LI CENTI ATE THES I S

Lule University of Technology

Department of Chemical Engineering and Geosciences
Division of Biochemmical and Chemical Process Engineering
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Succinic acid production
using metabolically engineered
Escherichia coli
Christian Andersson
Succinic acid production using metabolically
engineered Escherichia coli
Christian Andersson
Division of Biochemical and Chemical Process Engineering
Department of Chemical Engineering and Geosciences
Lule University of Technoogy
S-971 87 Lule
February 2007
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). To date, the primary emphasis has been placed on the
replacement of oil for transportation fuels. A highly significant subset of petroleum usage is the
production of chemicals, which represents 10-15% of the petroleum usage. White
biotechnology, also called industrial biotechnology, is a fast evolving technology with a large
potential to have a substantial impact on the industrial production of fuels and chemicals from
biomass. This work addresses the issue of chemical production by investigating the production
of bio-based succinic acid, which can be used in a wide range of applications to replace
petroleum based chemicals. Succinic acid can be produced by fermentation of sugar by a
number of organisms; one is Escherichia coli (E. coli). It is known that E. coli under anaerobic
conditions produces a mixture of organic acids. In order to obtain a cost-effective production
it is necessary to metabolically engineer the organism to produce succinic acid in greater yield
than the other acids. In the current work, E. coli mutant AFP184 was used. AFP184 originates
from a near wild type strain, the C600 (ATCC 23724), which can ferment both five and six
carbon sugars and has mutations in the glucose specific phosphotransferase system (ptsG), the
pyruvate formate lyase system (pfl) and in the fermentative lactate dehydrogenase system (ldh).
The previous studies using different organisms have all used cultivation mediums supplemented
to some degree with different nutrients like biotin, thiamine and yeast extract. In order to
apply the technology to large scale, production must be cost-effective and it is important to
minimise the use of additional supplements.
The first part of this work aimed to investigate the fermentation characteristics of AFP184 in a
medium consisting of corn steep liquor, inorganic salts and different sugar sources without
supplementation of other additional nutrients. It addresses questions regarding the effect of
different sugars on succinic acid kinetics and yields in an industrially relevant medium. In order
to gain a sustainable production of succinic acid from biomass feedstocks (sugar from biomass)
it is important to investigate how well the organism can utilise different sugars in the biomass.
The sugars studied were sucrose, glucose, fructose, xylose and equal mixtures of glucose-
fructose and glucose-xylose at a total initial sugar concentration of 100 g L
-1
. 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). Fructose resulted in a yield of 0.66 and xylose of 0.5. Using a high
i
initial sugar concentration made it possible to obtain volumetric productivities of almost 3 g L
-
1
h
-1
, which is above estimated values for feasible economic production. Succinic acid
production ceased at final concentrations greater than 40 g L
-1
. In order to further increase
succinic acid concentrations, this inhibitory effect was studied in the second part of the present
work. The inhibitory effects can be two-fold including pH-based inhibition and an anion
specific effect on metabolism. It has been reported that high concentrations of ammonia inhibit
E. coli growth and damage cell membranes. In order to limit toxic and inhibitory effects
different neutralising agents were tested. First the use of NH
4
OH was optimised with respect
to fermentation pH and it was found that the best results were obtained at pH 6.5-6.7.
Optimal pH was then used with NaOH, KOH, and Na
2
CO
3
as neutralising agents and it was
shown that NaOH, KOH, and Na
2
CO
3
neutralised fermentations could reach succinic acid
concentrations of 69 and 61 and 78 g L
-1
respectively without any significant decrease in
succinic acid productivity. It was observed that cells lost viability during the cause anaerobic
phase. It resulted in decreasing succinic acid productivities. It is believed that the viability
decrease is a combined effect of organic acids concentration and the osmolarity of the medium.
The work done in this thesis is aimed towards increasing the economical feasibility of a
biochemical succinic acid production.
Keywords: Industrial biotechnology, Fermentation, Succinic acid, Escherichia coli, Productivity
ii
Acknowledgements
First of all I would like to thank my supervisor Professor Kris A. Berglund. Your constant
good mood and undying enthusiasm is close to contagious. You are never too tired for a
discussion be it politics or fermentation technology. Thank you for including me in your team.
My deepest gratitude and thankfulness I would like to extend to my secondary supervisor Dr.
Ulrika Rova. You taught me the basics of microbiology. Without you there would be no
thesis. Your care and resourcefulness are the foundation of our division. Thank you for all the
work you have put in.
I extend a special thanks to my master thesis worker and fermentation engineer Jonas
Helmerius. You came when I needed you the most. It has been a pleasure supervising you.
Thank you for all your help.
I am also grateful to Josefine Enman, Magnus Sjblom, David Hodge, and all my friends and
colleges at the Department of Chemical Engineering and Geosciences. Working with you guys
is great fun. My earlier master thesis workers Andreas Lennartsson, Ksenia Bolotova, and Mario
Winkler are all acknowledged. You all did great jobs. I also thank all my students. Our
discussions and your curiosity help me to develop.
Min nrmste vn Ivan Kaic och min bror Fredrik Andersson, ni r speciellt tackade. Ert std
och er vnskap r ovrderlig.
Mina frldrar Jarl och Margaretha Andersson. Jag har inte ord att beskriva vad ni betyder. Ni
r mina frebilder. Mitt rttesnre.
Finally, my love Antonina Lobanova. You came to me. I still cannot believe it is true. You
have changed everything. .
Lule 2007,
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.
I 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, In press
II Impact of neutralising agent and pH on the succinic acid production by
metabolically engineered Escherichia coli in dual-phase fermentations
Christian Andersson, Jonas Helmerius, David Hodge, Kris A. Berglund, and Ulrika
Rova
Manuscript
v
vi
Contents
INTRODUCTION ..........................................................................................1
PRODUCTION OF BIO-BASED FUELS AND CHEMICALS ..........................................................2
THE INTEGRATED FOREST BIOREFINERY (IFBR) ................................................................4
CURRENT LEVEL OF SUCCINIC ACID PRODUCTION.............................................................7
THEORY ..................................................................................................... 11
BASIS FOR THE EXPERIMENTAL WORK ..............................................................................11
SUGAR METABOLISM IN AFP184 .......................................................................................11
Glucose..............................................................................................................12
Fructose .............................................................................................................16
Xylose ...............................................................................................................16
ACID RESISTANCE, INHIBITION, AND TOXICITY IN ESCHERICHIA COLI..............................16
Energy generation and the chemiosmotic theory ..............................................................16
Background to pH homeostasis..................................................................................17
Organic acid, metabolic uncoupling and anion accumulation...............................................18
Acid resistance systems............................................................................................20
OSMOTIC STRESS IN ESCHERICHIA COLI ...........................................................................23
Osmotic pressure, osmolarity and turgor pressure ............................................................23
The role of compatible solutes in the response of Escherichia coli to osmotic stress ......................25
RESULTS AND DISCUSSION....................................................................... 29
SUGAR UTILISATION .........................................................................................................29
IMPACT OF THE NEUTRALISING AGENT .............................................................................33
CONCLUSIONS ........................................................................................... 37
FUTURE WORK .......................................................................................... 39
REFERENCES .............................................................................................. 41
vii
viii
Introduction
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 main focus so far has been replacement of oil for
transportation fuels, but the utilisation of petroleum for production of chemicals could
represent 10-15% of the petroleum usage.
White biotechnology, also called industrial biotechnology, is a fast evolving technology with a
large potential to have a substantial impact on the industrial production of fuels and chemicals
from biomass. By definition, the technology uses the properties of living organisms such as
yeast, moulds, bacteria and plants to make products from renewable resources. Since
biotechnology processes utilise living systems the reactions involved generally occurs under
mild conditions with high product specificity. One well-known example of white
biotechnology is glucose fermentation by the yeast Saccharomyces cerevisiae for the production of
ethanol.
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 bio-
degradable fuels and chemicals with reduced formation of undesirable/harmful by-products. As
a first step, target fermentations producing chemicals that can compete with fossil based
alternatives must be identified. Consequently, 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. In a report from the U.S. Department of Energy (USDOE), succinic
acid was considered as one of twelve top chemical building blocks manufactured from biomass
(1). 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 (2). Therefore bio-based production of
succinic acid offers the opportunity for a widespread replacement of fossil fuel based chemicals.
The key for a successful production of succinic acid based products is the development of an
1
economically feasible, efficient manufacturing process. The present work addresses different
aspects of succinic acid production by fermentation using metabolically engineered Escherichia
coli (E. coli).
Production of bio-based fuels and chemicals
It has been estimated that the annual global production of plant biomass exceeds 10010
12
kg
dry matter (3). The generated biomass must however be shared between many interests, for
example human food, forage crops and raw material for industry. With the depletion of oil-
reserves, increased greenhouse-gas emissions and an interest in using locally available raw
materials, attention has shifted from fossil fuel based technologies to alternative technologies
using sun, wind, water and biomass to produce energy and chemicals. Even if the current
interest for bio-based fuels and chemicals production increases the demand for usable biomass,
the supply of biomass is certainly enough to sustain the present biomass consumers and the
growing bio-based fuel and chemical sectors (3). Although todays prices for biomass (e.g.
agricultural residues and forest products) are much lower than the price of crude oil,(3) bio-
based produced fuels and chemicals have problems competing with their oil-based
counterparts. The main reasons for the lack of viability of the bio-based production methods
are:
- High processing costs; the costs of converting the cheap biological feedstocks into
molecules that can be further processed to fuels and chemicals are high compared to
petrochemical alternatives.
- Low raw material usage; only parts of the raw material is used to produce the desired
products.
- Lack of process integration; while using the same raw material or different parts of the
same feedstock, the production sites are often not located close to each other.
- High capital costs; in order to produce bio-based products investments in new plants
have to be made.
The situation in the petrochemical industry is the reverse. Even though the feedstock (crude
oil) is traded at a higher price than agricultural residues or forest materials, the processing costs
are lower due to well implemented and mature processes and technologies. The raw material
2
usage is very high and different processes refining various parts of the crude oil into high-value
products are linked together in an oil refinery (3). Oil refineries are very large chemical plants
in which the investments were done decades ago. Oil refineries offer a wide spectrum of
products from a single feedstock, and if the biotechnical process industries are to be
competitive they need to develop in the same direction. The solution is an integrated
production of many bio-based products by what is called a biorefinery.
The main concept of an integrated biorefinery is efficient conversion of all feedstock
components into value-added products by thermochemical or biochemical processes (Fig. 1).
By product 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 like corn dry and wet milling plants and pulp and paper mills (3, 4). Biorefineries
typically use regionally available biomass; in the US biorefineries are constructed around corn
wet, and dry mills and in Brazil around sugar cane mills. In Sweden the main source of biomass
is the forest. The pulp and paper industry is well established, has the infrastructure and labour
force to handle and process forest biomass. For Sweden it would be a suitable path to generate
a biorefinery around a pulp and paper mill.
Feedstocks
Processing
Products
Fuels Chemicals Materials Foods
Biochemical
Chemical
Thermo-chemical
Physical
Grains Agricultural residues Forest biomass Municipal solid waste
Figure 1. Principal sketch of the biorefinery concept.
3
The integrated forest biorefinery (IFBR)
Pulp and paper mills process forestry materials into value added products; pulp, paper, lignin,
energy and could therefore already be considered as an example of a biorefinery. Besides the
primary purpose of converting cellulose to pulp, other products from alkaline pulping processes
include extractives such as tall oil and the generation of steam and electricity. The main
concept of the integrated forest product refinery is continuous production of pulp and paper
products in addition to manufacturing of fuels and chemicals from the raw material or waste
streams that is currently underutilised.
The hemicellulose and lignin constitutes approximately 40-50% of the dry weight of wood (5).
In the present kraft pulp mill hemicelluloses and lignin are combusted in the recovery boiler to
generate steam and recover pulping chemicals. Based on a heating value of 13.6 MJ kg
-1
the
hemicellulose value has been calculated to be $50 per oven dry metric ton (ODMT) (6).
Converting the biomass into bioproducts valued at $2000 ODMT
-1
or more provides a great
opportunity for a kraft mill to expand its business (6, 7). Due to the accumulating quantities,
lignocellulosic materials could potentially provide a large and less expensive sugar source for
bio-based industries in countries with large amount of wood compared to sugars from starch.
In the kraft pulp process (also called sulphate process or alkaline 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 burned in the recovery boiler to
generate steam and regenerate the pulping chemicals. If a present day kraft mill (Fig. 2) should
be converted to an IFBR the hemicellulose fraction of the wood chips must be liberated and
separated from the wood chips before pulping.
4
Flue gas
Steam
Recovery
boiler
Caustisation
Digester
Pulp
Wood chips
Pulping chemicals
Black liquor
Evaporation
Figure 2. Recovery of pulping chemicals in the kraft process.
The hemicellulose can be separated from the wood chips prior to pulping by leaching the
wood chips with a hot alkaline solution. The hemicellulose in softwoods is mainly acetyl-
galactoglucomannan and during hot alkaline treatment the glucomannan is degraded by the
peeling reaction (6, 8, 9). In hardwoods on the other hand, the main hemicellulose form is
glucoronoxylan which is dissolved in oligomeric form when treated in the same way, and hot
alkaline extraction would thus be a suitable method for extraction of hemicellulose from
hardwood (6, 8, 9). If pure water was used as extractant at high temperature the acetic acid
released from the hemicellulose would create acidic conditions and may cause unwanted
cellulose degradation due to acid hydrolysis at the high temperatures used. Extracting the
hemicellulose from the wood chips has a number of positive effects besides opening up
possibilities for bio-based chemicals productions. It will increase the delignification rate in the
digester, reduce the alkali consumption and thus also reduce the amount of produced black
liquor (6). The most important question that has to be addressed is whether the pulp quality is
negatively affected.
5
Further processing of the black liquor could be done by precipitating the lignin under acidic
conditions. The lignin could then be used to produce carbon fibres, fuels and other chemicals
as outlined in Figure 3.
Wood
Chemicals
Pulp and paper
Power
Syngas
Hemicellulose
Lignin
Fischer-Tropsch fuels
Methanol
Hydrogen
Ethanol
Carbon fibers
Binders
Fuel
Green chemicals
Bioplastics
Ethanol
Biorefinery
Figure 3. Products from an IFBR.
Present kraft mills use Tomlinson recovery boilers for regenerating pulping chemicals and
producing energy and steam for other mill activities. The cost of the generated energy, due to
low efficiency, is rather high (10). A significant increase in electricity production at a mill
could be provided by black liquor gasification, where the black liquor after evaporation is
gasified, generating green liquor containing the pulping chemicals and hot synthesis gas (10). A
simplified block-flow diagram of a chemical recovery system for a kraft process employing
hemicellulose extraction, lignin precipitation and black-liquor gasification is presented in
Figure 4. One important product from black liquor gasification is synthesis gas. The produced
synthesis gas is cooled in a gas cooler generating low and medium pressure steam (Fig. 4). The
syngas can after cooling be further processed into liquid fuels or chemicals at a price
comparable to that of gasoline (7). The next step before industrial implementation is to
demonstrate the black liquor gasification technique at a commercial scale.
6
LP Steam
MP Steam
Pulping
chemicals
Wood
chips
Digester
Pulp
Black liquor
Evaporation
Caustisation
Alkaline
solution
Extracted
hemicellulose
Gasifier
Gas
cooler
Green liquor
Syngas Water
Cooling water
Extractor
Recovered
Pulping
chemicals
Lignin
precipitation
Lignin
Black
liquor
Acid
Figure 4. Chemical recycling in a kraft mill with hemicellulose extraction, lignin precipitation
and black liquor gasification.
Current level of succinic acid production
Succinic acid, a dicarboxylic acid with the molecular formula C
4
H
6
O
4
, was first discovered in
1546 by Georgius Agricola when he dry distilled amber. Currently, succinic acid is
manufactured through oxidation of n-butane or benzene followed hydrolysis and finally
dehydrogenation. It is used in the production of esters, plastics and dyes. Succinic acid could
also be produced using biochemical conversion of biomass by fungal or bacterial fermentations.
Increased markets for bio-based production of succinic acid are expected to come from the
synthesis of biodegradable polymers; polybutylene succinate (PBS) and polyamides
(Nylonx,4) and various green solvents. Different reaction pathways for succinic acid
applications from fermentation are outlined in Figure 5.
7
Acidulants
Food and beverage
Agicultural
chemicals
Herbicides
Defoliants
Adjuvants
Polymer
additives
Latex polymers
Polymers
Chelating agents
Detergent additives
Water treatment
Corrosion
inhibitors
Thermoset resins
Deicing chemicals
Runway
Commercial,
residential, industrial
Succinic Acid
Succinate Esters Succinic Anhydride
Succinic Salts
Fermentation
Polymerization
Esterification Dehydration
Condensation
Specialty Chemicals Itaconic Acid
New applications
Cleaners
Food and feed
additives markets
Diesel fuel additives
Solvents/Surfactants
Processing
Cleaning
Paint industry
Cosmetics
Dyes/pigments
Separations
Figure 5. Value-added products that can be derived from succinic acid.
Production of succinic acid has been demonstrated in a number of organisms including
Bacteroides ruminicola and Bacteroides amylophilus, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Corynebacterium glutamicum, and Escherichia coli (11-23).
The work done so far have all used media supplemented to some degree with high-cost
nutrient sources like tryptone, peptone and yeast extract.
Manufacturing costs of succinic acid are affected by succinic acid productivity and yield, raw
material costs and utilisation and recovery methods. Hence, in order to develop a bio-based
industrial production of succinic acid, three main issues need to be addressed. First of all a low
cost medium must be used. Second, the producing organism must be able to utilise a wide
range of sugar feedstocks and produce succinic acid in good yields in order to make use of the
cheapest available raw material. The third and most important factor for economically feasible
succinic acid economy 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 a recent review by
Song and Lee the most promising of these organisms, including E. coli mutants, were compared
(24).
8
The most investigated organism is A. succiniciproducens, a strict anaerobe. A succiniciproducens has
been characterised in a number of studies both with regards to medium composition (25-29)
and processing conditions (30, 31). One of the achievements with A. succiniciproducens was
conversion of wood hydrolysates into succinate with a mass yield of 0.88 gram succinate per
gram glucose demonstrated by Lee et al. (32). The main drawback wit the organism is that it
does not seem to tolerate succinic acid concentrations higher than 30-35 g L
-1
.
Recently a facultative anaerobe, M. succiniciproducens, was isolated from bovine rumen and
shown to produce succinate as its main fermentation product at yields in the order of 0.7 gram
succinate per gram sugar consumed.(14). This organism has been demonstrated to ferment
both glucose and xylose from wood hydrolysates (16). The highest succinic acid concentration
obtained in M. succiniciproducens fermentations is 52.4 g L
-1
(21). The concentration was
achieved using LB growth medium. M. succiniciproducens seems promising, and future work
should focus on development of mutants able to produce succinate in higher yields.
A. succinogenes is also a facultative anaerobe inhabiting the bovine rumen. The organism has
been shown to produce succinic acid to impressive concentrations (105.8 g L
-1
) in good yields
(approximately 0.85 gram per gram glucose) (33-35). The fermentations that have reached
succinate concentrations of more than 80 g L
-1
have been neutralised with MgCO
3
. When
using NH
4
OH or sodium alkali final concentrations were in the range of 60 g L
-1
, which is
lower than the concentration some E. coli strains have been shown to produce (36). Finally, the
fermentations have been carried out in either vial flasks or 1 L reactors. Before applying the
organism to large-scale production, it is necessary to demonstrate the process at a production
scale achieving the excellent concentrations obtained in laboratory experiments.
Under anaerobic conditions E. coli is known to produce a mixture of organic acids and ethanol
(37). 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 mol glucose consumed. If the
objective is to produce succinic acid, the yield of succinic acid relative the other acids must be
increased. In the late 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 interesting mutants developed by the program were AFP111
and AFP184 (13, 22, 38). AFP111 is a spontaneous mutant with mutations in the glucose
specific phosphotransferase system (ptsG), the pyruvate formate lyase system (pfl) and in the
9
fermentative lactate dehydrogenase system (ldh) (13). The mutations resulted in increased
succinic acid yields (1 mol succinic acid per mol glucose) (22). 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 (38). Beside these organisms, a number of different E. coli
mutants have been developed and tested for succinate production including pioneering work
to produce succinate under aerobic conditions (17, 18, 20, 23, 39-43). 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
(15, 36).
10
Theory
Basis for the experimental work
The specific experimental details in this work are elucidated in the papers I and II. Here an
overview of the conditions for the fermentation procedures is presented.
The medium used in this thesis was developed to be cost effective and relevant for use in
industrial production of succinic acid. Corn steep liquor is a by-product from the corn wet-
milling industry and is an inexpensive source of vitamins and trace elements as opposed to yeast
extract and peptone. The composition of corn steep liquor can vary widely mainly depending
on the variables involved in starch processing and the type and condition of the corn, but
averages can be found in literature (44-46). In the present work AFP184 was used in 1-10 L
laboratory fermentations using a medium based on corn steep liquor (50% solids) supplemented
with inorganic salts and a sugar solution of glucose, fructose, or xylose or mixtures of
glucose/fructose or glucose/xylose. The initial sugar concentration in the fermentations was
100 g L
-1
.
The fermentations were conducted in dual-phase mode, where each fermentation consisted of
an aerobic phase and an anaerobic phase. This way of running fermentations takes advantage of
E. coli being a facultative anaerobe; i.e. can grow both in the prescence 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 CO
2
. The cells grown aerobically now consume sugar anaerobically and produce
a mixture of organic acids, with succinic acid as the main product for AFP184.
Sugar metabolism in AFP184
In E. coli different sugars are metabolised in more or less different ways depending on structural
similarities between the sugars and the properties of the enzyme systems responsible for uptake
and degradation. As outlined above biological succinic acid production must be able to utilise
11
different sugars. The present work deals with the glucose, fructose, and xylose metabolism of
AFP184. 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 the
following section the metabolism of each of the sugars will be described briefly. The impacts of
the mutations in AFP184 will be discussed in the next section.
Glucose
The first step in the glucose metabolism of E. coli is glycolysis (also called Embdern-Meyerhof-
Parnas (EMP) pathway). In glycolysis one mole of glucose is taken up by the cell,
phosphorylated and degraded into 2 moles of pyruvate generating small amounts of energy
(ATP, NADH). In the energy metabolism of E. coli the fate of pyruvate depends on the
environmental conditions. In the presence of oxygen, pyruvate molecules enter the citric acid
cycle (TCA-cycle or Krebs cycle) where together with oxidative phosphorylation the main
part of the cellular energy is produced. Under anaerobic conditions E. coli will instead undergo
mixed acid fermentation and produce a mixture of organic acids and ethanol (37).
However, when metabolising glucose under anaerobic conditions the mutations in the pfl and
ldh genes significantly influence the ratio of the products formed. It has been long known that
E. coli under anaerobic conditions produced a mixture of organic acids and ethanol (37). 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 Figure 6.
12
Glucose
Glucose 6-P
Fructose 6-P
Glyceraldehyde 3-P
PEP
Pyruvate
Oxaloacetate
Malate
Fumarate
Succinate
Acetate
Acetyl-CoA
Citrate
Isocitrate
Glyoxylate
Fructose
PEP
Pyruvate
Xylose
Xylulose
Xylulose 5-P Ribose 5-P
Glyceraldehyde 3-P Sedoheptulose 7-P
Erythrose 4-P Fructose 6-P
Glyceraldehyde 3-P
ATP
ADP
2H, ATP
CO
2
2H, CO
2
2H
2H
ATP
ADP
Ethanol
4H
2H, CO
2
ATP
ADP
ATP
ADP
ATP
PEP
Pyruvate
Lactate
Formate
2H
Figure 6. 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.
The main product from anaerobic fermentations by AFP184 is succinic acid. It is also the aim
of the present work to investigate and improve the succinic acid production using strain
AFP184. Anaerobic succinic acid production by AFP184 requires CO
2
. The enzyme
phosphoenolpyruvate carboxylase directs the carbon flow from PEP to oxaloacetic acid by
fixing CO
2
. 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
13
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 it (15). Based on the
similarities between AFP184 and AFP111 it is assumed in the present study that glyoxylate
shunt also is active in AFP184. The third mutation in AFP184 affects the uptake and
phosphorylation of glucose.
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 allow glucose and other small solutes to diffuse through the membrane into the
periplasm (the volume between the inner and outer membrane). The two main porins for
glucose uptake during concentrations above 0.2 mM are OmpC and OmpF (47), 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
(48, 49) that internalise glucose (Fig. 7). The diffusion of glucose across the outer membrane
into the cytoplasm is a passive process and the rate of transport is determined by the activity of
the transport system.
GalP
MglB MglA
HPr
HPr
EI
EI
Glucokinase
Glucose 6-P
Glucokinase
Glucose 6-P
IIAB
Man
IICD
Man
OmpC
OmpF
IIC
Glc
MglC
IIB
Glc
IIA
Glc
P
P
P
PEP
Pyruvate
Glucose 6-P
Glucose 6-P
Glucose + H
+
ATP ADP
Glucose
ATP ADP
ATP ADP
Outside
cell
Glucose
Periplasm
Glucose
H
+
Cytoplasm
Figure 7. Glucose uptake systems in E. coli.
14
The process starts with Enzyme I (EI) taking a phosphate group from glycolytic intermediate
phosphoenolpyruvate (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 for example both fructose, glucose, and mannose. 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 (48). The ones responsible
for glucose uptake and phosphorylation are II
Glc
and II
Man
where the later complex has lower
glucose uptake rate than II
Glc
. The II
Glc
complex is made up of the sugar-specific enzymes,
IIA
Glc
and IIBC
Glc
. IIA
Glc
is a soluble enzyme and IIBC
Glc
is an integral membrane protein
permease.
The gene encoding the glucose-specific permease IIBC
Glc
normally represses the genes
encoding the enzymes in the II
Man
complex. AFP184 was metabolically engineered to have the
glucose-specific permease knocked out, resulting in an organism where the genes for the II
Man
complex is no longer repressed and glucose uptake can commence through the complexes
IIAB
Man
and IICD
Man
. 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 II
Man
complex (50).
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. GalP is a galactose:H
+
symport that has the ability to transfer both galactose and glucose into the cytoplasm. However
the rate for internalisation is substantially lower than by the PTS enzymes. The Mgl system
consists of a galactose/glucose periplasmic binding protein (MglB), an integral membrane
transporter protein (MglC), and an ATP-binding protein (MglA) (47). Glucose imported via
GalP or Mgl are not phosphorylated. The phosphorylation necessary before entering glycolysis
is carried out 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 carried out by the PTS (51). Once phosphorylated the
aerobic metabolism of AFP184 is essentially the same as in wild-type E. coli.
15
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 glucokinase (52-54). The result is a
higher aerobic growth rate, but a lower succinate yield since instead of generating succinate
more PEP is converted to pyruvate during uptake and phosphorylation of fructose . An
electron balance for succinate production by fructose metabolism gives a maximum theoretical
molar yield of 1.20 (mass yield 0.79).
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 (55, 56). An initial increase of
pH has been reported as xylose is consumed by E. coli (56). 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 (55, 57).
Xylulose 5-phosphate then enters the pentose phosphate pathway and can after conversion to
either fructose 6-phosphate or glyceraldehyde 3-phosphate enter 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).
Acid resistance, inhibition, and toxicity in Escherichia coli
It is well known that a reduction in the pH of foods helps to prevent microbial contamination
and spoilage (58). This preservation utilises the impact of three major factors influencing cell
physiology, function and viability during acid conditions. These are energy generation,
intracellular pH homeostasis and protection of proteins and DNA (59).
Energy generation and the chemiosmotic theory
At the time when Mitchell proposed his chemiosmotic theory it was known that oxidation of a
substrate is coupled to the phosphorylation of ADP (60). The chemiosmotic theory outlines
16
that a proton concentration gradient across the cytoplasmic membrane is generated and used as
a pool of energy for ATP synthesis (60). An essential component for generating the
protonmotive force 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 and the
difference in charge between the inner and outer sides of the membrane. The difference in
proton concentration across the membrane is simply the difference in pH between the
cytoplasm and the surrounding medium (pH). The charge difference is called membrane
potential () and is given in V or mV.
Using R = 8.315 J K
-1
mol
-1
, F = 96.48 kJ V
-1
mol
-1
, and a standard temperature of 298.15 K
the protonmotive force can be written as:
pH PMF A A+ = 058 . 0 (1)
The protonmotive force in E. coli actively undergoing cell division, is in the range of -140 to -
200 mV (61). 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.
Background to pH homeostasis
The cytoplasmic pH plays an important role in bacterial cell physiology. The membrane
surrounding the cytoplasm is more or less impermeable to protons that instead are taken up by
the cells via active transport systems, in particular ATP synthase. There are two main factors
that affect the pH homeostasis system in E. coli; the pH of the cultivation medium and the
concentraion 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 (62). E. coli is a neutrophile and thus grows
best at near neutral pH. The cytoplasm usually has a pH somewhat above that of the
17
surrounding medium. For growth of E. coli in a near neutral medium the internal pH is in the
range of 7.4-7.9 (63, 64). When E. coli is subjected to extreme acid stress the pH gradient
across the membrane increases and creates a strong influx force of protons. The cytoplasmic
membrane of E. coli are close to impermeable to protons, 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 (61). It has been shown that E. coli
cannot maintain a pH of more than 2 pH units (61). The result is more or less a severe
acidification of the cytoplasm. At low cytoplasmic pH, the activity and function of cellular
enzymes will be affected. E. coli has been demonstrated to survive acid stress down to pH 2 for
several hours (63, 65). However, due to improper enzyme function, protein denaturation and
damage on the purine bases of DNA, the low pH environment will eventually kill the cells.
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 e.g.
glucose. Those acids have pK
a
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. This
mechanism has been used hundreds of years as a method for food preservation. 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 under the pK
a
of the organic acids, the acids remain
undissociated and hence uncharged. An uncharged organic acid can travel freely across the cell
membrane by passive diffusion (66). Once inside the slightly alkaline cytoplasm the acids
dissociate increasing the proton concentration inside the cell, lowering the cytoplasmic pH and
dissipating the trans-membrane proton gradient and the protonmotive force (67). The effect of
a pH reduction is thus most severe when present together with organic acids.
Organic acid, metabolic uncoupling and anion accumulation
Acetate negatively affects growth of E. coli cultures (68). By using the ethanologenic E. coli
strain LY01 the effects of different organic acids from hemicellulose hydrolysates on growth has
been demonstrated (69). It was shown that the acids reduced growth more at lower pH than at
neutral or slightly alkaline. The effect of organic acids on pH is not only due to the lowering
of the cytoplasmic pH, but also includes anion specific effects (70). It has been shown that as
acetate anions accumulate, glutamate is released from the E. coli cells. In E. coli the turgor
pressure (pressure on the cell wall from the cytoplasm) is produced by accumulation of mainly
18
potassium glutamate (71). E. coli uses the release of glutamate to keep the turgor pressure
constant during acetate accumulation (70). The growth inhibition caused by acetate anions is
proposed to be linked to methionine biosynthesis (72). 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
E. coli growth. The effects of the fermentation acids anions are thus not fully understood,
although some interesting theories and contributions have been made.
As explained above, the protons generated by dissociation of organic acids in the cytoplasm
affects the organisms both by reducing the cytoplasmic pH and thus damaging protein
structures and DNA, and by reducing pH and 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 the 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.
HX HX
X
-
X
-
H
+ H
+
Inside cell Outside cell
Figure 8. Mechanism of uncoupling. HX represents the undissociated acid.
There has been a debate if organic acids are able to act as true uncouplers. Russel argues against
organic acids as true uncouplers by pointing out their inability to diffuse through the cell
membranes, instead they will accumulate in the cytoplasm like protons (58). He is contradicted
by Axe and Bailey who propose that both acetate and lactate permeates through the
19
membranes at comparable rates in both undissociated and dissociated form and in this way they
catalytically dissipate the protonmotive force (73). This model also purports that since acetate
could freely traverse the cell membrane it would not accumulate in the cytoplasm. In their
work it was observed that the intracellular acetate concentrations did not reach levels predicted
by pH. In contrast, Diez-Gonzalez and Russel have shown that acetate accumulation
increases with increasing external acetate concentration and an equilibrium between the
intracellular acetate and pH occurs when the external acetate concentrations is 60 mM or
more (74). They also found that acetate accumulates as long as the intracellular pH and thus
the pH gradient is kept high (74, 75) However, as 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 (75). 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 proton extrusion is a typically
observed in the presence of an uncoupler (75).
Acid resistance systems
E. coli have a number of ways to control acid charge. The pH homeostasis of E. coli can be
divided into a passive and an active homeostatic system (76). 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 (62). 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 (77, 78).
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 (79, 80).
20
Potassium uptake on the other hand has a strong correlation with the regulation and
maintenance of an alkaline cytoplasmic pH. Kroll and Booth showed that E. coli cells
suspended in potassium-free medium quickly reduced their intracellular pH. Upon
introduction of potassium in the medium alkaline pH in the cytoplasm was restored (81, 82).
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 (61, 83-86). AR1 is activated
when cells are grown aerobically in LB 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 (63, 83). The AR1 system is repressed by
glucose and does not need any extracellular amino acids present in the medium. The system
provides stationary phase acid resistance down to pH 2.5 (85).
Two other stationary phase acid resistance systems were discovered when E. coli was cultivated
in glucose containing media (87). These systems depend on extracellular glutamate (for AR2)
and arginine (for AR3) (87-90). 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 CO
2
. 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 (89). 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 optima 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 (61).
21
Lysine
Cadaverine
H
+
H
+
rpoS (AR1)
Glutamate decarboxylase (AR2)
Arginine decarboxylase (AR3)
Lysine decarboxylase (AR4)
Glutamate
GABA
Arginine
Agmatine
Lysine
Glutamate
H
+
GABA
Arginine
H
+
Agmatine
H
+
Cadaverine
Figure 9. Acid resistance system in E. coli.
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 reverts 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 (91). 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
dissociates into monomers at acid pH. The dissociated subunits bind to unfolded proteins in
the periplasm and prevents unwanted protein aggregation (92). It is not known what happens
to the proteins once the acid stress is relieved, but mutants lacking hdeAB showed a dramatic
22
loss of viability at acid pH (93). The protection of periplasmic proteins thus seems crucial for
cell survival at low pH.
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 can be quantified using the vant Hoff
equation:
RT ic
B
= H (2)
where i is the vant Hoff factor that describes the degree of dissociation of a solute into ions,
e.g. NaCl into Na
+
and Cl
-
, c
B
is the total solute concentration in moles per liter, R is the
universal gas constrant and T is the temperature in Kelvin. Equation (2) holds for ideal dilute
solutions.
23
At higher solute concentration the quantity RT H diverges from c
B
. Instead the unit osmolarity
(Osm, effective molar concentration of the solute in solution) is introduced and defined as:
m
w
w
V
a
RT
Osm
ln
=
H
= (3)
where, is the water (solvent) activity and is the molar volume of water. The complete
derivation of the equations is given elsewhere (94, 95). Osmolarity is measured in osmoles per
liter solvent (an osmole is the number of moles of a substance that contributes to a solutions
osmotic pressure).
w
a
m
w
V
E. coli has been shown to be able to grow in osmolarities from 0.015 Osm to approximately
1.9 Osm (minimal media) or 3.0 Osm (rich media) (96-98). 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 (99).
The hydrostatic pressure difference between the cytoplasm and the cells environment is called
turgor pressure ().
( )
in w
out w
m
w
ex cyto ex cyto
a
a
V
RT
Osm Osm RT
,
,
ln = = H H = AH (4)
The turgor pressure calculated by equation (4) has a major impact on the functionality of the
cells. 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 range. E. coli cells could lose important cellular functions and
mechanisms (100). 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. Gram-negative cell walls can withstand pressures of up to 100 atm and hypoosmotic
treatment has limited effect on cells (101, 102). Hyperosmotic shock is the opposite. Cells are
24
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 (103-106).
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. glycinebetaine), 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 (100, 107). 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 compatible solutes, K
+
and glutamate
-
, and will thus
not 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 (107).
Potassium is the major intracellular cation and is accumulated as the first response of E. coli to
osmotic upshock (71, 107). 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
pressure (108, 109). Closely coupled with potassium accumulation is the increased de novo
synthesis of glutamate. Glutamate synthesis is strongly dependent of K
+
and if a medium
deprived of K
+
is used, glutamate synthesis is reduced to approximately 10 % of the synthesis
25
rate in the presence of K
+
(71). 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 (107).
Glycinebetaine 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 glycinebetaine and proline K
+
is excreted from the cells. Glycinebetaine was shown
by May et al. to be mediated by the transport systems ProP and ProU. The same systems are
used for transport of proline, but the affinity of t ProU is higher for glycinebetaine (110, 111).
Glycinebetaine also offers higher osmotolerance to E. coli than proline. ProP uses the energy
from the protonmotive force for active transport and ProU uses energy derived from hydrolysis
of ATP (107, 111). The first system to respond to an osmotic upshock is the ProP, which is a
low affinity transport system. The transport system is activated within seconds after the cells are
subjected to the high osmolarity environment. ProP is also the physiologically more important
system at high osmolarities, since cells under high osmolarity conditions without this system are
unable to take up proline and glycinebetaine from the medium. ProU, on the other hand, is
first activated after several minutes (107). 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
glycinebetaine and proline are present in low concentrations. Both systems however only
accumulate glycinebetaine under osmotic stress (111). Roth et al. demonstrated that cells
grown in a medium lacking compatible solutes lost their colony-forming activity and regained
it when glycinebetaine was added (112).
Most bacteria are unable to synthesise glycinebetaine de novo and to use glycinebetaine as an
osmoprotectants they are dependent on transport of this compound for accumulation (100).
Even though glycinebetaine cannot be synthesised by E. coli from glucose it has been shown
that choline under osmotic stress is converted to glycinebetaine (113). Therefore choline is also
an osmoprotectants for E. coli. In E. coli one enzyme is responsible for the conversion of
choline to glycinebetaine and its activity is dependent on electron transport and requires thus
some terminal electron acceptor, e.g. O
2
. Choline can therefore only be used as an
osmoprotectants during aerobic conditions (111, 113).
26
Like glycinebetaine, proline cannot be synthesised in E. coli and other gram-negative bacteria
and high intracellular concentrations of proline during osmotic stress can only be obtained by
increased uptake. It has been found that both proline and glycinebetaine uptake from
exogenous sources is proportional to the osmotic strength of the medium (100, 114). Nagata
et al. studied the combined effects of proline together with K
+
and found that the co-existence
of K
+
and proline in high osmolarity media resulted in greater recovery of growth than in the
presence of proline alone (115).
Ectoine, another amino acid that has been identified as a compatible solute in halophiles, was
shown to improve E. coli growth in high osmolarity media (116). Ectoine accumulates in E.
coli and has similar osmoprotective abilities as glycinebetaine. Both the ProP and ProU systems
are responsible for ecotine uptake, where ProP is the main uptake system (116).
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 (117). Synthesis of trehalose is under
control of the rpoS sigma factor, which accumulates when cells are grown at high osmolarity.
Trehalose can accumulate to intracellular concentrations equal to approximately 20 % of the
osmolar concentration of solutes in the cultivation medium (100) and synthesis also leads to
potassium efflux.
A last part of the osmoregulation of E. coli is the outer membrane porins. Porins are proton
channels incorporated in the outer membrane of E. coli that transport small hydrophilic
molecules into the periplasm by passive diffusion. 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, temperature and good carbon sources increase 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 the temperature high; 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 (100).
27
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 (118). 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 carried out
during 20 minutes (119). The results imply that cells are able to respond to the increase in
osmolarity if the increase is slow.
28
Results and discussion
In this section a brief overview of the experiments carried out in Papers I and II will be given
together with a discussion of the main results. For full descriptions of experimental procedures
and results, please refer to Papers I and II. Common for both studies are the use of a low-cost
medium, relevant for use in industrial succinic acid fermentations and the use of a high initial
sugar concentration. Since the fermentations are carried out with a high sugar concentration
the organism is not metabolically limited by the carbon source and it is possible to achieve high
succinic acid productivity. The importance of the volumetric productivity on succinic acid
production was stressed under Current level of succinic acid productivity and should
preferably be at least 2.5 g L
-1
h
-1
(1).
Sugar utilisation
The first study conducted addresses the effect of different sugars on succinic acid kinetics and
yields during fermentations with AFP184. 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 line with the concept of a biorefinery, i.e. to make use of the
cheapest available raw material, the producing organisms must be able to produce succinic acid
in good yields from a wide range of sugar feedstocks. In this study glucose, fructose, xylose and
equal mixtures of glucose:fructose and glucose:xylose were used as feedstocks in dual-phase
fermentations. The fermentation profiles are shown in Figure 10.
29
Glucose Fructose
Xylose
Glucose:Fructose
Glucose:Xylose
a b
c
d
e
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Time (hours)
0
2
4
6
8
10
12
14
16
D
r
y

c
e
l
l

w
e
i
g
h
t

(
g

L
-
1
)
0
20
40
60
80
100
120
0 5 10 15 20 25 30
Time (hours)
0
2
4
6
8
10
12
14
16
D
r
y

c
e
l
l

w
e
i
g
h
t

(
g

L
-
1
)
0
20
40
60
80
100
120
0 5 10 15 20 25 30
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
0
2
4
6
8
10
12
14
16
0
20
40
60
80
100
120
0 5 10 15 20 25 30
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
0
2
4
6
8
10
12
14
16
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g
/
L
)
C
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l
l
s
/
m
L

x

1
0
9
0
2
4
6
8
10
12
14
16
D
r
y

c
e
l
l

w
e
i
g
h
t

(
g

L
-
1
)
Figure 10. 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
)10
9
.
Symbols used: glucose (), fructose (), xylose (o), succinic acid (), pyruvic acid (), viable
cells (*), dry cell weight (). Staggered line indicates time of switch to the anaerobic phase.
The most important results are summarised in Table 1. It is concluded that glucose is the
preferred sugar, resulting in the highest productivities, final titres, and yields. The higher yields
30
obtained when glucose was used as the carbon source is due to the mutation in the PTS
(described under Sugar metabolism in AFP184). Fermentation of fructose uses PEP for
phosphorylation committing a larger fraction of the carbon to pyruvate (Fig. 6) and thus
reducing the succinate yield. Xylose metabolism uses the pentose phosphate pathway (Fig. 6)
and, as described under Sugar metabolism, 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. Further work is required to identify the remaining carbon and improve xylose
fermentation.
Table 1. Summary of fermentation results for AFP184
a
.
Sugar (h
-1
) Y
P/S
(g g
-1
) Final titer Q
P
(g L
-1
h
-1
) Q
P
(g L
-1
h
-1
)
anaerobic (g L
-1
) anaerobic, T
22
d
anaerobic, T
32
d
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
Glu:Fru
b
- 0.58 25.2 1.67 1.05
Glu:Xyl
c
- 0.60 27.6 1.48 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 Q
P
is
the volymetric productivity of succinic acid during the anaerobic phase.
b
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
A series of fermentations were also carried out to determine aerobic growth rates for the three
sugars (Table 1). 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 (48, 52), 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
31
the enzyme glucokinase (120). Glucokinase is not subjected to any mutations in AFP184.
According to literature glucose uptake and phosphorylation by glucokinase is slower than by
the PTS (51), 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 (55, 56). Interestingly growth on xylose was
initially very slow, while the use of glucose and fructose rapidly initiated exponential growth.
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 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 metabolised utilisation of other sugars is initiated, a phenomenon called catabolite
repression. From the sugar consumption (Fig. 10) it was observed that when fermenting the
sugar mixtures, fructose was metabolised slightly faster during the aerobic phase. This effect
was expected due to 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. The effect is attributed
to a mutation in the PTS, which in turn prevents glucose from repressing uptake of other
sugars (120-123).
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 describes in the section Acid resistance, inhibition,
and toxicity in Escherichia coli. The fermentations in this study were carried out with
ammonium hydroxide as base for neutralisation of end products and it has been reported that
ammonium in concentrations above 3 g L
-1
inhibits growth (124, 125). 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 changes in the growth
characteristics of E. coli under different ammonium sulphate concentrations was investigated
and ammonium sulphate concentrations of 5 % had to be used before any severe inhibition was
observed (126). When the fermentations carried out in the present work showed a
productivity decreases 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
32
by the achieved ammonium concentrations. To better understand the causes of the possible
organic acid and ammonia inhibition was further investigated in Paper II.
Impact of the neutralising agent
In this study the aim was to address the problems associated with decreased succinic acid
productivity and limited final succinic acid concentrations. If the technology is to be applied to
a large scale production facility it is important to identify and minimise the factors that
negatively influence and limit productivity and final titre. Succinic acid production by AFP184
was studied when NH
4
OH, KOH, NaOH or Na
2
CO
3
were used as neutralising agents. In
Paper I a decrease in succinic acid production was observed when the fermentations
accumulated organic acids. Organic acids have a pK
a
in the order of 4. The fermentations in
Paper I were carried out at a pH of pH 6.6-6.7. At this pH the produced acids are dissociated
and the cytoplasmic membrane should be relatively impermeable to the acid anions and the
protons (62, 66). However, other studies indicate that acid anions can traverse the cytoplasmic
membrane (73). If dissociated in the medium, the effect would be accumulation of the acid
anions in the cytoplasm. Acetate anions are known to negatively affect growth in E. coli by
affecting the methionine biosynthesis (72). The metabolic effects of succinate anions on
anaerobe succinic acid production are unknown. The neutralising agent used in Paper I,
NH
4
OH, is known to cause growth inhibition in E. coli at concentrations >3 g L
-1
(124, 125).
The aim of Paper II was to study the possible effects of changing neutralising agent in order to
improve the succinic acid productivity and final titres obtained in Paper I. Fermentations with
different bases are summarised in Table 2.
Common for all fermentations was the loss of viable cells during the anaerobic phase of the
fermentations. The rate of cell death was significantly higher when using NH
4
OH than any of
the other neutralising agents. When using NH
4
OH succinate production and glucose
consumption completely ceased after 32 hours of total fermentation time. For potassium and
sodium bases succinate production never stopped, but the volumetric productivity decreased
during the course of the fermentations (Fig. 11). The reason could be both due to the toxicity
of ammonia (127) and due to lower glucose consumption and thus resulting in a decreased
energy generation as the fermentations proceed. Productivities per viable cell were calculated
33
and neither sodium nor potassium bases showed any significant reduction in productivity per
viable cell (see Paper II) and the decrease in productivity was attributed to a loss of viable cells.
Table 2. Summary of fermentation results for different bases
a
Base Y
P/S
(g g
-1
)
anaerobic
Q
P
(g L
-1
h
-1
)
anaerobic, T
20
Q
P
(g L
-1
h
-1
)
anaerobic, T
40
Q
P
(g L
-1
h
-1
)
anaerobic, (max g L
-1
)
b
NH
4
OH 0.74 2.91 1.38 1.85*
KOH 0.76 2.61 1.33 0.65
NaOH 0.71 2.45 1.52 0.72
Na
2
CO
3
0.77 3.31 2.05 0.89
a
Y
P/S
is the mass yield of succinic acid based on the glucose consumed in the anaerobic phase,
and Q
P
is the volumetric productivity of succinic acid during the anaerobic phase after 20 h
(T
20
) or 40 h (T
40
) total fermentation time.
b
Anaerobic productivity calculated either when
fermentations are terminated or maximum succinic acid concentration is achieved.
*maximum concentration was achieved after 32 hours
Comparing the bases used in this study showed that Na
2
CO
3
generated the highest
productivities and final succinate concentrations. The reason why Na
2
CO
3
neutralisation
results in a higher succinate productivity than NaOH could be explained by an incomplete
saturation of the medium with CO
2
. Addition of extra carbonate from the neutralising agent
might increase the available levels of resulting in a higher amount of carbon that can be
channelled through the reductive arm of the TCA.-cycle instead of being committed to
pyruvate. Using Na

3
HCO
2
CO
3
also resulted in a higher succinate yield than NaOH. The differences
in the yield can be explained by an increased carbon flux down the reductive arm of the
TCA-cycle. However it does not explain the generally low yields (0.7-0.8 gram succinate per
gram glucose consumed anaerobically) achieved. The theoretical yield of succinic acid from
one gram of glucose during anaerobic conditions is 1.12 (128).
34
0
20
40
60
80
100
0 5 10 15 20 25 30 35 40
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
0
20
40
60
80
100
0 20 40 60 80 100
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
0
20
40
60
80
100
0 20 40 60 80 100
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
0
20
40
60
80
100
0 20 40 60 80 100
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
a b
c d
Figure 11. Succinic acid and glucose concentrations and viable cell density for fermentations
neutralised with KOH (a), NaOH (b), Na
2
CO
3
(c), and NH
4
OH (d). Symbols used in the
Figure: Succinic acid (g L
-1
), Glucose (g L
-1
), and Viable cells (cells mL
-1
x 10
9
).
The productivity is strongly correlated to the concentration of viable cells and to further
increase succinate productivity by AFP184 it is essential to reduce the loss of viability during
the anaerobic phase. The reason the cells lose viability is not clear. Elevated organic acid
concentration in the medium and possible accumulation of organic acid anions in the
cytoplasm may be important factors. Increased osmolarity due to organic acid production and
addition of neutralising agent is also assumed to have a negative effect on viability. During high
osmolarity E. coli accumulates compatible solutes, preferably glycinebetaine, proline, and
ectoine. Corn steep liquor does not contain these osmoprotectants and in the absence of these,
E. coli is known to synthesise and accumulate trehalose. Synthesis of trehalose and
accumulation of succinate could be part of the answer to why the yields are diverging from the
theoretical. A study elucidating intracellular concentrations of metabolites, in particular
succinate and trehalose, and the effect of supplementation of the medium with
osmoprotectants like glycinebetaine is being undertaken.
35
36
Conclusions
In Paper I we have demonstrated growth and succinate production of E. coli strain AFP184 in
a medium containing corn steep liquor, salts, and different monosaccharides. 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 hours and need to be improved for the process to become more
economical.
In Paper II we have 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 NH
4
OH it was possible to
achieve an almost 100 % increase in final succinic acid concentration using Na
2
CO
3
. It was also
observed that the cell viability decreased during the anaerobic phase irrespective of the base
used. The viable cell decrease is attributed to increased acid concentration coupled with
possible cytoplasmic succinate accumulation, high medium osmolarity and long time exposure
to a low energy generating environment and high maintenance demands during the course of
acid production in the anaerobic phase.
37
38
Future work
The two studies done in this work has greatly improved to possible production of biological
succinic acid. They have also generated a number of potential future studies.
The most important aspect to address is the loss of cell viability during anaerobic succinate
production. To unravel the probable reasons studies investigating intracellular concentrations
of metabolites such as succinate and trehalose should be undertaken. The impact of addition of
different osmoprotectants to the growth medium should also be investigated in conjunction
with determination of intracellular metabolite concentrations.
A thorough study of the fermentation parameters (pH, temperature, agitation, etc) for the
anaerobic phase should be carried out to determine optimal conditions for succinic acid
production.
AFP184 has been shown to be able to utilise a number of different monosaccharides. In order
to fit into a biorefinery concept it is important to demonstrate and investigate the fermentation
characteristics of AFP184 in industrial hydrolysates from e.g. hemicellulose extracted prior to
alkaline pulping in a kraft pulp mill or hydrolysed corn stover.
Following fermentation succinic acid must be separated from the fermentation broth. Very
little work has been done in this area and more attention needs to be directed to this field in
order to improve the economical feasibility of biochemical succinic acid production.
39
40
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51
52
Paper I
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 chemi-
cals, 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 succi-
nogenes, 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
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 interest-
ing 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,
* To whom correspondence should be addressed. Ph: +46920491315.
Fax: +46920491199. Email: ulrika.rova@ltu.se.
10.1021/bp060301y CCC: $37.00 xxxx American Chemical Society and American Institute of Chemical Engineers
PAGE EST: 7.2
Published on Web 01/25/2007
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
): K
2
HPO
4
, 1.4; KH
2
PO
4
, 0.6;
(NH
4
)
2
SO
4
, 3.3; MgSO
4
7H
2
O, 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 fer-
menter 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 NH
4
OH (15% NH
3
solution).
The dissolved oxygen concentration (%DO), measured by a pO
2
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 produc-
tion 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 CO
2
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 (OD
550
). Optical
densities were then converted to dry cell weight (DCW, g L
-1
)
using the following OD-DCW calibration curve developed in
our laboratory:
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 con-
centrations 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 KH
2
PO
4
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 concentra-
tions 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 concen-
trations 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
DCW) 0.324OD
550
+ 1.687
B
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
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
) 10
9
. 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 AFP184
a
sugar (h
-1
) YP/S (g g
-1
) anaerobic QP (g L
-1
h
-1
) overall QP (g L
-1
h
-1
) anaerobic, T22
d
QP (g L
-1
h
-1
) anaerobic, T32
d
glucose 0.42 0.83 1.27 2.89 1.76
fructose 0.50 0.66 1.01 1.79 1.26
xylose 0.57 0.50 0.78 1.49 1.08
glu/fru
b
0.58 0.79 1.67 1.05
glu/xyl
c
0.60 0.86 1.48 1.08
a
Specific growth rates were obtained from fermentations carried out 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 volumetric productivity of succinic acid during the anaerobic phase.
b
glu/fru is an abbreviation for glucose/
fructose.
c
glu/xyl is an abbreviation for glucose/xylose.
d
Anaerobic productivity at 22 and 32 h total fermentation time, respectively.
C
hence 32 h was selected as the final fermentation time. When
fructose and xylose were used, the final succinic acid concentra-
tions 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 (Q
P
) 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 fermenta-
tions 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 fermenta-
tion 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 combina-
tions 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 EIICB
glc
, 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. Interest-
ingly, growth on xylose was initially very slow, whereas the
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).
D
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 EIICB
glc
, 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 anaero-
bic 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 concen-
tration 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 fermenta-
tions 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
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).
E
(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 concentra-
tions 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 sugar-
limited. 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.
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.
F
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.
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and fed-batch fermentations. Appl. EnViron. Microbiol. 1990, 56,
1004-1011.
(43) Shiloach, J.; Fass, R. Growing E. coli to high cell density-A
historical perspective on method development. Biotechnol. AdV.
2005, 23, 345-357.
(44) Riesenberg, D. High-cell-density cultivation of Escherichia coli.
Curr. Opin. Biotechnol. 1991, 2, 380-384.
(45) Liu, Y. C.; Cheng, K. Y. Effect of ammonium concentration on
the cultivation of recombinant Escherichia coli producing penicillin
G acylase. J. Chin. Inst. Chem. Eng. 2000, 31, 601-607.
Received October 2, 2006. Accepted December 21, 2006.
BP060301Y
H PAGE EST: 7.2
Paper II
Impact of neutralising agent and pH on the succinic acid production by
metabolically engineered Escherichia coli in dual-phase fermentations
Christian Andersson, Jonas Helmerius, David Hodge, Kris A. Berglund, and Ulrika Rova*
Division of Biochemical and Chemical Process Engineering, Lule University of Technology,
SE-971 87 Lule
Abstract
Escherichia coli AFP184 has previously been reported to produce succinic acid in a low-cost
medium achieving volumetric productivities close to 3 g L
-1
h
-1
. At total organic acid
concentrations of 30 g L
-1
the productivity decreased drastically resulting in final succinate
concentrations of 40 g L
-1
. In order to improve the economical viability of biochemical
succinic acid production the final succinic acid concentration must be increased and the
volumetric productivity should be maintained at elevated values (>2.5 g L
-1
h
-1
) for an
extended period of time. In the present work the fermentation pH was optimised and the
effects of different neutralising agents (NH
4
OH, KOH, NaOH, and Na
2
CO
3
) on succinic acid
production by AFP184 were investigated. It was found that AFP184 was rather sensitive to
the fermentation pH and an optimum was found in the range of 6.5-6.7. Highest concentration
of succinic acid was obtained with Na
2
CO
3
, 78 g L
-1
. It was also found that the productivity
per viable cell for the K- and Na-bases was rather constant during the anaerobic phase.
However, loss of cell viability occurred, and decreased the volumetric productivities. In this
study it was demonstrated that by altering the neutralising agent it was possible to increase the
final succinic acid concentration by almost 100 %.
Keywords: Succinic acid, E. coli fermentation, Neutralising agent, Cell viability
* To whom all correspondence should be addressed
Ph: +46920491315. Fax: +46920491199. Email: Ulrika.Rova@ltu.se
1
Introduction
Increased costs of petroleum have motivated the search for cost effective alternatives for
transforming relatively inexpensive biomass into fuels and chemicals. The key to success in
the development of profitable industrial biotechnology is the choice of target fermentations
that can compete with the efficiency of the petrochemical industry. It is essential to develop
fermentations that produce molecular building blocks which can be used as precursors to
produce a number of high-value chemicals or materials. This building block concept follows
much of the same strategy 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) identified twelve sugar-derived high-value chemicals that could be
produced from both lignocellulose and starch and serve as an economic driver for a
biorefinery (1). Succinic acid is considered as one of the most interesting building blocks to
be produced in a biorefinery. In addition to its own use as a food ingredient and chemical,
succinic acid has the potential to produce a wide range of products and derivatives; 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. Succinate is traditionally manufactured from maleic anhydride through n-butane
using petroleum as raw material. Succinic acid could also be produced using biochemical
conversion of biomass by fungal or bacterial fermentations. Production of succinic acid has
been demonstrated in a number of microorganisms including Bacteroides ruminicola and
Bacteroides amylophilus, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Corynebacterium glutamicum, and
Escherichia coli (2-14). The Alternative Feedstock Program (AFP) initiated by the USDOE
resulted in the development of two very interesting metabolically engineered E. coli strains
with increased succinic acid production, AFP111 and AFP184 (4, 13, 15). 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) (4). In this strain the mutations resulted in succinic acid yields in the order of 1 mol
succinic acid per mol 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 (15). A number of different E. coli
2
mutants have been developed by different groups and tested for succinate production (8, 9, 11,
14, 16-20). Fermentations using recombinant AFP111 overexpressing heterologous pyruvate
carboxylase conducted in a complex media with the main components tryptone (20 g L
-1
) and
yeast extract (10 g L
-1
), resulted in improved succinate mass yield of 1.10 and productivities
(6, 21).
In a previous study AFP184 was used to produce succinic acid to final concentrations of 25-
40 g L
-1
and productivities in the range of 1.5-3 g L
-1
h
-1
from glucose, fructose, and xylose in
a low-cost industrially relevant medium (22). The productivities, although initially very high,
declined as the organic acid concentration increased. It was suggested that the loss of succinic
acid productivity and cell viability were due to inhibition and toxicity of the produced organic
acids or the increased addition of the neutralising agent. Organic acid toxicity resulting in
inhibition of cell growth and product formation is a well known phenomenon in E. coli (23-
26). The inhibitory effects can be both pH-based and include anion specific effects on
metabolism (27-29). Acetate for example has been shown to inhibit enzymes in the
methionine pathway leading to accumulation of homocysteine, which is inhibitory to growth
(25). E. coli cell membranes possess an inherently low proton permeability, which is of
importance both for pH homeostasis and for creating and sustaining the protonmotive force
(30, 31). The undissociated form of the organic acids can however diffuse freely over the cell
membrane of E. coli. At low pH the extracellular concentration of undissociated form of the
organic acid will increase, resulting in increased diffusion into to cytosol. Since the
intracellular pH (pHi) is slightly alkaline (~7.5) the acids, having pK
a
values of around 4, will
dissociate, resulting in an increased proton concentration in the cytoplasm (32). Organic acids
could be considered as protonophores which shuttles protons into the cytoplasm. Eventually,
accumulation of protons will result in a disruption of the pHi and hence an uncoupling of the
membrane pH gradient (pH) (24) which, during aerobic growth is essential for maintenance
of the protonmotive force and the cellular energy generation. However, during anaerobic
succinate production the protonmotive force does not play a major role, but a lowering of the
cytoplasmic pH can have detrimental effects on the function of cellular proteins and enzymes
as well as affecting the integrity of the DNA. E. coli counter the imposed acid stress by
different acid resistance (AR) systems described in detail elsewhere (33-43). The effects of
the acid anions have been investigated for some acids, e.g. acetate (25), but the specific
effects of the succinate anion is not known.
3
As fermentation acids are produced the pH of the medium decreases. It is countered by
addition of a neutralising agent. In earlier work NH
4
OH was used to neutralise the
fermentations (22). The neutralising agent, in addition to the organic acid being produced,
may negatively affect the fermentations. It has been demonstrated that ammonia challenged
the integrity of the outer membrane of E. coli reducing growth (44) and at concentration >3 g
L
-1
ammonia is known to inhibit growth (45, 46). The present study aims to address the
problems associated with decreased succinic acid productivity and limited final succinic acid
concentrations. Maintaining a high volumetric productivity and achieving higher final
succinic acid concentrations is very important for applying the technology to a large-scale
industrial facility. Different neutralising chemicals were tested in order to outline and limit
possible toxic and inhibitory effects. The fermentation pH was first optimised for succinate
production using NH
4
OH. To investigate the differences between ammonia, alkali hydroxides
and carbonates, fermentations at the optimal pH was conducted, using KOH, NaOH, and
Na
2
CO
3
as neutralising agents.
Materials and Methods
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 (15), was
used in this study. Cultures were stored at 80 C as 30% glycerol stocks. Seed cultures were
prepared by inoculating 100 mL sterile medium (same medium as used for the batch
fermentation, see below) in a 500 mL shake-flask with 200 L of the glycerol stock culture.
The seed culture was incubated at room temperature, 200 rpm, for 16 h.
Fermentations
Batch fermentations with an aerobic growth phase and an anaerobic production phase 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
: K
2
HPO
4
, 1.4;
KH
2
PO
4
, 0.6; (NH
4
)
2
SO
4
, 3.3; MgSO
4
7H
2
O, 0.4; corn steep liquor (CSL; 50% solids,
Sigma-Aldrich), 15; and 1 mL antifoam agent (Antifoam 204, SigmaAldrich). The bioreactor
was sterilised with the medium at 121 C for 15 min; thereafter 200 mL of a separately
4
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 was controlled at 37 C and the pH during the aerobic phase was
controlled at 6.65 by automatic addition of NH
4
OH (15% NH
3
solution). The dissolved
oxygen concentration (%DO), measured by a pO
2
electrode, was controlled during the aerobic
phase by varying the agitation speed between 500 and 1000 rpm. During the aerobic growth
phase (8 h) the culture medium was aerated with an air flow of 5 L min
-1
. When the optical
density at 550 nm reached a value of 30-35 the anaerobic production phase was initiated by
withdrawing the air supply and sparging the culture medium with CO
2
at a flow rate of 0.8 L
min
-1
. The agitation speed was set to 500 rpm for the anaerobic phase. Succinic acid was
produced during the anaerobic phase and to sustain acid production 50 mL sterilised glucose
solution (500 g L
-1
) was added when the glucose concentration was less than 20 g L
-1
,
typically after 20 h of total fermentation time. The anaerobic phase proceeded for
approximately 100 h, or until the succinic acid productivity ceased. During the fermentations,
samples were aseptically withdrawn for analysis of optical density, viable cells, sugars and
organic acids concentrations.
pH optimum
To establish pH optimum, fermentations were carried out with NH
4
OH as neutralising agent
at a controlled anaerobic pH of 5.5, 6.0, 6.5, 6.65, 7.0 and 7.5, respectively.
Impact of the neutralising agent
In order to evaluate the effect of a fast increase in succinate concentration on the subsequent
succinate productivity and cell viability, succinic acid was externally added at the onset of the
anaerobic phase to a final concentration of 50 g L
-1
. The added succinic acid solution, 400 g
L
-1
, was neutralised with KOH, NaOH or NH
4
OH respectively. To compare the effects of
altering the neutralising agents dual-phase fermentations were carried out with KOH (10 M),
NaOH (10 M) or Na
2
CO
3
(300 g L
-1
). The pH was controlled at 6.5 6.7.
5
Analysis
Cell Growth
Cell growth was monitored by measuring the optical density at 550 nm (OD
550
). Optical
densities were then converted to dry cell weight (DCW, g L
-1
) using the following equation
derived from an OD-DCW calibration curve:
8574 . 0 3447 . 0
550
+ OD
To establish the number of viable cells, ten-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 millilitre
fermentation broth. All counts were made in duplicate.
Organic acid and sugar analysis
Organic acid and sugars were detected and quantified by HPLC as previously described (22).
Results
pH optimum
In order to initiate the anaerobic phase at a constant cell density all fermentations were carried
out under the same aerobic conditions at pH 6.65. To determine the optimum pH for succinic
acid production, the anaerobic phase was controlled at pH 5.5, 6.0, 6.5, 6.65, 7.0, and 7.5,
respectively using NH
4
OH as neutraliser. The final concentration of succinic acid was highest
at pH 6.5 and pH 6.65 (Fig. 1a). The maximum succinic acid concentration obtained was 43 g
L
-1
, which corresponds well with previously reported results for this organism (22). In
contrast, fermentations conducted at pH 5.5 and 7.5 resulted in very low succinate
concentration (< 5 g L
-1
). At pH 6.0 the final succinate concentration was in the same range as
obtained for pH 6.65, but the productivity was considerably lower (Fig. 1b). Apart from
succinate the only other product formed was acetate with the highest concentration obtained at
pH 6.65 (5.3 g L
-1
). Acetate was not detected at pH 7.5 and thereafter the acetate
concentration was lowest at pH 5.5 (1.4 g L
-1
), followed by pH 6.0 (3.6 g L
-1
), and pH 7.0 (3.9
g L
-1
). For the following fermentations it was decided to use a pH between 6.5 and 6.7.
6
From the measurements of viable cells it was observed that at the cell viability at the two
higher pH values (pH 7.0 and 7.5) drastically decreased (Fig. 1c). The cell death was most
severe for pH 7.5. For pH 7.0 the cell death started first after 20 hours of total fermentation
time, whereas for pH 7.5 it started at the onset of the anaerobic phase. Fermentations at pH
6.65 and 6.0 showed similar decrease in cell viability as previously observed (22). At the
higher pH values the loss in cell viability was accompanied by a decrease in optical density
and hence cell lysis is a reasonable explanation (Fig. 1d).
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40 50
Time (hours)
O
D
5
5
0
0
5
10
15
20
25
30
35
40
45
50
0 10 20 30 40 50
Time (hours)
C
e
l
l
s
/
m
L

x

1
0
9
0
5
10
15
20
25
30
35
40
45
50
0 10 20 30 40 50
Time (hours)
S
u
c
c
i
n
i
c

a
c
i
d

(
g

L
-
1
)
0
5
10
15
20
25
30
35
40
45
50
5 5.5 6 6.5 7 7.5 8
pH
F
i
n
a
l

s
u
c
c
i
n
i
c

a
c
i
d

(
g

L
-
1
)
a b
c d
Figure 1. Fermentation characteristics for pH optimisation during succinic acid production.
Figure a) shows how the final succinic acid concentration depends on fermentation pH. Figure
b) shows succinic acid concentration as a function of time. Figure c) and d) show the viable
cell concentration and dry cell weight respectively. Symbols used in b), c), and d): pH 6,
pH 6.65, pH 7, pH 7.5
Impact of the neutralising agent
Succinic acid neutralised with NH
4
OH, KOH, or NaOH, was added to the bioreactors to a
final succinate concentration of 50 g L
-1
. After addition no succinate was produced and the
cell viability decreased by approximately 50% within 2 hours after the addition. All three
7
fermentations gave similar results and no distinction considering cell viability could be
observed.
Standard, dual-phase fermentations were carried out with KOH, NaOH, or Na
2
CO
3
as
neutralising agents. In order to maximise productivity and final titre the pH was controlled
between 6.5 and 6.7. All three bases resulted in similar fermentation profiles (Fig. 2) where
the highest final succinic acid concentration, 78 g L
-1
, was achieved when Na
2
CO
3
was used
as base. Using NaOH resulted in 69 g L
-1
and KOH in 61 g L
-1
. Again, the only by-product
formed was acetic acid and the lowest concentration, 3.7 g L
-1
, was obtained when Na
2
CO
3
was used. Fermentations with NaOH and KOH resulted in acetic acid concentrations of 5.7 g
L
-1
and 6.0 g L
-1
respectively. Yields achieved were in the range of 0.75 gram succinic acid
per gram glucose consumed in the anaerobic phase (1.14 mol moles
-1
) for all fermentations. It
should be pointed out that glucose was not limiting in any of the fermentations as glucose (25
g) was added when the glucose concentration was less than 20 g L
-1
.
Table 1. Summary of fermentation results for different bases
a
Base Y
P/S
(g g
-1
)
anaerobic
Q
P
(g L
-1
h
-1
)
anaerobic, T
20
Q
P
(g L
-1
h
-1
)
anaerobic, T
40
Q
P
(g L
-1
h
-1
)
anaerobic, (max g L
-1
)
b
NH
4
OH 0.74 2.91 1.38 1.85*
KOH 0.76 2.61 1.33 0.65
NaOH 0.71 2.45 1.52 0.72
Na
2
CO
3
0.77 3.31 2.05 0.89
a
Y
P/S
is the mass yield of succinic acid based on the glucose consumed in the anaerobic
phase, and Q
P
is the volumetric productivity of succinic acid during the anaerobic phase after
20 h (T
20
) or 40 h (T
40
) total fermentation time.
b
Anaerobic productivity calculated either
when fermentations are terminated or maximum succinic acid concentration is achieved.
*maximum concentration was achieved after 32 hours
Viable cell concentrations for fermentations using NaOH or KOH as the pH regulator resulted
in 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. 2a and b). No significant decrease in optical density was observed
during either of the fermentations (data not shown). Using Na
2
CO
3
resulted in viable cell loss
8
first after a total fermentation time of 20 hours, but like the fermentations with NaOH and
KOH the decrease in cell density was slow during the remaining time of the fermentation
(Fig. 2c).
0
20
40
60
80
100
0 20 40 60 80 100
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
0
20
40
60
80
100
0 20 40 60 80 100
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
0
20
40
60
80
100
0 20 40 60 80 100
Time (hours)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g

L
-
1
)
C
e
l
l
s
/
m
L

x

1
0
9
a b
c
Figure 2. Succinic acid and glucose concentrations and viable cell density for fermentations
neutralised with KOH (a), NaOH (b), and Na
2
CO
3
(c). Symbols used in the Figure: Succinic
acid (g L
-1
), Glucose (g L
-1
), and Viable cells (cells mL
-1
x 10
9
).
The use of sodium based neutralisers gave higher productivity than potassium based, and
Na
2
CO
3
gave higher productivity than NaOH. Productivities are presented as grams succinic
acid produced per viable cell as functions of fermentation time for fermentations neutralised
with NH
4
OH (pH 6.65), KOH, NaOH, and Na
2
CO
3
(Fig. 3a). In general, productivities were
initially high, but decreased in the beginning of the anaerobic phase. When using NH
4
OH the
succinate productivity completely stopped after 32 hours, whereas the other fermentations
showed stable productivities which remained constant or increased slightly during the rest of
the fermentations. At the onset of the anaerobic phase, volumetric productivities reached
initial values of more than 3-3.5 g L
-1
h
-1
, but decreased significantly after approximately 15
9
hours of total fermentation time (Fig. 3b). After roughly 30 hours of total fermentation time
the volumetric productivity only decreased slowly.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 10 20 30 40 50 60 70 80 90 100
Time (hours)
Q
P

(
g

L
-
1

h
-
1
)
0
2
4
6
8
10
12
14
16
0 10 20 30 40 50 60 70 80 90 100
Time (hours)
Q
P

(
g

c
e
l
l
-
1

h
-
1
)
a b
Figure 3. Productivity per viable cell x 10
14
(a) and volumetric productivity (b) as functions
of time. Symbols used in the Figure: NH
4
OH, KOH, NaOH, and Na
2
CO
3
. Viable
counts were made in duplicates and the difference is shown by the error bars. Where no error
bar is present the error was small enough to be covered by the Figure symbols.
Discussion
pH optimum
From NH
4
OH neutralised fermentations at different pH it can be concluded that pH has a
strong impact on succinic acid production. The optimal pH interval is also very narrow around
6.6 and diverging to higher or lower pH results in a significant reduction of succinic acid
productivity and considerably decreased final titres. It is well-documented that enzymes have
an optimal pH range outside which its activity is reduced (47, 48). It seems reasonable to
assume that the decrease in productivity and cell viability observed at higher and lower pH
values are correlated to generally impaired enzymatic activity. In case of cell viability, the
cells were significantly more sensitive to the higher than the lower pH values investigated.
Considerable loss in cell viability was observed during the anaerobic phase at pH 7.5. It is
known that E. coli cells lyse at alkaline pH (pH 9-12) and that ammonia concentrations in the
range of 80-100 mM damage the outer membrane and increase the leakage of periplasmic
proteins to the medium (44, 49). However, a pH of 7.5 is only slightly alkaline and it is
possible to grow E. coli aerobically to high cell density at that pH. Anaerobic acid production
imposes limits on the cells ability to generate energy. At pH 7.5 the glucose consumption was
10
very low compared to that of the other pH values investigated (data not shown) and cells
would be limited in their ability to divide and maintain viability. The slightly elevated pH
might also weaken the outer and cytoplasmic membranes (49), this together with moderate
agitation and the ammonia concentration could compromise the ability of the thin
peptidoglycan cell wall to stabilise the cells. The result would be disruption of the
cytoplasmic membrane resulting in cell lysis and death.
Organic acid production and inhibition
The major aim of the present study was to elucidate the effects of different neutralising agents
on succinic acid productivity. From the fermentation profiles (Fig. 2) it is clear that using
potassium or sodium bases for neutralisation would be beneficial for the final succinic acid
concentration. Compared to fermentations neutralised with NH
4
OH fermentations neutralised
with potassium or sodium bases obtained increased final titres by at least 50%. It should be
noted that unlike NH
4
OH fermentations succinate production in alkali fermentations never
ceased. With regards to productivity and final acid concentrations the difference between
using NaOH or KOH was small (Fig. 2 and 3). However, using Na
2
CO
3
resulted in increased
productivity and succinate concentration concurrently with a decreased acetic acid production.
The increased productivity might caused by an increased availability of hydrogen carbonate
( ) from the addition of the base chemical. The enzyme phosphoenolpyruvate (PEP)
carboxylase catalysing the carboxylation of PEP to oxaloacetic acid uses as a substrate
for the reaction (50). The higher productivity during neutralisation of Na

3
HCO

3
HCO
2
CO
3
indicates that
the growth medium is not saturated by the CO
2
from the sparging. The additional availability
of might help to direct the metabolic flow towards succinate which can explain the
lower acetate formation. The yield when using Na

3
HCO
2
CO
3
was 0.77 gram succinic acid produced
per gram glucose consumed in the anaerobic phase. The anaerobe succinate yield when using
NaOH was 0.71 g g
-1
. The difference in yield between the two sodium bases can be explained
by the increased succinate concentration as a result of a reduced flow of carbon to acetate.
The theoretical yield of succinic acid from glucose during anaerobic succinic acid production
is 1.12 g g
-1
(22). Synthesis and accumulation of compatible solutes as a response to osmotic
stress in the high osmolarity environment generated by the succinate production and base
addition could explain the divergence between the achieved and theoretical yield (see Viable
cells below for a more thorough discussion).
11
Productivities per viable cell as a function of time showed that the production of succinate
stopped after 32 hours when NH
4
OH was used as neutraliser. Fermentations using any of the
other three bases did not show similar results, instead a productivity decrease was observed
after approximately 60 hours for NaOH and KOH, and after 35 hours for Na
2
CO
3
(Fig. 3).
This corresponds to succinic acid concentration around 55-60 g L
-1
for the sodium bases and
around 50 g L
-1
for KOH. For the remaining time of the fermentations productivity per viable
cell remained constant or increased slightly. Given that the productivities per viable cell
remain more or less constant from approximately 50 hours of total fermentation time for
potassium and sodium base neutralised fermentations it seems like neither of these bases nor
the succinic acid concentration negatively affects or inhibits the succinic acid productivity of
AFP184 in the medium used. However, the viable cell concentration decreases throughout the
anaerobic phase and reduces the total succinic acid productivity, which also reflects the
volumetric productivities decrease during the anaerobic phase (Fig. 3b). This might be
attributed to the sodium/potassium or succinic acid concentration (see discussion regarding
viable cells below). When using NH
4
OH, productivity completely ceased after 32 hours. At
this time the viable cell count was still high and it is concluded that ammonium at the given
concentrations (~10 g L
-1
) severely inhibit succinic acid productivity in AFP184. The same
productivity repression was observed in a previous study (22).
It is clear from the results that the preferred neutralising agent would be Na
2
CO
3
, since it
generated the highest productivities, final titres and had the lowest by-product formation.
Viable cells
From the results with neutralisation by potassium or sodium base the cell viability seems to be
the limiting step in succinic acid production when using AFP184.
Organic acid toxicity is a well studied phenomena since it impairs cell growth and
productivity (24, 25, 27-29, 51). The most obvious aspect to consider is the total organic acid
load. Organic acids in their undissociated form can diffuse over the cell membranes. Once
inside the cytoplasm they dissociate and lower the slightly alkaline cytoplasmic pH, which
can result in protein denaturation and DNA damages (23, 28, 32, 33). The impact of different
bases on succinic acid productivity was studied at conditions where the external pH was
controlled between 6.5 and 6.7. At this pH interval succinic acid with pKa of 4.19 and 5.57
would be dissociated and not able to freely traverse the cell membranes. It has been indicated
that acetate and lactate can cross the cytoplasmic membrane in the dissociated form (24). This
12
implies that the anions of succinic acid might accumulate in the cytoplasm. Increased
cytoplasmic concentrations of the anions can have different metabolic affects depending on
which specific organic acid being studied (23, 25, 52). Experiments to quantify intracellular
succinate concentrations are currently being undertaken.
Another effect of the increased acid concentration is a subsequent increase in medium
osmolarity, originating both from organic acids and from the added neutralising agent. There
are numerous studies and reviews on osmotic stress and osmotolerance in bacteria (53-56). It
could be proposed that the viable cell loss could be due to the high osmolarity of the medium
that is generated during the anaerobic phase (>1.2 osmoles per litre from neutralised succinate
alone). When succinic acid (50 g L
-1
) was added at the beginning of the anaerobic phase, cell
viability decreased by approximately 50% irrespective of neutralising agent. However, when
the osmolarity was due to cellular succinate production the cells adapted to the conditions and
no sudden decreases in viability were observed. Even at high osmolarities (from e.g. 30-80
hours) viable counts remained relatively constant.
E. coli subjected to osmotic stress first respond by accumulating K
+
. At higher osmolarities
K
+
accumulation in the cytoplasm interfere with cellular functions and cells excrete K
+
and
instead accumulate other compatible solutes, like glycinebetaine, proline, and trehalose (57,
58). The solutes offering the highest osmotolerance in E. coli are glycinebetaine and proline.
Since glycinebetaine cannot be synthesise de novo in E. coli, it must be accumulated from the
medium. CSL does not contain glycinebetaine (or proline, which cannot be synthesised de
novo either). Choline, a quaternary amine present in CSL (~0.5 % of the dry weight) can be
converted into glycinebetaine by E. coli in the presence of a terminal electron acceptor like O
2
(59). During anaerobic succinic acid production by AFP184 choline can thus not be used as
an osmoprotectant. Under anaerobic conditions, or in media deprived of these
osmoprotectants, E. coli will instead synthesise trehalose intracellular (60). Trehalose can
accumulate to intracellular concentrations equal to approximately 20 % of the osmolar
concentration of solutes in the cultivation medium (53). Carbon balances over the anaerobic
phase can account for approximately 80-85 % of the carbon. Osmotically stressed AFP184
could hence synthesise trehalose during the anaerobic phase to substantial intracellular
concentrations, which could explain the divergence between the achieved and theoretical
yield and identify the unknown carbon fraction. We are currently conducting a study to
characterise intracellular concentrations of metabolic products in AFP184 during succinic
13
acid production and also investigate the effects of addition of osmoprotectants such as
glycinebetaine and proline to the medium.
Conclusions
In this study we have 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 NH
4
OH it
was possible to achieve an almost 100% increase in final succinic acid concentration using
Na
2
CO
3
. It was also observed that the cell viability decreased during the anaerobic phase
irrespective of the base used. The viable cell decrease is attributed to increased acid
concentration coupled with possible cytoplasmic succinate accumulation, high medium
osmolarity and long time exposure to a low energy generating environment and high
maintenance demands during the course of acid production in the anaerobic phase. Further
studies will be directed towards increasing the long term viability in the anaerobic phase by
outlining intracellular metabolite concentrations and investigating the impact of
supplementation of the medium with osmoprotectants.
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