ENHANCING SODIUM CHLORIDE TOLERANCE OF ESCHERICHIA COLI
by
XIANGHAO WU
(Under the Direction of Mark A. Eiteman)
ABSTRACT
The primary goal of this research is to enhance the sodium chloride tolerance of E. coli
in order to prolong the fermentation time and improve organic acid production. One means
examined was to overexpress nhaA encoding a native Na+/H+ antiporter, which led to severely
curtailed microbial growth. Adaptive evolution was then employed to generate NaCl-tolerant
random mutants. Four E. coli mutants with enhanced ability of NaCl tolerance, designated
ALS1184–ALS1187, were isolated after seventy three days of transfer. Several physiological
parameters of MG1655 and ALS1187 were calculated and compared using glucose-limited
chemostats. Strains ALS1317 (MG1655 pflB) and ALS1318 (ALS1187 pflB) were constructed
for lactate accumulation. ALS1318 generated 76.2 g/L lactate compared to 56.3 g/L lactate
produced by ALS1317. The same evolutionary approach used on ALS929, a
pyruvate-generating E. coli strain, resulted in four isolates with greater NaCl tolerance, but did
not improve pyruvate production under comparable conditions.
INDEX WORDS: Sodium chloride tolerance, Escherichia coli, Na+/H+ antiporter, Adaptive
evolution, Lactate production, Pyruvate production, Fed-batch, Chemostat
ENHANCING SODIUM CHLORIDE TOLERANCE OF ESCHERICHIA COLI
by
XIANGHAO WU
B.S., Jiangnan University, China, 2008
A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment
of the Requirements for the Degree
MASTER OF SCIENCE
ATHENS, GEORGIA
2011
© 2011
Xianghao Wu
All Rights Reserved
ENHANCING SODIUM CHLORIDE TOLERANCE OF ESCHERICHIA COLI
by
XIANGHAO WU
Major Professor: Mark A. Eiteman
Committee: Jenna R. Jambeck Yajun Yan
Electronic Version Approved:
Maureen Grasso Dean of the Graduate School The University of Georgia May 2011
iv
DEDICATION
Dedicated to my parents, Zihui Wu and Xiufang Hou for their love.
v
ACKNOWLEDGMENTS
My sincere gratitude goes to my major professor, Dr. Mark A. Eiteman, for his
encouragement, mentorship and technical guidance. I also thank him for sharing his philosophy
and wisdom and for his help with problems in and beyond the research lab. I would also like to
thank the members of my advisory committee, Dr. Jenna R. Jambeck and Dr. Yajun Yan, for
critically reviewing my work and providing valuable insights. I am very grateful to Dr. Elliot
Altman for his thoughtful suggestions and for providing me with various strains. I thank Sarah
Lee and Ronnie Altman for teaching me experiment skills and for their technical assistance. I
also thank my colleagues Dr. Yihui Zhu, Dr. Shiying Lu, Arun Shivkumar Lakshmanaswamy,
Yingjie Ma, Tian Xia, Rupal Prabu, and Eashwar Rajaraman for their cooperation and support.
Finally, I thank the Department of Biological and Agricultural Engineering at the
University of Georgia for providing the opportunity, research facilities and research
assistantship.
vi
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ...............................................................................................................v
LIST OF TABLES ........................................................................................................................ vii
LIST OF FIGURES ..................................................................................................................... viii
CHAPTER 1
Introduction ..........................................................................................................................1
Literature Review .................................................................................................................2
Objectives ..........................................................................................................................15
References ..........................................................................................................................16
CHAPTER 2
Introduction ........................................................................................................................27
Materials and Methods .......................................................................................................30
Results ................................................................................................................................36
Discussion ..........................................................................................................................48
References ..........................................................................................................................51
APPENDICES
A Summary of results from chemoststs ..........................................................................56
B Data of fermentation processes ...................................................................................58
vii
LIST OF TABLES
Table 1: Nongrowth-related energy metabolism (maintenance coefficient, mS) and the biomass
yield on glucose (YX/S) for MG1655 and ALS1187 at different NaCl concentrations ..................44
Table A.1: Summary of the results obtained from chemostat cultures using MG1655 and
ALS1187 at different growth rates and different Na+ concentrations ...........................................56
Table A.2: Summary of the results in this thesis study ..................................................................57
viii
LIST OF FIGURES
Figure 1: nhaA is regulated by Na+ and growth phase via separate promoters ...............................8
Figure 2: Growth rate of two E. coli strains at various NaCl concentrations ................................36
Figure 3: Evolution progress of ALS1187 .....................................................................................37
Figure 4: Growth rate of the evolved strains compared to that of the parent strain at various
NaCl concentrations .......................................................................................................................38
Figure 5. Growth rate of the evolved strains compared to that of the parent strain at various KCl
concentrations ................................................................................................................................39
Figure 6: Specific glucose consumption rate (qS) as a function of specific growth rates for E.
coli MG1655 and ALS1187 at 0.18 M and 0.68 M NaCl ..............................................................40
Figure 7: Specific oxygen uptake rate (qO2) at various specific glucose consumption rate (qS) for
E. coli MG1655 and ALS1187 at 0.18 M and 0.68 M NaCl .........................................................41
Figure 8: Specific carbon dioxide evolution rate (qCO2) at various specific glucose consumption
rates (qS) for MG1655 and ALS1187 at 0.18 M and 0.68 M NaCl ...............................................42
Figure 9: Lactate concentration and Na+ concentration in fed-batch fermentations using
MG1655 pflB and ALS1187 pflB ..................................................................................................44
Figure 10: Evolution progress of ALS1182 ...................................................................................45
Figure 11: Pyruvate production in fed-batch fermentations using ALS929 and ALS1182 ...........46
1
CHAPTER 1
Introduction
Over the last century, biotechnology has been applied to produce a wide variety of
biochemicals including organic acids, amino acids, antibiotics, chiral compounds, and
biopolymers. In comparison to traditional chemical processes which often use nonrenewable
mineral resources, bioprocesses can save energy and are environmentally friendly.
For bioprocesses which generate an organic acid, the accumulation of the acid product
reduces the pH, which affects cell growth. In order to maintain the pH at a desirable, optimal
level for continued growth and product formation, a base such as NaOH is added into the
bioreactor. However, addition of NaOH or KOH for pH control causes the accumulation of
cations such Na+ or K+. Ultimately, bacterial growth and acid production is hindered by the
increased osmotic pressure resulting from high ion concentration.
Several mechanisms are used by the bacteria Escherichia coli to regulate the
intracellular osmotic pressure to withstand changes in extracellular osmotic pressure. These
mechanisms permit E. coli to survive against osmotic fluctuations, and could potentially be
used to enhance acid production in strains that are engineered to accumulate specific organic
acids.
2
Literature Review
Pyruvate Production in E. coli
Pyruvic acid (pyruvate) is used in several specialty applications, including in
weight-control (Koh-Banerjee et al., 2005), as a nutraceutical (McCarty, 2000), and as an
antioxidant (Wang et al., 2007). Pyruvate also is a useful starting material in chemical,
pharmaceutical, and agrochemical industries because this chemical has both reactive ketonic
and carboxyl groups. For example, pyruvate is a raw material for the synthesis of the
pharmaceutical precursors L-tyrosine (Lütke-Eversloh et al., 2007), N-acetyl-D-neuraminic
acid (Lee et al., 2007), and (R)-phenylacetylcarbinol (Guanawan et al., 2007). Reflecting the
variety of application fields, the commercial demand for pyruvate has been increasing. The
classical chemical synthesis of pyruvate from tartaric acid is simple but energy-intensive, with
an estimated production cost of $8,650 per ton, while the estimated cost for pyruvate by
fermentative process is $1,255 per ton (Li et al., 2001a).
Pyruvate is a metabolic intermediate in all living cells. Because pyruvate is
biochemically located at the end of glycolysis, the flux through the glycolytic pathway should
be maximal to generate pyruvate at a high rate (Yokota et al. 1994a; Yokota et al. 1994b;
Schmid et al. 2001; Causey et al. 2004). Of course, pyruvate catabolism should also be reduced.
For example, the aerobic conversion of pyruvate into acetyl-CoA can be blocked by the
chromosomal deletion of genes encoding pyruvate dehydrogenase (aceEF) (Tomar et al.,
2003). Similarly, the conversion of pyruvate into phosphoenolpyruvate (PEP), acetate and
3
lactate may be blocked by the deletion of genes encoding PEP synthetase (pps), pyruvate
oxidase (poxB) and lactate dehydrogenase (ldhA) (Zelić et al., 2004).
Both yeast and bacteria have been examined for the fermentative production of
pyruvate. One approach involves a multivitamin auxotroph of the yeast Torulopsis glabrata.
For example, Chen and coworkers decreased the activity of pyruvate dehydrogensase (Liu et
al., 2004) and used a two-stage oxygen supply control approach (Li et al., 2002) to increase the
pyruvate. A NaCl-tolerant mutant, T. glabrata RS23 (CCTCC M202019), was isolated using
70.0 g/L NaCl as the selective criterion (Liu et al., 2007). With this mutant the pyruvate
production reached 94.3 g/L in 82 h with a yield of 0.635 g/g on glucose (Li et al., 2007).
The bacteria Escherichia coli may also be used for pyvurate accumulation. An
F1-ATPase-defective mutant strain produced more than 30 g/L pyruvate from 50 g/L glucose in
24 h, although this strain had a lower growth rate due to reduced energy metabolism (Yokota et
al., 1994b). Compared to the parent strain (with higher F1-ATPase activity), the specific rates of
glucose consumption and pyruvate production increased 1.9- and 2.8-fold, respectively. A
derivative strain was engineered for pyruvate production by combining mutations which
minimize ATP yield, cell growth, and CO2 production (ΔatpFH ΔadhE ΔsucA) with mutations
which eliminate acetate production [poxB::FRT (FLP recognition target) ΔackA] and other
fermentation products (ΔfocApflB ΔfrdBC ΔldhA ΔadhE). In a mineral salts medium containing
glucose as the sole carbon source, this strain converted glucose to pyruvate with a volumetric
productivity of 1.2 g pyruvate/L·h and a yield of 0.75 g pyruvate/g glucose (77.9% of
theoretical yield) (Causey et al., 2003). A maximum of 749 mM (66.7 g/L) pyruvate was
4
produced with excess glucose. Zelić (2004) constructed an ldhA knockout of E. coli ΔaceEF
ΔpflB ΔpoxB Δpps and obtained a yield of 0.86 g/g with a high volumetric productivity of about
6 g/L·h using a repetitive fed-batch process.
During pyruvate production using E. coli, extracellular pyruvate concentrations above
45 g/L may inhibit microbial pyruvate synthesis (Li et al., 2001b; Zelić et al., 2003; Zelić et al.,
2004). However, this reported microbial inhibition could instead be the result of the
accumulation of a cation. Because of the addition of a base (e.g., NaOH) to maintain the pH at
the optimal growth pH of 7.0, a cation (e.g., Na+) inevitably accumulates. Considering a
fed-batch process without the use of betaine, 56 g/L pyruvate accumulated using E. coli
ALS929 in a fed-batch process (Zhu et al., 2008). Thus, in this example case, the concentration
of Na+ reached about 0.64 M in the bioreactor. This high Na+ concentration and the associated
osmotic stress may be the more important factor in the inhibition of cell growth and organic
acid formation.
Tolerance to the Sodium Ion
Both Na+ and H+ ions play important roles in cell bioenergetics, and in the general
functioning of the cell and proteins. Either too low or high of a concentration of these ions
stresses cells (Padan and Krulwich, 2000). Hence, cells generally rely on a very efficient
homeostatic mechanism to maintain the necessary concentration range. E. coli maintains a
sodium concentration gradient across the cell membrane so that the intracellular Na+
concentration (Na+in) is less than the extracellular concentration (Na+
out). Also, within a wide
range of external pH levels, the intracellular pH is maintained at 7.6 (Padan et al., 1981).
5
E. coli has two Na+/H+ antiporter membrane proteins, NhaA and NhaB, and both play
important roles in osmotic homeostatis (West and Mitchell, 1974). These antiporters exchange
1 Na+ (or 1 Li+) for 2 H+ (Taglicht et al., 1993) and are driven by the electrochemical proton
gradient (i.e., H+out > H+
in) generated by the primary proton pumps. NhaA encoded by nhaA
(Niiya et al., 1982) in E. coli (previously designated ant) is the archetypal Na+/H+ antiporter
and is absolutely required for survival in the presence of 100 mM Li+ or 700 mM Na+ at a pH of
6.8 (Padan et al., 1989, 2001, 2004).
Corroborating its role in pH homeostasis, the activity of NhaA is tightly controlled by
pH. The protein acts as a pH sensor, with conformational changes converting the pH signal into
a change in activity (Padan et al., 2004). The protein is fully downregulated below a pH of 6.5.
Its activity increases by over three orders of magnitude with increasing pH, reaching a
maximum at a pH of 8.5 (Padan et al., 2004). The N-terminus and residues of helix IX at the
cytoplasmic funnel entrance transmit the pH signal required to activate NhaA (Tzubery et al.,
2004). Conformational changes induced by an increase in cytoplasmic pH from 6 to 7 occur at
these locations. Although an increase in pH from 6.5 to 8.5 does not increase nhaA expression,
this pH change markedly increases the sensitivity of expression to the Na+ ion. For example, at
a pH of 8.6 the relatively low concentration of 0.01 M Na+ elicited maximal gene expression,
whereas at a pH of 7.5, 0.1 M Na+ induced a similar response (Karpel et al., 1991;
Rahav-Manor et al., 1992).
The nhaA gene has been mapped at 0.1 min on the E. coli chromosome (Goldberg et al.,
1987; Karpel et al., 1988), cloned (Goldberg et al., 1987), sequenced (Karpel et al., 1988), and
6
encodes a membrane protein of 41,316 Daltons (Taglicht et al., 1991). The NhaA protein has
been purified and reconstituted in proteoliposomes in a functional form (Taglicht et al., 1991).
This membrane protein consists of 388 residues that traverse the inner membrane 12 times, with
both termini ending in the cytoplasm (Rothman et al., 1996). The structure of NhaA exhibits a
distinctive fold of 10 contiguous transmembrane helices and 2 antiparallel, discontinuous
helices (iv and xi) aligned end-to-end to span the membrane (Hunte et al., 2005). A model of
the transport mechanisms, pH regulation and cation selectivity of the NhaA protein has recently
been formulated (Arkin et al., 2007).
Several proteins regulate the expression of the nhaA gene. In the exponential growth
phase, nhaA responds to Na+ by increasing transcription via a Na+-specific regulatory system.
This regulatory system involves the positive regulator NhaR encoded by nhaR (Carmel et al.,
1997), a member of the LysR family of regulators. The expression of nhaR, which maps
downstream of nhaA, is induced specifically by Na+. Hence, NhaR appears to be both a sensor
and a transducer of the Na+ signal, and this protein regulates nhaA expression by undergoing a
conformational change upon Na+ binding, which modifies the NhaR-nhaA contact points
(Carmel et al., 1997). The global regulator hns encoding H-NS protein also plays a role in nhaA
regulation (Dover et al., 1996). In an hns+ strain in the absence of Na+, nhaA is repressed
whether or not nhaR is present. In the presence of Na+, however, NhaR functions as a positive
regulator of nhaA and overcomes the repressive effects of H-NS. Thus, H-NS and NhaR
interact directly or indirectly in the regulation of nhaA. When the hns gene is present, H-NS
7
represses NhaA, and a single copy of NhaR is required for induction of nhaA transcription by
Na+.
Two promoters have been identified for nhaA located at 30 and 72 bp upstream of the
initiation transcription codon (Karpel et al., 1991). The P1 promoter is an NhaR-dependent,
Na+-induced, and H-NS-affected promoter in both the exponential and stationary growth
phases. In contrast to the P1 promoter, the P2 promoter exhibits very low activity during the
exponential phase, but is induced to become the major promoter for the survival of
stationary-phase cells in the presence of high Na+, alkaline pH or the combination of high Na+
and alkaline pH (Dover and Padan, 2001). The regulation mechanism is shown in Figure 1
(Padan et al., 2001).
The other Na+/H+ antiporter found in E. coli is NhaB (Pinner et al., 1992a). This protein
has a molecular weight of 47,000 and is encoded by the nhaB gene. NhaB confers limited
sodium tolerance to cells (by itself) and becomes essential when the lack of NhaA expression
limits growth (Pinner et al., 1992b). The amino acid sequence is not similar to either the
sequence of the E. coli antiporter NhaA or the human antiporter (Sardet et al., 1989). Unlike
NhaA, the expression of NhaB shows no dependence on pH in the range 6.4–8.3.
8
Figure 1. nhaA is regulated by Na+ and growth phase via separate promoters.
9
Tolerance to the Potassium Ion
Potassium is another major monovalent intracellular cation of E. coli and other cells.
Potassium has four major roles: osmotic solute, activator of certain intracellular enzymes,
regulator of intracellular pH, and second messenger to stimulate accumulation of compatible
solutes (Brown, 1990; Epstein, 2003). Cytoplasmic pools of K+ are tightly regulated in bacteria
by several different transport systems that have different kinetics, energy coupling, and
regulation (Stumpe et al., 1996).
Intracellular K+ concentrations are regulated in E. coli through K+ uptake systems
(Epstein and Kim, 1971; Rhoads et al., 1976; Bossemeyer et al., 1989; Dosch et al., 1991), K+
efflux systems (Bakker et al., 1987) and other channels that transport small molecules
(including K+) across the membrane (Sukharev et al., 1994; Levina et al., 1999; Li et al., 2002).
However, K+ efflux systems have not been studied extensively because potassium is a major
monovalent intracellular cation and because of the belief that living cells accumulate K+ and
extrude the smaller Na+ ion. Recent results indicate that excess K+ can be toxic to cells and that
cells regulate levels via K+ efflux systems (Putnoky et al., 1998; Benito et al., 2002; Radchenko
et al., 2006). The only well-studied K+ efflux systems in E. coli involve KefB and KefC
(originally called TrkB and TrkC). However, neither efflux system appears to be a significant
path of K+ efflux produced by high turgor pressure, by alkalinization of the cytoplasm, or by
addition of the membrane decoupler 2,4-dinitrophenol (Bakker et al., 1987).
The ChaA protein encoded by chaA appears to serve as another sodium ion extrusion
system in E. coli (Ivey et al., 1993). ChaA was proposed as a calcium/proton antiporter that had
10
Na+/H+ antiport activity when its gene was encoded by a multicopy plasmid (Ivey et al., 1993).
Subsequently, ChaA was found to function at high pH and be induced by the addition of NaCl,
KCl or sucrose (Shijuku et al., 2002). More recent reports show that ChaA functions as a K+
extrusion system (Radchenko et al., 2006). Cells expressing ChaA mediated K+ efflux against
both outwardly and inwardly directed K+ concentration gradients. E. coli strains containing the
chaA gene were able to grow in an LB medium containing 600 mM KCl, whereas strains
lacking chaA were unable to grow under the same conditions (Radchenko et al., 2006).
Compatible Solutes
In E. coli, certain solutes accumulate during conditions of high osmolality but do not
accumulate during growth at low osmolality. In a medium having low osmolality, the
osmolality of the cytosol is controlled primarily as a result of ionic solutes. In a medium having
high osmolality, however, cytoplasmic osmolality is maintained by the accumulation of
numerous organic solutes called compatible solutes (or neutral solutes). Osmoregulation
modulates the synthesis, catabolism, uptake, and efflux of compatible solutes in response to
external osmolality changes. Several differences exist between the function and effectiveness of
the compatible solutes. For example, ionic solutes such as K+ or glutamate regulate cytoplasmic
osmolality only over low osmolality ranges, and these solutes inhibit some enzymes (Richey et
al., 1987). At high osmolality, neutral compatible solutes such as trehalose, proline and betaine
accumulate. Natural abundance 13C-NMR spectroscopy was employed to demonstrate that
trehalose, a nonreducing disaccharide of glucose accumulated in E. coli K10 in glucose-mineral
11
medium containing 0.45 M NaCl but no betaines or other osmoprotectants accumulated (Strøm
et al., 1986).
The medium composition affects which compatible solute cells accumulate. In
osmotically stressed cells growing in glucose-mineral medium, betaine is generated from
exogenous choline by betaine synthetase encoded by the betA gene (Strom, 1998). Cells also
accumulate betaine if it is present in the medium (Cayley et al., 1992). In a defined medium,
proline accumulates only if present in the medium. Two genes, proP and proU, encode two
permeases that mediate uptake of these betaine and proline in E. coli (Gowrishankar, 1986).
Trehalose is one of three neutral compatible solutes effective in E. coli. Trehalose may
protect against the deleterious effects of extreme dehydration (Crowe et al., 1992), and this
chemical increases the survival percentage of metabolic engineered E. coli cells after
desiccation (Miller and Ingram, 2008). In the absence of exogenously supplied
osmoprotectants, trehalose accounts for up to 20% of cellular osmolality in E. coli grown in a
medium having high osmolality (Larsen et al., 1987; Dinnbier et al., 1988; Cayley et al., 1991;
Welsh et al., 1991; Cayley et al., 1992). Entry into stationary phase also stimulates the
synthesis of trehalose (Hengge-Aronis, 1993; Kolter et al., 1993; Strøm and Kaasen, 1993).
Stationary phase and mild osmotic shock increase both the heat and the osmotic stress tolerance
of cells, which have been ascribed to the protective effects of trehalose (Hengge-Aronis, 1993).
Trehalose is synthesized by two enzymes encoded by the genes of the otsAB operon
(Giæver et al., 1988; Kaasen et al., 1992; McDougall et al., 1993; Ishida, 1996). The otsA
product, trehalose-6-phosphate synthase, catalyzes the condensation of glucose 6-phosphate and
12
UDP-glucose. Trehalose-6-phosphate phosphatase encoded by otsB generates free trehalose.
Mutations in both genes, as well as in galU (the gene encoding
glucose-1-phosphate-UTP-pyrophosphorylase, which catalyzes the formation of UDP-glucose),
block the synthesis of trehalose and result in increased sensitivity to heat and osmolarity
(Giæver et al., 1988, Hengge-Aronis et al., 1993). The osmosensitivity of these mutants can be
alleviated by adding exogenous betaine to the medium (Giæver et al., 1988).
Osmotic stress induces the otsAB operon 5- to 10-fold. The otsAB operon is also
induced upon entry of cells into stationary phase, but the levels of trehalose in stationary phase
cells are much lower than in cells under osmotic stress (Hengge-Aronis et al., 1991). Mutants in
which this operon is expressed constitutively do not make trehalose at low osmolality (Giæver
et al., 1988). These two examples show that trehalose synthesis is regulated not only at the level
of expression of the otsAB operon but also at the level of activity of the OtsA or OtsB enzyme.
Trehalose-6-phosphate synthase is stimulated in vitro by 0.1 to 0.6 M potassium
glutamate, suggesting that trehalose synthesis could be regulated at least in part by potassium
glutamate (Giæver et al., 1988). However, if K+ alone has any role, it is not very significant
because a kdp trkA (K+ uptake enzyme) double mutant, which accumulated less K+ and did so
more slowly than the wild type, was unimpaired in trehalose synthesis (Dinnbier et al., 1988).
The transcriptions of several genes including otsAB that are induced during entry into
stationary phase are dependent on the RpoS (σS) protein (Loewen and Hengge-Aronis, 1994),
an alternate sigma factor for RNA polymerase. Mutants that lack functional RpoS are defective
in osmotic stress-induced heat and H2O2 tolerance as well (Hengge-Aronis et al., 1993). The
13
level of RpoS increases moderately upon osmotic stress and markedly upon entry into
stationary phase (Lange and Hengge-Aronis, 1994). Recent work implicated homoserine
lactone as a major signal to increase levels of RpoS (Huisman and Kolter, 1994). UDP-glucose
seems to be a negative effector, since strains unable to make UDP-glucose have elevated levels
of RpoS (Böhringer et al., 1995). The increase in RpoS levels at high osmolality is due to
translational control (Lange and Hengge-Aronis, 1994).
Exogenous trehalose can also be used as a carbon and energy source through its
cleavage first into glucose 6-phosphate and glucose by trehalose-6-phosphate hydrolase
(encoded by the treBC operon) at low osmolality and periplasmic trehalase (encoded by treA) at
high osmolality.
Adaptive Evolution
Rational genetic alterations of a microorganism for a specific purpose are not possible in
many situations in which our knowledge of the relationship between phenotype and genotype is
limited. Adaptive evolutionary techniques can be useful when beneficial mutations cannot be
rationally predicted but can be encouraged by environmental conditions. Evolution for
improved and robust strains has been used to improve substrate consumption (Herring et al.,
2006), to perform complicated chemical syntheses (Hur et al., 1994; Carlson et al., 2005; Ro et
al., 2006), or to improve lignocellulosic ethanol production by increasing the tolerance to
lignocellulosic hydrolysates (Gorsich et al., 2006; Keating et al., 2006; van Maris et al., 2006).
Various approaches of adaptive evolution have been employed as tools for metabolic
engineering. A NaCl-tolerant mutant T. glabrata RS23 has been obtained using 70.0 g/L NaCl
14
as the selective criterion by Liu et al. (2007). The pH-controlled continuous culture was
initiated with the feeding medium containing 30 g/L NaCl. The NaCl concentration in the
feeding medium was manually increased to 70 g/L in 10 g/L increments. Adaptive evolution
can also be conducted in shake flasks instead of continuous stirred-tank reactor. For example, in
order to study the adaptation to glycerol minimal medium at the genetic level, E. coli cultures
were passed daily to a glycerol-based medium to maintain cultures in prolonged exponential
phase growth by adjusting the volume transferred (Herring et al., 2006). Five different strains
were monitored over the 44-day process. The same approach was adopted to select three E. coli
MG1655 mutants having increased growth rate to overcome the loss of phosphoglucose
isomerase activity (Charusanti et al., 2010).
15
Objectives
The overall goal of this research is to prolong the fermentation time and improve
organic acid production of E. coli. The underlying assumption is that tolerance limits to
monovalent cations such as Na+ hinders the growth and the production of organic acid during
fermentations using NaOH as pH-control. In order to accomplish this objective, we must
understand the physiological mechanism for high NaCl tolerance of E. coli. The objective is
based on the following hypotheses:
1. Elevating the expression of nhaA gene will increase the NaCl tolerance of E. coli, and
will increase the maximal pyruvate production by ALS929 or its derivatives when NaOH is
used for pH control.
2. Culturing wildtype E. coli strain MG1655 in a defined medium with a progressively
increasing NaCl concentration will confer NaCl resistance to genetic stable mutants. The isolate
strain will exhibit different physiological characters from the wildtype, such as specific growth
rate, maintenance coefficients and biomass yield.
3. Culturing the pyruvate-producing E. coli strain ALS929 in a defined medium with a
progressively increasing NaCl concentration will generate stable strains with increased NaCl
resistance, which also lead to greater final concentrations of pyruvate in the fed-batch
fermentation.
16
References
Amann, E., B. Ochs and K. J. Abel. 1988. Tightly regulated tac promoter vectors useful for the
expression of unfused and fused proteins in Escherichia coli. Gene. 69:301-15.
Arkin, I. T., H. Xu, M. Ø. Jensen, E. Arbely, E. R. Bennett, K. J. Bowers, E. Chow, R. O. Dror,
M. P. Eastwood, R. Flitman-Tene, B. A. Gregersen, J. L. Klepeis, I. Kolossváry, Y. Shan and
D. E. Shaw. 2007. Mechanism of Na+/H+ antiporting. Science. 317:799-803.
Böhringer, J., D. Fischer, G. Mosler and R. Hengge-Aronis. 1995. UDP-glucose is a potential
intracellular signal molecule in the control of expression of σS and σS-dependent genes in
Escherichia coli. J. Bacteriol. 177:413-422.
Brown, A. D. (1990) Microbial Water Stress Physiology: Principles and Perspectives, John
Wiley & Sons, Chichester, UK.
Carlson, R., A. Wlaschin and F. Srienc. 2005. Kinetic studies and biochemical pathway analysis
of anaerobic poly-(R)-3-hydroxybutyric acid synthesis in Escherichia coli. Appl. Environ.
Microbiol. 71:713-720.
Carmel, O., O. Rahav-Manor, N. Dover, B. Shaanan and E. Padan. 1997. The Na+-specific
interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+
antiporter of Escherichia coli. EMBO J. 16:5922-5929.
Causey, T. B., K. T. Shanmugam, L. P. Yomano and L. O. Ingram. 2003. Engineering
Escherichia coli for efficient conversion of glucose to pyruvate. Proc. Natl. Acad. Sci. U. S. A.
101:2235-2240.
17
Cayley, S., B. A. Lewis, H. J. Guttman and M. T. Record, Jr. 1991. Characterization of the
cytoplasm of Escherichia coli K-12 as a function of external osmolarity. J. Mol. Biol.
222:281-300.
Cayley, S., B. A. Lewis and M. T. Record, Jr. 1992. Origins of the osmoprotective properties of
betaine and proline in Escherichia coli K-12. J. Bacteriol. 174:1586-1595.
Charusanti, P., T. M. Conrad, E. M. Knight, K. Venkataraman, N. L. Fong, B. Xie, Y. Gao, B.
Ø. Palsson. 2010. Genetic basis of growth adaptation of Escherichia coli after deletion of pgi, a
major metabolic gene. PLoS Genet. 6(11):e1001186.
Crowe, J. H., and L. M. Crowe. 1992. Membrane integrity in anhydrobiotic organisms: toward
a mechanism for stabilizing dry cells, p. 87-103. In G. N. Somero, C. B.
Dinnbier, U., E. Limpinsel, R. Schmid and E. P. Bakker. 1988. Transient accumulation of
potassium glutamate and its replacement by trehalose during adaptation of growing cells of
Escherichia coli K-12 to elevated sodium chloride concentrations. Arch. Microbiol.
150:348-357.
Dover, N., C. F. Higgins, O. Carmel, A. Rimon, E. Pinner and E. Padan. 1996. Na+-induced
transcription of nhaA, which encodes an Na+/H+ antiporter in Escherichia coli, is positively
regulated by nhaR and affected by hns. J. Bacteriol. 178:6508-6517.
Dover, N. and E. Padan. 2001. Transcription of nhaA, the main Na+/H+ antiporter of
Escherichia coli, is regulated by Na+ and growth phase. J. Bacteriol.183:644-653.
Epstein, W. 2003. The roles and regulation of potassium in bacteria. Prog. Nucleic Acid Res.
Mol. Biol. 75:293-320.
18
Giæver, H. M., O. B. Styrvold, I. Kaasen and A. R. Strøm. 1988. Biochemical and genetic
characterization of osmoregulatory trehalose synthesis in Escherichia coli. J. Bacteriol.
170:2841-2849.
Goldberg, E. E., T. Arbel, J. Chen, R. Karpel, G. A. Mackie, S. Schuldiner and E. Padan. 1987.
Characterization of a Na+/H+ antiporter gene of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A.
84:2615-2619.
Gorsich, S., B. Dien, N. Nichols, P. Slininger, Z. Liu and C. Skory. 2006. Tolerance to
furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1,
RPE1, and TKL1 in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 71:339-349.
Gowrishankar, J. 1986. proP-mediated proline transport also plays a role in Escherichia coli
osmoregulation. J. Bacteriol. 166:331-333.
Guanawan, C., Satianegara, G., Chen, A. K., Breuer, M., Hauer, B., Rogers, P.L., and Rosche,
B. 2007. Yeast pyruvate decarboxylases: variation in biocatalytic characteristics for
(R)-phenylacetylcarbinol production. FEMS Yeast Res. 7: 33-39.
Hengge-Aronis, R., W. Klein, R. Lange, M. Rimmele, and W. Boos. 1991. Trehalose synthesis
genes are controlled by the putative sigma factor encoded by rpoS and are involved in
stationary-phase thermotolerance in Escherichia coli. J. Bacteriol. 173:7918-7924.
Hengge-Aronis, R. 1993. The role of rpoS in early stationary-phase gene regulation in
Escherichia coli K12, p. 171-200. In S. Kjellberg (ed.), Starvation in Bacteria. Plenum Press,
New York.
19
Hengge-Aronis, R., R. Lange, N. Henneberg and D. Fischer. 1993. Osmotic regulation of
rpoS-dependent genes in Escherichia coli. J. Bacteriol. 175:259-265.
Herring, C.D., A. Raghunathan, C. Honisch, T. Patel, M. K. Applebee, A. R. Joyce, T. J.
Albert, F. R. Blattner, D. van den Boom, C. R. Cantor and B. O. Palsson. 2006. Comparative
genome sequencing of Escherichia coli allows observation of bacterial evolution on a
laboratory timescale. Nat. Genet. 38:1406-1412.
Huisman, G. W. and R. Kolter. 1994. Sensing starvation: a homoserine lactone-dependent
signaling pathway in Escherichia coli. Science. 265:537-539.
Hunte, C., E. Screpanti, M. Venturi, A. Rimon, E. Padan and H. Michel. 2005. Structure of a
Na+/H+ antiporter and insights into mechanism of action and regulation by pH. Nature.
435:1197-1202.
Hur, H. G., M. J. Sadowsky and L. P. Wackett. 1994. Metabolism of chlorofluorocarbons and
polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic
pathway. Appl. Environ. Microbiol. 60:4148-4154.
Ishida, A., N. Otsuka, S. Nagata, K. Adachi and H.Sano. 1996. The effect of salinity stress on
the accumulation of compatible solutes related to the induction of salt-tolerance in Escherichia
coli. J. Gen. Appl. Microbiol. 42:331-336.
Ivey, D.M., A.A. Guffanti, J. Zemsky, E. Pinner, R. Karpel, E. Padan, S. Schuldiner and T.A.
Krulwich. 1993. Cloning and characterization of a putative Ca2+/H+ antiporter gene from
Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the
overexpressed gene. J. Biol. Chem. 268:11296-11303.
20
Kaasen, I., P. Falkenberg, O. B. Styrvold and A. R. Strøm. 1992. Molecular cloning and
physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of
Escherichia coli: evidence that transcription is activated by KatF (AppR). J. Bacteriol.
174:889-898.
Karpel, R., J. Olami, D. Taglicht, S. Schuldiner and E. Padan. 1988. Sequencing of the gene ant
which affects the Na+/H+ antiporter acitivity in Escherichia coli. J. Biol. Chem.
263:10408-10414.
Karpel, R., T. Alan, G. Glaser, S. Schuldiener and E. Padan. 1991. Expression of a sodium
proton antiporter (NhaA) in Escherichia coli is induced by Na+ and Li+ ions. J. Biol. Chem.
266:21753-21759.
Keating, J. D., C. Panganiban and S. D. Mansfield. 2006. Tolerance and adaptation of
ethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol. Bioeng.
93:1196-1206.
Koh-Banerjee, P. K., M. P. Ferreira, M. Greenwood, R. G. Bowden, P. N. Cowan, A. L.
Almada and R. B. Kreider. 2005. Effects of calcium pyruvate supplementation during training
on body composition, exercise capacity, and metabolic responses to exercise. Nutrition.
21:312-9.
Kolter, R., D. A. Siegle and A. Tormo. 1993. The stationary phase of bacterial life. Annu. Rev.
Microbiol. 47:855-874.
21
Lange, R. and R. Hengge-Aronis. 1994. The cellular concentration of the σS subunit of
RNA-polymerase in Escherichia coli is controlled on the levels of transcription, translation and
protein stability. Genes. Dev. 8:1600-1612.
Larsen, P. I., L. K. Sydnes, B. Landfald and A. R. Strøm. 1987. Osmoregulation in Escherichia
coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose. Arch.
Microbiol. 147:1-7.
Lee, Y. C., H. C. Chien and W. H. Hsu. 2007. Production of N-acetyl-D-newraminic acid by
recombinant whole cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase
and Escherichia coli N-acetyl-D-neuraminic acid lyase. J Biotechnol. 129: 453-460.
Li, Y., J. Chen and S. Y. Lun. 2001a. Biotechnological production of Pyruvic acid. Appl.
Microbiol. Biotechnol. 57:451-459.
Li, Y., J. Chen, S. Y. Lun and X. S. Rui. 2001b. Efficient pyruvate production by a
multi-vitamin auxotroph of Torulopsis glabrata: Key role and optimization of vitamin levels.
Appl. Microbiol. Biotechnol. 55:680-685.
Li, Y., J. Hugenholtz, J. Chen and S. Y. Lun. 2002. Enghancement of pyruvate production by
Torulopsis glabrata using a two-stage oxygen supply control strategy. Appl. Microbiol.
Biotechnol. 60:101-106.
Liu L., Y. Li, H. Li and J. Chen. 2004. Manipulating the pyruvate dehydrogenase bypass of a
multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production. Lett. Appl.
Microbiol. 39:199-206.
22
Liu, L. M., Q. L. Xu, Y. Li, Z. P. Shi, Y. Zhu, G. C. Du and J. Chen. 2007. Enhancement of
pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata. Biotechnol. Bioeng.
97:825-832.
Loewen, P. C. and R. Hengge-Aronis. 1994. The regulation of the sigma factor σS (KatF) in
bacterial global regulation. Annu. Rev. Microbiol. 48:53-80.
Lütke-Eversloh, T., C. N. Santos and G. Stephanopulos. 2007. Perspectives of biotechnological
production of L-tyrosine and its applications. Appl. Microbiol. Biotechnol. 77:751-762.
McCarty, M. F. 2000. Toward a wholly nutritional therapy for type 2 diabetes. Med
Hypotheses. 54:483-487.
McDougall, G., I. Kaasen and A. R. Strøm. 1993. A yeast gene for trehalose-6-phosphate
synthase and its complementation of an Escherichia coli otsA mutant. FEMS Microbiol. Lett.
107:25-30.
Miller, E. N. and L. O. Ingram. 2008. Sucrose and overexpression of trehalose biosynthetic
genes (otsBA) increase desiccation tolerance of recombinant Escherichia coli. Biotechnol. Lett.
30:503-508
Niiya, S., K. Yamasaki, T. H. Wilson and T. Tsuchiya. 1982 Altered cation coupling to
melibiose transport in mutants of Escherichia coli. J. Biol. Chem. 257:8902-8906.
Ogino, T., C. Garner, J. L. Markley and K. M. Herrmann. 1982. Biosynthesis of aromatic
compounds: 13C-NMR spectroscopy of whole Escherichia coli cells. Proc. Natl. Acad. Sci.
USA 79:5828-5832.
23
Padan, E., D. Zilberstein and S. Schuldiner. 1981. pH homesstasis in bacteria. Biochim.
Biophys. Acta. 650:151-166.
Padan, E., N. Maisler, D. Taglicht, R. Karpel and S. Schuldiner. 1989. Deletion of ant in
Escherichia coli reveals its function in adaptation to high salinity and an alternative Na+/H+
antiporter system(s). J. Biol. Chem. 264:20297-20302.
Padan, E. and T. Krulwich. Sodium stress, in: G. Storz, R. Hengge-Aronis (Eds.). 2000.
Bacterial Stress Responses, ASM Press, Washington, DC.117-130.
Padan, E., M. Venturi, Y. Gerchman and N. Dover. 2001. Na+/H+ antiporters. Biochim.
Biophys. Acta. 1505:144-157.
Padan, E., T. Tzubery, K. Herz, L. Kozachkov, A. Rimon and L. Galili. 2004. NhaA of
Escherichia coli, as a model of a pH-regulated Na+/H+ antiporter. Biochim. Biophys. Acta.
1658:2-13.
Pinner, E., E. Padan and S. Schuldiner. 1992. Cloning, sequencing and expression of nhaB
gene, encoding a Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli. J. Biol. Chem.
267:11064-11068.
Pinner, E., Y. Kotler, E. Padan and S. Schuldiner. 1992. Physiological role of NhaB, a specific
Na+/H+ antiporter in Escherichia coli. J. Biol. Chem. 268:1729-1734.
Radchenko, M. V., K. Tanaka, R. Waditee, S. Oshimi, Y. Matsuzaki, M. Fukuhara, H.
Kobayashi, T. Takabe and T. Nakamura. 2006. Potassium/Proton Antiport System of
Escherichia coli. J. Biol. Chem. 2006. 281:19822-19829.
24
Rahav-Manor, O., O. Carmel, R. Karpel, D. Taglicht, G. Glaser, S. Schuldiener and E. Padan.
1992. NhaR, a protein homologous to a family of bacterial regulatory proteins (LysR) regulates
nhaA, the sodium protein antiporter gene in Escherichia coli. J. Biol. Chem. 267:10433-10438.
Richey, B., D. S. Cayley, M. C. Mossing, C. Kolka, C. F., Anderson, T. C. Farrar and M. T.
Record, Jr. 1987. Variability in the intracellular ionic environment of Escherichia coli:
differences between in vitro and in vivo effects of ion concentrations on protein-DNA
interactions and gene expression. J. Biol. Chem. 262:7157-7164.
Ro, D., E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. Ho, R. A.
Eachus, T. S. Ham, J. Kirby, M. C. Y. Chang, S. T. Withers, Y. Shiba, R. Sarpong and J. D.
Keasling. 2006. Production of the antimalarial drug precursor artemisinic acid in engineered
yeast. Nature. 440:940-943.
Rothman, A., E. Padan and S. Schuldiner. 1996. Topological analysis of NhaA, a Na+/H+
antiporter from Escherichia coli. J. Biol. Chem. 271:32288-32292.
Sardet C., A. Franchi and J. Pouyssegur. 1989. Molecular cloning, primary structure and
expression of the human growth factor-activatable Na+/H+ antiporter. Cell. 56: 271-280.
Schmid, A., J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts and B. Witholt. 2001. Industrial
biocatalysis today and tomorrow. Nature. 409:258-268.
Shijuku, T., T. Yamashino, H. Ohashi, H. Saito, T. Kakegawa, M. Ohta and H. Kobayashi.
2002. Expression of chaA, a sodium ion extrusion system of Escherichia coli, is regulated by
osmolarity and pH. Biochim. Biophys. Acta. 1556:142-148.
25
Somero, G. N., C. B. Osmond and C. L. Bolis (ed.), Water and Life: Comparative Analysis of
Water Relationships at the Organismic, Cellular, and Molecular Levels. Springer-Verlag,
Berlin.
Strøm, A. R., P. Falkenberg and B. Landfald. 1986. Genetics of osmoregulation in Escherichia
coli: uptake and biosynthesis of organic osmolytes. FEMS Microbiol. Rev. 39:79-86.
Strøm, A. R. and I. Kaasen. 1993. Trehalose metabolism in Escherichia coli: stress protection
and stress regulation of gene expression. Mol. Microbiol. 8:205-210.
Strøm, A. R. 1998. Osmoregulation in the model organism Escherichia coli: genes governing
the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress
tolerance. J. Biosci. 23:437-445.
Taglicht, D., E. Padan and S. Schuldiner. 1991. Overproduction and purification of a functional
Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli. J. Biol. Chem. 266:11289-11294.
Taglicht, D., E. Padan, and S. Schuldiner. 1993. Proton-sodium stoichiometry of NhaA, an
electrogenic antiporter from Escherichia coli. J. Biol. Chem. 268: 5382-5387.
Tomar, A., M. A. Eiteman and E. Altman. 2003. The effect of acetatepathway mutations on the
production of pyruvate in Escherichia coli. Appl. Microbiol. Biotechnol. 62:76-82.
Trchounian, A. and H. Kobayashi. 1999. Kup is the major K+ uptake system in Escherichia coli
upon hyper-osmotic stress at a low pH. FEBS Letters. 447:144-148.
Tzubery, T., Rimon, A. and Padan, E. 2004. Mutation E252C Increases Drastically the Km
Value for Na+ and Causes an Alkaline Shift of the pH Dependence of NhaA Na+/H+ Antiporter
of Escherichia coli. J. Biol. Chem. 279:3265-3272.
26
van Maris, A. J. A., D. A. Abbott, E. Bellissimi, J. van den Brink, M. Kuyper, M. A. H. Luttik,
H. W. Wisselink, W. A. Scheffers, J. P. van Dijken and J. T. Pronk. 2006. Alcoholic
fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current
status. Antonie. Van. Leeuwenhoek. 90:391-418.
Welsh, D. T., R. H. Reed and R. A. Herbert. 1991. The role of trehalose in the osmoadaptation
of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during
osmoadaptation in continuous culture. J. Gen. Microbiol. 137:745-750.
West, I.C. and P. Mitchell. 1974. Proton/Sodium ion antiport in Escherichia coli. Biochem. J.
144:87-90.
Yokota, A., Shimizu, H., Terasawa, Y., Takaoka, N. and Tomita, F. 1994a. Pyruvic acid
production by a lipoic acid auxotroph of Escherichia coli W1485. Appl. Microbiol. Biotechnol.
41: 638-643.
Yokota, A., Terasawa, Y., Takaoka, N., Shimizu, H. and Tomita, F. 1994b. Pyruvic acid
production by an F1-ATPase-defective mutant of Escherichia coli W1485lip2. Bioscience.
Biotechnol. Biochem. 58: 2164-2167.
Zelić, B., T. Gerharz, M. Bott, D. Vasić-Rački, C. Wandrey and R. Takors. 2003. Fed-batch
process for pyruvate production by recombinant Escherichia coli YYC 202 strain. Eng. Life.
Sci. 3:299-305.
Zelić, B., S. Gostović, K. Vuorilehto, D. Vasić-Rački, R. Takors. 2004. Process strategies to
enhance pyruvate production with recombinant Escherichia coli: From repetitive fed-batch to in
situ product recovery with fully integrated electrodialysis. Biotechnol.Bioeng. 85:638-646.
27
Zhu, Y., M. A. Eiteman, R. Altman and E. Altman. 2008. High glycolytic flux improves
pyruvate production by a metabolically engineered Escherichia coli strain. Appl. Environ.
Microbiol. 74:6649-6655.
28
CHAPTER 2
Introduction
Escherichia coli is one of the most industrially important microorganisms because it can
be cultured to a high cell density at large scale with simple, defined medium. In addition,
central carbon metabolism of E. coli is well-understood (Sauer and Eikmanns, 2005), and
therefore the microbe serves in metabolic engineering to generate a wide range of projects
including amino acids, and both reduced (e.g. ethanol and lactate) and oxidized commodity
chemicals (e.g. acetate and pyruvate).
For many bioprocesses which generate an organic acid using E. coli, the accumulation
of the acid product without base titrant reduces the pH dramatically which affects cell growth.
Organic acid toxicity and associated tolerance of E. coli have been reviewed recently
(Warnecke and Gill, 2005). In order to maintain the pH at an optimal level for continued growth
and product formation, a base such as NaOH must be added into the bioreactor. However,
addition of NaOH or KOH for pH control causes the accumulation of cations such Na+ or K+.
For example, in a fed-batch process without the use of betaine, 56 g/L pyruvate accumulated
using E. coli in a fed-batch process (Zhu et al., 2008), at which point about 0.64 M Na+ was
achieved in the bioreactor. This high Na+ concentration and the associated osmotic stress could
be the most important factor in the inhibition of cell growth and further organic acid formation.
29
That is, bacterial growth and acid production may ultimately be hindered by the increased
osmotic pressure.
E. coli maintains a Na+ concentration gradient across the cell membrane so that the
intracellular Na+ concentration is less than the extracellular concentration. NhaA, the main
Na+/H+ antiporter in the inner membrane of E. coli, regulates the cytosolic concentration of
Na+ and H+ and the volume of cells (West and Mitchell, 1974). NhaA is crucial for E. coli to
survive at high salinity or alkaline pH by excreting Na+ in exchange for the flow of protons into
the cell (Taglicht et al., 1993). Corroborating its role in pH homeostasis, NhaA activity is
tightly controlled by pH, which involves a pH sensor and conformational changes that convert
the pH signal into a change in activity (Padan et al., 2004). The protein is fully downregulated
below pH 6.5, and its activity increases by more than three orders of magnitude upon shift to
alkaline pH, reaching a maximal level at pH 8.5 (Padan et al., 2004).
Rational genetic alterations of a microorganism for a specific purpose are not possible in
many situations in which our knowledge of the relationship between phenotype and genotype is
limited. Additionally, bacteria have a remarkable capability for adaptation to environmental
stress (Storz and Hengge-Aronis, 2000). Adaptive evolutionary techniques often become useful
when beneficial mutations cannot be rationally predicted but can be encouraged by
environmental conditions. For example, evolution for improved strains has been used to
improve substrate consumption (Herring et al., 2006), to perform complicated chemical
syntheses (Hur et al., 1994; Carlson et al., 2005; Ro et al., 2006), and to improve ethanol
production by increasing the tolerance to lignocellulosic hydrolysates (Gorsich et al., 2006;
30
Keating et al., 2006; van Maris et al., 2006). Various approaches of adaptive evolution have
been employed, including continuous cultivation with progressively increased NaCl
concentration in the feed (Liu et al., 2007) and shake flasks cultures with prolonged exponential
phase growth (Herring et al., 2006; Charusanti et al., 2010).
The objective of this study was to prolong the fermentation time and improve organic
acid production by enhancing the sodium chloride tolerance of E. coli. Since adaptive evolution
was employed to obtain mutant isolates, associating chemostats coupled with fed-batch
fermentation provides insights into the physiological mechanisms of NaCl tolerance strains.
31
Materials and Methods
Strains and Growth Media
E. coli MG1655 (F- λ- ilvG rfb-50 rph-1) and ALS929 (Hfr zbi::Tn10 poxB1 ∆(aceEF)
rpsL pps-4 pfl-1 ldhA::Kan) were used in this study (Zhu, et al., 2008).
The defined adaptation (DA) medium contained (per L): glucose, 10 g, citric acid, 1.70
g; KH2PO4, 13.30 g; (NH4)2HPO4, 4.50 g; MgSO4·7H2O, 1.2 g; Zn(CH3COO)2·2H2O, 13 mg;
CuCl2·2H2O, 1.5 mg; MnCl2·4H2O, 15 mg; CoCl2·6H2O, 2.5 mg; H3BO3, 3.0 mg;
Na2MoO4·2H2O, 2.5 mg; Fe(III) citrate, 100 mg; thiamine·HCl, 4.5 mg; Na2(EDTA) ·2H2O,
8.4 mg. For ALS929 this medium was supplemented with 3.75 g CH3COONa·3H2O, 0.2 g
L-isoleucine and 100 mg kanamycin. The NaCl concentration was calculated from Na+
contained in the medium, Na+ contained in the base NaOH which was used for pH adjustment,
and NaCl added separately.
TYA medium contained (per L): 10.0 g tryptone, 5.0 g NaCl, 1.0 g yeast extract, 1.36 g
Na(CH3COO)·3H2O. SF medium contained (per L): 10.0 g glucose, 2.3 g Na(CH3COO)·3H2O,
5.66 g Na2HPO4·7H2O, 1.5 g KH2PO4, 0.25 g NaCl, 0.5 g NH4Cl, 0.1 g MgSO4·7H2O, 0.013 g
CaCl2·2H2O, 0.02 g thiamine·HCl, 0.5 g L-isoleucine. GAM medium contained (per L): 30.0 g
glucose, 2.75 g Na(CH3COO)·3H2O, 1.5 g NaH2PO4·H2O, 3.25 g KH2PO4, 3.275 g
K2HPO4·3H2O, 0.2 g NH4Cl, 2.0 g (NH4)2SO4, 1.024 g MgSO4·7H2O, 0.01 g CaCl2·2H2O, 0.5
mg ZnSO4·7H2O, 0.25 mg CuCl2·2H2O, 2.5 mg MnSO4·H2O, 1.75 mg CoCl2·6H2O, 0.12 mg
H3BO3, 1.772 mg Al2(SO4)3, 0.5 mg Na2MoO4·2H2O, 18.29 mg FeSO4·7H2O, 0.02 g
thiamine·HCl, 0.75 g L-isoleucine.
32
Adaptive Evolution
To evolve the strains for increased NaCl tolerance, 10 mL of E. coli MG1655 and
ALS929 were each cultured in four independent 125 mL shake flasks in DA medium at 37°C
and 250 rpm (19 mm pitch). Every 24 h, the optical density (OD) was measured, and 1 mL of
the culture was transferred into 9 mL of a fresh medium. If the OD was greater than that had
been observed in the previous culture transfer, then the fresh medium contained a greater NaCl
concentration. This process was continued for 73 days. From each final culture, a single colony
was isolated by growing with 0.98 M NaCl for MG1655 derivatives or 0.92 M NaCl for
ALS929 derivatives. The four isolates evolved from MG1655 were designated
ALS1184–ALS1187, whereas the four strains isolates from ALS929 were designated
ALS1180-ALS1183. The isolates were then suspended in LB broth containing 25% glycerol
and stored at -80°C.
Strain stability was confirmed by growing each of the eight isolates in DA medium
without additional Na+, transferring once into the same medium, and then transferring to the
medium having a final concentration of 0.91 M Na+. Na+ tolerance was quantified by growing
MG1655 and each isolate in DA medium, and then transferring the culture into a series of
media having 0.77–1.05 M Na+, wherein the growth rate was calculated by OD measurement.
To construct MG1655 pflB and ALS1187 pflB, a P1vir lysate was prepared from
NZN111 (Bunch et al., 1997), and the pflB::Cam deletion was transduced into MG1655 and
ALS1187, respectively, and chloramphenicol resistant transductant colonies were selected.
33
NhaA overexpression
The E. coli nhaA gene was amplified by a research technician with PCR using E. coli
genomic DNA as the template with Pfu DNA polymerase. Primers were designed based on the
E. coli genome sequence (Blattner et al., 1997) and contained a Kpn I restriction site and a
Shine-Dalgarno sequence at the beginning of the amplified fragment and a Hind III restriction
site at the end of the amplified fragment (forward primer, 5’ TAC TAT GGT ACC CAG GAG
AAC AGC TAT GAA ACA TCT GCA TCG ATT CTT TAG C 3’; reverse primer, 5’ AGA
GAG AGA GAG AGA GAG AGA AGC TTT AAC AAT GAA AAG GGA GCC GTT TAT 3’,
the underlined sequences are the Kpn I, Shine-Dalgarno, ATG start, and Hind III sites,
respectively). The natural GTG start codon of the nhaA gene was replaced with an ATG start
codon. The resulting 1.2 Kb PCR product was gel isolated, digested with Kpn I and Hind III,
and ligated into the pTrc99A expression vector which had been digested with the same two
restriction enzymes. E. coli strains designated ALS1189 and ALS1192 were obtained by
transfecting pTrc99A-nhaA and pTrc99A to MG1655, respectively. The Na+ tolerance was
quantified by growing ALS1189 in DA medium in several shake flasks. When a culture reached
an OD of 0.1, 32 µM IPTG was added. The concentration of NaCl was increased progressively
by about 0.17 M every 30 min to different final Na+ concentrations in the range of 0.09–0.95
M, and the growth rate calculated by OD measurement.
Fed-batch processes
For pyruvate production, ALS929 or ALS1182 was first grown in a 250 mL shake flask
containing 30 mL TYA medium for about 8 h, then 5 mL transferred to 50 mL SF medium in a
34
250 mL shake flask. After 12 h of growth, the contents of this shake flask were used to
inoculate a 2.5 L bioreactor (Bioflo 310, New Brunswick Scientific Co. Edison, NJ, USA)
containing 1.0 L GAM medium. Cells grew at their maximum specific growth rate until the
initial acetate was nearly exhausted (OD about 3.0). At this time, the fed-batch mode
commenced with exponentially feeding a solution containing 600 g/L glucose and 30 g/L
acetate so that cell growth was controlled at a constant specific rate of about 0.15 h-1. The pH
was controlled at 7.0 using 5% (w/v) NH4OH/25% NaOH, the temperature was controlled at
37°C, and the agitation maintained at 400 rpm. Air and O2 were mixed as necessary at 1.0
L/min total flow rate to maintain a dissolved oxygen concentration (DO) above 40% of
saturation.
For lactate production, MG1655 pflB or ALS1187 pflB cells was first grown in a 250
mL shake flask containing 50 mL DA medium. After 12 h the contents were used to inoculate
the 2.5 L bioreactor (Bioflo 310) containing 1.0 L DA medium but with 20 g/L glucose. During
an initial aerobic phase of about 6 h the agitation was maintained at 400 rpm, and air and O2
were mixed as necessary at 1.0 L/min total flow rate to maintain the DO above 40% of
saturation until the OD reached 8.0. During a second, anaerobic phase the agitation was
maintained at 200 rpm, and a 9:1 mixture of N2 and CO2 was sparged at 0.5 L/min. During this
phase the glucose concentration was maintained at 2–4 g/L using a 600 g/L glucose solution
automatically fed in response to the measurement of an on-line glucose analyzer (YSI 2700
SELECT, YSI Life Sciences Inc. Yellow Springs, OH, USA). An experiment was terminated
when the glucose solution was not automatically fed for 3 h. For both phases, the pH was
35
controlled at 7.0 using 30% (w/v) NaOH, the temperature at 37°C, and the agitation at 400 rpm.
All the fermentations were run in duplicate.
Chemostats
Continuous fermentations of 900 mL volume at several dilution rates were operated as
carbon-limited chemostats and initiated in batch mode in a 2.5 L bioreactor (Bioflow 310). The
influent medium contained DA medium but with 5.0 g/L glucose and either high Na+ (0.68 M)
or low Na+ (0.18 M) concentration. A steady-state condition was assumed after four residence
times at which time the oxygen and CO2 concentrations in the effluent gas remained
unchanged. For dry cell weight measurement, three 25.0 mL samples were centrifuged (3287 g,
10 min), the pellets washed by vortex mixing with 30 mL 0.9% saline solution and then
centrifuged again. After repeating the washing step twice using DI water, the cell pellets were
dried at 60°C for 24 h and weighed. All fermentations were conducted at 37°C, with an air
flowrate of 0.5 L/min, an agitation of 400 rpm and a pH of 7.0. The DO in each chemostat
remained above 40%.
Analytical methods
The optical density at 600 nm (OD) (UV-650 spectrophotometer, Beckman Instruments,
San Jose, CA, USA) was used to monitor cell growth, and this value was correlated to dry cell
mass. The concentrations of oxygen and CO2 in the off-gas were measured by using a gas
analyzer (Innova 1313 gas monitor, Lumasense Technologies, Ballerup, Denmark).
Concentrations of soluble organic compounds were determined by high performance liquid
chromatography as previously described (Eiteman and Chastain, 1997).
36
The betaine concentration was measured at steady-state by withdrawing a 200-250 mL
sample, centrifuging (3287 g, 10 min), washing the pellets with 0.9% saline solution, washing
twice with DI water, and then mixing the cell pellet for 10 min in 0.5 M perchloric acid at 4°C.
The mixture then was recentrifuged (13147 g, 10 min), and the pellet extracted twice with 1 mL
cold 0.2 M perchloric acid. The pH of the three pooled supernatants was adjusted to 4.5 by
careful addition of 5 M KOH. The KClO4 was removed by centrifugation (131 g, 1 min at 0°C,
Sutherland and Wilkinson, 1971). The concentration of betaine was analyzed by refractive
index detection using HPLC (Gilson, Inc., Middleton, WI, USA) with an Coregel-87C column
(Transgenomic, Inc., Omaha, NE, USA) (Rajakylä and Paloposki, 1983).
37
Results
Effect of nhaA overexpression
NhaA, the main Na+/H+ antiporter within the inner membrane of E. coli, is crucial for E.
coli survival at high salinity or alkaline pH. In order to examine the effect of overexpressing
this membrane protein, the growth rates of two strains were compared at various Na+
concentrations (Fig. 2): ALS1189 (MG1655 with pTrc99A-nhaA) and the control strain
ALS1192 (MG1655 with pTrc99A). At each Na+ concentration examined, the overexpression
of nhaA typically resulted in 10-20% lower growth rate compared to the control strain. Neither
strain grew when the Na+ concentration exceeded 0.9 M.
NaCl (mol/L)0.0 0.2 0.4 0.6 0.8 1.0
Gro
wth
Rat
e (h
-1)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7ALS1189ALS1192
Figure 2. Growth rate of control strain and nhaA-overexpressing strain at various NaCl concentrations.
38
Evolution of MG1655
Four isolates (ALS1184-ALS1187) were obtained from MG1655 after 73 days of
transfer into progressively increasing NaCl concentration. The evolutionary progress of
ALS1187 is shown in Fig. 3, and the other three isolates had a similar evolutionary pattern (data
not shown). These four isolates were able to grow after 24 h in a defined medium with 0.98 M
NaCl and 10 g/L glucose.
Transfer (day)0 10 20 30 40 50 60 70
NaC
l (m
ol/L
)
0.0
0.3
0.6
0.9
1.2
1.5
24 h
OD
0.0
0.5
1.0
1.5
2.0
2.5NaClOD
Figure 3. Evolution progress of ALS1187.
39
Characterization of evolved strains
The genetic stability of the four isolates was verified after each isolate had been
preserved by freezing. After thawing each isolate was able to grow in DA medium with 0.91 M
NaCl after 24 h, but the parent strain MG1655 did not grow under these same conditions.
The specific growth rates of MG1655 and the isolates were compared at various NaCl
concentrations (Fig. 4). At low NaCl concentration, the growth rates of MG1655 and the
isolates were essentially the same. When the NaCl concentration exceeded 0.4 M, each of the
isolates showed similar specific growth rates significantly faster than MG1655.
NaCl (mol/L)0.1 0.3 0.5 0.7 0.9 1.1
Gro
wth
Rat
e (h
-1)
0.0
0.2
0.4
0.6
0.8MG1655 ALS1184 ALS1185 ALS1186 ALS1187
Figure 4. Growth rate of the evolved strains compared to that of the parent strain at various NaCl concentrations.
40
The specific growth rates of MG1655 and the isolates were also compared at various
NaCl concentrations (Fig. 5). ALS1185 - ALS1187 grew significantly faster than the MG1655
at KCl concentration above 0.5 M, which are similar to the NaCl results.
KCl (mol/L)0.2 0.4 0.6 0.8 1.0 1.2
Gro
wth
Rat
e (h
-1)
0.0
0.2
0.4
0.6
0.8MG1655 ALS1184 ALS1185 ALS1186 ALS1187
Figure 5. Growth rate of the evolved strains compared to that of the parent strain at various KCl concentrations.
Steady-State Growth
Carbon-limited chemostats were conducted at two different NaCl concentrations (0.18
M and 0.68 M) to compare the steady-state metabolism of MG1655 and ALS1187. The results
showed the expected linear relationship between the specific glucose consumption rate (qS) and
the specific growth rate (Fig. 5). However, the results demonstrated a significant difference in
41
the glucose consumption rates between high and low NaCl concentration and between MG1655
and the isolate ALS1187.
For MG1655, growth in medium having 0.68 M NaCl consistently required a 2–2.5-fold
greater glucose consumption rate than the identical growth rate in the same medium having
0.18 M NaCl. For ALS1187, growth in medium having 0.68 M NaCl required a similarly
greater glucose consumption rate than growth in medium having 0.18 M NaCl. Furthermore, for
any specific growth rate at either 0.18 M and 0.68 M NaCl, ALS1187 required a 5–40% greater
glucose consumption rate than MG1655. The difference between glucose consumption rates of
MG1655 and ALS1187 was greatest at 0.18 M NaCl. Since glucose consumption rate is directly
related to energy demand, these results suggest that some characteristic of the evolved isolate
ALS1187 leads to a greater ATP requirement than MG1655, particularly at 0.18 M NaCl.
42
Growth Rate (h-1)0.04 0.07 0.10 0.13 0.16
q s (g/
gh)
0.0
0.2
0.4
0.6
0.8
1.0MG1655 0.18 M NaClMG1655 0.68 M NaClALS1187 0.18 M NaClALS1187 0.68 M NaCl
Figure 6. Specific glucose consumption rate (qS) as a function of specific growth rates for E. coli MG1655 and ALS1187 at 0.18 M and 0.68 M NaCl.
The specific oxygen uptake rate (qO2) generally correlated proportionately with qS, with
no difference between MG1655 and ALS1187 or at different NaCl concentrations. The range of
glucose consumption rates studied were below the threshold glucose consumption rate for
overflow metabolism reported previously in MG1655 (Vemuri et al., 2006), and hence the
values for qO2 observed in this study are below the maximum plateau qO2 for wildtype E. coli.
43
qs(g/gh)0.0 0.2 0.4 0.6 0.8
q O2 (
mm
ol/g
h)
0
5
10
15
20MG1655 0.18 M NaClMG1655 0.68 M NaClALS1187 0.18 M NaClALS1187 0.68 M NaCl
Figure 7. Specific oxygen uptake rate (qO2) at various specific glucose consumption rate (qS) for E. coli MG1655 and ALS1187 at 0.18 M and 0.68 M NaCl.
The specific carbon dioxide evolution rate (qCO2) also increased proportionately with qS,
but did not depend on the strain or on the concentration of NaCl (Fig. 7). Note that in Fig. 7 the
independent variable is specific glucose consumption rate, not the specific growth rate.
Because qS showed significant differences with growth rates (Fig. 5), the values of qCO2 did
vary with growth rate. For example, at the highest growth rate studied (0.14 h-1) with 0.18 M
NaCl, MG1655 showed a qCO2 of 4.6 mmol/g·h while ALS1187 showed a qCO2 of 9.6 mmol/g·h.
However, the differences observed in qCO2 at any given growth rate can be attributed to
differences in qS between strains or NaCl concentrations. In other words, growth at a high NaCl
concentration caused a greater generation of CO2 entirely because at an elevated NaCl
concentration the glucose consumption rate was greater.
44
qs (g/gh)0.0 0.2 0.4 0.6 0.8
q CO
2 (m
mol
/gh)
0
5
10
15
20
25MG1655 0.18 M NaClMG1655 0.68 M NaClALS1187 0.18 M NaClALS1187 0.68 M NaCl
Figure 8. Specific carbon dioxide evolution rate (qCO2) at various specific glucose consumption rates (qS) for MG1655 and ALS1187 at 0.18 M and 0.68 M NaCl.
For each of the two strains and two NaCl concentrations, a value for the maintenance
coefficient (mS) and for the (true) biomass yield (YX/S) was obtained (Table 1). For both strains,
NaCl concentration strongly affected maintenance, or nongrowth-related energy metabolism.
The evolved strain ALS1187 and MG1655 showed similar maintenance coefficients at 0.18 M
NaCl. However, at 0.68 M NaCl the maintenance coefficient for ALS1187 was 30% lower than
the maintenance coefficient for MG1655. Furthermore, for either strain growth at 0.68 M NaCl
decreased the biomass yield by about 50% compared to the value at 0.18 M NaCl.
45
Table 1. Nongrowth-related energy metabolism (maintenance coefficient, mS) and the biomass yield on glucose (YX/S) for MG1655 and ALS1187 at different NaCl concentrations.
Strain NaCl (M)
mS (g/gh)
YX/S (g/g)
MG1655 0.18 0.031 0.591 0.68 0.111 0.287
ALS1187 0.18 0.033 0.445 0.68 0.078 0.233
Lactate Production
One potential benefit of increased salt tolerance in E. coli is the ability to accumulate a
higher concentration of an organic acid product. E. coli with a knockout in the pflB gene
encoding pyruvate formate lyase readily accumulates lactic acid (Zhu and Shimizu, 2004). In
order to determine whether the evolved isolate could accumulate a higher concentration of
lactate, two strains MG1655 pflB and ALS1187 pflB were compared in a two-phase
aerobic-anaerobic process. In both cases during the anaerobic phase, the glucose concentration
was automatically maintained at 2–4 g/L, and each process terminated when no additional
glucose was required. Lactate production called for the addition of NaOH to maintain the pH,
and therefore Na+ accumulated throughout either processes. For ALS1187 pflB the lactate
concentration achieved 76.2 g/L, 35% greater than the 56.3 g/L concentration achieved for
MG1655 pflB (Fig. 8). Moreover, for ALS1187 pflB the final Na+ concentration reached 0.97 M,
whereas for MG1655 pflB the Na+ concentration reached 0.88 M. The fermentation time usisng
ALS1187 pflB was also prolonged to 52 h, 8 h longer than fermentation using MG1655 pflB.
Based on the total glucose consumed, ALS1187 attained a lactate yield of 0.77 g/g, while
46
MG1655 attained a lactate yield of 0.64 g/g. A duplicate set of fed-batch fermentations with
MG1655 pflB and ALS1187 pflB attained essentially identical results.
Time (h)0 10 20 30 40 50 60
Lact
ate
(g/L
)
0
20
40
60
80
NaC
l (m
ol/L
)
0.0
0.5
1.0
1.5
2.0
MG1655 pflB LactateMG1655 pflB NaClALS1187 pflB LactateALS1187 pflB NaCl
Figure 9. Lactate concentration and Na+ concentration in fed-batch fermentations using MG1655 pflB and ALS1187 pflB. The anaerobic phase commenced at 6 h.
Evolution of ALS929
The E. coli ALS929 is a pyruvate-producing strain (Zhu et al., 2008). Similar to the
evolution from MG1655, four independent sets of shake flasks were exposed to increasing
concentrations of NaCl over 73 days, and the final isolates were denoted ALS1180-ALS1183.
The evolution progress of ALS1182 is shown in Fig. 9, and the other three strains showed a
similar evolution pattern (data not shown). The final isolates came from solutions having a
NaCl concentration of 0.92 M, a concentration which MG1655 could not tolerate (Fig. 4).
47
Three of the isolates showed genetic stability, while ALS1183 was not able to regrow in DA
medium with 0.84 M NaCl.
Transfer (day)0 10 20 30 40 50 60 70
NaC
l (m
ol/L
)
0.0
0.3
0.6
0.9
1.2
24 h
OD
0.0
0.5
1.0
1.5
2.0
2.5NaClOD
Figure 10. Evolution progress of ALS1182.
Pyruvate Production
In order to determine whether the evolved isolate ALS1182 could accumulate a higher
concentration of pyruvate, two strains ALS929 and ALS1182 were compared in an aerobic
process. The fed-batch fermentations were conducted with an exponential
glucose/acetate-limited feeding at a constant growth rate of 0.15 h-1 (Zhu et al., 2008). The lag
phase for ALS1182 varied but was consistently longer than the lag phase for ALS929 (Fig. 10).
For both strains, cell growth slowed when the OD reached about 20 and the pyruvate
48
concentration reached about 25 g/L. Also, for both strains the final pyruvate concentration
achieved was about 70 g/L. The pyruvate yield using ALS1182 was 0.69 g/g, only slightly
higher than the yield of 0.66 g/g attained using ALS929. Essentially identical results were
achieved in a duplicate set of fed-batch fermentations.
Time (h)0 10 20 30 40 50
OD
0
10
20
30
40
50
Pyru
vate
(g/L
)0
20
40
60
80ALS929, ODALS929, PyruvateALS1182, ODALS1182, Pyruvate
Figure 11. Pyruvate production in fed-batch fermentations using ALS929 and ALS1182.
49
Discussion
In order to enhance the NaCl tolerance of E. coli, the nhaA gene was overexpressed.
NhaA is a native membrane protein in E. coli, which mediates Na+ and Li+ efflux driven by the
electrochemical proton gradient (i.e. H+out > H+
in) (Taglicht et al., 1993). Wu et al. (2004)
reported that overexpression of the E. coli nhaA gene in rice plants enhanced the salt tolerance
and improved rice yield. In this study, an overexpression of nhaA was achieved in E. coli, but
the nhaA-expressing transformant exhibited no significant growth difference from the control at
a low NaCl concentration. Moreover, the specific growth rate of the transformant became lower
under high concentrations of NaCl. Unexpectedly, E. coli cells overexpressing the nhaA
antiporter were slightly more sensitive to NaCl than the control cells. A similar result was
observed when the nhaA antiporter was expressed heterologously in the yeast Saccharomyces
cerevisiae, which conferred only lithium but not sodium tolerance (Ros et al., 1998).
Since overexpression of nhaA showed no improvement in NaCl tolerance, adaptive
evolution was conducted with wildtype strain MG1655 and a pyruvate-producing strain
ALS929. Four independent isolates from MG1655 were characterized for improvement in
growth rates at elevated NaCl concentrations. One derivative, ALS1187, was further
characterized for steady-state growth parameters and, after deletion the pflB gene, for the
improved ability to accumulate the acid product lactate in fed-batch fermentations. The results
indicate that the adaptation of MG1655 did enhance NaCl tolerance compared to the wild-type
and this adaptation did improve lactate production. On the other hand, the isolate evolved from
ALS929 did not lead to increased pyruvate production.
50
MG1655 and ALS1187 showed markedly different glucose consumption rates and
maintenance coefficients under steady-state conditions. The maintenance coefficient is the
non-growth related substrate consumption (Pirt, 1965), caused by (1) shifts in metabolic
pathways, (2) proofreading of protein and turnover of mRNA, (3) cell death and lysis, and (4)
osmoregulation (Mason et al., 1986; Stouthamer et al., 1990; Russell and Cook, 1995). In this
study, MG1655 and ALS1187 exhibited similar maintenance coefficients at low NaCl
concentration (0.18 M). For both strains a high NaCl concentration (0.68 M) led to a much
greater maintenance coefficient. Moreover, the maintenance coefficient for ALS1187 was 40%
less than the maintenance coefficient for MG1655 at this higher NaCl concentration. A lower
maintenance implies that ALS1187 is much more efficient at non-growth metabolism under
conditions of high NaCl concentration than wild-type MG1655. Sachidanandham et al. (2005)
similarly reported that in a hyper-osmotic medium, a Bacillus strain isolated from the Kuwait
desert showed a lower maintenance than a wildtype Bacillus thuringiensis. Bacteria in the
desert might be expected to evolve to accommodate this environment. The maintenance
coefficient of course describes metabolism in the absence of growth and may not be directly
predictive of an elevated maximal growth rate or the ultimate tolerance of a strain to NaCl.
An important result of enhanced NaCl tolerance in ALS1187 was the ability to attain a
significantly greater final lactate concentration in fed-batch fermentation. At pH 7 lactic acid
exists as a dissociated form, in this case, sodium lactate. Since the Na+ ion must therefore in
tandem with the formation of lactate, NaCl tolerance was able to prolong the fermentation time
51
despite limited growth in the pflB mutants. Liu et al. (2007) similarly isolated an osmo-tolerant
Torulopsis glabrata mutant, which resulted in an enhanced pyruvate production.
In order to accumulate pyruvate, E. coli ALS929 contains multiple gene knockouts
which block pyruvate catabolism to acetyl-CoA, phosphoenolpyruvate, acetate, or lactate.
Despite its elevated NaCl tolerance, ALS1182 evolved from ALS929 did not show increased
pyruvate production after adaptive evolution in contrast to ALS1187. One explanation might be
long-term instability of the strain caused by the adaptive evolution. Zelić et al. (2004) reported
that E. coli YYC202 ldhA::Kan, which shared the same genotype, could not maintain stability
over approximately 40 h.
In summary, this study demonstrated that the inhibition in lactate-producing
fermentation could be diminished by increase the NaCl tolerance of E. coli. Additionally, the
strategy for enhancing NaCl tolerance by adaptive evolution may provide an alternative
approach to increase organic acid accumulation with E. coli.
52
References
Blattner, F. R., G. Plunkett 3rd, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J.
Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A.
Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau and Y. Shao. 1997. The complete genome
sequence of Escherichia coli K-12. Science. 277:1453-1462.
Bunch, P. K., F. Mat-Jan, N. Lee and D. P. Clark. 1997. The ldhA gene encoding the
fermentative lactate dehydrogenase of Escherichia coli. Microbiol. 143:187-195.
Carlson, R., A. Wlaschin and F. Srienc. 2005. Kinetic studies and biochemical pathway analysis
of anaerobic poly-(R)-3-hydroxybutyric acid synthesis in Escherichia coli. Appl. Environ.
Microbiol. 71:713-720.
Charusanti, P., T. M. Conrad, E. M. Knight, K. Venkataraman, N. L. Fong, B. Xie, Y. Gao, B.
Ø. Palsson. 2010. Genetic basis of growth adaptation of Escherichia coli after deletion of pgi, a
major metabolic gene. PLoS Genet. 6(11):e1001186.
Eiteman, M. A. and M. J. Chastain. 1997. Optimization of the ion-exchange analysis of organic
acids from fermentation. Anal. Chim. Acta. 338:69-75.
Gorsich, S., B. Dien, N. Nichols, P. Slininger, Z. Liu and C. Skory. 2006. Tolerance to
furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1,
RPE1, and TKL1 in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 71:339-349.
Hur, H. G., M. J. Sadowsky and L. P. Wackett. 1994. Metabolism of chlorofluorocarbons and
polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic
pathway. Appl. Environ. Microbiol. 60:4148-4154.
53
Herring, C.D., A. Raghunathan, C. Honisch, T. Patel, M. K. Applebee, A. R. Joyce, T. J.
Albert, F. R. Blattner, D. van den Boom, C. R. Cantor and B. O. Palsson. 2006. Comparative
genome sequencing of Escherichia coli allows observation of bacterial evolution on a
laboratory timescale. Nat. Genet. 38:1406-1412.
Keating, J. D., C. Panganiban and S. D. Mansfield. 2006. Tolerance and adaptation of
ethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol. Bioeng.
93:1196-1206.
Liu, L. M., Q. L. Xu, Y. Li, Z. P. Shi, Y. Zhu, G. C. Du and J. Chen. 2007. Enhancement of
pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata. Biotechnol. Bioeng.
97:825-832.
Mason, C. A, G. Hamer and J. D. Bryers. 1986. The death and lysis of microorganisms in
environmental processes. FEMS Microbiol. Rev. 39:373-401.
Padan, E., T. Tzubery, K. Herz, L. Kozachkov, A. Rimon and L. Galili. 2004. NhaA of
Escherichia coli, as a model of a pH-regulated Na+/H+ antiporter. Biochim. Biophys. Acta.
1658:2-13.
Pirt, S. J. 1965. The maintenance energy of bacteria in growing cultures. Proc. R. Soc. London.
B. 163:224-231.
Rajakylä, E. and M. Paloposki. 1983. Determination of sugars (and betaine) in molasses by
high-performance liquid chromatography: Comparison of the results with those obtained by the
classical lane-eynon method. J. Chromatogr. 282:595-602.
54
Ro, D., E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. Ho, R. A.
Eachus, T. S. Ham, J. Kirby, M. C. Y. Chang, S. T. Withers, Y. Shiba, R. Sarpong and J. D.
Keasling. 2006. Production of the antimalarial drug precursor artemisinic acid in engineered
yeast. Nature. 440:940-943.
Ros, R., C. Montesinos, A. Rimon, E. Padan and R. Serrano. 1998. Altered Na+ and Li+
homeostasis in Saccharomyces cerevisiae cells expressing the bacterial cation antiporter NhaA.
J. Bacteriol. 180:3131-3136.
Russell, J. B. and G. M. Cook. 1995. Energetics of bacterial growth: balance of anabolic and
catabolic reactions. Microbiol. Rev. 59:48-6.
Sachidanandham, R., Y. Al-Shayji, N. Al-Awadhi and K. Y. H. Gin. 2005 A cryptic Bacillus
isolate exhibited narrow 16S rRNA gene sequence divergence with Bacillus thuringiensis and
showed low maintenance requirements in hyper-osmotic complex substrate cultivations.
Biotechnol. Bioeng. 91:838-847.
Sauer, U. and B. J. Eikmanns. 2005. The PEP-pyruvate-oxaloacetate node as the switch point
for carbon flux distribution in bacteria. FEMS Microbiol. Rev. 29:765-794.
Storz, G. and R. Hengge-Aronis. 2000. Bacterial stress responses. ASM Press, Washington,
D.C.
Stouthamer, A. H., B. A. Bulthuis, H. W. van Verseveld. 1990. Energetics of growth at low
growth rates and its relevance for the maintenance concept. In: Poole, R. K., M. J. Bazin and C.
W. Keevil (Eds.) Microbial Growth Dynamics. Special Publications Society for General
Microbiology. Oxford: IRL Press. 28:85-102.
55
Sutherland, I. W. and J. F. Wilkinson. 1971. Chapter IV Chemical Extraction Methods of
Microbial Cells. Methods. Microbiol. 5:345-383.
Taglicht, D., E. Padan and S. Schuldiner. 1991. Overproduction and purification of a functional
Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli. J. Biol. Chem. 266:11289-11294.
Taglicht, D., E. Padan, and S. Schuldiner. 1993. Proton-sodium stoichiometry of NhaA, an
electrogenic antiporter from Escherichia coli. J. Biol. Chem. 268: 5382-5387.
van Maris, A. J. A., D. A. Abbott, E. Bellissimi, J. van den Brink, M. Kuyper, M. A. H. Luttik,
H. W. Wisselink, W. A. Scheffers, J. P. van Dijken and J. T. Pronk. 2006. Alcoholic
fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current
status. Antonie. Van. Leeuwenhoek. 90:391-418.
Vemuri, G. N., E. Altman, D. P. Sangurdekar, A. B. Khodursky, M. A. Eiteman. 2006.
Overflow Metabolism in Escherichia coli during steady-state growth: transcriptional regulation
and effect of redox ratio. Appl. Environ. Microbiol. 72:3653-3661.
Warnecke, T. and R. T. Gill. 2005. Organic acid toxicity, tolerance, and production in
Escherichia coli biorefining application. Microb. Cell Fact. 4:25.
West, I.C. and P. Mitchell. 1974. Proton/Sodium ion antiport in Escherichia coli. Biochem. J.
144:87-90.
Wu. L., Z. fan, L. Guo, Y. Li, Z. Chen and L. Qu. 2004. Over-expression of the bacterial nhaA
gene in rice enhances salt and drought tolerance. Plant Science. 168:297-302.
56
Zelić, B., S. Gostović, K. Vuorilehto, D. Vasić-Rački, R. Takors. 2004. Process strategies to
enhance pyruvate production with recombinant Escherichia coli: From repetitive fed-batch to in
situ product recovery with fully integrated electrodialysis. Biotechnol.Bioeng. 85:638-646.
Zhu, J. and K. Shimizu. 2004. The effect of pfl gene knowout on the metabolism for optically
pure D-lactate production by Escherichia coli. Appl. Environ. Microbiol. 64:367-375.
Zhu, Y., M. A. Eiteman, R. Altman and E. Altman. 2008. High glycolytic flux improves
pyruvate production by a metabolically engineered Escherichia coli strain. Appl. Environ.
Microbiol. 74:6649-6655.
57
APPENDIX A
Summary of results
Table A.1. Summary of the results obtained from chemostat cultures using MG1655 and ALS1187 at different growth rates and different Na+ concentrations.
Strain Na+ (M) D (h-1) DCW (g/L) qs (g/gh) qO2 (g/gh) qCO2 (g/gh) MG1655 0.18 0.05 1.65 0.11 N/A N/A
0.07 1.78 0.15 2.17 3.76 0.10 1.85 0.21 4.35 4.08 0.14 1.89 0.26 12.88 4.25 0.68 0.05 0.78 0.28 7.10 7.72 0.07 0.83 0.36 8.10 8.73 0.10 0.89 0.48 11.25 12.04 0.14 0.99 0.59 10.48 14.74
ALS1187 0.18 0.05 1.31 0.14 4.43 4.64 0.07 1.40 0.19 5.30 5.54 0.10 1.45 0.26 5.83 7.65 0.14 1.54 0.35 8.04 9.55 0.68 0.05 0.69 0.29 5.82 8.83 0.07 0.74 0.37 6.21 10.31 0.10 0.78 0.52 8.62 14.62 0.14 0.83 0.67 15.59 17.92
58
Table A.2. Summary of the results in this thesis study.
Study Results
Overexpressed NhaA in wildtype E. coli MG1655
No beneficial effect on sodium tolerance. Overexpression of nhaA reduced growth rate.
Adaptive evolution of the wildtype E. coli MG1655 to tolerate NaCl
Obtained four isolate strains. Enhanced NaCl tolerance by 15%.
Chemostat using MG1655 and ALS1187
Specific glucose consumption rate, specific oxygen uptake rate and specific carbon dioxide evolution rate of ALS1187 increased; maintenance coefficient and the biomass yield on glucose of ALS1187 decreased.
Lactate fermentation using MG1655 pflB and ALS1187 pflB
Fermentation time prolonged and lactate production increased when using ALS1187 pflB.
Adaptive evolution of ALS929 to tolerate NaCl
Obtained three isolate strains. Enhanced NaCl tolerance by 10%.
Lactate fermentation using ALS929 and ALS1182
Essentially same amount of pyruvate was accumulated.
59
APPENDIX B
Data of fermentation processes
Fermentation 1
Date 2/23-26/2010 Strain ALS929
Inoculum 30 mL SF Media GAM with 5mM betaine
Initial Vol 1.032 L Feed I 600 g/L Glucose, 30 g/L Acetate
II 125 g/L Acetate, manually control Glucose conc. over 10 g/L by using 600 g/L feed pH 7.0
Temp 37°C Flow rate 1.0 L/min Air and Oxygen Agitation 400 rpm
Base I 25% NaOH 5% NH4OH II 30% NaOH
Notes Use Feed I during OD = 3~15; use Feed II after OD = 15. Perform exponential feed by fed_batch_calculation excel file. Use Base I before OD = 10, then use Base II.
Time Sample OD Glucose Acetate Pyruvate Pyruvate Succinate Lactate Ethanol Sample Feed I Feed II Feed III Base I Base II Total ln(OD) (h) (g/L) (g/L) (g/L) (g) (g/L) (g/L) (g/L) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL)
0.00 1 0.01 23.11 0.76 3.15 3.23 0.00 0.00 0.00 8.0 0.0 0 0 0.0 0 1024.0 -4.61 12.00 2 1.84 26.83 0.50 1.36 1.40 0.00 0.00 0.00 2.5 0.0 0 0 5.5 0 1027.0 0.61 13.30 3 3.52 26.18 0.00 2.76 2.83 0.00 0.00 0.00 3.0 0.0 0 0 6.5 0 1025.0 1.26 16.30 4 6.25 22.81 0.00 8.93 9.31 0.00 0.00 0.00 2.5 10.4 0 0 16.0 0 1042.4 1.83 19.30 5 8.17 21.95 0.00 15.30 16.29 0.00 0.00 0.00 4.5 27.4 0 0 26.0 0 1064.9 2.10 22.30 6 8.45 28.27 0.00 17.74 19.36 0.00 0.00 0.00 3.5 53.1 0 0 30.0 0 1091.1 2.13 25.30 7 8.38 42.18 0.00 20.03 22.59 0.00 0.00 0.00 4.0 89.6 0 0 34.0 0 1127.6 2.13 28.30 8 7.59 55.07 0.00 20.40 24.01 0.00 0.00 0.00 3.5 89.6 4.38 30.00 34.0 8.5 1177.0 2.03
Feed I: 620.9 g/L glucose 33.2 g/L acetate pH 7.31 When OD reaches 8.17 at 19.30 h, the nitrogen source is Started at: 13.30 h 0.2 g NH4Cl, 2.0 g (NH4)2SO4 and 1.3 g NH4OH; Feed II: 127 g/L acetate pH 8.52 that is 2.49 g NH4OH. Started at: 25.30 h The dry cell weight is 2.70 g. Feed III: 625.8 g/L glucose pH 5.15 Started at: 26.30 h Total Glucose consumption (g): 33.25 @ 29 h 0.5 g betaine, in 10 mL, was added at 26.3 h. Pyruvate yield on glucose (g/g): 0.722 @ 29 h
60
Fermentation 2
Date 3/1-4/2010 Strain ALS929
Inoculum 30 mL SF Media GAM with 5mM betaine
Initial Vol 1.032 L Feed I 600 g/L Glucose, 30 g/L Acetate
II 125 g/L Acetate, manually control Glucose conc. over 10 g/L by using 600 g/L feed pH 7.0
Temp 37°C Flow rate 1.0 L/min Air and Oxygen Agitation 400 rpm
Base I 25% NaOH 5% NH4OH II 30% NaOH
Notes Replicate Yihui's feeding method. Use Base I before OD = 10, then use Base II.
Time Sample OD Glucose Acetate Pyruvate Pyruvate Succinate Lactate Ethanol Sample Feed I Feed II Feed III Base I Base II Total ln(OD) (h) (g/L) (g/L) (g/L) (g) (g/L) (g/L) (g/L) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL)
0.00 1 0.01 25.21 1.08 3.91 4.03 0.00 0.00 0.00 3.5 0.0 0.0 0.0 2.0 0 1030.5 -4.61 12.50 2 1.75 16.76 0.34 0.80 0.82 0.00 0.00 0.00 3.5 0.0 0.0 0.0 2.0 0 1027.0 0.56 14.00 3 2.76 17.74 0.00 1.69 1.73 0.00 0.00 0.00 2.5 0.0 0.0 0.0 2.0 0 1024.5 1.02 14.50 4 3.55 16.94 0.00 2.03 2.08 0.00 0.00 0.00 3.0 0.0 0.0 0.0 4.0 0 1023.5 1.27 17.50 5 7.88 14.46 0.00 5.75 6.08 0.00 0.00 0.00 3.5 23.1 0.0 0.0 19.0 0 1058.1 2.06 20.50 6 11.70 14.63 0.00 13.24 14.66 0.00 0.00 0.00 3.0 54.6 0.0 0.0 39.5 0 1107.1 2.46 23.50 7 15.78 11.77 0.00 20.66 23.89 0.00 0.00 0.00 3.0 80.3 0.0 0.0 66.0 0 1156.3 2.76 26.50 8 16.01 18.70 0.00 43.20 53.57 0.00 0.00 0.00 3.0 133.0 0.0 0.0 66.0 34.0 1240.0 2.77 29.50 9 18.28 36.30 0.00 58.80 78.88 0.00 0.00 0.00 3.5 210.0 0.0 0.0 66.0 62.0 1341.5 2.91 32.50 10 15.84 52.44 0.94 51.95 75.76 0.00 0.00 0.00 3.5 258.0 18.3 30.0 66.0 72.0 1458.3 2.76 33.50 11 15.74 50.96 0.55 51.87 75.67 0.00 0.00 0.00 4.0 258.0 20.8 30.0 66.0 74.0 1458.8 2.76
Feed I: 614.9 g/L glucose 32.3 g/L acetate pH 7.31 When OD reaches 18.28 at 29.50 h, the nitrogen source is Started at: 14.50 h 0.2 g NH4Cl, 2.0 g (NH4)2SO4 and 3.3 g NH4OH; Feed II: 127 g/L acetate pH 8.52 that is 4.49 g NH4OH. Started at: 31.25 h The dry cell weight is 6.03 g. Feed III: 625.8 g/L glucose pH 5.15 Started at: 31.50 h Total Glucose consumption (g): 106.41 @ 29.5 h 0.5 g betaine, in 14 mL, was added at 30 h. Pyruvate yield on glucose (g/g): 0.741 @ 29.5 h
61
Fermentation 3
Date 3/9-12/2010 Strain ALS929
Inoculum 30 mL SF Media GAM with 5mM betaine
Initial Vol 1.032 L Feed I 600 g/L Glucose, 30 g/L Acetate
II 125 g/L Acetate, manually control Glucose conc. over 10 g/L by using 600 g/L feed pH 7.0
Temp 37°C Flow rate 1.0 L/min Air and Oxygen Agitation 400 rpm
Base I 25% NaOH 5% NH4OH II 30% NaOH
Notes Replicate Yihui's feeding method. Measure glucose conc. by using offline analyser. Use Base I before OD = 10, then use Base II.
Time Sample OD Glucose Acetate Pyruvate Pyruvate Succinate Lactate Ethanol Sample Feed I Feed II Feed III Base I Base II Na+ conc. Total ln(OD) (h) (g/L) (g/L) (g/L) (g) (g/L) (g/L) (g/L) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) (mol/L) Vol. (mL)
0.00 1 0.01 28.42 0.59 2.69 2.77 0.00 0.00 0.00 3.0 0 0.0 0.0 2.0 0 0.012 1031.0 -4.61 12.50 2 2.64 16.39 0.00 1.11 1.14 0.00 0.00 0.00 4.0 0 0.0 0.0 4.0 0 0.024 1029.0 0.97 15.80 3 7.34 19.66 0.00 7.30 7.76 0.00 0.00 0.00 3.0 22.4 0.0 0.0 18.0 0 0.116 1062.4 1.99 18.80 4 11.61 23.03 0.00 20.13 22.41 0.00 0.00 0.00 2.5 56.0 0.0 0.0 38.0 0 0.238 1113.5 2.45 21.80 5 14.12 17.96 0.00 34.92 41.06 0.00 0.00 0.00 5.0 92.4 0.0 0.0 69.0 0 0.406 1175.9 2.65 24.80 6 17.74 14.77 0.00 42.14 53.67 0.00 0.00 0.00 4.0 144.0 0.0 0.0 105.0 0 0.572 1273.5 2.88 26.60 7 18.07 20.38 0.00 53.90 72.41 0.00 0.00 0.00 4.0 193.0 0.0 0.0 130.0 0 0.677 1343.5 2.89 27.80 8 21.43 9.85 0.00 47.97 65.33 0.00 0.00 0.00 5.5 193.0 9.8 0.0 144.0 0 0.747 1361.8 3.06 30.80 9 24.64 3.28 0.00 64.55 93.02 0.00 0.00 0.00 5.0 193.0 39.0 30.0 156.0 13.0 0.868 1441.0 3.20 31.80 10 22.87 12.94 0.00 70.65 105.23 0.00 0.00 0.00 7.5 193.0 52.0 50.0 156.0 36.0 0.973 1489.5 3.13 32.20 11 22.43 3.04 0.00 68.39 102.96 0.00 0.00 0.00 11.0 193.0 55.0 50.0 156.0 40.0 0.987 1505.5 3.11
Feed I: 616 g/L glucose 32.1 g/L acetate pH 7.31 When OD reaches 22.87 at 31.8 h, the nitrogen source is Started at: 12.80 h 0.2 g NH4Cl, 2.0 g (NH4)2SO4 and 7.8 g NH4OH; Feed II: 127 g/L acetate pH 8.52 that is 9.99 g NH4OH. Started at: 26.60 h The dry cell weight is 7.54 g. Feed III: 625.8 g/L glucose pH 5.15 Started at: 27.60 h Total Glucose consumption (g): 160.20 @ 31.8 h 0.5 g betaine, in 14 mL, was added at 24.5 h. Pyruvate yield on glucose (g/g): 0.657 @ 31.8 h
62
Fermentation 4
Date 3/15-20/2010 Strain ALS1182
Inoculum 30 mL SF Media GAM with 5mM betaine
Initial Vol 1.032 L Feed I 600 g/L Glucose, 30 g/L Acetate
II 125 g/L Acetate, manually control Glucose conc. over 10 g/L by using 600 g/L feed pH 7.0
Temp 37°C Flow rate 1.0 L/min Air and Oxygen Agitation 400 rpm
Base I 25% NaOH 5% NH4OH II 30% NaOH
Notes Replicate Yihui's feeding method. Measure glucose conc. by using offline analyser. Use Base I before OD = 10, then use Base II.
Time Sample OD Glucose Acetate Pyruvate Succinate Lactate Ethanol Sample Feed I Feed II Feed III Base I Base II Na+ conc. Total ln(OD) (h) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) (mol/L) Vol. (mL)
0.00 1 0.01 26.30 1.00 3.01 0.00 0.00 0.00 2.0 0 0.0 0.0 2.0 0 0.012 1032.0 -4.61 23.00 2 2.50 14.02 0.00 0.49 0.00 0.00 0.00 4.5 0 0.0 0.0 2.0 0 0.012 1027.5 0.92 26.25 3 7.10 23.20 0.00 6.60 0.00 0.00 0.00 3.5 21.1 0.0 0.0 13.0 0 0.087 1056.1 1.96 29.25 4 10.65 24.31 0.00 18.41 0.00 0.00 0.00 4.0 52.4 0.0 0.0 32.0 0 0.205 1102.4 2.37 32.75 5 14.74 19.35 0.00 34.63 0.00 0.00 0.00 4.0 97.4 0.0 0.0 66.0 0 0.392 1177.4 2.69 35.25 6 16.51 23.96 0.00 52.14 0.00 0.00 0.00 5.0 143.0 0.0 0.0 92.0 0 0.520 1244.0 2.80 37.25 7 20.10 11.70 0.00 45.63 0.00 0.00 0.00 4.0 152.0 3.5 0.0 96.0 5.2 0.572 1261.7 3.00 38.25 8 21.90 7.14 0.00 54.91 0.00 0.00 0.00 6.5 152.0 16.8 0.0 96.0 23.0 0.687 1286.3 3.09 42.25 9 21.70 4.76 0.00 58.27 0.00 0.00 0.00 6.0 152.0 60.0 40.0 96.0 55.0 0.861 1409.5 3.08 44.25 10 22.10 4.33 1.55 70.20 0.00 0.00 0.00 7.0 152.0 78.0 50.0 96.0 63.0 0.911 1438.5 3.10
Feed I: 616 g/L glucose 32.1 g/L acetate pH 7.31 When OD reaches 18.28 at 29.50 h, the nitrogen source is Started at: 23.25 h 0.2 g NH4Cl, 2.0 g (NH4)2SO4 and 3.3 g NH4OH; Feed II: 127 g/L acetate pH 8.52 that is 4.49 g NH4OH. Started at: 26.60 h The dry cell weight is 6.03 g. Feed III: 625.8 g/L glucose pH 5.15 Started at: 27.60 h Total Glucose consumption (g): 145.84 @ 44.25 h 0.5 g betaine, in 14 mL, was added at 42 h. Pyruvate yield on glucose (g/g): 0.692 @ 44.25 h
63
Fermentation 5
Date 3/30-4/02/2010 Strain ALS1182
Inoculum 30 mL SF Media GAM with 5mM betaine
Initial Vol 932 mL Feed I 600 g/L Glucose, 30 g/L Acetate
II 125 g/L Acetate, manually control Glucose conc. over 10 g/L by using 600 g/L feed pH 7.0
Temp 37°C Flow rate 1.0 L/min Air and Oxygen Agitation 400 rpm
Base I 25% NaOH 5% NH4OH II 40% NaOH
Notes Replicate Yihui's feeding method. Measure glucose conc. by using offline analyser. Use Base I before OD = 15, then use Base
II.
Time Sample OD Glucose Acetate Pyruvate Pyruvate Sample Feed I Feed II Feed III Base I Base II Na+ conc. Total ln(OD) (h) (g/L) (g/L) (g/L) (g) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) (mol/L) Vol. (mL)
0.00 1 0.01 32.23 1.15 5.20 4.84 1.5 0.0 0.0 0.0 1.0 0.0 0.007 931.5 -4.61 15.00 2 1.21 26.60 0.62 0.64 0.60 2.0 0.0 0.0 0.0 2.0 0.0 0.013 930.5 0.19 16.00 3 2.43 32.23 0.00 1.18 1.09 3.0 0.0 0.0 0.0 2.1 0.0 0.014 927.6 0.89 16.33 4 3.05 25.89 0.00 1.15 1.06 2.5 0.0 0.0 0.0 2.2 0.0 0.015 925.2 1.12 19.40 5 7.46 28.23 0.00 7.80 7.48 2.0 24.0 0.0 0.0 14.0 0.0 0.104 959.0 2.01 22.40 6 12.17 29.75 0.00 22.07 22.18 4.5 54.4 0.0 0.0 34.0 0.0 0.239 1004.9 2.50 25.40 7 15.71 23.67 0.00 35.38 37.71 4.0 91.4 0.0 0.0 62.0 0.0 0.406 1065.9 2.75 28.40 8 18.45 6.71 0.00 50.24 56.12 9.0 91.4 12.1 10.0 62.0 24.0 0.572 1117.0 2.92 31.40 9 20.17 4.12 0.00 58.54 68.85 9.0 91.4 32.2 40.0 62.0 42.0 0.693 1176.1 3.00 34.40 10 18.61 14.29 0.37 63.27 78.83 7.0 91.4 61.1 80.0 62.0 50.0 0.751 1246.0 2.92 35.10 11 19.31 12.07 0.59 61.89 77.26 8.0 91.4 69.4 80.0 62.0 52.0 0.775 1248.3 2.96
Feed I: 584 g/L glucose 17.6 g/L acetate pH 7.31 When OD reaches 15.71 at 25.40 h, the nitrogen source is Started at: 16.40 h 0.2 g NH4Cl, 2.0 g (NH4)2SO4 and 3.1 g NH4OH; Feed II: 127 g/L acetate pH 8.52 that is 4.29 g NH4OH. Started at: 25.40 h The dry cell weight is 5.19 g. Feed III: 602.3 g/L glucose pH 5.15 Started at: 27.90 h Total Glucose consumption (g): 113.78 @ 34.40 h 0.5 g betaine, in 14 mL, was added at 26 h. Pyruvate yield on glucose (g/g): 0.693 @ 34.40 h
64
Fermentation 6
Date 4/7-10/2010 Strain ALS1182
Inoculum 30 mL SF Media GAM with 5mM betaine
Initial Vol 922 mL Feed I 600 g/L Glucose, 30 g/L Acetate
II 125 g/L Acetate, manually control Glucose conc. over 10 g/L by using 600 g/L feed pH 7.0
Temp 37°C Flow rate 1.0 L/min Air and Oxygen Agitation 400 rpm
Base I 25% NaOH 5% NH4OH II 40% NaOH
Notes Replicate Yihui's feeding method. Measure glucose conc. by using offline analyser. Use Base I before OD = 15, then use Base II.
Time Sample OD Glucose Acetate Pyruvate Pyruvate Sample Feed I Feed II Feed III Base I Base II Na+ conc. Total ln(OD) (h) (g/L) (g/L) (g/L) (g) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) Vol. (mL) (mol/L) Vol. (mL)
0.00 1 0.01 25.44 0.93 4.15 3.82 3.5 0.0 0.0 0.0 1.0 0.0 0.007 919.5 -4.61 14.00 2 4.56 19.95 0.00 2.61 2.40 3.5 0.0 0.0 0.0 3.0 0.0 0.020 918.0 1.52 17.40 3 8.59 23.10 0.00 9.47 9.09 4.0 29.1 0.0 0.0 20.0 0.0 0.145 960.1 2.15 20.40 4 13.03 16.72 0.00 16.63 16.74 4.5 59.2 0.0 0.0 41.0 0.0 0.284 1006.7 2.57 23.40 5 16.01 18.14 0.00 32.65 34.99 6.0 97.8 0.0 0.0 73.5 0.0 0.474 1071.8 2.77 26.40 6 19.50 1.72 0.00 49.32 54.08 9.0 97.8 13.8 0.0 73.5 20.0 0.627 1096.6 2.97 29.40 7 20.91 2.96 0.00 54.81 63.40 6.0 97.8 35.9 30.0 73.5 34.0 0.725 1156.7 3.04 31.05 8 20.80 3.88 0.00 56.21 67.28 8.5 97.8 50.6 45.0 73.5 39.0 0.757 1196.9 3.03 32.40 9 19.40 3.01 0.59 54.18 65.27 9.5 97.8 59.8 50.0 73.5 42.0 0.787 1204.6 2.97 32.90 10 19.20 1.68 0.00 55.99 66.96 9.5 97.8 59.8 50.0 73.5 43.0 0.799 1196.1 2.95
Feed I: 584 g/L glucose 17.6 g/L acetate pH 7.31 When OD reaches 15.71 at 25.40 h, the nitrogen source is Started at: 14.25 h 0.2 g NH4Cl, 2.0 g (NH4)2SO4 and 3.1 g NH4OH; Feed II: 127 g/L acetate pH 8.52 that is 4.29 g NH4OH. Started at: h The dry cell weight is 5.19 g. Feed III: 602.3 g/L glucose pH 5.15 Started at: h Total Glucose consumption (g): 108.61 @ 32.90 h 0.5 g betaine, in 7 mL, was added at 26.8 h. Pyruvate yield on glucose (g/g): 0.617 @ 32.90 h
65
Fermentation 7 Date 4/15-16/2010 Strain ALS413 Inoculum 100 mL Media Park Medium Initial Vol 1000 mL Feed 604 g/L glucose pH 7.0 Temp 37°C Air Flow I aerobic, 1 L/min, air and/or oxygen II anaerobic, 0.4 L/min, CO2 and N2 (1:9) Agitation I aerobic, 400 rpm anaerobic, 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL)
0.00 1 0.10 19.5 0 0.0 0.0 0.0 1.4 0.0 0.008 3.5 1 0 997.5 8.33 2 11.63 5.2 2.3 0.0 0.4 0.0 1.7 0.0 0.097 8.5 13 0 1001.0 9.33 3 12.72 2.3 1.4 0.0 0.0 0.0 1.1 0.0 0.120 7.5 16 0 996.5
10.33 4 12.67 3.5 2.1 0.0 1.0 0.0 1.9 0.0 0.144 6.5 19 0 993.0 13.33 5 11.12 7.3 4.5 1.4 3.3 0.4 1.7 3.0 0.271 6.5 37 20 1024.5 16.33 6 9.31 7.0 6.3 2.1 4.8 1.5 1.7 4.4 0.381 7 53 10 1043.5 19.33 7 9.89 5.0 5.8 1.9 4.7 1.9 1.4 4.0 0.443 7 62 5 1050.5 22.33 8 8.96 3.7 7.8 2.5 6.2 3.0 1.6 5.2 0.487 9.5 68 0 1047.0 23.33 9 8.58 2.9 7.8 2.5 6.2 3.2 1.6 5.3 0.498 9 69 0 1039.0
The fermentation changed from aerobic to anaerobic at 10.33 h. The fermentation ended at 23.33 h. Total glucose consumption is 37.74 g.
66
Fermentation 8 Date 4/19-20/2010 Strain ALS1187 Inoculum 100 mL Media Park Medium Initial Vol 1000 mL Feed 604 g/L glucose pH 7.0 Temp 37°C Air Flow I aerobic, 1 L/min, air and/or oxygen II anaerobic, 0.4 L/min, CO2 and N2 (1:9) Agitation I aerobic, 400 rpm anaerobic, 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL) 0.0 1 0.10 19.8 0.0 0.0 0.0 0.0 1.7 0.0 0.008 4 1 0 997.0
10.0 2 10.06 1.8 2.8 0.0 0.0 0.0 1.2 0.0 0.105 6.5 14 0 1003.5 11.5 3 10.53 0.3 3.9 0.0 0.0 0.0 1.2 0.0 0.186 7 25 0 1007.5 14.5 4 8.93 3.2 6.2 1.2 2.3 0.8 1.3 1.6 0.318 8 44 20 1038.5 17.5 5 8.25 3.2 7.3 1.8 3.1 2.7 1.3 2.5 0.411 5 58 10 1057.5 20.5 6 8.03 1.3 7.3 1.8 3.2 4.9 1.2 3.0 0.485 6.5 69 5 1067.0 23.5 7 7.82 1.1 8.3 2.0 3.4 8.0 1.3 3.9 0.540 6.5 77.5 7 1076.0 26.5 8 7.63 0.3 9.5 2.1 3.8 11.8 1.4 5.1 0.590 7.5 85 5.3 1081.3 28.0 9 7.55 0.4 8.7 2.0 3.4 11.9 1.3 4.9 0.612 9 88 3.3 1078.6
The fermentation changed from aerobic to anaerobic at 11.5 h. The fermentation ended at 28.0 h. Total glucose consumption is 49.96 g.
67
Fermentation 9 Date 5/15-16/2010 Strain ALS413 Inoculum 100 mL Media Park Medium Initial Vol 1000 mL Feed 606 g/L glucose pH 7.0 Temp 37°C Air Flow I aerobic, 1 L/min, air and/or oxygen II anaerobic, 0.4 L/min, CO2 and N2 (1:9) Agitation I aerobic, 400 rpm anaerobic, 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL) 0.0 1 0.10 19.9 0.00 0.00 0.00 0.00 1.39 0.00 0.000 3 0 0 997.0 8.2 2 8.53 5.28 5.22 0.84 2.24 0.38 0.74 0.66 0.191 4.5 26 0 1018.5 9.5 3 10.49 0.14 7.32 1.02 2.54 0.40 0.70 0.90 0.250 5 34 0 1021.5
12.5 4 10.32 6.68 9.56 2.26 4.50 3.38 0.36 2.22 0.404 5.5 57 20 1059.0 15.5 5 9.89 0.28 10.64 3.02 5.38 6.02 0.34 2.58 0.499 6 71 0 1067.0 18.5 6 9.72 2.44 11.06 3.52 5.80 9.26 0.32 2.94 0.059 5.5 8 15 1013.5 21.5 7 9.60 2.86 11.30 3.88 5.94 12.12 0.36 3.20 0.632 5 93 10 1103.5 24.5 8 9.18 1.08 11.76 4.06 6.12 15.30 0.36 3.48 0.687 4.5 102 5 1113.0 26.0 9 8.95 1.68 11.76 4.18 6.02 16.62 0.50 3.56 0.706 5 105 5 1116.0
The fermentation changed from aerobic to anaerobic at 9.5 h. The fermentation ended at 26.0 h. Total glucose consumption is 51.36 g.
68
Fermentation 10 Date 5/15-16/2010 Strain ALS1187 Inoculum 100 mL Media Park Medium Initial Vol 1000 mL Feed 606 g/L glucose pH 7.0 Temp 37°C Air Flow I aerobic, 1 L/min, air and/or oxygen II anaerobic, 0.4 L/min, CO2 and N2 (1:9) Agitation I aerobic, 400 rpm anaerobic, 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL) 0.0 1 0.10 19.9 0.00 0.00 0.00 0.00 1.19 0.00 0.000 3 0 0 997.0 8.2 2 7.39 0.68 5.50 1.16 2.18 2.14 1.30 0.48 0.255 4.5 35 0 1027.5
10.2 3 9.14 0.10 8.78 1.34 2.32 2.40 1.54 0.28 0.317 5 44 10 1041.5 11.2 4 10.32 0.72 9.90 1.40 2.16 3.20 2.38 0.32 0.363 4.5 51 10 1054.0 14.2 5 10.15 0.24 10.48 2.06 2.30 10.28 2.16 2.04 0.496 5.5 72 20 1089.5 17.2 6 9.37 2.48 10.42 2.06 2.20 16.78 1.92 2.92 0.589 5 88 20 1120.5 20.2 7 8.91 0.62 10.44 2.10 2.12 22.90 1.78 3.68 0.665 5 101 10 1138.5 23.2 8 8.56 0.58 10.14 2.08 1.98 26.90 1.66 4.14 0.728 5 112 10 1154.5 26.2 9 8.42 1.86 10.04 2.06 1.94 30.26 1.58 4.54 0.771 5 120 10 1167.5
The fermentation changed from aerobic to anaerobic at 11.2 h. The fermentation ended at 26.2 h. Total glucose consumption is 72.27 g.
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Fermentation 11 #This strain was labeled ALS1318 when provided to us by Ronni Altman. However, we believe that it was mislabeled and is actually ALS1317. Date 7/16-19/2010 Strain #ALS1317 = ALS413 pflB::cam Inoculum 100 mL Media Park Medium Initial Vol 1237.5 mL Feed 605 g/L glucose pH 7.0 Temp 37°C Switch Change from aerobic phase to anaerobic phase when OD = 8 Air Flow aerobic 1.2 L/min, air and/or oxygen anaerobic 0.4 L/min, 10% CO2 and 90% N2 Agitation aerobic 400 rpm anaerobic 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL) 0.0 1 0.10 19.3 0.0 0.0 0.0 0.0 0.2 0.0 0.012 5 2 0 1234.5 6.0 2 8.30 9.3 1.9 0.5 0.0 0.6 1.5 0.1 0.143 5 22 0 1251.5 9.0 3 8.07 1.2 3.0 1.5 0.0 13.7 2.6 2.0 0.334 5 35 39 1310.5
12.0 4 7.11 1.7 2.4 1.4 0.0 18.9 2.1 2.3 0.448 5 23 29 1347.5 15.0 5 7.02 1.8 2.7 1.5 0.0 28.2 2.3 3.0 0.531 6 18 22 1371.5 18.0 6 6.49 2.1 3.0 1.8 0.0 36.6 2.4 3.9 0.602 5 16 17 1389.5 21.0 7 6.09 2.1 2.9 1.8 0.0 40.2 2.3 4.2 0.648 7 11 14 1397.5 24.0 8 5.14 3.0 3.0 2.0 0.0 43.4 1.5 5.0 0.691 6 11 16 1408.5 27.0 9 4.91 2.0 2.4 1.7 0.0 46.5 2.0 4.7 0.719 6 7 6 1405.5 30.0 10 5.02 2.1 2.7 1.8 0.0 49.7 2.0 5.4 0.745 6 7 8 1404.5 33.0 11 5.01 2.1 2.6 1.7 0.0 51.6 2.0 5.6 0.768 7 6 6 1399.5 36.0 12 4.95 2.0 2.6 1.8 0.0 54.0 2.0 5.9 0.780 7 4 6 1392.5 39.0 13 4.91 2.0 2.4 1.7 0.0 55.4 2.0 6.0 0.798 5 5 4 1386.5 42.0 14 4.89 2.1 2.3 1.8 0.0 56.1 2.0 6.1 0.808 5 3 2 1376.5 45.0 15 4.88 2.0 2.3 1.7 0.0 56.3 1.9 6.2 0.810 5 1 0 1362.5
Total (mL) 85 171 169 The fermentation changed from aerobic to anaerobic at 6 h. The fermentation ended at 45.0 h. Total glucose consumption is 118.68 g. Lactate yield on glucose is 0.646 g/g.
70
Fermentation 12 #This strain was labeled ALS1317 when provided to us by Ronni Altman. However, we believe that it was mislabeled and is actually ALS1318. Date 7/22-25/2010 Strain #ALS1318 = ALS1187 pflB::cam Inoculum 100 mL Media Park Medium Initial Vol 1237.5 mL Feed 605 g/L glucose pH 7.0 Temp 37°C Switch Change from aerobic phase to anaerobic phase when OD = 8 Air Flow aerobic 1.2 L/min, air and/or oxygen anaerobic 0.4 L/min, 10% CO2 and 90% N2 Agitation aerobic 400 rpm anaerobic 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL) 0.0 1 0.10 19.3 0.0 0.0 0.0 0.0 0.2 0.0 0.012 4 2 0 1235.5 6.0 2 7.93 10.8 1.0 0.0 0.0 0.2 1.3 0.4 0.084 9 12 0 1238.5 9.0 3 9.21 4.1 0.9 0.0 0.0 15.2 1.8 1.1 0.255 6 30 24 1276.5
12.0 4 9.00 3.2 0.9 0.0 0.0 28.5 1.8 1.7 0.402 5 28 30 1319.5 15.0 5 8.35 3.8 0.9 0.0 0.0 36.9 2.0 2.1 0.498 5 20 24 1348.5 18.0 6 8.34 3.3 0.9 0.0 0.0 43.1 2.0 2.6 0.570 9 16 22 1367.5 21.0 7 8.01 3.3 0.8 0.0 0.0 48.8 2.0 2.9 0.618 6 12 18 1381.5 24.0 8 7.77 3.2 0.6 0.0 0.0 54.0 2.0 3.2 0.669 8 12 13 1388.5 27.0 9 7.63 2.1 0.6 0.0 0.0 57.2 2.0 3.3 0.710 6 10 9 1391.5 30.0 10 7.33 2.2 0.5 0.0 0.0 61.1 2.0 3.5 0.750 6 10 10 1395.5 33.0 11 7.25 2.7 0.5 0.0 0.0 64.8 2.0 3.6 0.788 5 10 13 1403.5 36.0 12 7.13 2.5 0.5 0.0 0.0 67.5 2.0 3.8 0.827 5 10 11 1409.5 39.0 13 7.01 1.9 0.5 0.0 0.0 70.4 2.0 3.9 0.861 6 9 8 1410.5 42.0 14 6.87 2.1 0.5 0.0 0.0 73.0 2.0 4.0 0.888 6 7 5 1406.5 45.0 15 6.97 2.2 0.6 0.0 0.0 74.6 2.0 4.1 0.901 5 4 3 1398.5 48.0 16 7.01 2.1 0.5 0.0 0.0 76.0 2.0 4.2 0.906 5 2 1 1386.5 52.0 17 6.84 2.0 0.5 0.0 0.0 76.2 2.0 4.2 0.908 5 1 0 1372.5
Total (mL) 101 195 191 The fermentation changed from aerobic to anaerobic at 6.0 h. The fermentation ended at 52.0 h. Total glucose consumption is 131.86 g. Lactate yield on glucose is 0.763 g/g.
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Fermentation 13 Date 8/13-16/2010 Strain ALS1318 = ALS1187 pflB::cam Inoculum 100 mL Media Park Medium Initial Vol 1237.5 mL Feed 612 g/L glucose pH 7.0 Temp 37°C Switch Change from aerobic phase to anaerobic phase when OD = 8 Air Flow aerobic 1.2 L/min, air and/or oxygen anaerobic 0.4 L/min, 10% CO2 and 90% N2 Agitation aerobic 400 rpm anaerobic 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL) 0.0 1 0.01 19.5 0.0 0.0 0.0 0.0 0.0 0.0 0.024 4 4 0 1237.5 6.0 2 8.16 11.1 1.7 0.0 0.0 0.0 0.0 0.0 0.137 4 19 0 1252.5 9.0 3 8.01 3.9 0.5 0.0 0.0 13.2 0.0 2.3 0.327 6 35 45 1316.5
12.0 4 7.45 3.9 2.0 0.0 0.0 20.0 0.0 3.2 0.476 5 30 30 1361.5 15.0 5 6.94 3.2 1.8 0.0 0.0 22.1 0.0 3.6 0.576 5 22 25 1393.5 18.0 6 6.47 5.0 2.3 1.4 0.0 36.9 0.0 5.9 0.645 5 16 18 1412.5 21.0 7 6.06 4.8 2.3 1.4 0.0 40.5 0.0 6.3 0.703 5 14 15 1426.5 24.0 8 5.66 3.5 2.1 1.4 0.0 42.6 0.0 6.5 0.752 6 12 10 1432.5 27.0 9 5.47 3.6 2.1 1.5 0.0 47.0 0.0 7.1 0.786 7 9 10 1434.5 30.0 10 5.32 3.5 2.0 1.4 0.0 47.9 0.0 7.1 0.816 6 8 7 1433.5 33.0 11 5.25 11.3 1.8 1.4 0.0 51.8 0.0 7.5 0.842 6 7 5 1429.5 36.0 12 5.13 8.3 0.6 1.2 0.0 58.8 0.0 7.1 0.860 6 5 4 1422.5 39.0 13 5.13 3.7 1.8 1.2 0.0 63.4 0.0 7.5 0.878 5 5 2 1414.5 42.0 14 5.11 4.3 2.0 1.2 0.0 66.9 0.0 7.6 0.897 5 5 2 1406.5 45.0 15 5.05 4.4 1.8 1.4 0.0 70.5 0.0 8.6 0.912 5 4 1 1396.5 48.0 16 5.03 2.1 1.8 1.2 0.0 71.6 0.0 7.9 0.918 7 2 0 1381.5
Total (mL) 87 197 174 The fermentation changed to anaerobic at 6.0 h, ended at 48.0 h. Total glucose consumption is 127.84 g. Lactate yield on glucose is 0.773 g/g.
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Fermentation 14 Date 8/26-30/2010 Strain ALS1317 = ALS413 pflB::cam Inoculum 100 mL Media Park Medium Initial Vol 1237.5 mL Feed 615 g/L glucose pH 7.0 Temp 37°C Switch Change from aerobic phase to anaerobic phase when OD = 8 Air Flow aerobic 1.2 L/min, air and/or oxygen anaerobic 0.4 L/min, 10% CO2 and 90% N2 Agitation aerobic 400 rpm anaerobic 200 rpm Base 30% NaOH Notes Cells are grown in the 50/250 mL flask to OD above 2, then adjust the volume to 100 mL by using Park Medium. The final OD is 1 and inoculate the fermenter.
Time Sample OD Glucose Acetate Ethanol Formate Lactate Pyruvate Succinate Na+ conc. Sample Base Feed Total (h) No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (mol/L) Volume (mL) 0.0 1 0.10 20.0 0.0 0.0 0.0 0.0 0.0 0.0 0.012 10 2 0 1229.5 6.0 2 8.39 9.5 0.8 0.0 0.0 0.0 0.0 0.0 0.114 7 17 0 1239.5 9.0 3 7.76 6.2 0.6 0.0 0.0 10.5 0.0 1.2 0.276 8 29 32 1284.5
12.0 4 7.01 3.3 0.6 0.0 0.0 18.5 0.0 1.7 0.405 7 25 26 1318.5 15.0 5 6.89 3.3 0.6 0.0 0.0 25.5 0.0 2.1 0.483 7 16 16 1333.5 18.0 6 6.59 3.0 0.6 0.0 0.0 26.0 0.0 2.1 0.548 5 14 14 1346.5 21.0 7 5.88 3.6 0.5 0.0 0.0 34.1 0.0 2.7 0.603 7 12 11 1352.5 24.0 8 5.26 2.7 0.5 0.0 0.0 30.8 0.0 2.4 0.647 7 10 9 1354.5 27.0 9 4.90 3.6 0.6 0.0 0.0 41.6 0.0 3.2 0.684 6 9 10 1357.5 30.0 10 4.92 3.6 0.5 0.0 0.0 42.9 0.0 3.2 0.717 8 8 8 1355.5 33.0 11 4.78 3.9 0.5 0.0 0.0 45.3 0.0 3.3 0.769 8 12 6 1355.5 36.0 12 4.91 3.8 0.5 0.0 0.0 48.2 0.0 3.5 0.792 7 7 8 1358.5 39.0 13 4.89 5.4 0.9 0.0 0.0 52.1 0.0 3.5 0.812 10 6 2 1353.5 42.0 14 4.71 2.1 0.6 0.0 0.0 52.6 0.0 3.5 0.812 7 2 0 1345.5
Total (mL) 104 169 142 The fermentation changed from aerobic to anaerobic at 6 h. The fermentation ended at 42.0 h. Total glucose consumption is 108.51 g. Lactate yield on glucose is 0.642 g/g.
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