ENHANCING SODIUM CHLORIDE TOLERANCE OF …

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


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


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

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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

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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







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







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








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


Initial Vol 932 mL Feed I 600 g/L Glucose, 30 g/L Acetate




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



Initial Vol 922 mL Feed I 600 g/L Glucose, 30 g/L Acetate





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


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


67


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


68


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


69

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.

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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.


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.


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.


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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