184 to generate more metabolic energy in order to promote higher final
titres and better anaerobic viability.
50
Industrial Raw Materials and Detoxification (Paper IV)

Following the preliminary investigation of the fermentation characteristics of AFP184 on
glucose and xylose in Paper I the natural extension of that work was to implement the
technology using carbohydrates derived from industrial feedstocks. Hydrolysates of softwood
from a two-stage dilute acid hydrolysis process conducted at the Swedish ethanol pilot plant
in rnskldsvik were used (described under Industrial Raw Materials). The first stage
hydrolysed the hemicellulose (hydrolysate called H1) and the second stage the cellulose
hydrolysates (H2). Equal parts of H1 and H2 were then combined and evaporated to
approximately twice the sugar concentration of the mixed hydrolysates. Fermentation of
lignocellulosic hydrolysates to succinic acid by E. coli has been performed using fed-batch
fermentation of concentrated acid hydrolysates of rice straw [55] and cellulose enzyme
hydrolysates
of
steam
exploded
oak
by
M.
succiniciproducens
[48]
and
A.
succiniciproducens [44]. The present study is unique in that no other work so far has
demonstrated succinic acid production from softwood hydrolysate sugars, although a few
examples of bacterial conversions of softwood hydrolysate sugars to fermentation products,
including lactic acid by Bacillus species [160] and ethanol by metabolically engineered E. coli
strains [161].
As previously discussed (see Industrial Raw Materials) dilute acid hydrolysis of
lignocellulosic biomass generates apart from monomeric sugars also sugar degradation
products such as HMF, furfural, levulinic and formic acid, phenolic compounds from lignin
breakdown, and acetic acid released from acetyl groups in the hemicellulose; all of which are
inhibitory or toxic to microorganisms. Except these, inorganic ions and trace metals
accumulated from the biomass and/or pretreatment equipment also adds to the total
inhibitory potential of the hydrolysate. To make the hydrolysates amendable for microbial
conversion to succinate different procedures for detoxification can be employed. In the
present study treatment with activated carbon and/or overliming were utilised. Figure 18
shows the concentration of inhibitors and sugars in hydrolysates H1, H2 and H3. Figure 19
demonstrates the effects of overliming and activated carbon treatment on the inhibitor
concentrations in the hydrolysates.
51
60
-1
Concentration (g L )
80
Acetic Acid
HMF
Phenolics
Formic Acid
Levulinic Acid
Galactose
Mannose
Arabinose
Xylose
Glucose
100
-1
40
20
5
4
3
2
1
0
0
1st Stage
Hydrolysate
2nd Stage
Hydrolysate
1st Stage
2nd Stage Comb Evap
Hydrolysate Hydrolysate Hydrolysate
Comb Evap
Hydrolysate
Figure 18. Hydrolysate sugar (a) and inhibitor concentrations (b) in the three hydrolysate
types used.
Overliming was able to generate a fermentable hydrolysate for all three hydrolysates (Table
4). Compared to activated carbon treatment overliming was more effective for the first stage
hydrolysate, which is further supported by the failure of the cells to grow for several
replicates using 5% activated carbon treatment. From Fig. 18 the phenolics are the only
inhibitor in H1 that should pose an obvious barrier to growth. While these are significantly
reduced by treatment with activated carbon they are relatively unaffected by overliming. For
H2 and H3, the activated carbon treatment was apparently more effective when inhibition
was considered in terms of the time required for aerobic growth. Due to this, the changes in
concentration of HMF and phenolics (Fig. 19), which are the only inhibitors that were
quantifiably affected by the detoxifications, cannot be considered as the only factors affecting
the toxicity of the hydrolysate.
4.5
P h e n o l i c s (g L - 1 )
2nd stage hydrolysate
3.5
Comb evap hydrolysate
3
2.5
1st stage hydrolysate
2nd stage hydrolysate
3.5
H M F (g L - 1 )
4.5
1st stage hydrolysate
Comb evap hydrolysate
2.5
2
1.5
1.5
1
0.5
0.5
0
none
2.5% AC
5.0% AC
5% AC + OL
OL
none
2.5% AC
5.0% AC
5% AC + OL
OL
Figure 19. Effects of overliming (OL) and activated carbon (AC) detoxification on inhibitors.
52

It is proposed that differences in microbial response can be due to changes in the inorganic
ion content of the hydrolysates with proposed mechanisms including sulphate toxicity, total
ionic or osmotic stress, and toxicity of trace metal cations; all of which have been found to be
significantly decreased by overliming [140]. A summary of the main fermentation results is
given in Table 4. Yields and final titres were in the range expected for the present sugars used
(see Paper I) and the anaerobic volumetric productivities compare very positively to other
studies producing succinic acid from lignocellulosic biomass [44, 48, 55].
Table 4. E. coli AFP184 growth and fermentation after different detoxification methods of
softwood hydrolysates.
Hydrolysate
Time
(h)
x
Aerobic
Exp. Growth
(, h-1)
x
Max.
OD550
x
Time
(h)
x
Anaerobic
Final SA
(g L-1)
x
Yield
(g g-1)
X
5% AC + OL
9.3
0.36
24.0
11.0
19.7++
0.68
OL
7.0
0.45
36.4
6.5
18.0
0.62
2.5% AC
5% AC
15
0.36
35.5
4.0
9.9
0.86
OL
23
39.0
5.0
14.1
0.93
Neutralisation
24
33.2
23.0
32.8
0.72
37
31.6
28.0
35.0++
0.86
11.75
0.38
35.4
42.25
42.2
0.72
0.50
34.1
26.0
38.7
0.59
13.25
0.29
34.0
34.75
27.2++
0.64
Detoxification
Neutralisation
2.5% AC
1st stage
(H1)
2nd stage
(H2)
5% AC
2.5% AC
Combined
evaporated
(H3)
5% AC
5% AC + OL
OL
AC activated carbon, OL - overliming

* Not determined
x minimal growth
++ Fermentation terminated before sugar utilization was completed
53
54
Conclusions
In Paper I growth and succinate production of E. coli strain AFP184 in media containing corn
steep liquor, salts, and the monosaccharides glucose, fructose, and xylose were
demonstrated. The reduction of expensive complex components in the medium will have a
positive effect on the final price of the produced succinate due to a lower raw material cost;
however final concentrations were in the range of 30-40 g L-1 after 32 hours and need to be
improved for the process to become more economically attractive.
In Paper II it was demonstrated that replacing the neutralising agent can provide substantial
process improvements in the form of increased duration of high volumetric productivity and
increased final titre. Compared to fermentations neutralised with NH4OH it was possible to
achieve an almost 100% increase in final succinic acid concentration using Na2CO3. The
decrease in productivity could be attributed to accumulation of organic acids in the
fermentation broth and organic acid toxicity rather than osmolarity as the primary reason for
reduced productivity and limited final titres.
The results from Paper III show that removing succinic acid from the fermentation broth
makes it possible to maintain high anaerobic succinic acid productivities. It was also shown
that removing all complex nutrients did not have a major effect on productivity and yield.
Furthermore it was demonstrated that succinic acid has limited or no effect on the cellular
viability during the anaerobic phase. Instead low energy generation during succinic acid
production is thought to be the cause.
Paper IV demonstrates that AFP184 is able to ferment dilute acid hydrolysates to succinate
after detoxification with lime or activated carbon treatment. The presence of inorganic ions
not quantified added to the toxicity of the hydrolysates. The use of lime for detoxification was
efficient in removing the inorganic inhibitors as shown by the fermentability of the
hydrolysates after overliming pretreatment even though the quantity of organic inhibitors, in
particular phenolics, still was high.
55
56
Future work
In the current work major improvements towards realising commercial production of
biobased succinic acid have been made. The present technology is however limited in
productivity and final titre due to the presence of succinic acid. The biocatalyst AFP184 is
also limited in its anaerobic viability which puts an upper boundary on the duration of the
fermentations. In order to further improve the technology research should be focused on
genetic improvement of the biocatalyst, product separation, and process integration.
The biocatalyst
Genetic engineering and strain selection to improve acid tolerance and robustness
towards inhibitors in the raw material.
Metabolic engineering of AFP184 by for instance insertion of the pyruvate carboxylase

gene could potentially improve the yield and lower by-product formation during
succinic acid production. Lower by-product concentrations also results in lower
separation costs.
Investigate means to increase the anaerobic durability of the organism and improve
the excretion of produced acids. Mutations that can liberate more metabolic energy
might accomplish this.
The process
Integrating the current batch operation with continuous removal of succinic acid thus
relieving the problem of succinate accumulation in the fermentation broth and the
subsequent end-product inhibition.
Separation processes for succinic acid recovery and purification should be further
developed. Some work has been done in this area, but cost-efficient solutions to be
integrated with the continuous recovery of succinic acid outlined above should be
pursued in order to improve the economical feasibility of biochemical succinic acid
production. For this purpose adsorption or ion-exchange operations seem most
promising.
57
58
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72
Paper I
Biotechnol. Prog. 2007, 23, 381388
381
Effect of Different Carbon Sources on the Production of Succinic Acid Using

Metabolically Engineered Escherichia coli
Christian Andersson, David Hodge, Kris A. Berglund, and Ulrika Rova*
Division of Biochemical and Chemical Process Engineering, Lule University of Technology, SE-971 87 Lule, Sweden
Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity
and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli
strain AFP184 were performed using a medium suited for large-scale industrial production of
SA. The ability of the strain to ferment different sugars was investigated. The sugars studied
were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose
and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all
sugars and sugar combinations except sucrose for biomass generation and succinate production.
For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized.
The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed
anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a
carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite
repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1)
than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g
g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production.
The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations
were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected
and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid
decreased when organic acid concentrations reached approximately 30 g L-1. This study
demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium
from a variety of sugars with only small amounts of byproducts formed.
Introduction
Sustainable production of fuels and chemicals is driven by
the need to control atmospheric concentrations of greenhouse
gases such as carbon dioxide, depletion of existing oil reserves,
and increasing oil prices. In a report from the U.S. Department
of Energy (USDOE), succinic acid was considered as one of
12 top chemical building blocks manufactured from biomass
(1). Succinic acid has a wide range of applications and can be
a platform for deriving many commodity and specialty chemicals, for example, diesel fuel additives, deicers, biodegradable
polymers and solvents, and detergent builders (2).
Succinic acid can be produced by a number of organisms
including Bacteroides ruminicola and Bacteroides amylophilus,
Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, and Escherichia coli
(3-9). Corynebacterium glutamicum has also been shown to
produce mixtures of lactic and succinic acid (10). The work
done so far have all used media supplemented to some degree
with high-cost nutrient sources such as tryptone, peptone, and
yeast extract.
In order to develop a bio-based industrial production of
succinic acid, three main issues need to be addressed. First, a
low cost medium must be used. Second, the producing organism
must be able to utilize a wide range of sugar feedstocks,
producing succinic acid in good yields in order to make use of
* To whom correspondence should be addressed. Ph: +46920491315.
Fax: +46920491199. Email: ulrika.rova@ltu.se.
10.1021/bp060301y CCC: $37.00
the cheapest available raw material. Glucose and xylose are the
most abundant sugars in lignocellulosic biomass, while fructose
and glucose are the components of sucrose, a widely available
disaccharide. The third and most important factor for the
succinic acid economy, as described in the USDOE report, is
the volumetric productivity. To make succinic acid production
feasible, volumetric productivities above 2.5 g L-1 h-1 will be
necessary (1).
In 1949 Stokes demonstrated that E. coli under anaerobic
conditions produces a mixture of organic acids and ethanol (11).
Typical yields from such fermentations are 0.8 mol ethanol,
1.2 mol formic acid, 0.1-0.2 mol lactic acid, and 0.3-0.4 mol
succinic acid per mole glucose consumed. If the objective is to
produce succinic acid, the yield of succinic acid relative the
other acids must be increased. The USDOE initiated in the late
1990s the Alternative Feedstock Program (AFP) with the aim
to develop a number of metabolically engineered E. coil strains
with increased succinic acid production. The two most interesting mutants developed by the program were AFP111 and
AFP184 (9, 12, 13). AFP111 is a spontaneous mutant with
mutations in the glucose specific phosphotransferase system
(ptsG), the pyruvate formate lyase system (pfl), and the
fermentative lactate dehydrogenase system (ldh) (12). The
mutations resulted in increased succinic acid yields (1 mol
succinic acid per mole glucose) (13). AFP184 is a metabolically
engineered strain where the three mutations described above
were deliberately inserted into the E. coli strain C600 (ATCC
23724), which can ferment both five- and six-carbon sugars and
has strong growth characteristics (9). Beside these organisms,
2007 American Chemical Society and American Institute of Chemical Engineers

Published on Web 01/25/2007
Biotechnol. Prog., 2007, Vol. 23, No. 2
382
a number of different E. coli mutants have been developed and

tested for succinate production including pioneering work to
produce succinate under aerobic conditions (14-22). In order
to improve succinate yield, other studies used recombinant
AFP111 overexpressing heterologous pyruvate carboxylase in
complex media with the main components tryptone (20 g L-1)
and yeast extract (10 g L-1), which resulted in mass yields of
1.10 and productivities of 1.3 g L-1 h-1 (23, 24). In the present
work the aim was to investigate the fermentation characteristics
of AFP184 in a medium consisting of corn steep liquor,
inorganic salts, and different sugars without supplementation
of other additional nutrients. Corn steep liquor is a byproduct
from the corn wet-milling industry, but as opposed to yeast
extract and peptone it is an inexpensive source of vitamins and
trace elements. The composition of corn steep liquor can vary
widely depending mainly on the variables involved in starch
processing and the type and condition of the corn, but averages
can be found in the literature (25, 26). For the purpose of this
study sucrose, glucose, fructose, xylose, and mixtures of glucose
and fructose and glucose and xylose were used as carbon
sources. This work addresses the effect of different sugars on
succinic acid kinetics and yields in an industrially relevant
medium.
Materials and Methods

Strain and Inoculum Preparation. The E. coli strain
AFP184, which lacks functional genes coding for pyruvate
formate lyase, fermentative lactate dehydrogenase, and the
glucose phosphotransferase system (9), was used in this study.
Cultures were stored at -80 C as 30% glycerol stocks. The
inoculum was prepared by inoculating four 500-mL shake flasks
containing 100 mL sterile medium (same medium as used in
the biorector; see below) with 200 L of the glycerol stock
culture. The inoculated flasks were incubated for 16 h at 37 C
(200 rpm).
Fermentations. Batch fermentations were conducted in a 12
L bioreactor (BR12, Belach Bioteknik AB, Sweden) with a total
starting volume of 8 L (including 0.4 L inoculum and 2 L sugar
solution). The medium used for strain AFP184 contained the
following components (g L-1): K2HPO4, 1.4; KH2 PO4, 0.6;
(NH4)2SO4, 3.3; MgSO47H2O, 0.4; corn steep liquor (50%
solids, Sigma-Aldrich), 15; and 3 mL antifoam agent (Antifoam
204, Sigma-Aldrich). The medium was sterilized in the fermenter at 121 C for 20 min; thereafter 2 L of a separately
sterilized sugar solution (400 g L-1) and 400 mL inoculum were
aseptically added to a final volume of 8 L and a total sugar
concentration of 100 g L-1. The fermentation temperature was
controlled at 37 C, and the pH was maintained between 6.6
and 6.7 by automatic addition of NH4OH (15% NH3 solution).
The dissolved oxygen concentration (%DO), measured by a pO2
electrode, was regulated by varying the agitation speed between
500 and 1000 rpm. The total fermentation time was 32 h and
consisted of an aerobic growth phase and an anaerobic production phase. During the aerobic growth phase (7-9 h) the culture
medium was aerated with an air flow of 10 L min-1. When the
optical density at 550 nm had reached a value of 30-35, the
anaerobic production phase was initiated by withdrawing the
air supply and sparging the culture medium with CO2 at a flow
rate of 3 L min-1. The agitation speed was set to 500 rpm for
the anaerobic phase. Succinic acid was produced during this
anaerobic production phase, which continued for the remainder
of the fermentation. During the fermentations, samples were
aseptically withdrawn for analysis of optical density, viable cells,
and sugars and organic acids concentrations. Sucrose was tested
with strain AFP184 in a 1 L bioreactor (Biobundle 1L, Applikon
Biotechnology, The Netherlands) to determine if the strain could

grow and produce succinic acid when using sucrose as the
carbon source. The fermentation procedure was the same as in
the 12 L bioreactor. Three fermentations in the 1 L bioreactors
were also carried out to determine the specific growth rates of
AFP184 growing on glucose, fructose, and xylose, respectively.
Samples were taken during the aerobic phase and analyzed for
optical density. No anaerobic phase was carried out.
Analysis. Cell Growth. Cell growth was monitored by
measuring the optical density at 550 nm (OD550). Optical
densities were then converted to dry cell weight (DCW, g L-1)
using the following OD-DCW calibration curve developed in
our laboratory:
DCW ) 0.324OD550 + 1.687

To establish the number of viable cells, 10-fold serial dilutions
of the fermentation samples were plated on tryptone soy agar
plates and incubated overnight at 37 C. The number of colonies
was calculated, and the number of viable cells was expressed
as cells per milliliter fermentation broth.
Organic Acid and Sugar Analysis. The organic acid concentrations were measured by HPLC (Series 200 Quaternary
LC pump and UV-vis detector, Perkin-Elmer) equipped with
a C18 column (Ultra Aqueous, 5 m, 150 mm 4.6 mm,
Restek) using a 50 mM KH2PO4 buffer with 2% acetonitrile
(pH 2.5 adjusted by HCl) at a flow rate of 0.35 mL min-1 as
the mobile phase, with 20 L of sample injected manually for
analysis with UV detection at 210 nm. Samples for acid analysis
were centrifuged at 10 000 rpm for 10 min at 4 C. The
supernatant was diluted with the mobile phase and 37%
hydrochloric acid (volume sample:buffer:HCl ) 0.1:0.8:0.1 mL)
and filtered through a 0.2 m syringe filter. Sugar concentrations were determined using the same HPLC system as above
but equipped with a Series 200 refractive index (RI) detector
(Perkin-Elmer), guard column, and an ion exchange column
(Aminex HPX87-P, BioRad). The column was kept at 85 C in
a column oven for optimal performance. Prior to analysis, the
samples were centrifuged at 10 000 rpm for 10 min at 4 C.
The supernatant was diluted with water and filtered through a
0.2 m syringe filter, and 20 L of sample was injected
manually. Water at a flow rate of 0.6 mL min-1 was used as
the mobile phase. All data was collected and processed using
Perkin-Elmers TotalChrom analytical software. Peak areas
from the chromatograms were evaluated by comparison to
standard curves prepared from solutions with known concentrations of organic acids (formic, pyruvic, malic, lactic, acetic,
succinic, and fumaric acid), sucrose, glucose, fructose, and
xylose.
Results
Fermentations with strain AFP184 using sucrose, glucose,
fructose, xylose, and equal mixtures of glucose/fructose and
glucose/xylose were performed and analyzed.
Single-Sugar Fermentations. AFP184 was able to use
glucose, fructose, and xylose, but not sucrose, as a carbon source
for biomass and succinic acid production (Figure 1a-c). During
sucrose fermentation no sugar was metabolized, and sucrose
was therefore excluded from further analysis. Specific growth
rates during the aerobic phase were calculated using data
collected from experiments carried out with glucose, fructose,
and xylose in 1 L bioreactors (Table 1). It was observed that in
fermentations using glucose and fructose exponential growth
was initiated within 1 h after inoculation, but when xylose was
used exponential growth did not start until after 4 h. Once
383
Figure 1. Sugar, product, and cell concentration profiles for (a) glucose, (b) fructose, (c) xylose, (d) glucose/fructose, and (e) glucose/xylose
fermentations. Product and sugar concentrations are given in g L-1, dry cell weights in g L-1 and viable cells in (cells mL-1) 109. Symbols used:
glucose (4), fructose ()), xylose (O), succinic acid (b), pyruvic acid (9), viable cells (/), and dry cell weight (). Staggered line indicates time
of switch to the anaerobic phase.
Table 1. Summary of Fermentation Parameters for AFP184a
sugar
(h-1)
YP/S (g g-1) anaerobic
QP (g L-1 h-1) overall
QP (g L-1 h-1) anaerobic, T22d
QP (g L-1 h-1) anaerobic, T32d
glucose
fructose
xylose
glu/frub
glu/xylc
0.42
0.50
0.57
0.83
0.66
0.50
0.58
0.60
1.27
1.01
0.78
0.79
0.86
2.89
1.79
1.49
1.67
1.48
1.76
1.26
1.08
1.05
1.08
a Specific growth rates were obtained from fermentations carried out in 1 L bioreactors. Y
P/S is the mass yield of succinic acid based on the glucose
consumed in the anaerobic phase, and QP is the volumetric productivity of succinic acid during the anaerobic phase. b glu/fru is an abbreviation for glucose/
c
d
fructose. glu/xyl is an abbreviation for glucose/xylose. Anaerobic productivity at 22 and 32 h total fermentation time, respectively.
growing exponentially xylose resulted in the highest specific

growth rate and glucose in the lowest. It should be noted that
when glucose was used a slow increase in dry cell mass was
observed during the first 7 h of the anaerobic phase.
Viable cell counts were made for all of the different
fermentations (Figure 1a-c). During the first hours of the
anaerobic phase the number of viable cells was constant or
increased slightly as an effect of continued growth. After

approximately 20 h the viable cell density started to decrease.
Succinic, acetic, and pyruvic were the only acids produced
in concentrations over 1 g L-1. The highest succinic acid
concentration, 40 g L-1, was detected after 32 h when glucose
was used as the carbon source. After 32 h the succinate
production did not increase significantly (data not shown), and
384
Figure 2. Succinic acid productivities for single-sugar fermentations in grams succinate per viable cell as a function of (a) time and (b) succinic
acid concentration. Symbols used in the figure: glucose fermentation (4), fructose fermentation (]), and xylose fermentation (O).
hence 32 h was selected as the final fermentation time. When

fructose and xylose were used, the final succinic acid concentrations reached values of 32 and 25 g L-1, respectively. These
fermentations also resulted in lower acetate concentrations but
produced pyruvate. The highest yield, 0.83 g succinate per gram
sugar consumed in the anaerobic phase, was obtained when
glucose was used as a carbon source. The lowest yield was
obtained from xylose (0.50 g g-1). The yield from fructose was
0.66 g g-1.
The overall volumetric productivities (QP) are presented in
Table 1. Productivities per viable cell were calculated as
functions of time and succinic acid concentrations, respectively
(Figure 2). Productivities per viable cell as function of time for
fermentations using fructose and xylose were initially high but
decreased substantially during the first hours of anaerobic
conditions (Figure 2a). During the remainder of the fermentations productivities remained relatively constant. Glucose
fermentations can be distinguished from fermentations on the
other sugars as the productivity per viable cell showed only a
marginal productivity decrease up until 16 h, after which it
decreased significantly. When investigating productivities as a
function of succinic acid concentration, the fructose and xylose
fermentations resulted in, after a strong initial productivity
decrease, slightly decreasing productivities as the concentration
of succinic acid increased (Figure 2b). Glucose on the other
hand gave a constant productivity until a succinate concentration
of approximately 30 g L-1 was reached, after which the
productivity decreased in an almost linear manner.
Mixed-Sugar Fermentations. Strain AFP184 grew well in
both glucose/fructose and glucose/xylose mixtures. No increase
in dry cell weight was observed during the anaerobic phase
(Figure 1d and e). An interesting observation is that the substrate
consumption rate was higher for both fructose and xylose than
for glucose in the aerobic phase when AFP184 was grown on
mixed sugars. In the anaerobic phase glucose and fructose were
metabolized at similar rates, but when glucose was mixed with
xylose, xylose was metabolized faster during the first 16 h of
the fermentation. After 16 h the consumption rates of glucose
and xylose were approximately equal. The succinic acid yields
in the anaerobic phase were 0.58 and 0.60 g succinic acid per
gram sugar consumed for glucose/fructose and glucose/xylose
mixtures, respectively (Table 1). The final succinic acid
concentrations were in the range of 25-27 g L-1, and during
both mixed-sugar fermentations between 8 and 12 g L-1 of
pyruvate was formed (Figure 1d and e). Most of the pyruvate
was excreted during the first 2-4 h of the anaerobic phase and
at a rate similar to the succinic acid production rate. The
pyruvate concentration decreased in both cases to final con-
centrations of approximately 1-3 g L-1. Acetate levels increased

slowly during both fermentations. Less acetate was formed when
fructose was used, 2.7 g L-1 compared to 6.7 g L-1 when xylose
was used.
The viable cell densities increased during the aerobic phases
and were approximately constant during the anaerobic phases
up until 20 h of the fermentations had passed, where after they
decreased (Figure 1d and e).
Productivities during mixed-sugar fermentation are presented
in the same way as for single-sugar fermentations (Figure 3).
When using gluose/fructose the productivity per viable cell as
a function of time was initially high (Figure 3a). A major
productivity decrease occurred at a succinate concentration of
20 g L-1 (Figure 3b). For glucose and xylose the productivity
was constant throughout the anaerobic phase (Figure 3a and
b).
Discussion
The aim of the current work was to investigate the fermentation characteristics of E. coli strain AFP184 in a corn steep
liquor based medium using different mono- and disaccharides
as carbon source. From the cell densities obtained after 8 h of
aerobic growth and under the conditions used, it could be
concluded that AFP184 grew on all sugars and sugar combinations except sucrose. However, specific growth rate on glucose
was slower than for growth on xylose or fructose. This result is
most likely an effect of the mutation in the phosphotransferase
system (PTS) of AFP184. The PTS is a system of enzymes
responsible for uptake and phosphorylation of different sugars
in bacteria (27, 28). The PTS is sugar-specific, and the mutation
in AFP184 is affecting EIICBglc, a glucose specific permease
in the PTS (12). There is also a phosphotransferase permease
for fructose (27, 29), but in strain AFP184 this permease is not
affected by any mutations. Apart from the PTS, E. coli can also
transport glucose by galactose permease and phosphorylate it
by the enzyme glucokinase (30). Glucokinase is not subjected
to any mutations in AFP184. According to the literature glucose
uptake and phosphorylation by glucokinase is slower than by
the PTS (31), which explains why the growth rate on fructose
is higher than the growth rate on glucose. Unlike fructose, xylose
is not transported into the cells via the PTS. Instead xylose
uptake is facilitated by chemiosmotic effects (32, 33). Once
inside the cell it is isomerized by xylose isomerase to xylulose
and phosphorylated by xylulose kinase to xylulose 5-phosphate.
Xylulose 5-phosphate then enters the pentose phosphate pathway
and can enter the glycolytic pathway after conversion to either
fructose 6-phosphate or glyceraldehyde 3-phosphate. Interestingly, growth on xylose was initially very slow, whereas the
385
Figure 3. Succinic acid productivities for mixed-sugar fermentations in grams succinate per viable cells as a function of (a) time and (b) succinic
acid concentration. Symbols used in the figure: Glucose/fructose fermentation (]), and glucose/xylose fermentation (O).
use of glucose and fructose rapidly initiated exponential growth.

Because the inocula were grown in a glucose medium, glucose
and fructose fermentations may already have the enzymes
required for utilization of the respective sugars, whereas in the
case of xylose expression of the enzymes first needs to be
induced before exponential growth can start.
When glucose is mixed with other sugars, E. coli usually
consumes it first, and after all glucose has been metabolized
the utilization of other sugars is initiated, a phenomenon called
catabolite repression. From the sugar consumption (Figure 1)
it was observed that in glucose/fructose mixtures, fructose was
metabolized slightly faster during the aerobic phase. This effect
was expected as a result of the mutation strain AFP184 has in
the glucose PTS. More interesting is the observation that when
glucose and xylose were mixed, the sugars were fermented
simultaneously but xylose was consumed at a substantially
higher rate. Other studies of glucose/xylose fermentations by
E. coli PTS mutants show similar results where glucose and
xylose are co-metabolized. The effect is attributed to a mutation
in the PTS affecting EIICBglc, which in turn prevents glucose
from repressing uptake of other sugars (30, 34-36).
To estimate maximum yields and optimal metabolic flows
between intermediates, redox balances were carried out. The
anaerobic glucose, fructose, and xylose metabolism of AFP184
for maximal succinate production is presented in simplified form
in Figure 4. From a redox balance for the anaerobic phase the
maximum succinate yield from 1 mol of glucose is 1.71 mol
(1.12 g succinate per gram glucose), which is in accordance
with previous results (23).
The redox balance assumes activity of the enzyme isocitrate
lysase, a key enzyme in the glyoxylate shunt. It has previously
been reported that AFP111 shows isocitrate lyase activity after
aerobic growth (23). The yield obtained in this study from
glucose was 1.27 mol succinate per mole glucose, which is 74%
of the theoretical. If generation of new cells during the anaerobic
phase is considered, a carbon balance can account for more than
95% of the carbon.
The theoretical yield from fructose is 1.20 mol succinate per
mole fructose, which corresponds to a mass yield of 0.79,
although this requires another electron acceptor such as ethanol,
which was not detected. A redox balance without considering
ethanol yields 1 mol succinate and 1 mol pyruvate per mole
fructose. A molar yield of 1.00 was achieved (mass yield 0.66)
for succinate from pure fructose. Redox balances under anaerobic conditions indicate that 2.5 times greater flux through the
reductive arm is required to match the flux through the oxidative
TCA plus the glyoxylate shunt for either glucose or fructose as
a substrate. During fructose fermentation by AFP184, phos-
phoenolpyruvate (PEP) is converted to pyruvate by the PTS

(29, 37), with 1.0 mol fructose yielding 1.0 mol PEP and 1.0
mol pyruvate due to transport requirements. This metabolite
imbalance prevents a balanced redistribution of reducing
equivalents, and as a result pyruvate will accumulate and be
excreted (38) as was observed in Figure 1b. Glucose mixed with
fructose should provide an increased pool of PEP to balance
the pyruvate generated by fructose transport and result in higher
yields than fructose alone. However, experimental results do
not confirm this hypothesis (Table 1), although much more
pyruvic acid was excreted initially with fructose/glucose
mixtures relative to fermentation of pure fructose (12.4 vs 5.2
g L-1). The accumulation of higher concentrations of pyruvic
acid for the mixed substrate fermentation is suspected to be due
to the more rapid uptake of both sugars, resulting in a faster
intracellular accumulation of pyruvate with no available electron
sink for further metabolism. For both fermentations, most of
this excreted pyruvic acid was taken up again, requiring the
expenditure of reducing equivalents for active transport (39).
This excretion and reutilization of pyruvate can explain both
the low yields and the lower rates found for fructose/glucose
fermentation.
A redox balance for xylose gives a maximum theoretical yield
of 1.43 mol succinate per mole xylose. In the case of xylose
only about 45% of the theoretical yield was achieved (0.64 mol
mol-1). By carrying out a carbon balance, 60% of the carbon
can be accounted for. Further work is required to identify the
remaining carbon. When xylose was mixed with glucose, the
yield increased to 0.83 mol succinate per mole sugar, which
was expected because glucose gives higher yields.
When glucose was used as the carbon source, the productivity
per viable cell decreased drastically after 16 h (Figure 2a).
Succinic acid productivity as a function of succinic acid
concentration showed a sharp decrease in productivity at
approximately 30 g L-1 (Figure 2b). The succinic acid concentration at 16 h corresponds to almost 30 g L-1. From this result
it can be concluded that as long as the cells are viable, they
retain their productivity until the concentration of succinic acid
is not too high. Fructose and xylose did not show any drastic
decreases in productivity (Figure 2). Neither of these fermentations achieved succinate concentrations of more than 20-25 g
L-1 during the first 20 h of the fermentations (Figure 1b and
c). In the mixed-sugar fermentations glucose/xylose mixtures
did not result in very high succinate concentrations and no
decrease in productivity was observed (Figure 3c and d). When
glucose and fructose were mixed and fermented, a sharp
productivity decrease was observed after 16 h (Figure 3a) when
the concentration of succinate was approximately 20 g L-1
386
Figure 4. Anaerobic glucose, fructose, and xylose metabolism of AFP184 for maximal succinate production. Note that some metabolites are
excluded. The reactions blocked by the mutations in AFP184 are indicated by crosses.
(Figure 1d). When glucose was used as the carbon source,

productivities did not decline until concentrations of 30 g L-1
were reached. In the other fermentations the concentrations of
end products other than succinate were low, but in this
fermentation the concentration of pyruvic acid was 10 g L-1
after 16 h and the total concentration of end products exceeded
30 g L-1 (Figure 1d). From these results it seems likely that
high final concentrations of end products inhibit productivity.
Many investigations regarding the inhibition effects by organic
acids on E. coli growth and productivity have been carried out
(40-42). The effects of organic acid inhibition is greatest at
low pH when the acids are undissociated, but even at neutral
pH high concentrations of the acids have shown inhibitory
effects (42). The fermentations in this study were carried out
with ammonium hydroxide as base for neutralization of end
products, and it has been reported that ammonium in concentrations above 3 g L-1 inhibits growth (43, 44). In the present
work ammonium concentrations of approximately 10 g L-1 were
obtained at the end of the fermentations with high succinate
production. In other studies changes in the growth characteristics
of E. coli under different ammonium sulfate concentrations was
investigated, and ammonium sulfate concentrations of 5% had
to be used before any severe inhibition was observed (45). When
the fermentations carried out in the present work showed
productivity decreases, they were in the anaerobic phase and
only very little growth would take place even without any
inhibition; it is not clear if productivity would be affected by
the achieved ammonium concentrations. Possible ammonia

inhibition is being further investigated in studies where different
bases are compared to better understand the causes of the
observed productivity decrease.
Finally, a comment should be made regarding the volumetric
productivities achieved. As mentioned in the Introduction,
biological production of succinic acid should preferably reach
volumetric productivities of 2.5 g L-1 h-1 to be feasible. In
this study volumetric productivities during anaerobic production
in the range of 1.5-2.9 g L-1 h-1 were demonstrated. The
reason the productivities reach almost 3 g L-1 h-1 is because
the cells after the switch to the anaerobic phase are not sugarlimited. A high initial sugar load was used, but the same results
could be achieved by feeding a high concentration sugar stream
after the end of the aerobic phase. The total volumetric
productivity is directly dependent on the cell density, and to
achieve a high cell density requires the use of substantial
amounts of the carbon source. This of course could compromise
the overall process economics, and the necessary cell density
for feasible production of succinic acid must be considered if
the technology is to be applied to a large scale production
facility.
Conclusions
In this paper we have demonstrated growth and succinate
production of E. coli strain AFP184 in a medium containing
corn steep liquor, salts, and different mono- and disaccharides.
The reduction of complex components in the medium has a

positive effect on the final price of the produced succinate due
to lower cost of raw materials; however, final concentrations
are in the range of 30-40 g L-1 after 32 h and need to be
improved for the process to become more economical. Studies
will be directed to increase the final titer and to further improve
the process.
Acknowledgment
This research was supported by Diversified Natural Products
Inc. and the Research Council of Norrbotten. Equipment was
provided by the Kempe Foundation.
References and Notes

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Received October 2, 2006. Accepted December 21, 2006.
BP060301Y
Paper II
Inhibition of Succinic Acid Production in Metabolically Engineered

Escherichia Coli by Neutralizing Agent, Organic Acids, and Osmolarity
Christian Andersson, Jonas Helmerius, David Hodge, Kris A. Berglund, and Ulrika Rova
Div. of Biochemical and Chemical Process Engineering, Lulea University of Technology, SE-971 87, Lulea, Sweden
DOI 10.1021/bp.127
Published online February 5, 2009 in Wiley InterScience (www.interscience.wiley.com).
The economical viability of biochemical succinic acid production is a result of many processing parameters including nal succinic acid concentration, recovery of succinate, and the
volumetric productivity. Maintaining volumetric productivities [2.5 g L1 h1 is important
if production of succinic acid from renewable resources should be competitive. In this work,
the effects of organic acids, osmolarity, and neutralizing agent (NH4OH, KOH, NaOH,
K2CO3, and Na2CO3) on the fermentative succinic acid production by Escherichia coli
AFP184 were investigated. The highest concentration of succinic acid, 77 g L1, was
obtained with Na2CO3. In general, irrespective of the base used, succinic acid productivity
per viable cell was signicantly reduced as the concentration of the produced acid
increased. Increased osmolarity resulting from base addition during succinate production
only marginally affected the productivity per viable cell. Addition of the osmoprotectant glycine betaine to cultures resulted in an increased aerobic growth rate and anaerobic glucose
consumption rate, but decreased succinic acid yield. When using NH4OH productivity completely ceased at a succinic acid concentration of 40 g L1. Volumetric productivities
remained at 2.5 g L1 h1 for up to 10 h longer when K- or Na-bases where used instead of
NH4OH. The decrease in cellular succinic acid productivity observed during the anaerobic
phase was found to be due to increased organic acid concentrations rather than medium
C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 116
osmolarity. V
123, 2009
Keywords: succinic acid, neutralizing agent, product inhibition, cell viability, osmotic stress
Introduction
Increased environmental concern and cost of petroleum
has motivated the search for cost-effective alternatives
for transforming relatively inexpensive biomass into fuels
and chemicals through thermochemical and biochemical
conversion. The key to success in the development of
protable industrial biochemical conversion technologies
is the choice of target fermentations that can compete
with the efciency of the petrochemical industry. For this
purpose, it is essential to develop fermentations that produce molecular building blocks, which can be used as
precursors for the production of a number of high-value
chemicals or materials. This building block concept follows much of the same strategy that is used by the petrochemical industry, i.e., production of high-value
chemicals from a limited number of chemical intermediates. In 2004, U.S. Department of Energy (USDOE) identied 12 sugar-derived chemicals that could be produced
from both lignocellulose and starch and serve as an economic driver for a biorenery.1 Succinic acid is consid-
Correspondence concerning this article should be addressed to U.

Rova at ulrika.rova@ltu.se.
116
ered as one of the more promising of these building

blocks. In addition to its direct use as a food ingredient
and chemical, succinic acid has the potential to produce
a wide range of products and derivatives, e.g., green solvents and biodegradable plastics.2 Succinate is traditionally manufactured from maleic anhydride through nbutane using petroleum as raw material.
Succinic acid could also be produced by biochemical
conversion of biomass using fungal or bacterial fermentations. Production of succinic acid has been demonstrated
in a number of bacteria.37 A number of Escherichia coli
(E. coli) mutants has recently been developed for succinate production.3,810 In this study, E. coli AFP184, a
metabolically engineered strain with mutations in the glucose specic phosphotransferase system (ptsG), the pyruvate formate lyase system (p), and in the fermentative
lactate dehydrogenase system (ldh), was used.11 AFP184
has been shown to produce succinic acid to nal concentrations of 2540 g L1 with productivities in the range
of 1.53 g L1 h1 from glucose, fructose, and xylose
using a low-cost industrially relevant medium.12 The productivities, although initially very high, declined during
the anaerobic phase of the fermentations. This loss of
productivity and cell viability during the anaerobic production phase might be caused by organic acid inhibition,
a well-known phenomenon in E. coli.13,14 The inhibitory
C 2009 American Institute of Chemical Engineers
V
effects can be due to both the difference between external and internal pH and specic effects on the metabolism caused by the organic acid anion.1517 Acetate, for
example, has been shown in E. coli to inhibit enzymes in
the methionine pathway leading to accumulation of homocysteine, which is inhibitory to growth.18 Another factor known to affect cell growth and product formation is
high medium osmolaritiy due to unfavorable salts and
sugar concentrations.19 Ethanologenic E. coli have, for
example, been shown to divert more carbon into production of osmolytes when osmotically stressed.20 Addition
of neutralizing agent to maintain a neutral fermentation
pH might result in an osmotic environment unfavorable
for succinic acid production. If biobased production of
succinic acid should be economically feasible, the volumetric productivity should be kept higher than 2.5 g L1
h1 for as long as possible. A high nal titre, although
not as integral to the process as the volumetric productivity, is important for reducing the cost of downstream
processing.1
Comprehensive summaries of rates and yields for different
strains have been published.10,21 In general, work done with
E. coli or other succinic acid producing organisms all show
similar trends; high initial productivities that decline during
the fermentation. The efciency of the fermentations with
respect to production rate, yield, and nal acid titer in batch
fermentations depends on a number of factors including organism robustness, media components, and genetic engineering of the organism. Most of the work done so far has
focused on metabolic engineering or optimization of the
growth medium used, thus temporarily postponing but not
solving the inhibitory problem of the production method. For
example, a metabolically engineered E. coli mutant with the
Rhizobium etli pyruvate carboxylase gene was able to produce up to 99.2 g L1 of succinate in 70 h of anaerobic fermentation when rich media was used.22 During the rst 25 h
of anaerobic production, this strain achieved a succinate concentration of 70 g L1, corresponding to a volumetric productivity of 2.8 g L1 h1. However, over the course of the
fermentation the productivity decreased, resulting in a total
volumetric productivity of 1.4 g L1 h1. In a recent study,
an E. coli strain was developed to grow and produce succinate in minimal salt media.10 Here, a volumetric productivity
of 1.2 g L1 h1 was achieved for the rst 48 h resulting
in 60 g L1 of succinate. The maximum concentration
obtained, 86 g L1, was reached after 120 h giving a productivity of 0.7 g L1 h1. This raises the question whether organic acid toxicity, osmotic stress generated by the produced
acids and added base, or the chemical properties of the base
reduces the productivity.
The toxicity of succinic acid has not been reported before
for E. coli, and therefore the objective of this work was to
uncouple the effect of succinic acid vs. base and osmolarity
to demonstrate quantitatively how increased production of
succinate effects productivity. Differences in succinic acid
productivity, titer, yield, and cell viability when using ammonia, alkali hydroxides, and alkali carbonates as neutralizers were studied by conducting fermentations with
NH4OH, KOH, NaOH, K2CO3, and Na2CO3. Effects of organic acid toxicity and osmotic stress were studied in fermentations with externally added succinic acid, sodium
buffer, or by supplementing the growth medium with the
osmoprotectant glycine betaine.
117

Strain and seed culture preparation
The E. coli strain AFP184, which lacks functional genes
coding for pyruvate formate lyase, fermentative lactate dehydrogenase, and the glucose phosphotransferase system,11 was
used in this study. Cultures were diluted to 70% with glycerol and stored at 80 C. Seed cultures were prepared by
inoculating 100 mL sterile medium (same medium as used
for the batch fermentation, see below) in a 500-mL shakeask with 200 lL of the stock culture. The seed culture was
incubated at room temperature (22 C) in an orbital shaker at
200 rpm for 16 h.
Fermentations
Media and Growth Conditions. All batch fermentations,
consisting of an aerobic growth phase (89 h) and an anaerobic production phase (30100 h), were conducted in 1 L
bioreactors (Biobundle 1L, Applikon Biotechnology, the
Netherlands) with a total starting volume of 700 mL (including 35 mL seed culture and 200 mL glucose solution). The
growth medium contained the following components in
g L1: K2HPO4, 1.4; KH2PO4, 0.6; (NH4)2SO4, 3.3; MgSO4
7H2O, 0.4; corn steep liquor (CSL; 50% solids, SigmaAldrich), 15; and antifoam agent (Antifoam 204, SigmaAldrich). The bioreactor was sterilized with the medium at
121 C for 15 min; thereafter, 200 mL of a separately sterilized glucose solution (350 g L1) and 35 mL seed culture
were aseptically added, resulting in a nal volume of 700
mL and a total glucose concentration of 100 g L1. The fermentation temperature and pH were controlled at 37 C and
6.56.7, respectively. During the aerobic phase pH control
was achieved by automatic addition of NH4OH (15%, v/v;
NH3 solution). The dissolved oxygen concentration (%DO)
was measured by a pO2 electrode. The agitation speed was
varied between 500 and 1000 rpm. During the aerobic
growth phase, the culture medium was aerated with an air
ow of 5 L min1. When the optical density at 550 nm
(OD550) reached a value of 3035 (after 89 h), the anaerobic production phase was initiated by withdrawing the air
supply and sparging the culture medium with CO2 at a ow
rate of 0.8 L min1. The agitation speed was set to 500 rpm
for the anaerobic phase, during which succinic acid was produced. To sustain acid production, 40 mL sterilized glucose
solution (600 g L1) was added after 23 and 40 h of total
fermentation time. The anaerobic phase proceeded for 100 h,
or until the succinic acid production ceased. During the fermentations, samples were aseptically withdrawn for analysis
of optical density, viable cells, sugars, and organic acids
concentrations.
Fermentations with Different Neutralizing Agents. To
compare the effects of different neutralizing agents on succinic acid productivity, nal titer, and cell viability, different
bases NH4OH (15%, v/v; NH3 solution), KOH (10 M),
NaOH (10 M), K2CO3 (4 M), or Na2CO3 (2 M), were used
for pH control during the anaerobic phase. All fermentations
were carried out in triplicate except for the fermentations
using NH4OH or K2CO3.
Effects of Increased Osmolarity, Succinic Acid
Concentration, and Glycine Betaine. During the anaerobic
phase, succinic acid or Na in the form of a sodium phosphate buffer, pH 6.6, were added to fermentations, which
used 2 M Na2CO3 as the anaerobic neutralizing agent.
118
Figure 1. Succinic acid (g L21) (a), glucose (g L21) (b), and viable cell (cells per liter 3 1012) (c) concentrations for fermentations neutralized with KOH, NaOH, K2CO3, Na2CO3, and NH4OH.
Symbols used are as follows: ^, KOH; &, NaOH; , K2CO3; D, Na2CO3; *, NH4OH. The broken lines indicate the transition to the anaerobic
phase. Fermentations were done in triplicate and the values are averages of the data range (error bars).
A stock solution of succinic acid, 140 g L1, was prepared

and neutralized with KOH. Addition of succinic acid or
Na, 30 mL, respectively, were made 4 h into the anaerobic
phase and then every 2 h until a total of 150 mL had been
added, corresponding to 30 g L1 of succinic acid or a sodium concentration of 12.5 g L1. In the later case, this represents the amount of sodium required to neutralize 30 g
L1 of succinic acid.
Fermentations with an initial glucose concentration of 100
or 150 g L1, respectively, and neutralized with 2 M
Na2CO3 were supplemented with glycine betaine to a nal
concentration of 50 mM, which based on cell concentration
is on the same order of magnitude as previous work.19 Three
glucose additions (3 24 g) were done during the anaerobic
phase of the fermentation with a starting glucose concentration of 100 g L1. A standard fermentation without glycine
betaine addition and with an initial glucose concentration of
100 g L1 and two glucose feedings (24 g) was included as
a reference.
Analysis
Cell concentration was monitored by spectrophotometry
using OD550 correlated to dry cell weight.12 To establish the
number of viable cells, 10-fold serial dilutions of the fermentation samples in 0.9%, w/v NaCl were plated on tryptone
soy agar plates and incubated overnight at 37 C. The number
of colonies was calculated and the number of viable cells
was expressed as cells per liter fermentation broth. All dilutions were made in duplicate. Organic acids and sugars were
detected and quantied by HPLC as previously described.12
Results
Fermentations with different neutralizing agents
Standard dual-phase fermentations were carried out with
NH4OH, KOH, NaOH, K2CO3, or Na2CO3 as neutralizing
agents. The total cell mass (gram dry cells in the culture)
generated was similar for all neutralizing agents (data not
shown) and no growth was detected in the anaerobic phase
in any of the fermentations carried out. Instead, the optical
density continuously decreased due to dilution and cell lysis.
All four alkali-bases resulted in similar fermentation proles
(Figures 1a,b), where the highest nal succinic acid concentration, 77 g L1, was achieved when Na2CO3 was used as
base. Using NaOH resulted in 69 g L1, K2CO3 in 64 g L1,
and KOH in 61 g L1. The only by-product formed was acetic acid and the lowest concentration, 4.6 g L1, was
obtained when Na2CO3 was used. Fermentations with
NH4OH, NaOH, KOH, and K2CO3 resulted in acetic acid
concentrations of 5.5, 5.7, 6.0, and 5.1 g L1, respectively.
All acetic acid was produced during the anaerobic phase.
When NH4OH was used for neutralization a maximum succinic acid concentration of 43 g L1 was obtained and succinic acid production completely stopped after a total
fermentation time of 32 h (Figure 1a). Yields were in the
range of 0.8 g succinic acid per gram glucose consumed in
the anaerobic phase (1.22 mole mole1) for all fermentations
(Table 1).
Viable cell concentrations for fermentations using NaOH
or KOH as the pH regulator showed similar trends. During
the rst 20 h of anaerobic conditions, the viability of the
cultures decreased signicantly, but for the remainder of
the fermentations only small losses in viability occurred
119
Table 1. Summary of Fermentation Results for Different Bases

Parameters*
Base
Yp/s (g g1)
anaerobic
Qp (g L1 h1)
anaerobic, T20
Qp (g L1 h1)
anaerobic, T40
Qp (g L1 h1)
anaerobic, (max g L1)
NH4OH
KOH
NaOH
K2CO3
Na2CO3
0.75
0.84 0.09
0.78 0.09
0.82
0.75 0.03
2.91
2.62 0.01
2.47 0.09
3.02
2.95 0.14
1.38
1.46 0.08
1.61 0.06
1.76
2.04 0.07
1.85
0.67 0.02
0.76 0.03
0.72
1.05 0.17
* Yp/s is the mass yield of succinic acid based on the glucose consumed in the anaerobic phase. Qp is the volumetric productivity of succinic acid during the anaerobic phase after 20 h (T20) or 40 h (T40) total fermentation time, calculated as grams succinic acid produced during the anaerobic phase per
litreliter fermentation medium per hour. Anaerobic productivity calculated either when fermentations are terminated or maximum succinic acid concentration is achieved. Maximum concentration was achieved after 32 h.
Figure 2. Productivity per viable cell (g cell21 h21) 3 1014 (a) and volumetric productivity (g L21 h21) (b) as functions of time.
Productivities are calculated for the anaerobic phase. Symbols used are as follows: ^, KOH; &, NaOH; , K2CO3; D, Na2CO3; and *, NH4OH.
Fermentations were done in triplicate and the values are averages of the data range (error bars).
(Figure 1c). Using K2CO3 and Na2CO3 resulted in improved

cell viability during the anaerobic phase relative to KOH and
NaOH (Figure 1c).
The use of alkali carbonates for neutralization gave higher
productivities than the alkali hydroxides (Table 1). In general, productivities per viable cell were initially high, but
decreased after 20 h of total fermentation time (Figure 2a).
When using NH4OH the succinate productivity completely
stopped after 32 h, whereas the other fermentations showed
gradually decreasing productivities during the remaining anaerobic phase (Figure 2a). At the onset of the anaerobic
phase, volumetric productivities reached initial values of 3
3.5 g L1 h1, but decreased signicantly after 2025 h of
total fermentation time for fermentations neutralized with
KOH, NaOH, K2CO3, and Na2CO3 (Figure 2b). At this time,
the productivity in NH4OH fermentations was well below 1
g L1 h1.
Effects of increased osmolarity, succinic acid
concentration, and glycine betaine
Fermentations in which 150 mL of either a 140 g L1 succinic acid solution or a sodium phosphate buffer were added
gradually during the anaerobic phase were carried out. The
amount of succinic acid produced when the buffer was added
was signicantly higher than when the succinic acid solution
was added (Figures 3a,b). In neither of the fermentations
were the viable cell concentration negatively affected (Figure
3c). Externally added succinic acid resulted in a decreased
anaerobic productivity per viable cell as the total succinic
acid concentration increased (Figure 3d).
Fermentations with varying initial glucose concentration
(100 or 150 g L1) with or without addition of the osmopro-
tectant glycine betaine to a concentration of 50 mM were

carried out using Na2CO3 for neutralization. A standard fermentation without glycine betaine addition and with an initial glucose concentration of 100 g L1 and two intermittent
glucose feedings (2 24 g) was included for comparison
(described above under the section, fermentations with different neutralizing agents). Compared with the reference run
the fermentation with glycine betaine added and glucose at
100 g L1 resulted in faster growth and consequently a
shorter growth phase (7 h), a higher glucose consumption rate,
similar succinic acid productivities, but a signicantly lower
yield (Table 2). The nal succinic acid concentration was 65 g
L1 with a yield of 0.57 g g1. Using an initial glucose concentration of 150 g L1 with or without glycine betaine
resulted in nal succinic acid concentrations of 59 and 65 g
L1, respectively, (Table 2). When glycine betaine was added,
the aerobic growth time needed to obtain an optical density of
35 was 8.5 h, whereas it was 11.5 h without. The sugar consumption rate was considerably higher when glycine betaine
was added and all glucose was metabolized in less than 50 h.
The anaerobic glucose consumption rate was signicantly
higher in the presence than in the absence of glycine betaine
(2.5 g L1 h1 compared with 1.5 g L1 h1). Viable cell
concentrations and productivities per viable cells were not
affected by the osmoprotectant (data not shown). The acetate
level in all fermentations was low 3.33.9 g L1.
Discussion
Effects of neutralizing agent on succinic acid production
When constructing processes for biobased production of
fuels and chemicals it is important to consider how changes
120
Figure 3. Fermentations with succinic acid or salt-buffer addition.

(a) Total (^), produced (~), and added (n) succinic acid concentration when additional succinic acid was added. (b) Succinic acid concentration
equivalent to the amount of added sodium ions (^) and produced succinic acid (~). (c) Viable cells per liter 1012 for fermentation with succinic
acid (^) and Na-buffer (&) addition. (d) productivity per viable cell (g cell1 h1) 1014 for fermentation with succinic acid (^) and Na-buffer
(&) addition. Viable cell counts were done in duplicate and the values are averages of the data range (error bars). The broken lines indicate the transition to the anaerobic phase. In all gures, () represents a control using 2 M Na2CO3.
Table 2. Summary of Fermentations Parameters for Fermentations with Glycine Betaine and/or Higher Initial Glucose Concentration
Parameters*
Fermentation
SA
(g L1)
Yp/s
(g g1)
Time
(hours)
Qp (g L1 h1)
anaerobic, T20
Qp (g L1 h1)
anaerobic
Qc (g L1 h1)
anaerobic
Reference
Betaine
Glucose 150 g L1
Glucose 150 g L1 and betaine
72
65
65
59
0.73
0.57
0.79
0.66
80
72
80
48
2.95
2.98
2.41
2.48
1.22
1.20
1.07
1.72
1.62
2.11
1.36
2.62
* Yp/s is the mass yield of succinic acid based on the glucose consumed in the anaerobic phase, and Qp is the volumetric productivity of succinic acid
during the anaerobic phase after termination of fermentation, 20 h (T20) total fermentation time. Qc is the volumetric glucose consumption rate during
the anaerobic phase after termination of fermentation. Calculations are done for 80 hours fermentation time in order to be comparable with the other
experiments in the series.
in the fermentation phase might affect downstream processing operations. Separation of the products as well as recovery and recycling of chemicals used in the process is
essential in order to obtain good plant economics. An efcient downstream process conguration for the recovery of
succinic acid that permits internal recycle of chemicals has
previously been demonstrated.23 The method involves formation of diammonium succinate, which is accomplished by
using NH4OH as neutralizing agent during the fermentation.
High concentration of ammonia has been shown to negatively effect E. coli growth,24 and it is possible that ammonia
can account for the observed decrease in succinate productivity by E. coli AFP184 during the anaerobic phase.12 The
effects on succinate production when replacing the neutralizing agent were studied. The main requirements of the
selected bases were that they should be low-cost and be
compatible with the proposed recovery process; hence, different monovalent alkali-bases were selected. As a macronutrient for E. coli growth, potassium is involved in a number
of fundamental biological processes including maintaining
the osmotic balance of cells.25 NaOH is a widely available
low-price commodity chemical, and E. coli has been
reported to grow in media containing high concentrations of
sodium.26 Na2CO3 and K2CO3 were used to investigate if
additional carbonate would increase the carbon ux to succinate. Other options regarding choice of neutralizing chemicals exist; divalent bases of alkaline earth metals such as
calcium hydroxide or carbonate could be considered. The
use of calcium bases, however, would interfere with the recovery and recycling operations. Therefore, divalent alkaline
earth metal bases were excluded from the scope of this
investigation.
From the fermentation proles (Figure 1), it is clear that

using potassium or sodium bases for neutralization would be
benecial for the nal succinic acid concentration. Compared
with fermentations neutralized with NH4OH, fermentations
neutralized with potassium or sodium bases obtained
increased nal titers by at least 50% and in the case of
Na2CO3 almost 100%. It should be noted that unlike
NH4OH fermentations, succinate production in alkali neutralized fermentations never ceased. Other studies have demonstrated that ammonia challenges the integrity of the outer
membrane of E. coli, reducing growth24 and at concentrations [3 g L1 ammonia is known to inhibit growth.27
Using NH4OH as a neutralizing agent generated ammonia
concentrations of more than 10 g L1 after 16 h anaerobic
succinate production. Both the concentration of viable cells
and succinate productivity (Figures 1c and 2) decrease rapidly, indicating that concentrations of this magnitude are
toxic to the organism.
With regards to productivity and nal acid concentrations
the difference between using NaOH or KOH was marginal
(Figures 1 and 2). However, using Na2CO3 and K2CO3
resulted in an increased volumetric productivity compared
with NaOH and KOH. The increased productivity is likely
caused by an increased availability of hydrogen carbonate
(HCO
3 ) from the addition of the base chemical. The enzyme
phosphoenolpyruvate (PEP) carboxylase catalyzing the carboxylation of PEP to oxaloacetic acid uses HCO
3 as a substrate for the reaction.28 The higher productivity during
neutralization with CO3 bases indicates that the medium is
not saturated by the sparged CO2. The additional availability
of HCO
3 seem to result in an increased metabolic ux toward succinate. It is clear from the results that the preferred
neutralizing agent would be Na2CO3, because it generated
the highest productivities, nal titers and had the lowest byproduct formation. However, if considering the yield, the
choice of base is not as obvious as all fermentations resulted
in an average yield of 0.8 g succinate per gram glucose consumed during the anaerobic phase, which constitutes 71% of
the theoretical maximum (1.12 g g1).3,12
Effects of organic acids and osmotic stress on succinic

acid production
From the experiments carried out with addition of either
succinic acid or sodium buffer, it is clear that the main reason for the decrease in productivity per viable cell is the
increase in succinic acid concentration during the anaerobic
production phase (Figure 3). The osmolarity of the medium
appears to have only marginal effects on succinate productivity. This conclusion is further substantiated by the results
from the fermentations supplemented with glycine betaine
(Table 2). There are numerous studies and reviews on osmotic stress and osmotolerance in bacteria.26,2931 E. coli
subjected to osmotic stress respond by accumulating compatible solutes, such as glycine betaine, proline, and trehalose.32,33 Of these, the solute offering the highest
osmotolerance in E. coli is glycine betaine.19 E. coli can
only accumulate glycine betaine or proline if supplemented
in the medium or by a two-step oxidation of externally provided choline. CSL contain some proline, but no glycine betaine. In the fermentations supplemented with glycine
betaine, the cellular succinic acid productivity was not
affected (data not shown), but the anaerobic glucose consumption rate was increased resulting in a decreased succi-
121
nate yield suggesting synthesis of other fermentation

products (Table 2). However, the acetic acid concentration
was not increased and no other fermentation products were
detected. Using a higher initial glucose concentration (150 g
L1) and hence a higher medium osmolarity, increased the
duration of the aerobic growth phase necessary to reach an
OD550 of 35. With glycine betaine added to the growth medium this effect was cancelled. Although the medium osmolarity does not seem to affect succinate productivity, it could
still be proposed that the viable cell loss could be due to the
high osmolarity of the medium that is generated during the
anaerobic phase ([1.5 osmoles per liter from neutralized
succinate alone). The repressed growth observed when high
glucose concentrations were used also point to osmolarity
having a negative impact on the cellular metabolism. Under
anaerobic conditions, or in media deprived of other osmoprotectants, E. coli synthesize trehalose intracellularly.33,34 It
has been shown that increased intracellular concentrations of
trehalose in ethanologenic E. coli did not improve growth in
the presence of formate, lactate, or acetate, suggesting that
another mechanism than osmotic stress is responsible for
growth inhibition in cultivations with weak organic acids.20
The same result was observed in this study, i.e., glycine betaine did not improve the anaerobic cell viability, suggesting
that in this study the reduced viability in the anaerobic phase
is related to the succinic acid concentration and not the medium osmolarity. Studies with ethanologenic E. coli in high
osmolaritiy CSL media with xylose as the sugar source have
shown that addition of betaine improved growth, but did not
signicantly affect ethanol production.19 In contrast, studies
with E. coli engineered for lactic acid production has shown
that betaine greatly increased the volumetric lactic acid productivity.35 In this investigation glycine betaine improved
the aerobic growth at high osmolarities, but affected the anaerobic succinic acid yield negatively. Response and effectiveness of betaine as an osmolyte has been reported to be
dependent on the nature of the fermentation process, i.e.,
media composition and growth conditions,19 which might
explain the different results obtained.
It can be concluded that addition of glycine betaine and
most likely any other agent increasing the organisms osmotolerance does not benet succinic acid production by
AFP184. These results suggest that the osmolarity and ionic
strength (salt concentration) of the medium are of little importance for cellular succinic acid productivity. Not even at
osmolarities of the same magnitude as when succinic acid
concentrations are 60 g L1 ([1.5 Osmoles) does the productivity per viable cell decrease, instead the produced organic acids are responsible for the reduced productivity.
Organic acid toxicity is well known to affect cell growth and
limit product formation in E. coli36 and if the main product
is the organic acid itself, it also limits any industrial production. During anaerobic succinate production organic acids
can affect cells both by lowering the cytoplasmic pH (pHi),
which can have detrimental effects on the function of cellular proteins and enzymes36,37 and by the increased intracellular concentration of the acid anion.18 The extracellular pH in
this study was controlled between 6.5 and 6.7. In this pH
interval, succinic acid with pKa of 4.19 and 5.57 will be
present in its dissociated form ([99.6%) and will thus not
channel protons into the cytoplasm lowering the pHi. In contrast, it has been suggested that dissociated lactate and acetate can traverse the E. coli cell membranes catalytically
dissipating the proton motive force.14 If this also applies for
122
succinate it purports that accumulation of the succinate anion

in the cytoplasm would be responsible for the observed
decrease in productivity. Although acetate is a potent inhibitor of E. coli growth,38 the concentrations obtained in this
study are not high enough to solely be the cause of the
observed decrease in glucose consumption and succinate productivity. Rather the total load of organic acids must be considered. The inhibitory potential of different organic acids
varies and is related to the hydrophobicity of the acid.13
Acetic acid would thus be a stronger inhibitor than succinic,
but the data obtained in this work does not indicate that the
acetate concentrations achieved would be detrimental to succinate production. Nevertheless, in an effort to maximize
succinate productivity and yield as well as from a downstreaming perspective, it is desirable to minimize the amount
of produced acetate.
Conclusions
In this study we have demonstrated that replacing the neutralizing agent can provide substantial process improvements
in the form of increased duration of high volumetric productivity and increased nal titer. Compared with fermentations
neutralized with NH4OH, it was possible to achieve an
almost 100% increase in nal succinic acid concentration
using Na2CO3. The decrease in cellular succinic acid productivity observed during the anaerobic phase was found to be
due to increased organic acid concentrations rather than medium osmolarity. It was also observed that the cell viability
decreased during the anaerobic phase irrespective of the base
used. The viable cell loss is attributed to increased acid concentration coupled with a possible cytoplasmic accumulation
of succinate. Further studies will be directed toward increasing the duration of high anaerobic succinic acid production
by investigating different methods to circumvent the impairing effects of the generated organic acid load.
Acknowledgment
This research was supported by Diversied Natural Products
Inc. and the Research Council of Norrbotten. Equipment was
provided by the Kempe Foundation.
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Paper III
Maintaining High Anaerobic Succinic Acid Productivity by Cell

Resuspensions
Christian Andersson1, Ekaterina Petrova1, Kris A. Berglund1,2, and Ulrika Rova1*

1
Division of Biochemical and Chemical Process Engineering, Lule University of

Technology, SE-971 87 Lule, Sweden
Departments of Forestry and Chemical Engineering, Michigan State University, East

Lansing, MI 48824 USA
* To whom all correspondence should be addressed

Ph: +46920491315. Fax: +46920491199. Email: Ulrika.Rova@ltu.se
Abstract
Dual-phase fermentations with Escherichia coli AFP184, a strain engineered for succinic
acid production, is a standard approach to succinate production having initially very high
productivity. However, the productivity and viable cell concentration decrease during the
fermentation as the concentration of succinic acid increases. The productivity decrease is
a direct effect of the product organic acids. To investigate the impact of succinic acid
concentration on succinate productivity and cell viably during AFP184 fermentations,
cells were harvested and resuspended in fresh media after selected fermentation times
corresponding to different succinic acid concentrations. Resuspending the cells restored
the cellular succinic acid productivity, but had no effect on cell viability, which
continuously decreased during the fermentations and thus reduced the volumetric
productivity. By resuspending the cells, the amount of succinic acid produced during a
100 hour fermentation was increased by more than 60%. The results demonstrate that by
product removal succinic acid productivity can be maintained at high levels for extended
periods of time.
Keywords: Succinic acid, fermentation, cell resuspension, productivity, cell viability
Introduction
The versatility of organic acids has rendered them highly interesting as building block
chemicals for the bio-based chemicals industry. Succinic acid can be produced by
fermentation and converted into a variety of commercially valuable products including:
diesel fuel oxygenates for particulate emission reduction; biodegradable, glycol free, low
corrosion deicing chemicals for airport runways; glycol free engine coolants;
polybutylene succinate (PBS), a biodegradable polymer that can replace polyethylene and
polypropylene; and non-toxic, environmentally safe solvents that can replace chlorinated
and other VOC emitting solvents [27, 29]. Currently, as most of the potential applications
for succinic acid are covered by fossil-based carbon in the form of maleic anhydride, new
development for succinic acid has been limited. The primary reason for the use of maleic
anhydride, which is produced by oxidation of n-butane, has been lower manufacturing
costs. However, due to increased environmental awareness, improvements in biological
succinic acid production techniques, including genetic refining and process optimisation,
fermentation-based production is currently considered as having feasible economics.
Technologies for biological production of succinic acid from renewable resources have
seen massive improvements in the past five to ten years. Yields and productivities have
increased [5, 15], the span of possible feedstocks has been extended [1, 11, 18], new
strains have been identified and existing strains and organisms have been developed and
improved [6, 10, 12, 14, 24]. Irrespective of the organism used fermentations carried out
in batch mode follow similar trends; productivities are initially high and decline as the
process proceeds. In a previous study we demonstrated that for a succinic acid producing
Escherichia coli mutant, AFP184, it is the product succinic acid that is responsible for the
decrease in productivity [2]. Organic acids are known to inhibit growth and productivity
in E. coli [26], and in order to maintain the initially high productivity it appears essential
to remove the succinic acid from the cells. Many techniques for recovery and purification
of succinic acid exists [9, 21, 28], but to improve the production these operations should
be used simultaneously with the fermentation stage. This approach has proven very
successful in fumaric acid production by immobilised Rhizopus oryzae [4]. Recently, the
use
of
electrodialysis
coupled
with
cell
recycling
was
employed
using
Anaerobiospirillum succiniciproducens for succinic acid production resulting in a

volumetric productivity of 10.4 g L-1h-1 that was maintained for 15 days [17]. Removing
the succinic acid thus assists in sustaining high productivities. With respect to succinic
acid production by E. coli further process improvement is likely to come from the
combination of fermentation with continuous or step-wise removal of the product. Since
the inhibitory effects of succinic acid observed for AFP184 becomes evident first at
concentrations of approximately 40 g L-1, carrying out the fermentations continuously
with a succinate concentration kept below 40 g L-1 could also be a valid solution for
limiting the exposure of the cells to the produced succinic acid. A number of bioreactors
connected in series with product recovery after the last reactor offers the possibility of
operating each reactor and the following separations under optimal conditions once
identified. In this way the amount of complex nutrients, often in the form of corn steep
liquor, yeast extract or a hydrolysate, containing a number of nutrients, salts, and
colouring substances, could be progressively decreased throughout the series of reactors
benefiting the downstream processing of the fermentation broth [20]. Since succinic acid
production with AFP184 is based on dual-phase fermentations (aerobic growth followed

by anaerobic production) this scheme would also allow for the first reactor to be operated
as a seed reactor feeding fresh cells to the first anaerobic reactor.
Previous studies with AFP184 also showed a loss of cell viability during the
fermentations [2, 3]. The loss of viability could be due to poor anaerobic growth
characteristics of the strain in question, but could also be caused by high organic acid
concentrations. The objective of this study was to investigate the effects of succinic acid
on the fermentation kinetics, yield, and cell viability and if it is possible to restore the
initially high succinic acid productivities in cultures exposed to high concentrations of
succinic acid. Succinic acid fermentations were interrupted at pre-determined times, as
different time points correlate to different succinic acid concentrations and varying
physiological states of the cells. The cells were harvested and resuspended in fresh
succinate free media for continued fermentation. As mentioned above, decreasing the
quantities of complex nutrients would benefit downstreaming. On the other hand it could
have detrimental effects on the fermentation. In order to address this possibility, cells
were resuspended in media completely deprived of complex nutrients and their succinate
producing capacity evaluated. This work presents how E. coli AFP184 cells respond to
succinic acid with respect to nutritional requirements, productivity, yield, and cell
viability. The results will assist in the design of future processes with continuous removal
of succinic acid for maximum production.

Strain and Seed Culture Preparation
The E. coli strain AFP184, which lacks functional genes encoding pyruvate formate
lyase, fermentative lactate dehydrogenase, and part of the glucose specific
phosphotransferase system [7], was used in this study. Fermentation seed cultures were
prepared as previously described [2].
Fermentations
Media and the Aerobic Growth Phase
All batch fermentations, consisting of an aerobic growth phase (8-9 h) and an anaerobic
production phase (approximately 50-100 h), were conducted in 1 L bioreactors
(Biobundle 1L, Applikon Biotechnology, the Netherlands) with a total starting volume of
700 mL (including 35 mL seed culture and 200 mL glucose solution). The growth
medium contained the following components in g L-1: K2HPO4, 1.4; KH2PO4, 0.6;
(NH4)2SO4, 3.3; MgSO47H2O, 0.4; corn steep liquor (CSL; 50% solids, Sigma-Aldrich),
15; and antifoam agent (Antifoam 204, Sigma-Aldrich). The bioreactor was sterilised
with the medium at 121 C for 15 min; thereafter 200 mL of a separately sterilised
glucose solution (350 g L-1) and 35 mL seed culture were aseptically added, resulting in a
final volume of 700 mL and a total glucose concentration of 100 g L-1. The fermentation
temperature and pH were controlled at 37 C and 6.5 - 6.7 respectively. During the
aerobic phase pH control was achieved by automatic addition of NH4OH (15% (v/v) NH3
solution). The dissolved oxygen concentration (%DO) was measured by a pO2 electrode.
The agitation speed was varied between 500 and 1000 rpm. During the aerobic growth
phase the culture medium was aerated with an air flow of 5 L min-1. When the optical
density at 550 nm (OD550) reached a value of 30-35 (after 8-9 h) the anaerobic production
phase was initiated.
Anaerobic Succinic Acid Production Phase

Anaerobic conditions were created by withdrawing the air supply and sparging the
culture medium with CO2 at a flow rate of 0.8 L min-1. The neutralising agent was
changed from NH4OH to 2M Na2CO3 and the agitation speed was set to 500 rpm. Cells
were harvested, washed in phosphate buffered saline (PBS) and resuspended in a reactor
filled with fresh, sterilised medium after 23, 40 or 60 hours of total fermentation time. If
cells were not harvested after 23 hours of total fermentation time, 40 mL sterilised
glucose solution (600 g L-1) was added in order to sustain acid production. Another 40
mL was added after 40 hours if cells were harvested after 60 hours. In control
fermentations without cell resuspension, glucose was added as described above after both
23 and 40 hours of total fermentation time. During the fermentations, samples were
aseptically withdrawn for analysis of optical density, viable cells, sugars and organic
acids concentrations.
Resuspension of Cells
Cells were harvested at different times (23, 40, and 60 hours of total fermentation time)
and centrifuged at 5000 rpm for 5 minutes. The cell pellet was washed in PBS,
centrifuged again, resuspended in PBS and used to inoculate a second reactor (as
described above). All the cells from the first reactor were used to inoculate the second
reactor. The inoculum volume was approximately 100 mL (10 mL). The second reactor
was only operated under anaerobic conditions (0.8 L min-1 CO2 flow, temperature 37 C,
and pH 6.5-6.7). Multiple resuspensions were also investigated by harvesting and
resuspending the cells from the first reactor into a second after 23 hours then
resuspending the cells from the second reactor in a third reactor after another 22 hours
(45 hours total time) and finally resuspending the cells from the third reactor in a forth
after an additional 25 hours (70 hours total time). To investigate the impact of removing
complex nutrients on the fermentation characteristics cells were resuspended after 23
hours of total fermentation time in a medium consisting only of glucose (100 g L-1),
KH2PO4/K2HPO4 (0.6/1.4 g L-1) and demineralised water, hereafter called glucose media
(see Media and Aerobic Growth Phase).
Analysis
Cell concentration was monitored by spectrophotometry using OD550 correlated to dry
cell weight [3]. To establish the number of viable cells, ten-fold serial dilutions of the
fermentation samples were plated on tryptic soy agar plates [2]. Organic acids and sugars
were detected and quantified by HPLC using a Series 200 refractive index detector
(PerkinElmer). The column used (Aminex HPX-87H, Biorad) was maintained at 65 C
using a flow rate of 0.6 mL min-1 and 0.005 M H2SO4 as mobile phase.

Producing succinic acid in microbial batch fermentations is a well investigated
technology and there are numerous studies done mainly with the goal of developing
powerful succinate producing biocatalysts [10, 13, 16, 19, 24, 25]. However, performing
batch fermentations that produce succinic acid or any other organic acid has an inherent
drawback due to the inhibiting nature of the acids. Organic acid inhibition has long been
an active research area and it has been shown that organic acids inhibit both growth and
production [8, 26]. Indeed all batch schemes used so far show the same tendencies;
decreasing productivities as the end-product is generated. In order to implement the
developed biocatalysts in industrial installations it is necessary to couple the fermentation
with a recovery process and maintain succinic acid concentrations at a non-inhibiting
level.
In the present study the aim was to investigate the effect of removal of the product
succinic acid on the fermentation kinetics and cell physiology. Removal was achieved by
harvesting the cells and resuspending them in fresh media. The loss in productivity
observed during fermentations does not originate from osmotic stress or the neutralising
agent used, but rather the produced succinic acid [2]. At neutral pH with the organic acids
in their deprotonated form it seems plausible that the inhibitory effects are due to
accumulation of the succinate anion in the cytoplasm. In the same study it was also
observed that the anaerobic viability of the cells was limited and after 100 hours of
anaerobic succinic acid production the viable cell mass was reduced by approximately
80%. The cells where resuspended after 23, 40 or 60 hours. These times where chosen
since they correlate to 40, 55, and 65 g L-1 respectively. At 40 g L-1 there are little or no
inhibitory effects [2] while at higher succinate concentrations the productivity decreases
as a result of organic acid inhibition. Using the simple technique of resuspending succinic
acid producing cells in fresh media it is possible to study the effects of succinic acid
concentration and media composition on the fermentation characteristics of AFP184 and
hence address the questions if succinic acid in concentrations of 40 80 g L-1 negatively
affects cell viability, if complex nutrients are necessary for achieving high succinate
yields and supporting anaerobic viability, and most importantly if removing the acid will
recover or maintain high anaerobic productivity during the early parts of the anaerobic
phase.
For fermentations conducted without resuspension of the cells, the rate of succinate
production decreased after approximately 25 hours of total fermentation time (Fig. 1).
The succinate concentration at this time was about 40 g L-1. Resuspending the cells after
23 hours of total fermentation time resulted in both maintained cellular and volumetric
productivities (Fig. 1a and b). Cellular and volumetric productivities were calculated as
the amount of succinate produced per cell or litre fermentation medium and hour of the
anaerobic phase. For fermentations with cell resuspension after 40 or 60 hours, the
cellular productivity decreased as a result of the accumulated succinic acid (Fig. 1a and
c). The decrease in cellular productivity follows very closely that of the control
fermentation (without resuspension). After resuspending the cells both the 40 and 60 hour
resuspensions regained full cellular productivity. Hence, the cells as such can recover
from the inhibition, as long as the product succinic acid is removed. This result shows
10
that succinic acid does not permanently damage the cells, and that the inhibitory effect of
the organic acid is reversible. In contrast, the volumetric productivity did not recover to
the same extent as the cellular. Resuspending the cells after 40 hours nearly reached the
volumetric productivities achieved in the beginning of the anaerobic phase, whereas
resuspension after 60 hours only recovered to approximately half of the initial volumetric
productivity. The reason is that volumetric productivity as opposed to cellular
productivity is dependent on the concentration of viable cells. Cellular productivity is
only affected by the succinate concentration and once that is reduced the productivity
increases. Since the volumetric productivity is the sum of what all cells in the culture
produce and it does not recover to the initial values it means that the concentration of
viable cells has decreased. The concentration of viable cells, as shown in Figure 2c,
decreases with fermentation time irrespective of whether the cells are resuspended or not.
It also seems that the cell viability is not connected to succinic acid concentration, being
dependent only on the time the cells spend in the anaerobic environment. This raises the
question of the strains anaerobic reproducibility and enforces the importance to
investigate this further as a possible improvement of the process. Another aspect in the
production of organic acids is the transport processes responsible for product export. It
has been suggested that Saccharomyces cerevisiae engineered for lactic acid production
cannot sustain growth under anaerobic conditions, due to the energy requirements of the
lactate export system and the inability of the engineered strain to generate ATP
anaerobically [23]. Organic acids can be transported out of the cell using different
mechanisms, depending on the ratio between the concentration of the acid inside and
outside the cell [22]. For high external acid concentrations, passive or facilitated diffusion
11
will not be possible due to the absence of a concentration driving force. Instead active
transport against a concentration gradient using ATP-driven excretion will be necessary.
The fermentative glucose metabolism of AFP184 generates zero ATP during maximal
succinic acid production [3]. One ATP is generated if phosphoenol pyruvate is converted
to pyruvate. However, the only end-product for AFP184 from pyruvate is acetic acid,
which is undesirable. There is thus an energy-related problem during anaerobic succinic
acid production with AFP184. Two possible effects could result from this. The anaerobic
viability could be limited since no energy is generated for cell growth or maintenance.
Insufficient energy supply is also likely to be responsible for decreased succinic acid
productivity during strict batch fermentations. Since ATP is necessary for export of the
produced acids from the cells at elevated external acid concentrations an ATP deficiency
would cause a build-up of succinate inside the cell and as a result restrain the
productivity.
12
10
8
6
4
2
3.5
P r o d u c t iv ity
-1 -1
(g L h )
P r o d u c t iv ity
-1 -1
14
( g c e ll h ) x 1 0
12
3
2.5
2
1.5
1
0.5
0
0
20
40
60
80
20
Time (hours)
60
80
12
10
8
6
4
2
3.5
P r o d u c tiv it y
(g L - 1 h - 1 )
P r o d u c tiv it y
-1
-1
14
(g c e ll h ) x 1 0
40
Time (hours)
3
2.5
2
1.5
1
0.5
0
0
0
20
40
60
Time (hours)
80
100
20
40
60
80
100
Time (hours)
Figure 1. Productivity as a function of time; a) and c) cellular productivity (g cell-1 h-1 x

1014); b) and d) volumetric productivity (g L-1 h-1). Symbols used in the figures represent
when resuspensions were done: - 23 hours, - 40 hours, - 23 hours (glucose media),
- 60 hours, - 23/45/70 hours, - control without harvesting/resuspension.
The volumetric productivities both overall and for each reactor during the fermentations
with cell resuspension are summarised in Table 1. Removing the succinic acid greatly
assists in maintaining high productivity. Irrespective of the time of the cell resuspension,
the result is an improvement over the control fermentation. Resuspending the cells at 23
hours maintains the overall productivity above 2.5 g L-1 h-1, a value recommended for
financially viable succinic acid production from biomass [27]. The yields achieved do not
13
seem to depend much on when the cells were resuspended (Table 1); however it appears
that the yield improves after the media is changed.
60
50
40
30
20
10
100
-1
70
-1
80
80
60
40
20
0
0
20
40
60
80
100
20
Time (hours)
60
80
100
Time (hours)
140
60
40
30
20
10
120
Succinic acid (g)
50
Concentration
-1
12
(cells L ) x 10
40
100
80
60
40
20
20
40
60
80
100
Time (hours)
20
40
60
80
100
Time (hours)
Figure 2. Fermentation profiles for fermentations with cell harvesting and resuspension.
a) Succinic acid (g L-1), b) glucose (g L-1), c) viable cells (cells L-1 x 1012), d) total
produced succinic acid (g). Symbols used in the figures represent when resuspensions
were done: - 23 hours, - 40 hours, - 23 hours (glucose media), - 60 hours, 23/45/70 hours, - control without resuspension.
Fermentation profiles are shown in Figure 2 and if the desire is to produce the maximum
amount of succinate in the shortest time the fermentations should be resuspended when
the succinic acid concentration is around 40 g L-1 (~23 hours) before the negative
14
inhibitory effects occur (Fig. 2d). To investigate how long the productivities could be
maintained, fermentations were subjected to up to three repeated resuspensions of the
cellular biomass when the succinate concentration was close to 40 g L-1. Cell viability
and volumetric productivity decreased over the course of the fermentation, but the
cellular productivity always recovered to high values (Fig. 1c and d).
Table 1. Volumetric productivities and yields for cell resuspensions at selected times.
Volumetric productivitya (g L-1 h-1) / Yieldb (g g-1)
Time of
resuspension
23
40
60
23 (glucose)
23/45/70
Controlc
Overall
2.52 / 0.80
1.74 / 0.83
1.30 / 0.75
2.00 / 0.77
1.95 / 0.75
1.05 / 0.75
Prior resusp
3.11 / 0.67
2.23 / 0.80
1.39 / 0.73
2.93 / 0.67
2.80 / 0.68
2.95
1st resusp
2.23 / 0.93
1.44 / 0.86
1.21 / 0.77
1.61 / 0.87
2.27 / 0.82
2.04
2nd resusp
1.72 / 0.79
1.22
3rd resusp
1.40 / 0.70
Volumetric productivity in gram succinic acid produced per litre fermentation medium
and hour during the anaerobic phase for fermentations with cell harvesting and
resuspension at the times given in the table columns. bYield in gram succinic acid per
gram glucose consumed during the anaerobic phase. cThe control is carried out without
harvesting/resuspension and the productivities cited are those obtained after the entire
fermentation, 20, 40, and 80 hours respectively.
In the present study complex nutrient in the form of corn steep liquor was used in the
medium. It is important to establish if complex nutrients are important during the
15
succinate production phase, since decreasing the amount or removing them would reduce
the cost of downstream processing of the fermentation products. Resuspending the cells
in a medium consisting of only glucose and a potassium phosphate buffer resulted in
cellular productivities (Fig. 1a) and succinate yields (Table 1) of the same order as
fermentations with CSL, however the cell viability was reduced faster (Fig. 2c) and
although the complex nutrients are not necessary for efficient succinate productivity they
might be significant for prolonging the anaerobic viability. The decrease in cell viability
compared to fermentations with CSL resulted in a relatively lower volumetric
productivity.
In this work the succinic acid concentration was decreased to a level that could sustain a
high productivity by transferring the cells to a new medium. Removing the succinate
continuously, after certain times, performing the fermentations continuously or
resuspending the cells in fresh medium all translate into interrupting the fermentation
before a maximum succinic acid concentration is achieved. In all cases one would have to
work with dilute succinic acid solutions and most separation operations benefit from
highly concentrated solutions. Utilising any of these techniques on a large scale would
require optimisation of when and to what extent the succinate should be recovered in
order to sustain maximal productivity and optimal downstream processing efficiency.
16
Conclusions
The inhibitory effects observed at high succinate concentrations are likely to depend on
problems with exporting the produced succinate from the cells resulting in increased
intracellular concentrations and diminished productivity. AFP184 generates minimal
amounts of energy during anaerobic succinic acid production, limiting its anaerobic
growth and viability, as well as the possibility of using energy dependent transport
systems for acid excretion. By resuspending the cells in fresh media when the succinic
acid concentration started to negatively affect productivity, it was shown that succinic
acid does not permanently decrease the cells succinate producing capacity, since after
being transferred into new media cellular succinic acid productivity was fully recovered.
The present work also demonstrates that sugar is the only component necessary in the
anaerobic media in order to achieve rapid succinate production. These results are
important, assisting the design of integrated processes for succinic acid production.
Acknowledgements
The authors acknowledge the financial support of Norrbottens forskningsrd (The
Research Council of Norrbotten) and the Kempe Foundation.
17
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Paper IV
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ARTICLE IN PRESS
Enzyme and Microbial Technology xxx (2009) xxxxxx
Contents lists available at ScienceDirect
Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/emt
Detoxication requirements for bioconversion of softwood dilute acid

hydrolyzates to succinic acid
David B. Hodge , Christian Andersson, Kris A. Berglund, Ulrika Rova
Department of Chemical Engineering, Lule University of Technology, SE-971 87 Lule, Sweden
a r t i c l e
i n f o
Article history:
Received 26 September 2008
Received in revised form
25 November 2008
Accepted 26 November 2008
Keywords:
Softwood hydrolyzate
Succinic acid
Detoxication
Lignocellulose Fermentation
Biomass
a b s t r a c t
In this work an Escherichia coli metabolically engineered to ferment lignocellulosic biomass sugars to succinic acid was tested for growth and fermentation of detoxied softwood dilute sulfuric acid hydrolyzates,
and the minimum detoxication requirements were investigated with activated carbon and/or overliming
treatments. Detoxied hydrolyzates supported fast growth and complete fermentation of all hydrolyzate
sugars to succinate at yields comparable to pure sugar, while untreated hydrolyzates were unable to support either growth or fermentation. Activated carbon treatment was able to remove signicantly more
HMF and phenolics than overliming. However, in some cases, overliming treatment was capable of generating a fermentable hydrolyzate where activated carbon treatment was not. The implications of this are
that in addition to the known organic inhibitors, the changes in the inorganic content and/or composition
due to overliming are signicant to the hydrolyzate toxicity. It was also found that any HMF remaining
after detoxication was completely metabolized during aerobic cell growth on the hydrolyzates that were
capable of supporting growth.
2008 Elsevier Inc. All rights reserved.
1. Introduction
Analogous to a petrochemical renery, which produces a variety of fuels and intermediate chemicals used for plastics, bers,
solvents, detergents, and adhesives from raw hydrocarbons, a biorenery can be envisioned using lignocellulosic biomass as the
feedstock to generate a similar range of products. Lignocellulosic
biomass as a feedstock is advantageous in that it is renewable and
environmentally benign with respect to greenhouse gas emissions.
Softwoods are an abundant, renewable source of lignocellulose in
the Northern Hemisphere. While use of the cellulosic portion of
this resource as a feedstock for a forest biorenery could compete
with the traditional pulp and paper industry, the introduction of
new higher value products from hemicellulose and even cellulose
can provide a more diverse range of income sources to improve the
competitiveness of the forest products industry. The cellulose and
hemicellulose fractions of lignocellulose can be hydrolyzed through
either chemical or enzymatic processes to pentoses and hexoses.
These carbohydrate feedstocks are ideally suited for conversion via
biochemical transformations because of their central role in cellular
metabolism. Microbial metabolism is capable of converting these
carbohydrates to a wide range of metabolites that have useful appli-
Corresponding author at: Department of Chemical Engineering and Material

Science, Michigan State University, East Lansing, MI 48824, United States.
Tel.: +1 517 353 4508.
E-mail address: hodgeda@egr.msu.edu (D.B. Hodge).
cations as transportation fuels and chemical intermediates that can

be upgraded through further chemical processing to higher value
products. One of these metabolites, succinic acid, is a dicarboxylic
acid that has been identied as one of the top 12 chemicals derived
from lignocellulosic biomass [1]. Existing applications for succinic
acid are as diesel fuel additives, deicers, solvents, and detergent
builders, and additionally there is great interest in its potential as
a 4-carbon building block for a number of higher value polymers
[2,3]. While currently, the majority of succinic acid is produced from
fossil n-butane or benzene via maleic anhydride, the bio-based
production route for succinic acid using microbial fermentation
of biomass carbohydrates has the potential of generating both an
economically competitive [3] and renewable platform chemical.
While many yeast strains are capable of converting mixtures of
hexose and pentose sugars that are found in lignocellulosic biomass,
bacteria often give higher yields or productivities. Additionally,
bacteria often have fewer metabolic pathways with less layers
of metabolic regulation than yeast, making them more susceptible to articial alterations in metabolic pathways through genetic
engineering. The metabolically engineered bacterium Escherichia
coli AFP 184 [4], which is used in this work can both grow
aerobically and anaerobically ferment the sugars contained in
lignocellulosic biomass including: glucose, mannose, xylose, arabinose, and galactose to succinate [5]. Fermentation of lignocellulosic
hydrolyzates to succinic acid by E. coli has been carried out using
fed-batch fermentation of concentrated acid hydrolyzates of rice
straw [4] and cellulose enzyme hydrolyzates of steam exploded
oak by Mannheimia succiniciproducens [6] and Anaerobiospirillum
0141-0229/$ see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2008.11.007
Please cite this article in press as: Hodge DB, et al. Detoxication requirements for bioconversion of softwood dilute acid hydrolyzates to succinic
acid. Enzyme Microb Technol (2009), doi:10.1016/j.enzmictec.2008.11.007
G Model
EMT-7895;
No. of Pages 8
ARTICLE IN PRESS
D.B. Hodge et al. / Enzyme and Microbial Technology xxx (2009) xxxxxx
succiniciproducens [7]. No work has demonstrated succinic acid

production from softwood hydrolyzate sugars, although a few
examples of bacterial conversions of softwood hydrolyzate sugars
to fermentation products, including lactic acid by Bacillus species
[8] and ethanol by metabolically engineered E. coli strains [9,10].
It is notable that these studies used either overliming or activated
carbon (AC) treatment to yield a hydrolyzate that is not toxic to the
bacteria.
Dilute acid pretreatments of lignocellulose do not generate a
pure liquor of fermentable sugars, but rather a complex mixture containing many compounds that are toxic to microbial
growth including furans, phenolics, and aliphatic acids [11,12].
Because of this, a detoxication step may be required to make
a lignocellulosic hydrolyzate amenable to bioconversion. Physical
detoxication methods are based on the principle that inhibitors
can be removed, either through phase equilibria-based separations
based on solubility or volatility using for example liquidliquid
extraction or evaporation [13] or separations based on physical
adsorption onto a solid substrate as for example with AC treatment
or ion exchange [14,15]. Chemical detoxications such as overliming, are based on chemical modications of the inhibitors to a less
toxic or non-toxic product. Overliming with a combination of high
pH and temperature [16] has for a long time been considered a
promising detoxication method for dilute sulfuric acid-pretreated
hydrolyzates of lignocellulosic biomass. AC treatment should also
be ideally suited for the detoxication of softwood dilute acid
hydrolyzates, since it is used as a common processing step for the
removal of color- and avor-conferring aromatic compounds (i.e.
phenolics and furans) in industries like water treatment and high
fructose corn syrup production [15]. For the present work, both
overliming and AC detoxication were performed, with the primary goal of demonstrating the successful conversion of softwood
dilute acid hydrolyzates to succinate by E. coli AFP 184. Concurrent with this goal it is also necessary to understand the effects
the inhibitors in the hydrolyzate have on growth and fermentation in order to determine the detoxication requirements for the
system.
2. Materials and methods
2.1. Hydrolyzate preparation and detoxication
Acid-pretreated spruce hydrolyzates were provided by SEKAB and produced in
the pilot plant they manage (rnskldsvik, Sweden). The spruce chips were rst
pretreated with dilute H2 SO4 (170 C, pH 2, 7 min residence time) to hydrolyze hemicellulose, which was separated in a membrane lter press (Fig. 1). The remaining
solids were subjected to a second dilute acid hydrolysis step (200 C, pH 2, 10 min
residence time) to hydrolyze cellulose. After separation, from the remaining insoluble solids, the second stage liquor was recombined with an equal volume of the rst
stage liquor and concentrated by a factor of 2 by evaporation.
All three hydrolyzates were treated using either Ca(OH)2 (for neutralization
only), overliming, AC, or AC followed by overliming. AC detoxication was carried out
using wood-derived steam-activated decolorizing AC powder (ColorSorb G5, Jacobi
Carbons, Kalmar, Sweden) and at the initial pH of the hydrolyzate (1.42.0) based
on the ndings of Rodrigues et al. [17]. Overliming detoxication was performed by

adjusting the pH to 10.5 with Ca(OH)2 . Both detoxication treatments used an incubator to maintain the temperature at 60 C for 1 h with orbital shaking of the AC or
overliming-treated hydrolyzates at 200 rpm. The detoxied hydrolyzates were ltered (Whatman no. 1 lter paper) and the overlimed hydrolyzates were readjusted
to a pH of 6.6 using 2 M H2 SO4 , while the AC treated samples were adjusted to a pH of
approximately 5.0 with Ca(OH)2 . The detoxication component concentration data
are averages of multiple samples and are plotted with error bars that indicate the
data range. Data plotted without error bars represent measurements from a single
sample.
2.2. Fermentation
The microorganism E. coli AFP 184 used in this study is derived from strain C600
and contains mutations in the genes ptsG, pB, ldhA as described by Donnelly et
al. [4]. Basal cultivation media consisted of mineral salts K2 HPO4, 1.4 g/L; KH2 PO4 ,
0.6 g/L; (NH4 )2 SO4 , 3.3 g/L; MgSO4 7H2 O, 0.4 g/L), and 15 g/L corn steep liquor (50%
solids, SigmaAldrich). For hydrolyzates, an additional 15 g/L yeast extract (Oxoid)
was used. Cells were stored as stock cultures, preserved in 10% glycerol, 90% basal
media at 80 C. Inocula were prepared by reviving frozen stock cultures in 200 mL
sterile basal media with 5 g/L glucose and cultivated in 500 mL shake asks (200 rpm
orbital shaking) at 37 C for 16 h.
Fermentations were performed in 1 L fermenters (Applikon, Schiedam, The
Netherlands) with a working volume of 700 mL. Temperature was controlled at
37 C and pH controlled at 6.60 by automatic addition of 10 M KOH. Air (3 L/min)
was sparged into the reactor during the aerobic cell growth phase with an agitation
rate increasing from 500 to 800 rpm as oxygen transfer requirements increased.
When cells reach an OD550 of approximately 35 the air was turned off and CO2 was
sparged into the reactor (0.7 L/min) with 500 rpm agitation initiating the anaerobic succinate production phase. The hydrolyzate was used at 80% strength based on
reactor volume (lter sterilized). The other media components at a volume of 20%
of the reactor were autoclaved in the reactor. After autoclaving the lter sterilized
hydrolyzate was added to yield a nal concentration equivalent to the basal media.
Fermentations using pure glucose, xylose, and mannose were performed using basal
media which was autoclaved separately from the sugar solution. The inoculum
(140 mL) is centrifuged (2795 g) and the pellets were resuspended the in reactor
media before inoculation yielding an initial dry cell concentration of approximately
0.30 g/L.
2.3. Analysis
Cell densities were estimated by measuring the optical density at 550 nm (OD550 )
in a spectrophotometer (Genesys). Because initial OD550 values for hydrolyzates varied between approximately 1 and 5 due to soluble and insoluble solids in the liquor,
this value was plotted as a characteristic value for cell density rather than correlating the OD550 to the dry cell weight. Samples for HPLC analysis were centrifuged
(11180 g) for 5 min, diluted 10-fold with water, and ltered through a 0.2 m
syringe lter. An HPLC system (Perkin Elmer) equipped with a refractive index (RI)
detector was used for sugar, furan, and carboxylic acid quantication using either
a BioRad Aminex HPX 87-H column (0.005 M H2 SO4 mobile phase; analysis of glucose, acetic acid, succinic acid, formic acid, levulinic acid, 5-hydroxymethylfurfural,
and 2-furfural) or a BioRad HPX 87-P column (pure water mobile phase; analysis of
glucose, xylose, mannose, arabinose, and galactose). A ow rate of 0.6 mL/min was
used with a sample injection volume of 20 L, and column temperatures of 65 C
and 85 C, respectively.
Phenolics were estimated using a FolinCiocalteu assay modied from Singleton
et al. [18] using vanillin, the aldehyde of the guaiacyl lignin monomer that forms the
majority of softwood lignin, as a standard. For this, the sample was diluted 1:10 in
water and 0.2 mL of the diluted sample was mixed with 2.6 mL water and 0.2 mL
of 2 N FolinCiocalteu reagent (SigmaAldrich) and incubated at room temperature
for 6 min. Next, 2 mL of 7% Na2 CO3 was added with mixing. After 90 min incubation
at room temperature, the samples were syringe ltered (0.45 m pore size) and the
absorbance measured at 750 nm by spectrophotometer (Genesys).
Fig. 1. The two-stage dilute acid hydrolysis used for the treatment of spruce chips and the source of the three hydrolyzate liquors used in this work.
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2.4. Growth and fermentation parameters

Aerobic specic growth rates () were determined by regression of an exponential function to a plot of optical density versus time during exponential growth using
at least 3 data points. The succinate yields were determined by calculating the total
succinate (g) per total sugar consumed (g) during the anaerobic phase. Because of
the large volume of base required to neutralize the succinic acid, the accurate determination of the reactor volume is necessary for calculating the yields [5]. The total
reactor volume at any sampling time used for the yield calculation was estimated
by subtracting the volume of the samples and adding the volume of base to the initial reactor volume. Fermentation kinetic plots are based on the data from a single
fermentation. Yields and specic growth rates are based on at least 2 experiments
for glucose and xylose, and only 1 run for mannose.
3. Results and discussion

3.1. Hydrolyzate
The two-stage dilute acid hydrolysis process from the ethanol
pilot plant used for this work is shown in Fig. 1, with the compositions of sugars and inhibitors from the three hydrolyzates shown in
Fig. 2. The rst hydrolysis stage is primarily a hemicellulose hydrolysis whereby the majority of the hemicellulose is hydrolyzed to
monomeric sugars and most of the acetate contained in the hemicellulose is solubilized. This hydrolyzate (H1) contains a mixture
of all the hemicellulose sugars, primarily mannose, xylose, and
glucose with minor amounts of galactose and arabinose. In the
second stage, the cellulose is hydrolyzed to glucose under more
severe hydrolysis conditions, which leads to higher concentrations
of the sugar degradation products HMF, levulinic acid, and formic
acid in the hydrolyzate H2. Equal volumes of the two liquors are
combined and evaporated to yield hydrolyzate H3 with the sugars
concentrated two-fold, and the phenolics, HMF, and aliphatic acids
increased approximately in proportion to their relative volatility
with respect to water.
3.2. Detoxication
When working with a process for converting lignocellulose to
chemicals via microbial processing, the considerations of feedstock, fractionation method, detoxication, microbial conversion,
and product recovery all need to be considered in tandem. For
a dilute sulfuric acid pretreatment process, the hydrolysis conditions can be considered as a balance between high hemicellulose
and cellulose conversions and the production of sugar degradation
products requiring the added burden of increased detoxication.
In addition to generating high cellulose and hemicellulose conversions it is necessary to generate high sugar titers by pretreatment
and/or secondary cellulose hydrolysis to give high product con-
centrations which economically translate into lower separation,

operating, and capital costs [19]. These high sugar concentrations,
however, usually come at the cost of higher inhibitor concentrations that pose a challenge for microbial conversion and require a
potentially expensive set of unit operations for detoxication.
Detoxications of the three hydrolyzates were performed to
determine both the effects of detoxication methods on the
inhibitors and the degree of detoxication required to permit
growth and fermentation. A comparison of the effectiveness of
HMF and phenolics removal for the various treatments is given in
Fig. 3. This shows that the AC treatments are particularly effective at removing furans and phenolics, which is understandable
considering that the powdered AC used in this work is employed
commercially for removing these compounds from rened sugar
streams such as high fructose corn syrup. Based on this data, the
adsorption capacity of the AC can be estimated between 0.06 and
0.12 g HMF/g AC and a similar range for simultaneous phenolic
adsorption of 0.050.12 g phenolics/g AC. Overliming removed only
a portion of the HMF from each of the hydrolyzates, ranging from
48% to 72% removal, whereas the AC treatments removed more
than 85% in all cases as shown in Fig. 3. The formic, levulinic, and
acetic acid were unaffected by either treatment which is consistent
with the ndings of Martinez et al. [16] for overliming. Overliming
performed at increasing severity of pH and temperature has been
shown to degrade sugars to formic acid [20], with sugar degradation
increasing approximately in proportion to sugar concentration. In
the present work, overliming resulted in losses of up to 11% of total
sugars, while total sugar concentrations were relatively unaffected
by AC treatment (data not shown).
The phenolics in the hydrolyzate are a heterogenous mixture of compounds, and it is expected that these can contribute
signicantly to the toxicity. Phenolic compounds from softwood
hydrolyzates have been shown to be particularly toxic to bacterial growth [21] at even very low concentrations. The reactions
of lignin-derived phenolic compounds are more complex and
the interpretation of the results depends strongly on the quantication method used for phenolics. The current work shows
minimal changes in the concentrations of phenolics as quantied
by the FolinCioucalteu assay for overliming detoxication (Fig. 3),
whereas AC treatments showed signicant decreases in the range
of 8698%. Generalizations of phenolics behavior during overliming are difcult to make since some studies have found decreases
in phenolics using quantication by 13 C NMR [22] and absorbance
at 282 nm [20], or up to several fold increases in phenolics with
quantication by HPLC (C18 column with UV detection) and the
FolinCioucalteu assay [20]. Other work, also using HPLC (C18 column and UV detection) has shown conversion between phenolic
Fig. 2. Hydrolyzate sugar and inhibitor concentrations.
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Fig. 3. Effects of overliming and activated carbon detoxication on inhibitors.
structures with in some cases overall increases in phenolic concentrations [23].

Both AC and overliming treatments can be considered to have
drawbacks or challenges associated with the processing costs or
equipment requirements. For overliming the difculties could be
in slurrying the lime at high concentrations, controlling lime addition, disposal of the lter cake, and equipment scaling. Recent work
has found that 3040% of the calcium can remain soluble after overliming leading to problems with equipment scaling [24]. Because of
the relatively high adsorbant requirements, on the order of 10 g AC/g
HMF or g phenolics, the application of AC as a detoxication may
be cost prohibitive unless either cheap sources of AC or economic
regeneration methods are used. Because aromatic adsorption to AC
is primarily irreversible [25], thermal reactivation of the adsorbent
is practiced in the sugar industry [26]. AC treatment is of particular
interest as a detoxication treatment since char is a low-value byproduct of the thermochemical conversion of biomass by pyrolysis,
and AC derived from biomass char may represent a large potential
resource from a future biorening industry.
3.3. Fermentation
Fermentations using E. coli AFP 184 were rst performed to
demonstrate characteristic kinetics and yields on pure glucose,
xylose, and mannose (the primary softwood sugars) and are given in
Table 1 and Fig. 4. With this strain, succinate can be produced at high
productivities and nal titers through a two-phase batch cultivation
in the same reactor vessel [5]. An added advantage of cultivation
with the same media under aerobic conditions is that cells are typically more tolerant of inhibitors under aerobic conditions and are
Table 1
Growth and fermentation on pure sugars.
Sugar
Cell growth (exponential h1 )
Succinate ferm. (anaerobic YP/S )
Glucose
Xylose
Mannose
0.49
0.59
0.35
0.660.90
0.500.62
0.64
even capable of detoxifying hydrolyzate inhibitors such as acetic

acid and HMF, which we have demonstrated to be co-metabolized
with sugars during aerobic growth (data not shown).
The reason that this aerobic cultivation is possible at high sugar
concentrations is that this strain of E. coli does not exhibit acetate
overow metabolism (Fig. 4), whereby the high catabolic load generated by glycolysis at high sugar concentrations leads to carbon
being redirected to acetate instead of entering the TCA cycle [27].
The mutation of the glucose-specic permease (EIICBglc ), a component of the glucose phosphotransferase system (PTS), means that
glucose uptake and phosphorylation is via the enzymes galactose
permease and glucokinase [28] rather than with the PEP-mediated
transporter protein. This mutation in the PTS system interferes
with the regulation of catabolite repression, so that simultaneous utilization of multiple sugar substrates is possible [4]. Another
consequence of the PTS mutation is that glucose uptake is slower,
resulting in a slower specic growth rate, 0.49 h1 on glucose compared to 0.77 h1 by K-12 using complex media [29] or 0.59 h1
for xylose (Table 1). The result of this decrease in the rate of glucose uptake as characterized by the specic growth rate is that
overow metabolism is avoided for this strain, even at high glucose concentrations. This result is very signicant in that cells can
Fig. 4. Characteristic kinetics for succinic acid production by E. coli AFP 184 on pure sugar substrates for glucose, xylose, and mannose.
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Table 2
Growth and fermentation on softwood hydrolyzate sugars.
Hydrolyzate
First stage (H1)
Second stage (H2)
Combined evaporated (H3)
Detoxication
Anaerobic
Exp. growth ( h1 )
Max. OD550
Time (h)
Final SA (g/L)
Final YP
5% AC + OL
OL
9.3
7.0
0.36
0.45
24.0
36.4
11.0
6.5
19.7++
18.0
0.68
0.62
2.5% AC
5% AC
OL
15
23
0.36
35.5
39.0
4.0
5.0
9.9
14.1
0.86
0.93
Neutralization
2.5% AC
5% AC
24
37
11.75
5
13.25
33.2
31.6
35.4
34.1
34.0
23.0
28.0
42.25
26.0
34.75
32.8
35.0++
42.2
38.7
27.2++
0.72
0.86
0.72
0.59
0.64
Neutralization
2.5% AC
5% AC
5% AC + OL
OL
*
Aerobic
Time (h)
*
*
0.38
0.50
0.29
Missing data; minimal growth; ++ fermentation terminated before sugar utilization was complete.
be cultivated to high cell densities in a batch reactor with high

initial sugar concentrations without the need for rigorously controlled fed-batch conditions to keep sugar concentrations low. It
also makes it possible to carry out both aerobic cell cultivation and
anaerobic fermentation with the same media in batch mode.
Microbial growth can be strongly and quantiably inhibited by
a number of compounds in lignocellulosic dilute acid hydrolyzates
such as various phenolic compounds, acetic acid, and the sugar
degradation products HMF, furfural, levulinic acid, and formic
acid [30,31]. Much research on the conversion of lignocellulosic
hydrolyzate sugars by E. coli in the presence of these inhibitors
has focused on the ethanol producing strain KO11 its derivative
strains [9,10,3033]. A comparison of the undetoxied hydrolyzate
inhibitor concentrations with those concentrations necessary to
inhibit anaerobic growth by 50% (IC50 ) of one of these ethanologenic E. coli strain [30,31] after 24 h can give insight into the level
of toxicity posed by the individual toxins in the hydrolyzates. It
should be kept in mind, however, that the literature data is for a
different strain for anaerobic growth, which would be more sensitive to inhibitors than aerobic growth. Based on this toxicity data,
it can be seen that each hydrolyzate should be unique in the detoxication requirements in that for H1, only the phenolics (based on
vanillin, IC50 of 0.5 g/L) should be signicant, H2 should be a challenge in terms of formic acid (IC50 of 2.5 g/L), HMF (IC50 of 2.7 g/L),
and potentially the phenolics, while in H3, the HMF and phenolics should be problematic. For all of the hydrolyzates the levulinic
(IC50 of 7.5 g/L) and acetic acid (IC50 of 9.0 g/L) are not expected to
be present at high enough concentrations to be the limiting component, although additive or synergistic effects with other inhibitors
can be a factor contributing to inhibition in that combined inhibition of two components can be either equal or greater than the sum
of the two individual contributions, respectively [21].
Succinate fermentation studies were next performed with
detoxied and undetoxied hydrolyzates. A summary of all of the
experiments and results are listed in Table 2, which is broken down
into data relevant to the aerobic growth phase and the anaerobic fermentation phase. Selected data for fermentation kinetics
from each hydrolyzate are plotted in Fig. 5, which shows growth
and fermentation of the mixed sugars and relatively rapid and
complete utilization of these sugars after detoxication. Robust
aerobic growth is rst necessary to generate the high concentrations of biomass that are needed to ferment the sugars to succinate
with high volumetric productivity. Samples taken from hydrolyzate
cultivation experiments that showed no apparent growth were cultured on agar plates and indicated that viable cells were still present
in the reactor after long periods of cultivation (data not shown).
Because of this it is clear that inhibitory hydrolyzates strongly suppress growth.
Much of the literature on microbial inhibition by lignocellulosic
hydrolyzates has focused on anaerobic growth and metabolism. It
is expected that the negative effects of the inhibitors are less under
aerobic conditions, and it has been shown that microaeration
for E. coli cultures can be used to improve the fermentability of
overlimed dilute acid hydrolyzates of softwood [10]. Additionally,
Fig. 5. E. coli AFP 184 fermentation of the three detoxied hydrolyzates.
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under aerobic growth conditions, some detoxication can take

place. In this case, the low concentrations of acetic acid in the
hydrolyzates (less than 3 g/L) are decreased even further during
the aerobic growth phase due to uptake and metabolism by the
cells. Formic and levulinic acid were unaffected, and it has been
demonstrated that formate is not reabsorbed by E. coli under aerobic conditions [34]. It is also known that furfural can be reduced in
situ by ethanologenic E. coli strains to the less toxic furfuryl alcohol
[35], and enteric bacteria have been shown to anaerobically reduce
HMF to less toxic compounds [36]. Even more promising than the
acetic acid removal is the HMF removal that was demonstrated
in this work. All experiments where aerobic growth was achieved
resulted in complete removal of HMF due to cellular metabolism.
This is signicant in that concentrations of HMF greater than 1.5 g/L
for the case of overlimed hydrolyzate H2 (Fig. 3A) are completely
removed during aerobic growth.
The time listed for aerobic growth in Table 2 is the time required
to reach the nal or maximum OD550 . This value of OD is set at
35 absorbance units and is the maximum cell density that can be
maintained aerobically by aeration and agitation in the present
laboratory setup. There are signicant differences in the aerobic
time and even in the fermentability of duplicate experiments which
shows that uncontrolled factors such as cell adaptation to the media
can play a deciding role in the success of a cultivation run. The
general trend, however, is that increasing detoxication generally
decreases the time required to reach the desired reactor cell density.
The specic growth rates on the various detoxied hydrolyzates
were determined where more than 3 data points were available
after exponential growth began. The general trend is that long lag
phases typically correlate to lower specic growth rates, which provides an indication of the toxicity of the hydrolyzates and that for
all cases, the specic growth rates are signicantly lower than for
mixtures of pure sugars (Table 1).
For the anaerobic data given in Table 2 differences between
yields for the same hydrolyzate (particularly for H3) under different
detoxication conditions can be proposed to be due to for example
the amount and type of sugar consumed in the aerobic phase, since
differing percentages of sugars were used in every case, and the
yields seem to be within the range expected for the mixture of sugars in each hydrolyzate compared to Table 1. A second possibility
is that yields are affected by differences in the availability of either
external redox acceptors or the redox state of the cells. This is based
on the idea that reduced compounds found in the media, such as
yeast extract or the hydrolyzate such as formate or phenolics, could
act as external electron donors and drive the ux of carbon through
the reductive arm of the TCA cycle to succinate with a resultant
increase in the yield. This mechanism has been proposed by Carvalheiro et al. [37] as a mechanism for lowering the xylitol yield
in fungal fermentations. The nal concentration of succinic acid in
Table 2 is a function of the yield and the sugar concentration at the
aerobic/anaerobic switch and it should be noted that not all of the
fermentations were carried out to completion. The anaerobic volumetric productivities that can be estimated from the nal titer and
time compare very positively to other studies producing succinic
acid from lignocellulosic biomass [4,6,7].
One key result from comparison of the data in Table 2 is
that Ca(OH)2 -neutralized hydrolyzates from all stages were unable
to support growth or fermentation of E. coli AFP 184, however,
all hydrolyzates were eventually capable of supporting strong
growth and fermentation with sufcient detoxication. Overliming was able to generate a fermentable hydrolyzate for all three
hydrolyzates, and similar results have been found in the literature
[9,10] for the fermentation of softwood acid hydrolyzates of glucose, mannose, and xylose to ethanol using the KO11 strain of E. coli
at total sugar concentrations comparable to the maximum used in
this work (Fig. 2).
The AC fermentation results are a little more difcult to interpret

in terms of only the quantied organic inhibitors. Clearly overliming
treatment is more effective for the rst stage hydrolyzate relative
to AC treatment, and is supported by the failure of the cells to grow
for several replicates using 5% AC treatment. From Fig. 2 the phenolics are the only inhibitor in H1 that should pose an obvious barrier
to growth. While these are signicantly reduced by AC treatment
they are relatively unaffected by overliming. For H2 and H3, the
AC treatment is apparently more effective when inhibition is considered in terms of the time required for aerobic growth. Due to
this, the changes in concentration of HMF and phenolics (Fig. 3),
which are the only inhibitors that are quantiably affected by the
detoxications, cannot be considered the only factors affecting the
toxicity of the hydrolyzate. For this it is proposed that differences
in toxicity can be due to changes in the inorganic ion content of
the hydrolyzates with proposed mechanisms including sulfate toxicity, total ionic or osmotic stress, or toxicity of trace metal cations
(originally picked up from the lignocellulosic biomass or pretreatment equipment); all of which have been found to be signicantly
decreased by overliming [38]. The differences between the effectiveness of AC treatment on the toxicity of H1 versus H3 indicate
that the process of evaporation plays a role in this such as through
precipitation of inorganics. The observation that the overliming
treatment alone is not effective for one of the replicates of H3 indicates that the HMF and/or phenolics concentration may still be
above the inhibition threshold for growth, while the application
of both 5% AC and overliming is clearly better than either of these
treatments independently for all cases.
These results suggest that the optimum detoxication method
is strongly dependent on the hydrolyzate and the conditions under
which it is generated. For example, in the work of Carvalheiro et
al. [37] it was found that AC treatment was the best detoxication method for hydrolyzates from hot water pretreated brewers
spent grain relative to treatment with overliming or ion exchange
resins. One key implication of this is that under these conditions
using hot water pretreatment, there would be minimal inorganics in the hydrolyzate, so that the primary inhibitors would be
the phenolics and furans, which AC treatment is ideally suited to
remove. However, for dilute sulfuric acid pretreated hydrolyzates,
as is demonstrated in the current work, the toxicity can be considered to be both a function of the inorganic ion concentrations
and/or species prole in addition to the organic inhibitors. Interestingly, in another work using AC detoxication of dilute acid
pretreated bagasse [39], the pH is adjusted to 8.0 with NaOH and ltered before lower the pH again indicating that modication of the
inorganic ion content through precipitation is an important step
for dilute sulfuric acid pretreatments. Similarly, in previous overliming studies it was found that only one third of the toxicity of
dilute sulfuric acid bagasse hydrolyzates to ethanologenic E. coli
could be attributed to furans while the other toxicity effects were
proposed to be soluble salts [16,40] The original proposed mechanism of overliming detoxication was metal cation removal [41],
and this change in the inorganic ion content and prole by overliming potentially contributes more to improving the fermentability
of the hydrolyzates than the decreases in HMF and minimal apparent changes in the phenolics. However, the conditions under which
overliming is effective at degrading HMF at a pH greater than 10.0
and elevated temperature is also conducive to sugar degradation
and can result in increased raw materials and separation costs for
pH adjustments and precipitate removal.
Besides detoxication, a number of other factors affecting the
fermentability of the hydrolyzates that were not tested in depth
were the inoculum size and complex media supplementation which
both the literature and initial work have demonstrated to be signicant factors in the fermentability of a hydrolyzate [21]. Still
unanswered questions include whether the protein added in the
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form of a complex media source acts in itself as a detoxication

method by physically adsorbing inhibitors such as lignin-derived
phenolics. Along these same lines, the use of large inocula have
been shown in the past to overcome inhibition in hydrolyzates, due
to either cells biologically degrading inhibitors such as HMF and
furfural [13] or presumably by physically adsorbing these inhibitors
onto dead cells [42]. Initial work in our laboratory (not shown) using
smaller inocula and less completemedia supplementation showed
decreased tolerance to the same hydrolyzates that are capable of
supporting growth under the conditions presented in Table 2.
4. Conclusions
While minimal or no detoxication would be the best process conguration either through the development of more
robust microorganisms or decreased inhibitor formation through
improved pretreatment methods, some detoxication is necessary
for the combination of hydrolyzate and microorganism used in
this study. Growth and fermentation to succinic acid of all sugars
was demonstrated for a detoxied softwood hydrolyzate that could
be considered relatively concentrated in terms of both sugars and
inhibitors. It is difcult to draw general conclusions on the exact
effect of the detoxications and individual inhibitors on growth
and fermentation due to the all or nothing way in which the
fermentation results were obtained and the wide variability in aerobic growth behavior due to differing media adaptation by the cells.
Overall, the concentrations of inhibitors in all of the hydrolyzates
were clearly above the threshold for growth and fermentation for E.
coli AFP 184 when considering the combined and synergistic effects
of the inhibitors. One important result is that with aerobic cell
cultivation, the HMF remaining after detoxication is completely
removed by aerobic cellular metabolism, although only when the
overall growth-inhibiting capacity of the hydrolyzates is below a
toxic threshold.
By considering the inhibitor effects in terms of both total inorganic load and organic inhibitors the effectiveness of both the
detoxication methods can be more clearly understood. Based on
the detoxications using AC treatment, the removal of HMF and
phenolics alone could not be considered to account for all of the
detoxication, but rather changes in the inorganic ion content and
prole are suspected to contribute to a considerable portion of the
detoxication of the hydrolyzates. This work suggests that detoxications of dilute sulfuric acid hydrolyzates can be optimized for
both the removal of inorganics only through pH adjustments with
lime (although not reaching the pH range where sugar and furans
are degraded) and aromatics removal with, for example, AC or ion
exchange. This is quite similar to existing processes used in the
cane sugar industry, whereby inorganics are precipitated with lime
assisted by occulating agents in a clarication step, while phenolics and furans are removed in a decolorizing step by activated
carbon [26]. These types of process developments, coupled with
more robust and industrially adapted microorganisms have the
potential to develop into an economically competitive biorening
industry based on renewable raw materials.
Acknowledgements
The authors would like to thank Torbjrn van der Meulen at
SEKAB and Peter Blomquist at Processum AB for providing the
hydrolyzates and SEKAB for providing funding for this work. Dr.
Hodge was supported by the Kempestiftelsen.
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DOC TOR A L T H E S I S

ISSN: 1402-1544 ISBN 978-91-86233-17-4

Christian Andersson Biobased Production of Succinic Acid by Escherichia coli Fermentation

Department of Chemical Engineering and Geosciences

Biobased Production of Succinic Acid

Lule University of Technology

Biobased Production of Succinic Acid

Division of Chemical Engineering

Tryck: Universitetstryckeriet, Lule

Effects of Different Carbon Sources on the Production of Succinic Acid

Inhibition of Succinic Acid Production in Metabolically Engineered

Maintaining High Anaerobic Succinic Acid Productivity by Cell

Detoxification Requirements for Bioconverision of Softwood Dilute

Other Publications by the Author

Process for Producing Succinic Acid from Sucrose

PRODUCTION OF BIOBASED FUELS AND CHEMICALS ................................... 3

ESCHERICHIA COLI AS A PLATFORM FOR INDUSTRIAL PRODUCTION OF

Energy Generation and pH Homeostasis ....................................................................... 20

Osmotic Pressure, Osmolarity, and Turgor pressure.....................................................30

Pretreatment and Hydrolysis.......................................................................................... 34

undesirable/harmful by-products. One well-known example of white biotechnology is glucose

Scope of the Present Work

Production of Biobased Fuels and Chemicals

Production of Biobased Fuels and Chemicals

The Integrated Biorefinery

Municipal solid waste

Figure 1. Principal sketch of the biorefinery concept.

Production of Biobased Fuels and Chemicals

Production of Biobased Fuels and Chemicals

Figure 2. An LCF biorefinery based on ethanol and succinic acid fermentation.

Production of Biobased Fuels and Chemicals

Figure 3. An LCF biorefinery based on a Kraft pulp mill.

Fermentative Production of Organic Acids

Production of Biobased Fuels and Chemicals

Figure 4. Organic acids as intermediates/end-products of microbial carbohydrate

Production of Biobased Fuels and Chemicals

Production of Biobased Fuels and Chemicals

Biobased Succinic Acid Production

Production of Biobased Fuels and Chemicals

Figure 5. Value-added products that can be derived from succinic acid.

Production of Biobased Fuels and Chemicals

Production of Biobased Fuels and Chemicals

Production of Biobased Fuels and Chemicals

Escherichia coli as a Platform for Industrial Production of

Escherichia coli Sugar Metabolism

Escherichia coli as a Platform for Industrial Production of Succinic Acid

phosphoenolpyruvate carboxylase directs the carbon flow from phosphoenolpyruvate (PEP)

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Figure 7. Glucose uptake systems in E. coli.

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Effects of pH and Organic Acids on Escherichia coli Metabolism

Energy Generation and pH Homeostasis

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Organic Acids Metabolic Uncoupling and Anion Accumulation

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Acid Resistance Systems

Escherichia coli as a Platform for Industrial Production of Succinic Acid

Glutamate decarboxylase (AR2)