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Dynamics of Microbial Growth in Single Substrate Culture

Permanent Link: http://ufdc.ufl.edu/UFE0021341/00001

Material Information

Title: Dynamics of Microbial Growth in Single Substrate Culture
Physical Description: 1 online resource (80 p.)
Language: english
Creator: Noel, Jason Todd
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bacteria, carbon, chemostat, dynamics, escherichia, flux, glucose, limitation, starvation
Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In this study the results of continuous to batch mode experiments conducted to characterize the initial transient response of carbon limited microbial cells growing in a chemostat to a pulse of the growth limiting substrate are presented. Changes in cellular rates of growth, substrate uptake, carbon dioxide evolution, and organic carbon excretion were recorded for three different preculture conditions. To ensure the validity of the data, ninety percent of added carbon was accounted for in generated biomass, evolved carbon dioxide, and in excreted organic products before and after the substrate pulse. The continuous shifts revealed that values of growth and respiration were proportional to the preculture dilution rate while the capacity to increase the respiration and growth rate was inversely proportional to the preculture dilution rate. Saturation of respiration and biosynthetic capacity led to a high amount of excretion at the intermediate and highest preculture dilution rates tested. Only the highest preculture dilution rate was able to utilize the excreted carbon before the specific carbon dioxide evolution rate fell considerably due to lack of substrate. The identity of the biosynthetic growth limitation was also explored in this study using similar continuous to batch shift experiments. Evidence was found to support the hypothesis that the biosynthetic limitation is an amino acid supply limitation and not a protein production capacity limitation. Continuous to batch mode shifts followed by a pulse of growth limiting carbon substrate show the transient response of the biosynthetic enzyme glutamate dehydrogenase qualitatively matched the transient response of the intracellular RNA concentration suggesting the enzyme was more likely to be the cause of the biosynthetic growth limitation rather than the intracellular ribosome concentration.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jason Todd Noel.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Svoronos, Spyros.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021341:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021341/00001

Material Information

Title: Dynamics of Microbial Growth in Single Substrate Culture
Physical Description: 1 online resource (80 p.)
Language: english
Creator: Noel, Jason Todd
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bacteria, carbon, chemostat, dynamics, escherichia, flux, glucose, limitation, starvation
Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In this study the results of continuous to batch mode experiments conducted to characterize the initial transient response of carbon limited microbial cells growing in a chemostat to a pulse of the growth limiting substrate are presented. Changes in cellular rates of growth, substrate uptake, carbon dioxide evolution, and organic carbon excretion were recorded for three different preculture conditions. To ensure the validity of the data, ninety percent of added carbon was accounted for in generated biomass, evolved carbon dioxide, and in excreted organic products before and after the substrate pulse. The continuous shifts revealed that values of growth and respiration were proportional to the preculture dilution rate while the capacity to increase the respiration and growth rate was inversely proportional to the preculture dilution rate. Saturation of respiration and biosynthetic capacity led to a high amount of excretion at the intermediate and highest preculture dilution rates tested. Only the highest preculture dilution rate was able to utilize the excreted carbon before the specific carbon dioxide evolution rate fell considerably due to lack of substrate. The identity of the biosynthetic growth limitation was also explored in this study using similar continuous to batch shift experiments. Evidence was found to support the hypothesis that the biosynthetic limitation is an amino acid supply limitation and not a protein production capacity limitation. Continuous to batch mode shifts followed by a pulse of growth limiting carbon substrate show the transient response of the biosynthetic enzyme glutamate dehydrogenase qualitatively matched the transient response of the intracellular RNA concentration suggesting the enzyme was more likely to be the cause of the biosynthetic growth limitation rather than the intracellular ribosome concentration.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jason Todd Noel.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Svoronos, Spyros.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021341:00001


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DYNAMICS OF MICROBIAL GROWTH IN SINGLE SUB STATE CULTURE


By

JASON NOEL













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007




































O 2007 Jason Noel




























To my Parents David and Tracey Noel









ACKNOWLEDGMENTS

I would first like to acknowledge my advisor Dr. Atul Narang for his guidance and support

during this research. Without his ideas and insight none of this would have been possible. I

would also like to thank my committee members Dr. Spyros Svoronos, Dr. Ben Koopman, Dr.

Ranganathan Narayanan, and Dr. Lewis Johns for their support.

I would also like to thank some individuals whose technical expertise and personal

assistance made this research possible. Dr. Thomas Egli from the Swiss Federal Institute of

Aquatic Science and Technology was extremely helpful in troubleshooting problems found in

operating a chemostat growing microbial cells. Dr. Tommaso Cataldi from the Universita degli

Studi della Basisilicate was instrumental in developing a reliable procedure for HPLC

quantification of low sugar concentrations in samples extracted from bacterial cultures. Lastly I

would like to thank Dr. Max Teplitski from the University of Florida for his help providing a

reliable mechanical disruption technique for starved bacteria.

I would like to thank my lab members Dr. Shaki Gupta, Dr. Eric May, Dr. Karthik

Subramanian, Ved Sharma, and Brenton Cox. Your technical support and companionship helped

make the sometimes frustrating experimental work tolerable. Lastly I would like to thank my

friends and family for being there for me through the good times and the bad.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............7................


LI ST OF FIGURE S .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 GENERAL INTRODUCTION .............. ...............12....


1.1 M otivation for Research .............. ...............12....
1.2 Conceptual Model of the Cell ................. ...............13.............
1.3 Steady state behavior of microbial cells ................. ...._.__........ ...............14
1.4 Cell density and Substrate Concentration ................. ...............15...............
1.5 Cellular Content and Excreted Metabolites .............. ...............18....
1.6 Transients Controlled by the Transport Enzymes ................. .......... ................1 8
1.7 Transient Dynamics Controlled by a Biosynthetic Limitation .................. ...............20
1.8 Transient Response to Dilution Rate Shift Ups ................ .............................25

2 MATERIALS AND METHODS .............. ...............27....


2.1 Growth Medium ................. ...............27........... ....
2.2 Bacterial Strain............... ...............27.
2.3 The Chemostat Setup .............. ...............28....
2.3.1 Wall Growth Limitation............... ...............2
2.3.2 Glucose Adaptation Limitation............... ...............2
2.4 Cell Density Measurement and Growth Rate ............... ...... ...............30.
2.5 Sugar Measurement, Yield Determination, and Uptake Rate ................. ................. 31
2.6 Total Cell Protein Extraction and Measurement............... ..............3
2.6.1 Extraction ................. ...............32.................
2.6.2 M easurement ............... ... .... ...... .. ...............32.......
2.7 Total Cell RNA Extraction and Measurement ................. .............................33
2.7.1 Extraction ................. ...............33.................
2.7.2 M easurement ................. .......... ...............33.......
2.8 Carbon Dioxide Evolution Measurement .............. ...............33....
2.9 Total Organic Carbon Measurement............... ..............3
2.10 Elemental Content Measurement ................. ...............35................
2.11 Cell Disruption Techniques .............. ...............35....
2.11.1 Chemical Disruption .............. ... ...............36.......... .....
2.11.1 Chemical Disruption Drawbacks .............. ...............37....
2.11.1 Mechanical Disruption................ ..............3
2. 11.2 Mechanical Disruption Advantages ................. ...............38........... ..












2.12 Enzymatic Assay of Glutamate Dehydrogenase............... .............3
2.13 Deactivation and Disposal of Microorganisms ......____ ........_ ...............40

3 PROTOCOL VALIDATION AND SYSTEM CHARACTERIZATION .............................41


3.1 Goals of Verification and Characterization ........._.__........_. ....._.__............41
3.2 Steady State Biomass Yield ............ ...... ..._. ...............41...
3.3 Carbon Dioxide Evolution Rate ........._....._ ...._.._....._._ ....... ....4
3.4 Protein and RNA Dry Weight Fractions ........._.._.. ....._.. ...._... ..........4
3.5 Maximum Specific Growth Rate on Minimal Media.........._.._.. .......__. ........._.45
3.6 Initial Growth and Uptake Rate Response to Nutrient Excess .................. ...............46
3.7 Concluding Remarks ................. ...............48........... ....

4 STEADY STATE AND TRANSIENT CARBON FLUX .............. ...............49....


4.1 Introduction ................. ...............49........... ....
4.2 M materials and M ethods ............... ... ............ ...............50......
4.2.1 Organism and Cultivation Conditions .............. ...............50....
4.2.2 Carbon Measurement Methodology .............. ...............51....
4.2.3 The Continuous to Batch Shift Experiment ................. ......... ................53
4.3 Results ................. ...............54.................
4.4 Discussion ................. ...............58.................


5 ATTEMPT TO IDENTIFY THE BIO SYNTHETIC LIMITATION ................. .................6 1


5.1 Introduction ................. ...............61........... ....
5.2 M materials and M ethods ............... ... ............ ...............64.....
5.2.1 Organism and Cultivation Conditions .............. ...............64....
5.2.2 The Continuous to Batch Shift............... ...............65.
5.3 Results and Discussion .............. ...............66....


6 C ONCLUDING REMARK S ................ ...............69........... ...


APPENDIX


A VISUAL BASIC PROGRAM FOR THE VAISALA GMT222 CO2 ANALYZER .............71


B RESULTS OF CARLO ERBA-1106 ELEMENTAL ANALYSIS OF E. COLI ML308......75


LIST OF REFERENCES ............_...... .__ ...............76...


BIOGRAPHICAL SKETCH .............. ...............80....










LIST OF TABLES


Table page

2-1 Minimal medium recipe used for all experimentation. ................... ...............2

2-2. Volumes and concentrations of reagents used in the GDH assay. .............. ................. 40

4-1 Frequency of data collection and approximate reactor volume taken during
experim ents. .............. ...............53....

4-2 Growth rate, uptake rate, carbon dioxide evolution rate, and biomass yield before and
during a batch mode shift. ................. ...............57............

5-1 Frequency of sample measurement / collection and approximate volume taken... ......... .65










LIST OF FIGURES


Fiare page

1-1 Example of a dilution rate shift up experiment............... ...............1

1-2 Conceptual model of a microbial cell. ............. .....................14

1-3 Dilution rate dependence of biomass yield of microorganisms. ................ ................16

1-4 Dilution rate dependence of the steady state glucose concentration. ............. ...............16

1-5 Dilution rate dependence of the cellular RNA and protein content of
microorganisms ................. ...............17.................

1-6 Dilution rate dependence of the carbon dioxide evolution rate of microorganisms ..........17

1-7 Dilution rate dependence of the glutamate dehydrogenase activity of
microorganisms ................. ...............18.................

1-8 Evidence for the transport enzyme limitation ................. ...............19........... .

1-9 Growth response of glucose limited cells exposed to glucose excess. ............. ...... ..........21

1-10 Substrate uptake response of glucose limited cells exposed to glucose excess. ................22

1-11 RNA content response of continuously grown Azobacter vinelan2dii to substrate
excess. ............. ...............24.....

1-12 The GDH activity response of continuously grown E. coli W to substrate excess. ..........25

1-13 Response of a chemostat growing E. coli K-12 to a dilution rate shift up from D=0.2
to 0.6 hrl ............. ...............26.....

2-1 Adaptation of E coli ML308 to the glucose limited chemostat environment. ..................30

2-2 Overall reaction mechanism of the enzyme glutamate dehydrogenase. ................... .........39

3-1 Steady state biomass yield of E coli grown on glucose minimal media. ..........................42

3-2 Steady state carbon dioxide evolution rate of E coli grown on glucose minimal
m edia ................. ...............43.................

3-3 Steady State protein dry weight fraction of E coli grown on glucose minimal media .....44

3-4 Steady state RNA dry weight fraction of E coli grown on glucose minimal media.........45

3-5 Initial growth rate response of E coli grown on glucose minimal media. ........................47










3-6 Initial uptake rate response of E cobi grown on glucose minimal media. .........................47

4-1 Steady state carbon mass fraction of dry weight measured in E. cobi grown on
glucose minimal media. ............. ...............52.....

4-2 Results of a continuous to batch shift of a bioreactor growing E. cobi continuously
precultured at D=0. 1 hrl ............. ...............54.....

4-3 Results of a continuous to batch shift of a bioreactor growing E. coli continuously
precultured at D=0.3 hr-1 ................ ...............55......_.._...

4-4 Results of a continuous to batch shift of a bioreactor growing E. coli continuously
precultured at D=0.6 hr-1.............. ...............55..

4-5 Comparison of the cell density evolution of the three preculture dilution rates. ...............56

4-6 Comparison of the specific CO2 CVOlution rate of the three preculture dilution rates.......56

4-7 A comparison of cellular carbon utilization pattern during steady state and transient
grow th. ............. ...............58.....

5-1 Dilution rate shift up from the literature tracking growth rate and RNA level transient
response............... ...............62

5-2 Data from the literature showing that amino acids limit the growth of carbon limited
cells exposed to excess glucose. ............. ...............63.....

5-3 Data obtained from a continuous to batch shift of glucose limited E. cobi precultured
at D=0.3 hrl to excess glucose. ............. ...............66.....

5-4 Data from Figure 5-3 converted to a per liter basis and scaled by their steady state
value for the purpose of trend comparison. ................ ...............67........... ..

5-5 Oscillations present in growth rate of glucose limited cells exposed to excess
glucose. ............. ...............68.....









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DYNAMICS OF MICROBIAL GROWTH IN SINGLE SUB STATE CULTURE

By

Jason Noel

August 2007

Chair: Spyros A. Svoronos
Major: Chemical Engineering

In this study the results of continuous to batch mode experiments conducted to

characterize the initial transient response of carbon limited microbial cells growing in a

chemostat to a pulse of the growth limiting substrate are presented. Changes in cellular rates of

growth, substrate uptake, carbon dioxide evolution, and organic carbon excretion were recorded

for three different preculture conditions. To ensure the validity of the data, ninety percent of

added carbon was accounted for in generated biomass, evolved carbon dioxide, and in excreted

organic products before and after the substrate pulse.

The continuous shifts revealed that values of growth and respiration were proportional to

the preculture dilution rate while the capacity to increase the respiration and growth rate was

inversely proportional to the preculture dilution rate. Saturation of respiration and biosynthetic

capacity led to a high amount of excretion at the intermediate and highest preculture dilution

rates tested. Only the highest preculture dilution rate was able to utilize the excreted carbon

before the specific carbon dioxide evolution rate fell considerably due to lack of substrate.

The identity of the biosynthetic growth limitation was also explored in this study using

similar continuous to batch shift experiments. Evidence was found to support the hypothesis that

the biosynthetic limitation is an amino acid supply limitation and not a protein production










capacity limitation. Continuous to batch mode shifts followed by a pulse of growth limiting

carbon substrate show the transient response of the biosynthetic enzyme glutamate

dehydrogenase qualitatively matched the transient response of the intracellular RNA

concentration suggesting the enzyme was more likely to be the cause of the biosynthetic growth

limitation rather than the intracellular ribosome concentration.










CHAPTER 1
GENERAL INTRODUCTION

1.1 Motivation for Research

The chemostat is the best laboratory approximation to natural water bodies and industrial

bioreactors. Naturally the transient response of a chemostat is a problem of significant biological

and engineering interest. The simplest dynamics are obtained when a single microbial species is

grown on a single growth limiting substrate. The dynamics are complex despite the simplicity of

the system. The complexity arises from the ability of cells to adapt to changes in the environment

around them. Certain intracellular components adapt to this change on a slow time scale of hours

and even days. An example of the effects of this slow adaptation to environmental change can be

seen in Figure 1-1.


81 14

71 12

3 6 -10
8

3;/ C (monod fit)
d -e- C "(experimental)- 4 1
2 S (monod fit) ti
1 -A-S (experimental) -C 2
0 -"4~rff 0
0 5 10 15 20 25
Time (hr)


Dilution rate shift up from D=0.004 to 0.240 hrl of continuously grown K.
aerogenes. [Reprinted without permission. Phipps, D. W. T. a. P. J. 1967.
Studies on the growth of Aerobacter aerogenes at low dilution rates in a
chemostat. Microbial Physiology and Continuous Culture. Her Maj esty's
Stationary Office:240-253. (Pages 360, Figure 2).] A least squares Monod Model
(including death rate) fit of the cell density data and substrate concentration has
been added. The error in the predicted cell density and actual cell density was
minimized with non-zero parameter value constraints and a upper limit of 0.8 for
the yield. The Monod Model predicts the decrease in cell density but does so with
unrealistic parameters values and a large amount of error in the reactor substrate
concentration. Clearly better models are needed to predict these kinds of
transients.


Figure 1-1.










Upon an increase in the flow rate of feed in to the chemostat there is a pronounced

transient decrease in the cell density and increase in the glucose concentration that ends fifteen

hours after the dilution rate shift up. Transients such as these can lead to product deterioration in

industrial bioreactors and regulatory violations in waste water treatment facilities as such

systems can be disturbed with perturbations to their feed flow rate. As seen in Figure 1-1 the

estimated cell density time evolution based on the well known Monod Model does a poor j ob of

predicting the transient response in microbial cells associated with an increase in the feed flow

rate in to the chemostat. Better model based control of such systems would be necessary to

lessen the impact of such transients in industrial systems.

Based on a review of the experimental literature, conducted by Dr. Atul Narang, two slow

variables were hypothesized to control the dynamics of the chemostat: transport enzyme levels

and biosynthetic capacity. Under certain operating conditions, the dynamics of the chemostat are

controlled by only one of these slow variables. The goals of this research were two fold. The first

was to characterize how microbial cells grown at a range of carbon limited growth rates respond

to environmental conditions brought about by a feed rate increase perturbation. The second goal

was to identify the intracellular cause of this biosynthetic limitation.

1.2 Conceptual Model of the Cell

Figure 1-2 is the conceptual model of growth used to design the experiments completed in

this work. The dashed line corresponds to the boundary between the external and internal

environments of a cell. Carbonaceous substrate, denoted S, enters the cell through transport

enzymes, denoted E. Typically microbial cells will modify sequestered substrate to an

internalized form denoted by X. There is possible positive feedback as internalized substrate can

induce the synthesis of more of the transport enzyme responsible for the uptake of the substrate

as seen with the lac operon. The internalized substrate is converted to a pool of precursor










catabolites denoted by P. There is negative feedback inhibition possible here as precursor

catabolite excess could inhibit the uptake of more substrate. The precursor catabolites typically

have three possible fates. These products of substrate catabolism can be either channeled into

additional cell mass, fully oxidized to carbon dioxide, or excreted as a partially catabolized

organic molecule. Storage of internalized carbohydrates such as glycogen is denoted by Ps and

excretion of partially catabolized substrate is denoted by Px. Synthesis of protein is denoted by

C-. The designations GDH, na, and RNA stand for glutamate dehydrogenase, amino acids, and

RNA, respectively.




(biosythertc product@
S(storage abohydrate) -
Ps R
GDH (sbosome3






CO
(exeseted COd
Px
(exczeted product)


Figure 1-2. Conceptual model of a microbial cell.

1.3 Steady state behavior of microbial cells

For a given microbial species, growth limiting substrate, media composition, pH and

temperature the steady states of a chemostat depend on two parameters: the substrate feed

concentration and the residual substrate concentration of the reactor bulk. If the substrate feed

concentration is increased at a fixed dilution rate, there is no change in the steady state substrate

concentration. An increase in the feed concentration is compensated by a proportional increase in

the cell density (41). Since the cellular steady state is completely determined by the state of the

environment, the steady state values of the cellular variables must also be independent of the









feed concentration. Variation of the steady states with respect to the dilution rate produces more

complex results. Located in Figure 1-3, Figure 1-4, Figure 1-5, Figure 1-6, and Figure 1-7 are

experimental data from the literature illustrating the dilution rate dependence of some common

cellular variables in a carbon limited chemostat.

1.4 Cell density and Substrate Concentration

Cells wash out at both small and large dilution rates. The lower washout dilution rate,

called the minimum growth rate, has been argued to be caused by the specific uptake of glucose

not being able to meet the minimum maintenance requirement of the cells. Tempest et al. found

steady states of glycerol-limited cultures of K. aerogenes at dilution rates as low as 0.004 hr- ,

and found that the specific growth rate of the viable cells approached a minimum value of 0.009

hr- (33).

The cell density passes through a maximum between the two washout dilution rates and

since the steady state substrate concentration equals the substrate feed concentration at dilution

rates above and below the two washout dilution rates the substrate concentration would pass

through a minimum value. The data from Figure 1-4 shows the substrate concentration appears

as an increasing function of the dilution rate.













-0 Biomass Yield


rn0.5-



E 0.1

S0.0
u,0.0


0.2 0.4 0.6
Dilution Rate (1/hr)


0.8 1.0


Figure 1-3.


Example of dilution rate dependence of biomass yield of microorganisms. The
biomass yield data was from K. aerogenes grown on minimal media. [Reprinted
without permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of
Aerobacter aerogenes at low dilution rates in a chemostat. Microbial Physiology
and Continuous Culture. Her Majesty's Stationary Office:240-253.1965 (Pages
361, Figure 3), Microbiological Research Establishment, Porton, Salisbury,
Wilks, UK.]


3 2.0

S1.6

1.2

g 0.8



(30.0


|o Glucose Level


0.0 0.2 0.4 0.6 0.8 1 .0
Dilution Rate (1/hr)


Figure 1-4.


Example of dilution rate dependence of the steady state glucose concentration.
The glucose data was from E. coli ML308 grown on minimal media. [Reprinted
without permission. Senn, H., U. Lendenmann, M. Snozzi, G. Hamer, and T.
Egli. 1994. The growth of Escherichia coli in glucose-limited chemostat cultures:
a re-examination of the kinetics. Biochim Biophys Acta 1201:424-36. (Page 428,
Table 2). EAWAG, Dubendorf, Switzerland.]












|+RNA Fraction -0-Protein Fraction
0.20

S0.16

S0.12 -


S0.08 -

S0.04 -

0.00 ,I,
0.0 0.2 0.4 0.6
Dilution Rate (1/hr)


0.8

0.6
o
0.4 E

0.2

0.0


Figure 1-5.


Examples of dilution rate dependence of the cellular RNA and protein content of
microorganisms. Both sets of data were from A. aerogenes grown on minimal
media. [Reprinted without permission. Phipps, D. W. T. a. P. J. 1967. Studies on
the growth of Aerobacter aerogenes at low dilution rates in a chemostat.
Microbial Physiology and Continuous Culture. Her Maj esty's Stationary
Office:240-253. (Pages 361, Figure 3).]


|+CO2 Evolution Rate


0.0 0.2 0.4
Dilution Rate (1/hr)


0.6 0.8


Figure 1-6.


Example of dilution rate dependence of the carbon dioxide evolution rate of
microorganisms. The carbon dioxide evolution rate data was from E. coli K-12
grown on minimal media. [Reprinted without permission. Han, K~, H. C. Lim,
and J. Hong. 1992. Acetic-Acid Formation in Escherichia-Coli Fermentation.
Biotechnology and Bioengineering 39:663-671. (Page 666, Figure 2).]












-aGDH Activity
0.6-

$0.5-


E 0.3-

o 0.2-
cu 0.1-
0.0
0.0 0.2 0.4 0.6 0.8
Dilution Rate (1/hr)


Figure 1-7. Example of dilution rate dependence of the glutamate dehydrogenase activity of
microorganisms. The glutamate dehydrogenase activity was from E. coli W
grown on minimal media. [Reprinted without permission. Senior, P. J. 1975.
Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes:
studies with the continuous-culture technique. J Bacteriol 123:407-18. (Page 413,
Figure 5).]

1.5 Cellular Content and Excreted Metabolites

RNA and protein are the maj or cellular constituents comprising 80-85% of the dry weight

of the cell. As the dilution rate increases, the RNA concentration increases at the expense of the

protein concentration. Carbohydrate concentration varies markedly with the identity of the

microbial species and the carbon source. For a given microbial species and carbon source, the

carbohydrate content is almost independent of the dilution rate (29). Excreted metabolite

concentrations also vary significantly depending on identify of the microbial species and the

carbon source. Glycerol limited cultures of K. aerogenes show no measurable excretion of

acetate at all dilution rates between 0.004 and 0.85 hrl (33). In contrast glucose and pyruvate

limited cultures of E. coli show a high degree of excretion at high dilution rates (1 1, 16, 1 7).

1.6 Transients Controlled by the Transport Enzymes

The role of transport enzymes in chemostat dynamics is seen by switching the growth

limiting substrate identity in the reactor feed. In this experiment a chemostat is allowed to reach











a steady state at some dilution rate and substrate feed concentration. After steady state is reached


the growth limiting substrate is abruptly switched. If the transport enzymes for the new substrate

are synthesized inducibly, their levels will be small when the switch occurs. Located in Figure 1-

8 is an example of a transport enzyme controlled transient taken from the literature.


-o NTA Level -0 Cell Density
Washout Curve Trans. Enzyme Activity
1.41 1.0
S1.2 r
4 0.8 E


S0.6- 0.4

0.4 t
-0.2
0.2

0 20 40 60
Time (hr)


Figure 1-8. The results of glucose to nitrilotriacetate substrate switch experiment performed
on a continuous culture of Chelatobacter heintzii grown on minimal media.
[Reprinted without permission. Bally, M., and T. Egli. 1996. Dynamics of
Substrate Consumption and Enzyme Synthesis in Chelatobacter heintzii during
Growth in Carbon-Limited Continuous Culture with Different Mixtures of
Glucose and Nitrilotriacetate. Appl Environ Microbiol 62:133-140. (Page 135,
Figure 2).] A theoretical washout curve was added to illustrate the predicted cell
density change assuming no cell growth.

When the carbon source is switched from glucose to nitrilotriacetate (NTA) there is little to


no growth for nearly twenty hours as shown by the cell density matching the theoretical washout

curve during this time. The cells simply cannot take up the NTA present in the environment as

shown by the lack ofNTA transport enzyme activity seen during the first twenty hours. This

initial lack of transport enzyme causes the substrate NTA to accumulate in the reactor until the

transport enzyme reaches a level that can support growth. The transport enzyme for NTA is

produced at extremely low rates under glucose limited conditions hence the low rate of transport










enzyme activity seen at the start of the experiment. The transport enzyme level is nearly zero for

twenty hours and reaches fifty percent of its final value in the following twenty hours.

The rapid buildup of transport enzyme after forty hours suggests the synthesis of the

enzyme is autocatalytic. Evidence from molecular biology shows that enzyme induction is not

only autocatalytic, but also cooperative, since inhibition of the repressor requires that two

inducer molecules to bind to it in the case of the lac operon repressor (46). Switching the growth

limiting substrate from NTA to glucose produces a different transient behavior. Rapid

consumption of glucose begins immediately after the switch occurs because glucose transport

enzymes are constitutively produced keeping these transport enzymes at high levels even in the

absence of glucose. Still little to no growth was seen for the first two to three hours of the switch

from NTA to glucose (2). An appropriate question might be what is preventing the specific

growth rate from adjusting instantaneously as the cell is taking up substrate yet not growing?

Similar results were found when continuous cultures ofE. coli (43) and K. aerogenes (36)

were subj ected to a substrate switch from glucose to xylose. For many hours there was a

dramatic increase in the level of xylose and a pronounced decline in the cell density. These

experiments imply that when the initial level of the inducible transport enzyme is low, the

transient behavior is controlled by the transport enzyme synthesis rate. There is little substrate

uptake, hence little growth, until the enzyme level reaches a sufficiently high level.

1.7 Transient Dynamics Controlled by a Biosynthetic Limitation

Continuous to batch shifts of glucose limited cells reveal the role of biosynthesis in

limiting microbial growth. In the experiments shown in Figure 1-9 and Figure 1-10, a sample of

glucose limited cells precultured in a chemostat are immediately exposed to supersaturating

concentrations of glucose in a batch reactor and the initial rates of various processes were

measured. As seen in Figure 1-9 and 1-10, the growth rate and substrate uptake rate both increase











upon exposure to excess glucose regardless of the preculture dilution rate at which the cells had

been growing (22). A similar response has been seen in other studies as well (28, 32). This rapid

increase occurs because the transport enzymes for glucose are constitutive so they are always

present at high levels. The data from the figures shows the substrate uptake rate increases to a

maximal level regardless of preculture dilution rate whereas the growth rate increase is highly

dependant on the preculture dilution rate. Appropriate questions here would be what is limiting

the growth rate as the cell has excess substrate available and how is the cell utilizing excess

substrate if it is not being channeled in to growth?


+ SS Growth Rate +~ Trans Growth Rate


0.8


S0.6


S0.4


(30.2

0.0


0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Dilution Rate (1/hr)


Results of a continuous to batch shift where cells of E coli ML308 grown in a
glucose limited chemostat on minimal media were exposed to excess glucose. The
transient growth rates were measured after thirty minutes of growth. [Reprinted
without permission. Lendenmann Phd dissertation (Page 72, Figure 8.1),
EAWAG, Dubendorf, Switzerland.]


Figure 1-9.












2.0" -SS Uptake Rate +~Trans Uptake Rate









0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Dilution Rate (1/hr)


Figure 1-10. Results of a continuous to batch shift where cells of E coli ML308 grown
in a glucose limited chemostat on minimal media were exposed to excess
glucose. The transient substrate uptake rates were measured during the
first 5-7 minutes after the glucose pulse was administered. [Reprinted
without permission. Lendenmann Phd dissertation (Page 72, Figure 8.1),
EAWAG, Dubendorf, Switzerland.]

The specific biosynthesis rate (of RNA and protein) depends on the dilution rate at which

the cells are growing before their withdrawal from the chemostat (18). This was shown in

experiments where glycogenless mutants of E coli B were exposed to saturating concentrations

of glucose and the initial specific growth rate were measured. Since these mutants were unable to

synthesize their maj or storage carbohydrate glycogen, the observed specific growth rate was

roughly equal to the specific biosynthesis rate of only RNA and protein. Based on the

measurements of the initial growth rates in these mutants it was concluded that at low dilution

rates (D<0.3 hr ) the specific biosynthesis rate of microbes increases rapidly but does not reach

the maximum value for the strain and for high dilution rates the specific biosynthesis rate does

not improve at all immediately.

It stands to reason that in wild type cells there is an absence of rapid improvement in

RNA and protein synthesis at the higher dilution rates. When wild type cells of E coli drawn

from a chemostat are exposed to saturating concentrations of glucose the specific growth rate,









increases rapidly regardless of the dilution rate at which the cells have been growing implying

that the increase is solely due to glycogen synthesis.

The synthesis of glycogen in wild type cells is not surprising. The above experiments

imply that when cells growing at large dilution rates are exposed to saturating concentrations of

glucose, the substrate instantly enters the cells, but cellular metabolites derived from it cannot be

completely channeled in to biosynthesis of RNA and protein. A large portion must therefore be

respired, excreted, or stored. The specific carbon dioxide evolution rate rapidly increases to

maximal levels (levels seen at near washout dilution rates) (18, 30). The specific excretion rate

(10, 12) and, if applicable, the specific glycogen synthesis rate (10, 19) increase to high levels

when exposed to saturating glucose conditions. These experiments imply that if the initial level

of transport enzyme is high, the transients are controlled by biosynthesis rather then by a

transport limitation. There is a pronounced substrate uptake, respiration, storage, and excretion

but limited or no biosynthesis until more biosynthetic capacity has been synthesized.

So what prevents the biosynthesis rate from instantly increasing to maximal levels upon

exposure to excess substrate? There are two possibilities: either one or more of the biosynthetic

enzymes is saturated with their substrate(s) or the ribosomes are saturated with amino acids.

Substrate saturation of a biosynthetic enzyme whose products) are required for growth would

represent a rate limiting step. Biochemical pathways necessary for growth that require the

products of the saturated biosynthetic enzyme will be forced to adjust their rate of reaction based

on the rate of supply of products from the saturated biosynthetic enzyme. Similarly ribosomes

saturated with their amino acid substrates would also limit growth as ribosomes generate the

proteins that comprise the maj ority of cell mass. It is generally believed that the ribosome

concentration is what limits biosynthesis (5, 31); however, there is evidence to suggest that










saturation of biosynthetic enzymes, rather than ribosome concentration, prevents the biosynthesis

rate from instantly increasing to the maximum level. It has been seen that the addition of amino

acids to a culture results in the rapid acceleration of protein synthesis (6, 20, 24) which implies

that the ribosomes are in fact not saturated with amino acids. This would suggest the limitation

lies with the supply of amino acids to the ribosomes and not the ribosomes themselves.

A possible explanation for this response to increased protein synthesis upon exposure to

excess amino acids could be the substrate saturation of the biosynthetic enzyme glutamate

dehydrogenase (GDH). Under carbon limited conditions this enzyme is the maj or pathway for

incorporation of inorganic nitrogen in to the cell (26, 40) and could be a limiting step in the rate

of amino acid synthesis explaining why the ribosomes are not saturated. Figure 1-11 and Figure

1-12 show the transient response of RNA level and GDH activity, respectively in continuously

grown microbial cells exposed to excess substrate.

+ D=0.10 + D=0.15 -&- D=0.20
0.40-


0.30-


S0.20-


0.10-


0.00
0 5 10 15 20
Time (hr)


Figure 1-11. The RNA content response of continuously grown Azobacter vinelan2dii to
substrate excess conditions. The bacteria were precultured on minimal media at
three different dilution rates: 0.10, 0.15, and 0.20 hr [Reprinted without
permission. Nagai, S., Y., Nishizawa, I. Endo, and S. Aiba. 1968. Response of a
chemostatic culture of Azobacter vinelandii to a delta type pulse of glucose. J.
Gen. Appl. Microbiol. 14:121-134. (Page 125, Figure 2).]













cn 0.06-





I 0.04-


0.03
0 50 100 150 200
Time (min)


Figure 1-12. The GDH activity response of continuously grown E. coli W to substrate excess
conditions. The bacteria were precultured on minimal media. [Reprinted without
permission. Harvey, R. J. 1970. Metabolic regulation in glucose-limited
chemostat cultures of Escherichia coli. J Bacteriol 104:698-706. (Page 704,
Figure 10).]

The slow accumulation of RNA (18, 27, 48) and GDH (18) after a continuous to batch

shift follows sigmoidal kinetics. This suggests that the synthesis of GDH and RNA is

autocatalytic. Such kinetics probably occur because an increase in the activity of GDH increases

the level of amino acid monomers. This stimulates the production of RNA and ribosomes

resulting in the synthesis of even more GDH and RNA.

1.8 Transient Response to Dilution Rate Shift Ups

In practice, chemostats regularly experience dilution rate and feed concentration shift ups.

Laboratory studies have mostly been concerned almost exclusively with dilution rate shift ups. In

these experiments the chemostat is allowed to reach steady state at some dilution rate and then is

abruptly increased. There is substantial literature on this topic (1, 4, 9, 13, 33, 42, 43, 48), but the

cellular variables were measured in relatively few studies (4, 33, 48). The cellular studies suggest

that when dilution rate shift ups are small, the transients are limited by the biosynthetic

machinery, and when the shift ups are large the transients are limited by transport enzymes.










Located in Figure 1-13 is the response of a glucose limited culture growing inside a chemostat to

a large dilution rate shift up.

Herbert exposed a glycerol-limited culture of K. aerogenes to a dilution rate shift up from

D=0.004 to D=0.24 hr- which corresponds to a sixty fold increase in the substrate input rate.

The transient response suggests that in the first few hours, there was a significant increase in the

transport enzyme level. The initial rapid jump in the substrate uptake rate is due to saturation of

the substrate uptake enzymes and the relatively slow increase after the initial jump is due to the

gradual buildup of transport enzyme level because the specific substrate uptake rate is

proportional to the transport enzyme level whenever the substrate concentration is saturating.

The data suggests that the transport enzyme levels reach a maximum fiye hours after the shift up.

During this time the RNA level is nearly constant. It increases significantly after the transport

enzymes have peaked, and reaches the maximum more then ten hours after buildup (19).


|+ Cell Density + Glucose +~ Ribosome Content
2.01 0.40

1.8- -0.36

8 1.6 -I -C 0.32

~1.4--- 0.28

1.2 ---0.24

1.0 1 0.20
0 5 10 15 20
Time (hr)


Figure 1-13. Response of a chemostat growing E. coli K-12 to a dilution rate shift up from
D=0.2 to 0.6 hr- Minimal media was used during the experiment. [Reprinted
without permission. Yun, H. S., J. Hong, and H. C. Lim. 1996. Regulation of
ribosome synthesis in Escherichia coli: Effects of temperature and dilution rate
changes. Biotechnology and Bioengineering 52:615-624. (Page 619, Figures 4
and 5).]









CHAPTER 2
MATERIALS AND METHODS

2.1 Growth Medium

The growth medium was prepared with deionized water in 20 L polypropylene bottles. The

medium used for these experiments was a modification of a recipe used by Lendenmann et al

(22). The recipe is shown in Table 2-1. Before sterilizing the medium, it was supplemented with

glucose, and the pH was adjusted to 3 with concentrated H2SO4. The low pH prevents

caramelization of the glucose during sterilization and back-contamination of the feed line during

operation of the chemostat.



Table 2-1. Minimal medium recipe used for all experimentation.
Component Conc. (M) 1L (g)
Potassium Phosphate (mono) 2.00E-02 2.7218
Ammonium Chloride 1.40E-02 0.7489
EDTA 2.20E-04 0.0819
Magnesium Sulfate Heptahydrate 2.3 0E-04 0.0567
Sodium Molybdate Dihydrate 1.00E-05 0.0024
Calcium Chloride Dihydrate ** 1.00E-04 0.0147
Manganese Chloride Tetrahydrate 2.50E-05 0.0049
Zinc Chloride 1.25E-05 0.0017
Cupric Chloride Dihydrate 5.00E-06 0.0009
Cobalt Chloride Hexahydrate 5.00E-06 0.0012
Ferric Chloride Hexahydrate 7.50E-06 0.0020
Note: Original source was Lendenmann 1995. PhD dissertation (Page 35, Table 4.1)
Note: Adjusted reactor (not shake flask) media to pH 3 with reagent grade sulfuric acid
Note: Replaced calcium carbonate with calcium chloride due to carbonate / acid reaction
Note: Removed boric acid from media because borate adheres to the HPLC column


2.2 Bacterial Strain

The microorganism used in this study was Escherichia coli ML308 (ATCC 15224)

obtained from the American Type Culture collection. Stock cultures were prepared by freezing

cells grown on complex media that had just entered stationary phase growth. The strain was

preserved at -20C in 30 ml vials containing 50% of the culture, 50% glycerol cryoprotectant, and









glass beads. The culture was resuscitated for experimental work by sterilely removing a glass

bead from the stock culture and adding it to a shake flask containing the growth medium

described below supplemented with 1 g/L glucose (35).

2.3 The Chemostat Setup

The chemostat cultures were grown in a 1.5 L Bioflow III fermenter (New Brunswick

Scientific Co.) with a working volume of 1.2 L. The agitation speed was 1000 rpm and the

aeration rate was 1.2 L/min. The bioreactor was equipped with automatic pH and temperature

control. The pH was maintained at 7.0 +/- 0.1 by addition of IM KOH / IM NaOH solution and

10% H3PO4 SOlutions. The temperature was maintained at 370C. The feed was pumped in by a

Masterfiex L/S peristaltic pump (7523-70) equipped with a Masterfiex EZ Load pump head

(7534-04).

2.3.1 Wall Growth Limitation

The glucose concentration in the reactor feed was kept at 200 mg/L or below. Higher feed

concentrations resulted in significant wall growth within a week of inoculating the reactor. Under

higher feed conditions, the initial specific growth rates after continuous mode to batch mode

shifts were observed to be significantly lower than the dilution rate at which the cells had been

growing in the chemostat. This probably reflects the fact that in the presence of wall growth, the

steady state specific growth rate is lower than the dilution rate, since cells shearing off the wall

become an additional source of cells inside the bioreactor (21). The reason for this lowering of

the growth rate due to wall growth can easily be seen in the steady state cell mass balance, seen

in Equation 2-1, with an extra cell source term a representing the shearing off of wall bioailm

cells.

D*C = rg* C +a (2-1)









The variables D, C, rg, and a are the reactor dilution rate, the cell density of the reactor volume,

the growth rate of the cells in the bulk, and the rate of cell input in to the bulk from wall growth,

respectively. If the value of a is non-zero then rg must be depressed to keep the chemostat

operating at steady state. At feed concentrations of 100 mg/L of glucose, the reactor could be

operated for up to month without significant wall growth.

2.3.2 Glucose Adaptation Limitation

The glucose concentration is one of the last variables to achieve a steady value inside of a

glucose limited chemostat. The time scale of this transient is on the order of days suggesting a

genetic adaptation to low glucose levels. This makes sense from a natural selection point of view

as those cells which possess a higher capacity for uptake for the growth limiting nutrient would

grow faster then cells with a lower capacity for uptake in the chemostat environment. Eventually

the better adapted mutant cells would out compete and take over the reactor bulk given enough

time. One such study found in the literature supported this idea (45). A deregulation mutation

was found inside glucose limited E. coli cells grown in a chemostat that affected the transcription

of a cell membrane glucose porin. Given enough time and selective pressure the cells would start

producing additional glucose porin proteins that are normally not expressed under glucose

limited conditions (45). These additional porins would facilitate glucose transport in to the cell

and could confer a selective advantage over non-adapted cells.

A glucose adaptation experiment was conducted using the laboratory chemostat to

characterize this transient in the laboratory's chemostat. Daily samples were taken from a

glucose limited chemostat post a shift to D=0.6 hr The glucose concentration was measured

and compared with a similar study found in the literature using the same strain of bacteria and a

similar media composition (22). The results of this comparison are shown in Figure 2-1.










Both glucose evoluion trend lines look extremely similar with a monotonic decrease in

the concentration of glucose down to the same steady value. The time it took the lab system to

achieve a steady glucose concentration was four days at D=0.6 hr- or 85 cell doublings. The

experiments conducted in this research were sensitive to the glucose uptake capacity thus this

transient behavior introduced variation into collected data. This reproducibility problem was

corrected by giving newly inoculated reactors four days time at D=0.6 hr- before any

experiments were attempted to allow for microbial adaptation to low glucose levels. This

operational change increased the quality of the experimental data gathered immensely.



1600 -0 Lendenmann 1994 + Narang Lab


;- 1 200-

S800-

.= 400-


0 20 40 60 80 100 120
Volume Changes since shift to D=0.6


Figure 2-1 Adaptation of E coli ML308 to the glucose limited chemostat environment. The
cultivation used minimal media and a glucose concentration of 100 mg/L.
Substrate measurements were made daily post shifting the chemostat to D=0.6 hr-
.The comparison data was measured using E. coli ML308 grown on glucose
minimal media. [Comparison data reprinted without permission. Lendenmann Phd
dissertation. (Page 44, Figure 5.1). EAWAG, Dubendorf, Switzerland.]

2.4 Cell Density Measurement and Growth Rate

The concentration of cells inside the reactor at any time was determined by an absorbance

measurement at 546 nm using a Spectronic Genesys 10UV spectrophotometer. In order to

ensure an accurate reading the 1 ml cuvette was given approximately 45 seconds to equilibrate

before the value was recorded (15). Specific growth rates of cells growing exponentially were









determined by fitting exponential curves over the time frame of interest. The fitted equation

takes the form seen in Equation 2-2.

C= Co~exp *t]t (2-2)

The variables Co, C, CI, and t are the initial cell density, final cell density, the growth rate, and

time, respectively.

2.5 Sugar Measurement, Yield Determination, and Uptake Rate

Sugar concentrations were measured with a Dionex-500 HPLC equipped with an anion

exchange column (CarboPac PA10) and a pulsed electrochemical detector. In this method, the

sugars are ionized by using a strong base as eluent (10 mM NaOH), separated based on their

differential affinity for the anion exchange column, and detected by the pulsed amperometric

detector. The manufacturer's protocol resulted in a rapid increase of retention times, presumably

due to adsorption of bicarbonate on the column. The addition of 1 mM Ba(OAc)2 to the eluent,

as recommended by (7, 8), precipitated the bicarbonate as Ba(CO3)2. The modified eluent

dramatically improved the reproducibility and precision of the laboratory' s sugar measurements.

The sugar biomass yield of a culture is simply the amount of cells produced per amount of

sugar consumed. The biomass yield was determined for a time interval during exponential

growth by plotting the cell density versus sugar concentration and using the slope of the linear

fit. The final equation for yield determination during a given time interval presented as Equation

2-3.

Y = -(dC /dS) (2-3)

The variables Y and (dC/dS) correspond to the biomass yield and the slope of the cell density

versus sugar concentration plot. This method assumes that the biomass yield is constant over the

time interval of interest. The slope of the linear fit trend line corresponds to the biomass yield









during that time interval. The specific uptake rate of sugar during the time interval was

determined by dividing the specific growth by the biomass yield. The Einal equation for the

substrate uptake rate is presented as Equation 2-4. This equation assumes that the death or decay

rate of cells is negligible for the purposes of calculating the specific substrate uptake rate.

rs =rg /Y (2-4)

The variables rs, rg, and Y are the specific uptake rate of sugar, the specific growth rate, and the

biomass yield, respectively.

2.6 Total Cell Protein Extraction and Measurement

2.6.1 Extraction

Total cell protein was extracted using a high temperature / low pH extraction method (15).

These conditions denature cellular protein and therefore the protein product could not be used for

enzymatic assays. Cells were harvested via low temperature centrifugation, washed with 0. 15M

sodium chloride sodium citrate solution (SSC), and digested with 0.5M sodium hydroxide

(NaOH) at 1000C. The harsh conditions of the digestion step solubilize the cellular protein. After

centrifugation, the supernatant was retained and an equivalent volume and molar strength of

trichloroacetic acid (TCA) was added to adjust the pH to a neutral value.

2.6.2 Measurement

Protein detection and quantification was preformed with colorimetric detection using a

bicinchchoninic acid (BCA) kit available from Pierce Biotech (34). The color change reaction

occurs from amino acid (cysteine, cystine, tryptophan, and tyrosine) catalyzed reduction of Cu2+

to Cul+. The resulting BCA-Cu complex exhibits a strong absorbance at 562 nm that shows a

near linear response with protein concentration over a broad range. Bovine serum albumin was

used as a standard to generate a calibration curve for cell protein quantification.










2.7 Total Cell RNA Extraction and Measurement


2.7.1 Extraction

Total cell RNA was extracted using a cold / hot TCA treatment (15). Cells were

harvested via low temperature centrifugation, washed with 0. 15M SSC, and digested with 0.25

TCA at OoC. These digestion conditions lyse the cell and cause cellular RNA to precipitate as a

solid. The insoluble fraction of the lysate was retained and resuspended in 0.5M TCA and placed

in a 700C water bath. The treatment solubilizes RNA but leaves other macromolecules such as

proteins and lipids in a solid form.

2.7.2 Measurement

RNA detection and quantifieation was performed with colorimetric detection using a

slightly modified acidified orcinol method (23). Solutions of ferric chloride dissolved in HCI and

orcinol dissolved in ethanol are combined in a 50/50 ratio and added to the extracted RNA

solution generated by the cold / hot TCA treatment. The RNA in the extract reacts with acidified

orcinol producing a green chromogen that exhibits a strong absorbance at 665 nm that shows a

near linear response with RNA concentration. Yeast RNA was used as a standard to generate a

calibration curve for cell RNA quantifieation.

2.8 Carbon Dioxide Evolution Measurement

The carbon dioxide evolution of the chemostat contents was measured with a Vaisala

GMT222 Infrared CO2 analyzer. The device was connected to a computer for continuous data

acquisition. This was especially useful for detecting any steady state perturbations to ensure the

initial conditions of the chemostat and bacteria growing within were acceptable before

experiments were attempted. Special software for the CO2 analyZeT WaS written to allow for this

continuous acquisition and recording. The code for this software appears in Appendix A.









To ensure a rapid analyzer response time to the state of the chemostat all effluent air from

the reactor system was passed through the CO2 analyzer to ensure a rapid analyzer response time

to changes in the chemostat condition. The air and liquid overflow outlets were the same

overflow line in the chemostat setup so an air/liquid separator was created to isolate the two

phases. The overflow line was connected to a 50 mL Erlenmeyer flask with two outlets. The air

phase was forced up through an exit line at the top of the flask to the analyzer while the liquid

phase fell to the bottom of the flask. The liquid phase was actively pumped out to a waste tank to

prevent the flask from overflowing. The setup allowed for detector response times on the order

of seconds rather then minutes to changes in the chemostat carbon dioxide level. The detector

output was recorded as a concentration of carbon dioxide in parts per million. The device came

from the manufacturer precalibrated but was checked periodically with specially made

calibration gas.

2.9 Total Organic Carbon Measurement

Total organic carbon measurements (TOC) were made with a Phoenix 8000 TOC Analyzer

to quantify the amount of carbon excreted by cells in the form of organic acids or alcohols.

Samples taken from the chemostat during an experiment contained excreted organic carbon as

well as organic carbon from residual sugars and from the chelating agent EDTA present in the

original media. The carbon contribution of the residual sugars was accounted for by first

measuring the sugar concentration in the extracted sample via HPLC measurement and then

subtracting the equivalent sugar carbon from a sample's TOC measurement. The carbon

contribution from EDTA was accounted for by measuring the TOC of the original media (before

sugar was added) and then subtracting the measured carbon from a sample's TOC. The

remaining TOC of a sample was modified forms of organic carbon because there were no other

known sources of organic carbon input in to the chemostat bulk.









The Phoenix 8000 TOC Analyzer used UV initiated peroxydisulfate free radical chemistry

to fully oxidize organic carbon to carbon dioxide (47). A nitrogen carrier gas transports the

evolved carbon dioxide to an infrared probe for detection. TOC calibration curves were

generated using a glucose standard.

2.10 Elemental Content Measurement

Elemental analysis of dry cell mass was performed on a Carlo Erba-1106 Elemental

Analyzer to determine the carbon content of cells for the purpose of conducting carbon balances

around the laboratory chemostat. Cell mass was collected and concentrated with low temperature

(4oC) centrifugation. The resulting cell pellet was washed with cold deionized water to remove

excess salts from the cell mass that could skew the elemental analysis results. The washed cell

pellet was incubated overnight at 800C to fully desiccate the cell biomass. The resulting dry mass

was pulverized to a coarse powder prior to elemental analysis.

The Carlo Erba-1 106 Elemental Analyzer determined the H, N, and C content of the dry

cell mass by using high temperature oxidation to convert organic H, N, and C to H20, NO2, and

CO2, respectively. The evolved gases were quantified and compared with the initial dry cell mass

to determine the percent content of the three elements.

2.11 Cell Disruption Techniques

In order to test the enzymatic activity of intracellular proteins, cells must be disrupted in a

way that does denature or inhibit the activity of cellular proteins. Two methods were used in this

research to extract proteins with little deviation from the original media pH, non-denaturing

temperatures, and protease inhibitors to keep enzymatic activity as close to harvest conditions as

possible.









2.11.1 Chemical Disruption

The Chemical Disruption method uses a surfactant and chicken egg white lysozyme to

disrupt cells without the need for excessive mechanical force (15). First extracted cells were

concentrated and washed using low temperature (4oC) centrifugation and 0.15M SSC. The cell

pellet was resuspended in 2 mL ice cold 50 mM Tris lysing buffer (pH=7.6) containing 100 mM

NaC1, and 1 mM EDTA. This buffer has an osmolarity and pH at a level similar to chemostat

conditions and more importantly will keep the pH constant during the extraction of prevent

denaturing of extracted proteins. The EDTA is present to scavenge any trace heavy metals that

can inactivate enzymes by adhering to active or cofactor sites on the protein. The temperature of

the disruption vial was kept at OoC for the duration of the disruption procedure using an ice-water

bath. A small volume (20 Cl) of surfactant (10% NP-40) was then added to the suspension and

vortexed vigorously (30 seconds) to permeabilize the outer membrane of the bacterial cells. The

cell suspension was again centrifuged (4oC) and resuspended in 0. 11 ml of Tris lysing buffer. A

small volume (20 Cl~) of sucrose solution (1.6M) was added to the disruption vial. The vial was

vortexed and placed in the ice-water bath for 20 minutes. After the sucrose equilibration a small

volume (43 Cl) of chicken egg white lysozyme suspension (10 mg/mL) was added to the

disruption vial. The vial was vortexed and placed in the ice-water bath for 20 minutes. The

chicken egg white lysozyme weakens bacterial cells by degrading the peptidoglycan present in

their cell walls. The sucrose added to the cell suspension prior to the lysozyme treatment helps

force the lysozyme into the cell membrane holes made by the initial surfactant treatment. After

the lysozyme digestion, cold deionized water (0.535 mL) was added to the cell suspension and

vortexed. Next more of the surfactant, 10% NP-40 (0. 179 mL), was then added to the disruption

vial. Upon vortexing the suspension clears and the non-soluble components of the cell are









removed with low temperature (4oC) centrifugation. The supernatant was saved and contained

the solubilized cell proteins in a non-denatured state (3 8).

2.11.1 Chemical Disruption Drawbacks

The supernatant product from this technique is extremely viscous due to nucleic acid

entanglement of liberated DNA. The viscosity of the supernatant made extracting accurate

volumes with a pipette extremely difficult. Literature sources recommended sheering the crude

lysate by drawing it through a syringe but this proved to be a less then ideal solution (15). The

enzyme DNAse was recommended as an alternate remedy for this problem but was never

implemented as a solution as there was a larger problem with the chemical disruption technique.

Of the dilution rates tested, significant cell lysis only occurred for dilution rates of 0.3 hr-

or greater. When the chemical lysis technique was attempted with cells growing at D=0. 1 hr- ,

little to no cell lysis was observed. The reason for this resistance to chemical lysing was thought

to be due to the starvation response ofE. coli (25). As the dilution rate of a carbon limited

chemostat decreases the carbon substrate available to growing cells at steady state decreases as

seen in Figure 1-4. Depending on the degree of starvation the cell may start activating stationary

phase genes to help the organism persist in the nutrient limited environment in the hope that

conditions conducive to growth will occur in the future. Formation of an extensive glycocalyx

capsule around the starved cell is thought to be the source of resistance to chemical disruption

(25, 29). It was hypothesized that the extensive capsule created around a starved cell provides a

barrier around the cell protecting the outer membrane and cell wall from surfactant and lysozyme

attack effectively preventing chemical disruption. A more effective technique was found for the

disruption of highly starved cells.









2.11.1 Mechanical Disruption

The Mechanical Disruption method uses sheer forces generated by vigorously vortexing

cells with micron size glass beads (3, 39). The shear force generated by the rapidly moving glass

beads leads to cell disruption in even the most robust of microorganisms (3 7). First extracted

cells are concentrated and washed using low temperature (4oC) centrifugation and 0. 15M SSC.

Cells are resuspended in 0.3 mL of ice cold 50 mM Tris disruption buffer (pH=7.6) containing

100 mM NaC1, and 1 mM EDTA. Ice cold glass beads (100-200 Cpm diameter) are added to the

cell suspension on a w/v basis. The cell suspension was disrupted by vortexing it vigorously for

six minutes at -200C. This was achieved by placing a table top vortex inside the lab freezer and

modifying the device to hold disruption vials for the purpose of hands free vortexing. The

subzero temperature ensured there was no increase in temperature due to mechanical energy

input that might denature the cellular proteins. The cell suspension was diluted with 0.7 mL of

ice cold disruption buffer after the vortexing step. The non-soluble components of the cell were

removed from the disrupted cell suspension with low temperature (4oC) centrifugation. The

supernatant was saved and contained the solubilized cell proteins in a non-denatured state (44).

2.11.2 Mechanical Disruption Advantages

The mechanical disruption technique solved many problems associated with the chemical

disruption technique. Significant cell disruption was possible at all tested dilution rates using the

mechanical technique. The resulting crude lysate from the mechanical technique had a much

lower viscosity then the crude lysate from the chemical technique due simply to the shearing

action of the rapidly moving glass beads allowing for more accurate volume extraction.

Additionally the mechanical technique required the addition of nothing other then glass beads

and a protease inhibitor solution to the cells being disrupted. The addition of surfactant,

lysozyme enzyme, and other reagents in the chemical disruption technique affected the results of










the protein assay and may have had an effect on cellular enzyme activity measurements. Overall

the mechanical disruption technique was superior to the chemical disruption technique for the

experiments being conducted in the laboratory.

2.12 Enzymatic Assay of Glutamate Dehydrogenase

The enzyme glutamate dehydrogenase (GDH) is the protein responsible for the maj ority of

inorganic nitrogen incorporation in E. coli under carbon limited conditions (26). Specifically the

enzyme converts the TCA cycle intermediate a-ketoglutarate to the amino acid glutamate. The

exact reaction can be seen in Figure 2-2.




COOH COOH

C =O + NADPH + H+ + NH5 4-GDHS H2N-C-H + NADP + fHzO

CHz CH,

COOH CHz

COOH


Figure 2-2 Overall reaction mechanism of the enzyme glutamate dehydrogenase. The
reaction can proceed in the forward or reverse reaction depending on the
concentrations of the reagents and products.

The enzymatic activity of GDH in the crude lysate extracted with the chemical and

mechanical disruption methods was measured by recording the consumption of NADPH (340

nm) over 15 minutes using a Beckman DU7500 spectrometer with all reagents in excess. The

enzymatic assay was performed in 1 mL cuvettes at a temperature of 250C and a pH of 7.6. The

contents of the 1 mL reaction volume are shown below in Table 2-2. All reagents were made in

50 mM Tris Buffer and adjusted to pH=7.6 with 1 M HCI and 1 M NaOH.









Table 2-2. Volumes and concentrations of reagents used in the GDH assay.
Vol. (mL) Reagent (pH=7.6)
0.6 50 mM Tris Buffer
0.1 50 mM alpha-ketoglutarate
0.1 2.5 mM NADPH
0.1 40 mM NH4C
0.1 Lysate from Cell Disruption


The crude lysate showed background NADPH activity that could not be attributed to the

GDH activity of the sample. Control trials, where all reagents were present except NH4C1, WeTO

run alongside experimental trials to account for this excess NADPH consumption activity. The

net rate of NADPH consumption was used to calculate the activity of the protein samples. The

activity was expressed in micromoles of NADPH consumed per minute per milligram of protein.

2.13 Deactivation and Disposal of Microorganisms

By requirement of the Environmental Health and Safety Department of the University of

Florida all used media and cellular material had to be deactivated before disposal. Reactor

effluent was collected in a 50 gallon waste tank and subj ected to overnight chlorination before

being drained in to the building water disposal system. All other cellular material was autoclaved

for 15 minutes at 121oC before being disposed. Any accidental spills or contamination were first

deactivated with a 50% v/v ethanol/water solution before cleaning (14).









CHAPTER 3
PROTOCOL VALIDATION AND SYSTEM CHARACTERIZATION

3.1 Goals of Verification and Characterization

Before novel experiments were attempted the laboratory model system and measurements

were compared with similar literature data to give credibility to the laboratory results. The steady

state of a microbial chemostat provides a reproducible initial condition for the cells growing

inside the reactor bulk so data collected at different reactor dilution rates was used as the basis

for comparison. Collecting this steady state data also was done to characterize the initial

conditions of chemostat grown cells for the purposes of future experiments. The cellular

variables quantified at a range of dilution rates were the biomass yield, the protein weight

percent, the RNA weight percent, the carbon dioxide evolution rate, and the initial growth and

substrate uptakes rates upon exposure of the cells to excess glucose. The maximum growth rate

of E coli ML308 on the laboratory medium was also measured during the characterization

experiments. Data from the literature that was used for comparison purposes was from systems

with similar operating conditions to those of the laboratory.

3.2 Steady State Biomass Yield

The biomass yield on glucose was measured at different dilution rates and compared with

data obtained using the same strain and similar media compositions (21). The two sets of data

show comparable yields for matching dilution rates. The biomass yield results and comparison

data appear in Figure 3-1. For the lower dilution rates (0.03-0.1 hr ) there is a sharp decrease in

the yield as the dilution rate approaches zero. This trend makes sense assuming microbial cells

have a minimum energy requirement for maintaining themselves. As the dilution rate decreases

the energy input to the microbial cells will also decrease leaving less energy available to the cell

for growth. If the energy maintenance requirement of the cell remains constant with decreasing










dilution rate then the yield must decrease as a larger percentage of consumed substrate must be

used for maintenance. At the extreme case of the low washout dilution rate the cells are utilizing

all consumed substrate for maintenance energy causing the yield at this point to be zero as the

cells at this condition are not generating cell mass.

At intermediate dilution (0. 1-0.6 hr- ) rates the biomass yield keeps a relatively steady

value. The maximum biomass yield occurred at a D=0.3 hr- Beyond D=0.6 hr- the biomass

yield started decreasing markedly. This trend makes sense as a chemostat will eventually

washout at sufficiently high dilution rates due to the inability of cells to grow at a rate matching

or exceeding the dilution rate.


*E. coli ML308 SS Yield Lendenmann 1995
0.6 -0 E. coli ML308 SS Yield Narang Lab

0.5-

J 0.4-


j; 0.2-

0.1-

0.0
0.0 0.2 0.4 0.6 0.8
Dilution Rate (1/hr)


Figure 3-1. Steady state biomass yield of E coli ML308 grown on 100 mg/L glucose minimal
media. The biomass yield was determined by dividing the cell density by the
amount of glucose utilized at different dilution rates. Specific details about
cultivation and measurement appear in the Materials and Methods section.
[Comparison data reprinted without permission. Lendenmann Phd dissertation
(Pages 158-160, Tables Bl, B2, and B3). EAWAG, Dubendorf, Switzerland.]

3.3 Carbon Dioxide Evolution Rate

The specific carbon dioxide evolution rate of E coli on glucose was measured at different

dilution rates and compared with similar data found for the same species grown on glucose

minimal media (16). The carbon dioxide evolution rate results and comparison data used appear










in Figure 3-2. The two sets of data show similar qualitative trends. There is a linear increase in

the rate of carbon dioxide evolution with respect to the dilution rate from 0. 1 to 0.4 hr- At

dilution rates greater then 0.4 hr- the carbon dioxide evolution rate slows considerably.




~14


S8-


O 41 E. coli K12D1 qCO2 Han 1992 et al.
Cr2 -1 E. coli ML308 qCO2 Narang Lab

0.0 0.2 0.4 0.6 0.8
Dilution Rate (1/hr)


Figure 3-2. Steady state carbon dioxide evolution rate of E coli ML308 grown on 100 mg/L
glucose minimal media. Specific details about cultivation and measurement
appear in the Materials and Methods section. The effluent air carbon dioxide
concentration, the chemostat bulk cell density, and the air flow rate were used to
calculate a specific evolution rate. [Comparison data reprinted without
permission. Han, K, H. C. Lim, and J. Hong. 1992. Acetic-Acid Formation in
Escherichia-Coli Fermentation. Biotechnology and Bioengineering 39:663-671.
(Page 666, Figure 2).]

3.4 Protein and RNA Dry Weight Fractions

The protein content of E coli ML308 was measured at different dilution rates and

compared with the results of a similar study done with a different strain of bacteria grown on

glucose minimal media. Unfortunately no data was found for the dilution rate dependence of E

coli protein content for similar conditions. The results of the protein dry weight fraction

measurements and the comparison data used appear in Figure 3-3. The protein fraction was a

decreasing function of the dilution rate ranging from 76% (at D=0. 1 hr- ) to 61% (at D=0. 7).

The data used for comparison matched the trend observed in the laboratory extremely well

despite being collected using a different species of bacteria.











+~A. aerogenes Protein Tempest et al. 1965, 1967
-0 E. coli ML308 Protein Narang Lab
1.0-

S0.8

E 0.6-





0.0
0.0 0.2 0.4 0.6 0.
Dilution Rate (11hr)


Figure 3-3.


Protein dry weight fraction of E coli ML308 grown on 100 mg/L glucose
minimal media. Specific details about cultivation and measurement appear in the
Materials and Methods section. The comparison data was measured using A.
aerogenes grown on glucose minimal media [Comparison data reprinted without
permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of Aerobacter
aerogenes at low dilution rates in a chemostat. Microbial Physiology and
Continuous Culture. Her Majesty's Stationary Office:240-253. (Pages 361, Figure
3).]


The RNA content of E coli ML308 was measured at different dilution rates and compared

with data from the same comparison data set. The RNA fraction results appear in Figure 3-4. The

RNA fraction was an increasing function of the dilution rate ranging from 12% (at D=0. 1 hr- ) to

16% (at D=0. 7). The data used for comparison was a monotonic increasing trend as well but had

a low RNA content at lower dilution rates that increased at faster rate. At a dilution rate of 0.7 hr-

Sthe RNA level of both data sets had similar values. The difference in RNA trends could be a

result of the fact the two trends collected were from different species of bacteria.











+ A. aerogenes RNA Tempest et al. 1965, 1967
0.20 -0 E. coli ML308 RNA Narang Lab

~-0.16-

0.12-

S0.08

a 0.04-

0.00
0.0 0.2 0.4 0.6 0.8
Dilution Rate (1/hr)


Figure 3-4. RNA dry weight fraction of E coli ML308 grown on 100 mg/L glucose minimal
media. Specific details about cultivation and measurement appear in the Materials
and Methods section. The comparison data was measured using A. aerogenes
grown on glucose minimal media. [Comparison data reprinted without
permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of Aerobacter
aerogenes at low dilution rates in a chemostat. Microbial Physiology and
Continuous Culture. Her Majesty's Stationary Office:240-253. (Pages 361, Figure
3), Microbiological Research Establishment, Porton, Salisbury, Wilks, UK.]

3.5 Maximum Specific Growth Rate on Minimal Media

The maximum specific growth rate is the highest speed of cell division a microbial species

can obtain on a given media at a specific pH, temperature, etc. All nutrients in the medium are at

saturating concentrations with respect to the growing cells. The maximum growth rate on a

particular medium is generally unique for different microbial species. Exposure of nutrient

limited cells to excess nutrients will not result in an immediate adjustment of the cell specific

growth rate to the maximal value. There is an adaptation period, on the order of days, for the

cells to achieve their maximum speed of cell division (21).

The maximum specific growth rate of E coli ML308 was determined and compared with a

value published for the same strain and similar media composition (21). A population ofE. coli

was constantly exposed to a nutrient surplus (1 g/L glucose) for five days by sterile subculturing.

The cells were never allowed to enter stationary phase for the duration of the experiment. After









five days the culture was growing at a growth rate of~-0.91 hr- which compared well with the

value 0.92 hr- found in the literature.

3.6 Initial Growth and Uptake Rate Response to Nutrient Excess

Chemostat grown glucose limited cells rapidly increase their growth rate and substrate

uptake rate upon exposure to saturating glucose concentrations. The level of increase for the

growth rates and uptake rates were dependant on the preculture dilution of the chemostat grown

cells. The initial growth rate and uptake rates of E coli ML308 growing at different dilution rates

were determined by measuring the cell density and glucose concentration of a culture exposed to

excess glucose. Reactor volume (50 mL) from a glucose limited chemostat was extracted and

transferred to a pre-warmed shake flask (250 mL) and inoculated with a saturating concentration

of glucose (50 mg/L). The shake flask was then placed in a tabletop shaker (2000 RPM, 370C)

and the cell density and glucose level were measured every five minutes for thirty minutes. The

growth and uptake rates were calculated as described in the Materials and Methods section.

The results of the initial rate experiments for three different dilution rates as well as

comparison data from similar experiments taken from the literature (21) appear in Figure 3-5 and

Figure 3-6. The arrows indicate the increase in growth or uptake rate upon the shift from glucose

limited continuous mode growth to glucose surplus batch mode growth. At all dilution rates

tested both the substrate uptake rate and growth rate increased upon exposure to excess glucose.

The growth rates compare well with the results of similar experiments done with the same

species. The initial growth rates of cells growing at dilution rates less then 0. 1 hr- have never

been explored to the best of our knowledge. The increase in the growth rate upon exposure to

excess glucose dropped considerably as the dilution rate approached zero. The highly starved

cells were unable to maintain the capacity for increased growth upon exposure to nutrient excess.













-- Continuous Mode -0 Batch Mode
-o Lendenmann


S0.8

S0.6


0.4


0.0 0.1 0.2 0.3 0.4
Dilution Rate (1/hr)


0.5 0.6


Figure 3-5.


Initial growth rate increase of E coli ML308 grown on 100 mg/L glucose
minimal media upon exposure to excess glucose (50 mg/L). The growth rate
under steady state conditions appears as the solid line. Specific details about
cultivation and measurement appear in the Materials and Methods section. The
comparison data was measured using E. coli ML308 grown on glucose minimal
media. [Comparison data reprinted without permission. Lendenmann Phd
dissertation (Page 72, Figure 8.1), EAWAG, Dubendorf, Switzerland.]


-- Continuous Mode -0 Batch Mode
-o Lendenmann


2.0

C 1.6
-cr
,"


c~ 0.8

~L 0.4


0.0 0.1 0.2 0.3 0.4 0.5 0.6
Dilution Rate (1/hr)


Figure 3-6.


Initial uptake rate of E coli ML308 grown on 100 mg/L glucose minimal media
upon exposure to excess glucose (50 mg/L). The uptake rate under steady state
conditions appears as the solid line. Specific details about cultivation and
measurement appear in the Materials and Methods section. The comparison data
was measured using E. coli ML308 grown on glucose minimal media.
[Comparison data reprinted without permission. Lendenmann Phd dissertation
(Page 72, Figure 8.1), EAWAG, Dubendorf, Switzerland.]









The substrate uptake rates of the two different data sets had noticeable differences. The

collected substrate uptake rates were lower then the comparison data but it has been

hypothesized that the reason for this discrepancy is the timescale on which the substrate uptake

rates were measured. Lendenmann (1995) collected glucose samples over a seven minute period

as opposed to over a thirty minute period. The reasoning for this discrepancy could be the

specific growth rate oscillations discussed in Chapter 5 and shown in Figure 5-5. If the specific

growth rate of the bacterial cells oscillates with time then the specific substrate uptake rate could

be oscillating as well.

3.7 Concluding Remarks

These characterization experiments were necessary to ensure the validity of the new

protocols and our model system. In all cases the results collected were comparable to those from

similar experiments done by other researchers. In addition the data from these experiments

provided a benchmark for the future experiments. The chemostat steady state data collected

characterized the initial state of cells of E. coli growing at different glucose limited growth rates.

These benchmarks were checked routinely before conducting experiments to ensure the state of

the chemostat grown cells were correct so reproducible data would be collected.









CHAPTER 4
STEADY STATE AND TRANSIENT CARBON FLUX

4.1 Introduction

The transient response of a microbial chemostat is a challenge facing biology and

engineering today. Continuously run bioreactors are inevitably disturbed with fluctuations in

their feed flow rate that can lead to lengthy transients involving massive cell loss and an

overshoot in the limiting substrate concentration. These long transients can lead to product

deterioration in industrial bioreactors and violations at wastewater treatment facilities.

Characterizing the initial carbon flux of these continuously cultivated microorganisms after one

of these perturbations is the goal of this research. This knowledge could help in the development

of a better model that could mitigate the effects of these reactor transients. A literature search

yielded no systematic study of the transient carbon flux of continuously grown microorganisms

at a range on carbon limited growth rates.

Exposure of glucose limited cells to saturating concentrations of glucose will rapidly

increase their specific growth rate and specific substrate uptake rate. As seen in Figure 3-5 and

Figure 3-6 the substrate uptake rate increase appears to be independent of the preculture dilution

rate at D=0.3 hrl and above but the increase in growth rate appears to change linearly with

dilution rate. The cells are consuming increased amounts of substrate at all dilution rates tested

yet grow at different rates. An appropriate question here would be how are cells utilizing this

excess sub state if it is not being channeled into production of more cells?

Microorganisms growing under aerobic conditions generally have three ways they can

utilize consumed carbonaceous substrate. They can channel the carbon in to synthesis of cell

biomass (biosynthesis), fully oxidize the organic carbon to carbon dioxide for energy

(respiration), or partially catabolize the substrate and discharge the product in to the environment









around the cell (excretion). All three of these cellular carbon sinks for consumed carbonaceous

substrate were measured during steady state growth and during transient growth immediately

following removal of the substrate limitation. The measurements were conducted at D=0. 1, 0.3,

and 0.6 hr- to see the change in carbon flux response at a range of glucose limited preculture

growth rates.

4.2 Materials and Methods

4.2.1 Organism and Cultivation Conditions

The organism used in this work was E. coli ML308 (ATCC 15224) obtained from the

American Type Culture collection. Cells were resuscitated from a frozen stock culture overnight

on glucose (1 g/L) minimal media. The minimal media used in this work was described in the

Materials and Methods Chapter and the components of which appear in Table 2-1. The

chemostat cultures were grown in a 1.5 L Bioflow III fermenter (New Brunswick Scientific Co.)

with a working volume of 1.2 L. The agitation speed was 1000 rpm and the aeration rate was 1.2

L/min. The bioreactor was equipped with automatic pH and temperature control. The pH was

maintained at 7.0 '/- 0.1 by addition of IM KOH / IM NaOH solution and 10% H3PO4 SOlutions.

The temperature was maintained at 370C. The feed was pumped in by a Masterfiex L/S

peristaltic pump (7523-70) equipped with a Masterfiex EZ Load pump head (7534-04). The

reactor was equipped with a Vaisala GMT222 CO2 analyZeT for continuous monitoring of the

carbon dioxide output of the reactor.

Chemostat cultures were precultured on glucose (100 mg/L) for four days at D=0.6 hr-

before shifting to the trial dilution rate to ensure adaptation of the E. coli cells to low glucose

conditions for reasons described in the Materials and Methods Chapter. The chemostat was then

allowed to equilibrate for ten additional residence times to ensure the cells were adjusted to the

new growth conditions (21). The cellular carbon dioxide evolution rate of the reactor was









monitored during the adaptation and equilibration phases of preculturing to ensure the history

and consequently the initial state of the chemostat grown cells were correct before attempting an

experiment.

4.2.2 Carbon Measurement Methodology

The carbon content of cell mass inside the reactor at any time was estimated using a carbon

mass fraction of cell dry weight conversion factor. The concentration of cells inside the reactor at

any time was determined by an absorbance measurement at 546 nm using a Spectronic Genesys

10UV spectrophotometer. This carbon content conversion factor was found by measuring the

steady state carbon mass fraction using a Carlo Erba-1106 Elemental Analyzer at six different

dilution rates (0.1-0.6 hr ). Cell mass was collected, concentrated in a centrifuge (5,000 RPM,

4oC), desiccated overnight in an 800C oven, and then pulverized to a fine powder prior to

analysis. The carbon mass fraction of the cell dry weight was assayed three times with this

method to ensure an accurate conversion factor. The steady state carbon fraction of the dry cell

weight for the different steady states appears in Figure 4-1. This conversion factor was used to

calculate the carbon content of cell biomass of the chemostat at any time as shown in Equation 4-



Cb = CF C (4-1)

The variables Cb, CF, and C in Equation 4-1 correspond to the cell biomass carbon content of

the chemostat (units mg C/L), the carbon weight fraction of cell mass of steady state cells at the

preculture dilution rate (units g C/mg), and the cell density of the chemostat volume (units mg

/L), respectively.














S0.45-


0.40-


B 0.35-


0.30
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Dilution Rate (1/hr)


Figure 4-1. Carbon mass fraction of dry weight E. coli ML308 grown at different glucose
limited dilution rates. The analysis was performed on a Carlo Erba-1106
Elemental Analyzer.

The carbon content of the effluent air was determined with output from the Vaisala GMT-

222 CO2 analyzer and associated software as described in the Materials and Methods Chapter.

The cell specific respiration rate calculated using Equation 2-1 was a five minute average of CO2

values centered on the time a cell density measurement was taken. The rate of evolution was

converted to milligrams of carbon respired per liter of reactor volume by the conversion shown

in Equation 4-2.

Cr = 12* qCO2*C Cdt (4-2)

The variables Cr, qCO2, C, and dt in Equation 4-2 correspond to the respired carbon evolved

over the dt time period (units mg C/L), the cell specific CO2 CVOlution rate (units mmoles C/mg-

hr), the cell density of the chemostat (units mg/L), and the time interval (unit hr), respectively.

The carbon content glucose and excreted products in the chemostat bulk were determined

with a Dionex DX-500 HPLC and a Phoenix 8000 TOC Analyzer. The details of how these

measurements were performed appear in the Materials and Methods Chapter. Little manipulation





































Table 4-1. Frequency of data collection and approximate reactor volume taken for each
measurement during the continuous to batch shift experiments. Sample extraction for
the total organic carbon measurement only applied during batch mode growth as
significant reactor volume was required. Steady state TOC samples were only taken
immediately before the shift from continuous to batch mode.

Variable Vol. (ml) Freq. (min)
Cell Density 6 5
CO2 Conc. 1
Glucose 2 10
TOC 20 10


To observe the change in carbon utilization pattern during a pseudo dilution rate shift up,

most of the steady state variables were monitored for an hour prior to the continuous mode to

batch mode shift. After the 50 mg/L pulse of glucose was added to the reactor the indicated

variables were monitored until exhaustion of glucose denoted by a sudden, rapid decrease in the

carbon dioxide concentration and an unchanging reactor cell density.


was needed to convert the glucose concentration and excreted carbon concentration to the correct

form (units mg C/L) necessary for analysis.

4.2.3 The Continuous to Batch Shift Experiment

The continuous to batch shift of a chemostat was achieved by simple switching off the

feed pump. To simulate the environment a microbial cell experiences during a dilution rate shift

up, a 50 mg/L pulse of glucose was inj ected in to the reactor bulk immediately following the

shift from continuous to batch mode. Shifting the chemostat to a batch reactor was necessary for

data collection as extraction of large sample volumes were necessary for measuring the total

organic content of the reactor bulk as kinetics of batch growth are independent of the active

volume of the reactor. Frequency of variable measurement, sample collection, and approximate

volumes taken for measurement appear in Table 4-1.











To ensure the validity of collected data at least 90% of the added glucose carbon was

accounted for in either the generated cell biomass, respired carbon dioxide, and in excreted

organic carbon both during continuous mode growth and at the end of batch mode growth. Each

dilution rate was tested at least twice to ensure a reproducible trend.

4.3 Results

Figure 4-2, Figure 4-3, and Figure 4-4 show example data from each of the three preculture

dilution rates tested. Figure 4-5 shows a comparison in the cell density trends among the three

different preculture dilution rates and Figure 4-6 shows a comparison in the specific CO2

evolution rate trends among the three dilution preculture dilution rates. Table 4-2 shows

numerical values for growth rate, substrate uptake rate, CO2 CVOlution rate, and biomass yield

before and during batch mode growth.


+ Cell Density +~ Glucose
10 o Excretion -0 CO2 Evolution

f2 80
1.2 E
a 60 i ~ ~ T~~~T
W W -C 0.8 (
S40
0.4
5 20

0 I^~~ -L 0.0
0.0 0.5 1.0 1.5 2.0 2.5
Time (hr)


Figure 4-2. Results of a continuous to batch shift of a bioreactor growing E. coli ML3 08
continuously at D=0. 1 hrl on 100 mg/L glucose minimal media. Batch mode shift
and inj section of ~50 mg/L glucose occurred at I hour.













+ Cell Density
+ Excretion


+ Glucose
+ CO2 Evolution


1.2

o

0.8$




-6 0.0
2.0 2.5


0.0 0.5 1.0 1.5
Time (hr)


Figure 4-3.


Results of a continuous to batch shift of a bioreactor growing E. coli ML3 08
continuously at D=0.3 hr- on 100 mg/L glucose minimal media. Batch mode shift
and inj section of ~50 mg/L glucose occurred at I hour.


+ Cell Density
+ Excretion


+ Glucose
+ CO2 Evolution


100

S80

60 s

40


S0


2.5

2.0
o
1.5
o
1.0 O




0.0


0.5 1.0 1.5
Time (hr)


Figure 4-4.


Results of a continuous to batch shift of a bioreactor growing E. coli ML3 08
continuously at D=0.6 hr- on 100 mg/L glucose minimal media. Batch mode shift
and inj section of ~50 mg/L glucose occurred at I hour.


At every dilution rate tested there was a seemingly immediate increase in substrate uptake


rate and growth rate. The dilution rate D=0.6 hr- attained higher overall growth rates and carbon


dioxide evolution rates as seen in Figure 4-5 and Figure 4-6. The greatest increase in growth rate,











substrate uptake rate, and carbon dioxide evolution rate were found at the dilution rate D=0.1 hrl


as seen in Table 4-2.


+ D=0.1 +~ D=0.3 + D=0.6


90-



-70-



S50



30
0.0 0.5 1.0 1.5
Time (hr)


2.0 2.5 3.0


Figure 4-5.


Comparison of the cell density evolution of the three preculture dilution rates
before, during, and after the continuous to batch shift.


+ D=0.1 +~D=0.3 O D=0.6
14


~10






014


0.0 0.5 1.0 1.5 2.0 2.5
Time (hr)


Figure 4-6.


Comparison of the cell density evolution of the three preculture dilution rates
before, during, and after the continuous to batch shift.










Table 4-2. Growth rate, uptake rate, carbon dioxide evolution rate, and biomass yield before
and during a batch mode shift for the six different trials.
Cont. Batch Cont. Batch Cont. Batch Cont. Batch
D (1 hr) rg (1 hr) rg (1 hr) rs (1 hr) rs (1 hr) Y (gdiv g) Y (gdiv g) qCO2 (mm g-hr) qCO2 (mm g-hr)
0.10 0.10 0.38 0.26 0.61 0.40 0.54 4.85 8.06
0.10 0.10 0.33 0.25 0.70 0.39 0.54 4.42 7.73
0.30 0.30 0.52 0.60 1.17 0.54 0.45 6.93 7.53
0.30 0.30 0.59 0.55 1.11 0.54 0.53 7.70 11.44
0.60 0.60 0.81 1.08 1.38 0.55 0.59 10.93 11.45
0.60 0.60 0.81 1.13 1.58 0.53 0.52 11.96 10.20



It is worth mentioning that at D=0. 1 hr- there is an increase in yield upon exposure to

excess glucose while at D=0.3 and 0.6 hr- the batch mode yield stays relatively constant. This

was unexpected as other studies looking into transient yields after a dilution rate shift up show a

marked drop in the yield (13). The experiments in question had transients that operated on a

much longer time scale then the transients in this study and was thought to be the cause of the

yield discrepancy. Simple shake flask experiments done at the three different dilution rates using

1 g/L glucose confirmed that the yield does decrease at all dilution rates on a longer time scale.

As stated earlier at least 90% of the inj ected glucose was accounted for in carbon balances

done at steady state and upon the exhaustion of glucose during batch mode growth. Carbon

utilization by cells does change under substrate excess conditions and varies with preculture

dilution rate. A comparison of carbon utilization between steady state growth and transient

growth appears in Figure 4-7.

During steady state growth at D=0. 1 hr- about 50% of the consumed glucose went to

respiration and 50% is going to biosynthesis. During excess glucose conditions most of the

consumed carbon is channeled towards biosynthesis resulting in the much higher yield seen

during batch growth. Little excretion is seen during steady state and transient growth at D=0. 1 hr

1. At D=0.3 hr- the split between biosynthesis and respiration was 64% and 39%, respectively.

During substrate excess conditions the fraction of consumed carbon being used for growth stays










relatively constant while the fraction being excreted increases immensely. At D=0.6 hr- the

trend is different still. This dilution rate shows the least amount of change upon exposure to

excess glucose. The fraction of carbon being utilized during steady state and transient growth

stays roughly the same before and after the shift at 66% being used for biosynthesis and 27%

being respired to carbon dioxide. While the cells did excrete organic compounds during batch

growth, they were completely consumed by the end of the transient (including some leftover

excretory products from steady state growth).



O Biosynthesis O Respiration 0 Excretion
1.2-






0.4-


LL0.0 o
0 1 SS 0 1 Trans 03SS 0 3Trans 06SS 0 6 Trans
-0.2-


Figure 4-7. A comparison of cellular carbon utilization pattern during continuous mode
growth and during batch mode growth for D=0.1i, D=0.3, and D=0.6.



4.4 Discussion

The most glucose starved dilution rate, D=0.1 hr- was extremely efficient at incorporating

consumed substrate into biomass. A sizable increase in the amount of carbon going toward

biosynthesis is observed upon exposure to excess glucose leading to a higher yield. Little to no

excretion was seen during batch growth. For the intermediate dilution rate, D=0.3 hr- the

bacteria were less starved during the preculturing phase which proved to make them more

wasteful in terms of carbon utilization. Cells growing at this dilution rate utilized the same









percentage of consumed substrate in biosynthesis during glucose limited growth and glucose

excess growth. There was an increase in the carbon dioxide evolution rate, but a large percentage

of the consumed substrate during transient growth was excreted back in to the medium. This

suggests saturation of respiration and biosynthetic capacity forcing the cell to excrete the excess

consumed substrate as partially catabolized organic molecules. The cells precultured at D=0.3 hi

Sdid not utilize the excreted organic products before exponential growth ended. Cells growing at

the highest dilution rate tested, D=0.6 hi were least affected by the excess glucose conditions.

Carbon accounting at the end of the exponential growth phase showed the excess carbon was

utilized by the cell in percentages proportional to those seen at steady state. At D=0.6 hrl there

was little or no increase in the carbon dioxide evolution rate leading to the same trend seen at

D=0.3 hrl where the cells tend to excrete a large fraction of the consumed substrate. The

excreted organic products were completely consumed by the end of the exponential growth

phase signifying that cells grown at D=0.6 hrl have a higher capacity to utilize their excretory

products than the intermediate dilution rate of D=0.3 hr In conclusion as the preculture growth

rate (and consequently degree of glucose availability) increases there tends to be less excess

capacity for respiration and growth leading to more excretion of excess consumed carbon. Only

at the highest dilution rate of D=0.6 hrl were the bacteria able to utilize the excreted compounds

before the transient ended.

The steady state and transient yields support the idea that as preculture dilution rate

increases the capacity for excess growth and respiration decreases. At D=0. 1 hrl the steady state

yield is significantly lower then those seen at D=0.3 hrl and D=0.6 hr The higher dilution rates

show an equivalent and higher yield. This suggests that cells growing at D=0. 1 hrl inside a

chemostat have to use more of their consumed substrate for the production of energy









(respiration) than in biosynthesis unlike the higher dilution rates. This idea is also supported by

the fact that little to no excretion is seen during steady state growth at D=0. 1 hr- as the cell is

either fully oxidizing carbon for energy or incorporating it in to cell biomass. Upon exposure of

cells growing at D=0. 1 hr- to excess glucose, the cells increase the fraction of consumed carbon

going to biosynthesis to levels seen at D=0.3 hr- and D=0.6 hr- This suggests that cells keep an

excess capacity to grow at their desired yield under starved conditions. This trend probably does

not hold at dilution rates less then D=0. 1 hr- There is a drastic decrease in the glucose excess

growth rate seen at extremely low dilution rates, as seen in Figure 4-5, as more substrate is

needed for energy production just to maintain the cell. The capacity for excess growth starts

decreasing markedly below D=0. 1 hr- as the microbial cells become increasingly starved.

In all cases the initial transient yield on the time scale measured increased or remained

constant upon exposure to excess glucose. This is in contrast to what has been seen in the

literature. Others have seen that exposure of substrate limited microorganisms to excess substrate

leads to an overall decrease in the yield during transient growth. Shake flask experiments done

with higher concentrations of inoculated glucose revealed that the yield does indeed decrease on

a much longer time scale. So there appears to be an initial yield increase on a fast time scale and

a decrease on a longer time scale. The cause of this yield variation with time is yet to be

determined









CHAPTER 5
ATTEMPT TO IDENTIFY THE BIOSYNTHETIC LIMITATION

5.1 Introduction

The inability of microbial cells, growing under carbon limited conditions, to instantly

adjust their growth rates to maximal levels has been seen in the literature and from data collected

in previous experiments. Currently the concentration of ribosomes inside microbial cells is

thought to be the cause of the intracellular growth limitation and is used as such for modeling

purposes (5). At different preculture dilution rates the initial increase in the growth rate upon

exposure to excess substrate has been found to be a linearly increasing function of the dilution

rate much like the ribosome level. Since ribosomes are necessary for the production of protein;

which constitutes the majority of cell biomass, one can easily draw the conclusion that ribosome

levels control the growth rate during a dilution rate shift up. Experimental data collected where

the growth rate and ribosome level have been monitored simultaneously certainly suggest this is

the case. Figure 5-1 is a dilution rate shift up experiment conducted where the cell specific

growth rate and the RNA level of the cell were recorded during transient growth.

The maj ority of cellular RNA (97%) is ribosomal RNA making total RNA content a good

indirect measure of ribosome content (29). Clearly the cell specific growth rate and RNA level

are a good qualitative match; however, there are lines of evidence to suggest that the ribosome

content of a cell is not the true intracellular biosynthetic limitation.












+ Glucose --Growth Rate -6-RNA Fraction


1.0

S0.8



; 0.4

0.2

0.0


0.12

0.10
0.08

0.06

0.04 u..

0.02 c2
0.00


0 2 4 6 8 10 12 14
Time (hr)


Example dilution rate shift up (0.075 to 0.409 hr ) from the literature where the
growth rate and RNA level were measured versus time. The microorganism used
was Lactococcus cremoris grown on glucose minimal media. [Reprinted without
permission. Benthin, S., and J. Villadsen. 1991. Growth energetic of
Lactococcus cermoris FD1 during energy-carbon and nitrogen limitation in steady
state and transient cultures. Chem. Eng. Sc. 49:589-609. (Page 4237, Figure 5).]


Figure 5-1.


The well known fact that microbial cells grow much faster on complex media than on

minimal media should already be enough evidence to show that the ribosome content of a cell

cannot be the true biosynthetic growth limitation seen in carbon limited microbial cells. Analysis

of data from the literature seems to suggest that the biosynthetic limitation is actually a result of

a lack of amino acids. Figure 5-2 is a comparison of transient growth rates among cultures of E.

coli grown under different preculture conditions and transient conditions. Cells extracted from a

carbon limited chemostat and exposed to excess glucose and excess amino acids produced


growth rates that exceeded even the maximum growth rate attainable on minimal media alone.

The ribosomes of microbial cells exposed to excess glucose are most likely unsaturated with

their amino acid substrates making the supply of amino acids to the ribosomes and not the

ribosome level itself the cause of the biosynthetic limitation.











-0 D=0.37 (no improvement) -o D=0.37 + glc
+~ Max Induction + D=0.34 + glc + aa
2.0-


$1.8


1.4-



1.0
0.0 0.2 0.4 0.6 0.8 1 .0 1 .2
Time (hr)


Figure 5-2. Example data from the literature showing that amino acids limit the growth of
carbon limited cells exposed to excess glucose. The microorganism used was E.
coli B precultured on glucose limited minimal media. The open box trend refers to
cells growing at D=0.37 that would show no improvement in the growth rate if
exposed to excess glucose. The open circle trend refers to the growth rate trend
observed when cells grown at D=0.37 were exposed to excess glucose. The open
triangle trend refers to cells growing at their maximum growth rate on glucose.
The open diamond trend refers to cells growing at D=0.34 exposed to excess
glucose and excess amino acids. All data was scaled by their initial cell densities
for trend comparison. [Reprinted without permission. Harvey, R. J. 1970.
Metabolic regulation in glucose-limited chemostat cultures of Escherichia coli. J
Bacteriol 104:698-706. (Pages 669 and 702, Figures 1 and 6).]

Since amino acids are clearly a limiting factor in the growth rate of microbial cells the

enzyme glutamate dehydrogenase (GDH) was thought to be a potential cause of the biosynthetic

growth limitation. This enzyme is responsible for the maj ority of inorganic nitrogen assimilation

in to cell biomass under carbon limited conditions and its amino acid product glutamate is

required for the production of many other amino acids (26). For these reasons the enzyme was

thought to be a better candidate for the intracellular growth limitation of microbial cells growing

with substrate excess. To test this hypothesis cells precultured under glucose limited conditions

were exposed to excess glucose and the cell density, RNA level, and GDH activity were all

monitored during the transient. If the GDH enzyme was the cause of the intracellular limitation

then the change in the enzyme's activity during transient conditions would mirror the change









seen in the RNA level as the production rate of ribosomes would be indirectly limited by the

cell's production rate of glutamate.

5.2 Materials and Methods

5.2.1 Organism and Cultivation Conditions

The organism used in this work was E. coli ML308 (ATCC 15224) obtained from the

American Type Culture collection. Cells were resuscitated from a frozen stock culture overnight

on glucose (1 g/L) minimal media. The minimal media used in this work was described in the

Materials and Methods Chapter and the components of which appear in Table 2-1. The

chemostat cultures were grown in a 1.5 L Bioflow III fermenter (New Brunswick Scientific Co.)

with a working volume of 1.2 L. The agitation speed was 1000 rpm and the aeration rate was 1.2

L/min. The bioreactor was equipped with automatic pH and temperature control. The pH was

maintained at 7.0 +/- 0.1 by addition of IM KOH / IM NaOH solution and 10% H3PO4 SOlutions.

The temperature was maintained at 370C. The feed was pumped in by a Masterfiex L/S

peristaltic pump (7523-70) equipped with a Masterfiex EZ Load pump head (7534-04). The

reactor was equipped with a Vaisala GMT222 CO2 analyZeT for continuous monitoring of the

carbon dioxide output of the reactor.

Chemostat cultures were precultured on glucose (200 mg/L) for four days at D=0.6 hrl

before shifting to the trial dilution rate to ensure adaptation of the E. coli cells to low glucose

conditions for reasons described in the Materials and Methods Chapter. The chemostat was then

allowed to equilibrate for ten additional residence times to ensure the cells were adjusted to the

new growth conditions (21). The cellular carbon dioxide evolution rate of the reactor was

monitored during the adaptation and equilibration phases of preculturing to ensure the history

and consequently the initial state of the chemostat grown cells were correct before attempting an

experiment.









The cell density, RNA concentration, and GDH activity were measured by the methods

covered in the Materials and Methods Chapter.

5.2.2 The Continuous to Batch Shift

Continuous to batch shifts with a subsequent glucose pulse were again employed to

simulate the environment cells experience during a dilution rate shift up. The reactor media was

pulsed with 1 g/L of glucose to ensure substrate level never became limiting during for length of

the experiment. Towards the end of the experiment the cell density is rather high compared to the

starting cell density and consequently the rate of substrate consumption would be high at this

point as well. The duration of the experiment was decided to be two and a half hours long after

finding an example GDH transient in the literature (18). The frequency of variable measurement

and sample collection can be found in Table 5-1.


Table 5-1. Frequency of sample measurement / collection and approximate volume taken.
Vol. Freq.
Sample (mL) (min)
Cell Density 6 5
RNA Level 2 10
GDH Activity 25 10
Note: Samples taken for GDH activity ranged from 5-25 mL depending on the cell density.


For qualitative comparison of the time evolution of all three variables the measured

values were converted to a per liter of reactor volume basis and then scaled by their steady state

value. The purpose of this analysis was to visualize the improvement in the ribosomal capacity

and GDH biosynthetic capacity as it compared to the increase in cell density. Unfortunately the

experiment was conducted only once at the intermediate dilution rate D=0.3 hrl due to time

constraints.











5.3 Results and Discussion

The results of the measured trial conducted are displayed in Figure 5-3 and the scaled


comparison of the three different variables appears in Figure 5-4. As hypothesized the scaled

GDH activity level and RNA level of the reactor volume are qualitatively similar initially upon


exposure of the culture to excess glucose. This data coupled with the fact that amino acids

instantly increase the growth rate of E coli beyond the maximum attainable growth rate supports

the idea that GDH and not ribosome level is the better candidate for the biosynthetic growth

limitation. Unfortunately one trial at one preculture dilution rate is not sufficient to make this a

substantial conclusion.


+ Cell Density -0 RNA -6- GDH Activity


400 -


S300-


S200 -




~100 o

0.0 0.5 1.0 1.5
Time (hr)


-0.8



0.7




2.0t 2.5 3.


Figure 5-3.


Data obtained from a continuous to batch shift of glucose limited E. coli ML308
precultured at D=0.3 hrl to excess glucose. The feed concentration of glucose
was 200 mg/L and the glucose pulse at half an hour was to 1 g/L glucose.













+ Cell Density -o RNA Density
aGDH Activity
O 3.0-



2.0-



1.0
0.5 1 .0 1 .5 2.0 2.5 3.0
Time (hr)


Figure 5-4. Data from Figure 5-3 converted to a per liter basis and scaled by their steady state
value for the purpose of trend comparison.

Another interesting discovery was found when trying to find a way to analyze the data

from this experiment was the presence of maj or oscillations in the growth rate with time upon

exposure of carbon limited microbial cells to excess carbon. Fifteen minute growth rate fits were

calculated for each cell density measurement collected using the cell density measurements five

minutes before and five minutes after. The seemingly large amount of scatter seen in the

collected data was first attributed to spectrometer noise, operator error, or a mechanical forcing

influence brought about by the reactor itself. All three of these sources of oscillation were

eliminated by a simple shake flask experiment where nothing but the cell density was measured.

The magnitude and time interval of the oscillations from the reactor trial and the shake flask trial

were similar. The properties of the mechanical shaker were much different then those of the

reactor setup supporting the idea that the observed oscillations were a cell driven phenomenon.

The results of the reactor and shake flask growth rate trials appear in Figure 5-5. The cause of

these oscillations is still an unknown up to this point.











-o Inside Reactor -6- Iside Shake Flask


1.0

~0.8

0.6



O 0.2

0.0


0 20 40 60
Time (min)


80 100


Figure 5-5.


Oscillations present in growth rate of glucose limited cells exposed to excess
glucose. Cells were precultured at D=0.3 hrl on 200 mg/L glucose minimal
media. Upon extraction or a switch to batch mode the reactor volume was pulsed
with glucose to a concentration of 1 g/L. The experiment was done in a shake
flask as well as in a reactor to ensure mechanical oscillations were not forcing the
cell growth rate oscillations.









CHAPTER 6
CONCLUDING REMARK S

The study was a success from the point of view that new knowledge is available for

understanding how microorganisms respond to the environment around them. Even the time

spent on protocol validation and system characterization provided information on E. coli not

published in the literature: protein and RNA content versus glucose limited dilution rate and low

dilution rate growth response of glucose limited cells exposed to excess glucose. The

experiments completed provide further insight in to how microorganisms respond to removal of

the nutrient growth limitation.

The carbon flux experiments provided a lot of insight in to how the cell utilizes substrate

before and after exposure to excess glucose at different glucose limited growth rates. Cells

grown at all dilution rates tested were able to instantly improve their growth rate and substrate

uptake rate upon removal of the glucose growth limitation. How the cells utilize this excess

glucose is highly dependent on the preculture glucose limited growth rate.

Unfortunately the biosynthetic limitation experiments remain incomplete due to time

constraints but it still remains that ribosomes are not the key cause of biosynthetic limitation as

excess amino acids will greatly speed the growth rate of carbon limited microbial cells. The data

that was collected did support the hypothesis that the enzyme glutamate dehydrogenase mirrors

the RNA concentration well and could be the cause of biosynthetic limitation rather then the

ribosome level. The detection of oscillations in growth rate of glucose limited cells exposed to

excess glucose was not the purpose of the experiments here but was found regardless. This is a

prime example of how research can answer questions but in the end can pose more. Finding the

source of this specific growth rate oscillation would be the potential next step in this research.
































































70









APPENDIX A
VISUAL BASIC PROGRAM FOR THE VAISALA GMT222 CO2 ANALYZER

CODE


Public CO2, X As Single
Public CollectingData As Boolean
Dim CO2Array(1 To 720, 1 To 2) As Single
Public ClockSeconds, ClockMinutes, Clock Integer

Sub GetCO2()
'Queries the detector for a value and rounds the value to the nearest ppm
CO2 = MSComml.Input
CO2 = Round(Val(CO2), 0)
End Sub

Private Sub FormLoad()
'Initialization subroutine. Setup for COM Port, array for data storage, and main chart.
MSComml1.InBufferSize = 6
MSComml1.CommPort = 6
MSComml1.PortOpen = True

For X = 0 To 39
RealTime.List(X) = ""
Next X
For X = 1 To 720
CO2Array(X, 1)= X
CO2Array(X, 2) =0
Next X
With CO2Chart
.chart Type = VtChChartType2dXY
.Plot.UniformAxis = False
.Plot. SeriesColl ecti on( 1). ShowLine = F al se
.Plot. SeriesCollection(1 ). SeriesMarker.Auto = False
.Plot. SeriesCollection(1).DataPoints(- 1).Marker. Style = VtMarkerStyleFilledCircle
.Plot. SeriesCollection(1 ).DataPoints(-1 ).Marker. Size = 60
.Plot. SeriesCollection(1).DataPoints(- 1).Marker.Vrisible = True
.Plot.AutoLayout = False
.Plot.Axis(VtChAxisId Y). Value Scale. Auto = False
.Plot.Axi s(VtChAxi sIdY).Value Scal e.Maximum = 1 000
.Plot.Axi s(VtChAxi sIdY). ValueScale.Minimum = 0
.Plot.Axi s(VtChAxi sIdY).Value Scal e.Maj orDivi si on = 10O
.Plot.Axis(VtChAxisIdX). Value Scale. Auto = False
.Plot.Axi s(VtChAxi sIdX).Value Scal e.Maximum = 720
.Plot.Axi s(VtChAxi sIdX). ValueScale.Minimum = 0










.Plot.Axi s(VtChAxi sIdX).Value Scale.Maj orDivi si on = 1 2
.Plot.Axi s(VtChAxi sIdY).Axi sTitle = CO2 Concentration (ppm)"
.Plot.Axi s(VtChAxi sIdX).Axi sTitle = "Data Point Taken Every Minute "
.ChartData = CO2Array
End With
End Sub


Private Sub Updategraph()
'Updates the data array and adjusts the chart accordingly.
If MinuteTimer = 60 Then MinuteTimer = 0
MinuteTimer = MinuteTimer + 1
If MinuteTimer Mod 60 = 0 Then
For X = 1 To 719
CO2Array(721 X, 2) = CO2Array(720 X, 2)
Next X
CO2Array(1, 2) = CO2
CO2Chart.ChartData = CO2Array
End If
End Sub

Private Sub SendCommandButton_Click()
' Sends text input directly to the Vaisala GMT222 CO2 Analyzer
MSComm l.Output = Command.Text
End Sub

Private Sub StartCollectionButtonClick()
' Starts a text file with the entered name and starts recording time stamped data to the file.
'Also starts the data collection clock.
If Interval.Text = "" Or Filename.Text = "" Then
Else
Open Filename.Text For Output As #1
Print #1, "Time (sec),CO2 (ppm)"
CollectingData = True
Interval.Locked = True
Filename.Locked = True
StartCollectionButton.Enabled = False
SendCommandButton.Enabled = False
End If
End Sub

Private Sub StopCollectionButton_Click()
' Stop recording to the data file and shuts the data collection clock off.
Close #1
CollectingData = False
Interval.Locked = False










Filename.Locked = False
StartCollectionButton.Enabled = True
SendCommandButton.Enabled = True
Hours.Caption = 0
Minutes.Caption = 0
Seconds.Caption = 0
End Sub

Private Sub Timerl_Timer()
' Subroutines called every time interval to collect data and update the chart.
Call RealTimeCO2
Call DataCollectionClock
Call Updategraph
Call DataCollection
End Sub

Private Sub RealTimeCO2()
'Updates the real time CO2 to the left of the chart. Values shown every second.
Call GetCO2
For X = 0 To 39
RealTime.List(39 X) = RealTime.List(3 8 X)
Next X
RealTime.List(0) = CO2
End Sub

Private Sub DataCollectionCl ock()
' Subroutine for the Data Collection Clock seen to the right of the chart.
If CollectingData = True Then
Clock ClockMinutes = Val(Minutes.Caption)
Clock Seconds = Val(Seconds.Capti on)
Clock Seconds = Clock Seconds + 1
If ClockSeconds = 60 Then
ClockMinutes = ClockMinutes + 1
Clock Seconds = 0
End If
If ClockMinutes = 60 Then
Clock ClockMinutes = 0
End If
Hours.Caption = Clock Minutes.Caption = ClockMinutes
Seconds.Caption = Clock Seconds
TotalCollectionTime = Clock End If
End Sub







































o02 cocentraton versu


rrrm~AXIIA~AI~ I~


- ~y0110bb*OCn-


00
I0

00

I





I a IL 1*1 +4 m La ul 4 Ya aa LI r~il
ormrresrrr,


Private Sub DataCollection()

Subroutine that prints the current value to the data file if data recording is active.

If CollectingData = True Then

If TotalCollectionTime Mod Val(Interval.Text) = 0 Then

Print #1, Str(TotalCollectionTime) + "," + Str(CO2)

End If

End If

End Sub

FORM SETUP



CO2 Data Collection


USER INTERFACE


CO32 Data Collection


8. I
t~irira~ro rl~

OU~ddnCI~


Ohn Om~r Orrw





I


0 rs 0 mins 0 sees



SeaommanatoM22















APPENDIX B
RESULTS OF CARLO ERBA-1106 ELEMENTAL ANALYSIS OF E. COLI ML308





Elemental Analysis Done on the Carlo Erba-1 106 Elemental Analyzer


D (1/hr) N (g/gdwl C (g/gdwl H g/gdw}
0.1 0.12 0.46 0.07
0.11 0.43 0.07
0.11 0.43 0.07
0.11 0.43 0.07
0.11 0.43 0.07
0.2 0.12 0.46 0.07
0.11 0.42 0.07
0.11 0.42 0.07
0.12 0.47 0.07
0.12 0.47 0.07
0.3 0.12 0.46 0.07
0.12 0.46 0.07
0.12 0.45 0.07
0.12 0.45 0.07
0.12 0.45 0.07
0.4 0.12 0.46 0.07
0.11 0.43 0.07
0.11 0.43 0.07
0.12 0.45 0.07
0.12 0.45 0.07
0.5 0.12 0.46 0.07
0.11 0.39 0.07
0.11 0.38 0.07
0.12 0.45 0.07
0.12 0.45 0.07
0.6 0.11 0.39 0.07
0.11 0.39 0.07
0.12 0.41 0.07
0.11 0.41 0.07
0.12 0.44 0.07
0.12 0.43 0.07


Elemental Analysis Statistics


D (1/hr) Ave N Ave C Ave H StDev N StDev C StDev H RSD N RSD C RSD H
0.1 0.11 0.44 0.07 0.01 0.01 0.00 0.06 0.03 0.03
0.2 0.12 0.45 0.07 0.01 0.02 0.00 0.05 0.05 0.01
0.3 0.12 0.46 0.07 0.00 0.00 0.00 0.02 0.01 0.01
0.4 0.12 0.44 0.07 0.00 0.01 0.00 0.04 0.03 0.02
0.5 0.12 0.43 0.07 0.01 0.04 0.00 0.06 0.08 0.03
0.6 0.12 0.41 0.07 0.01 0.02 0.00 0.05 0.05 0.02










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continuous-culture transients for two substrate systems. Appl Microbiol 23:354-9.

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transition from glucose-excess to glucose-limited growth conditions in continuous culture
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46. Yagil, G., and E. Yagil. 1971. Relation between Effector Concentration and Rate of
Induced Enzyme Synthesis. Biophysical Journal 11:11-&.

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Escherichia coli: Effects of temperature and dilution rate changes. Biotechnology and
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BIOGRAPHICAL SKETCH

Jason Noel received his B.S. from Virginia Commonwealth University in Richmond,

Virginia in August of 2003. Thereafter he has worked as a graduate student in the Chemical

Engineering Department of the University of Florida studying the growth kinetics of bacteria and

received his doctorial degree in August of 2007.





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1 DYNAMICS OF MICROBIAL GROWTH IN SINGLE SUBSTRATE CULTURE By JASON NOEL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOS OPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Jason Noel

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3 To my Parents David and Tracey Noel

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4 ACKNOWLEDGMENTS I would first like to acknowledge my advisor Dr. Atul Narang for his guidance and support during this research. Without his ideas and insight none of this would have been possible. I would also like to thank my committee members Dr. Spyros Svoronos Dr. Ben Koopman, Dr. Ranganathan Narayanan and Dr. Lewis Johns for their support. I would also like to thank some individuals whose technical expertise and personal assistance made this research possible. Dr. Thomas Egli from the Swiss Federal Institute of Aquatic Science and Technology was extremely helpful in troubleshooting problems found in operating a chemostat growing microbial cells. Dr. Tommaso Cataldi from the Universita degli Studi della Basisilicate was instrumental in developing a re liable procedure for HPLC quantification of low sugar concentrations in samples extracted from bacterial cultures. Lastly I would like to thank Dr. Max Teplitski from the University of Florida for his help providing a reliable mechanical disruption techniq ue for starved bacteria. I would like to thank my lab members Dr. Shaki Gupta, Dr. Eric May, Dr. Karthik Subramanian, Ved Sharma, and Brenton Cox. Your technical support and companionship helped make the sometimes frustrating experimental work tolerable. Lastly I would like to thank my friends and family for being there for me through the good times and the bad.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 7 LIST OF FIGURES ................................ ................................ ................................ ......................... 8 ABSTRACT ................................ ................................ ................................ ................................ ... 10 CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ .............................. 12 1.1 Motivation for Research ................................ ................................ .............................. 12 1.2 Conceptual Model of the Cell ................................ ................................ ...................... 13 1.3 Steady state behavior of microbial cells ................................ ................................ ...... 14 1.4 Cell density and Substrate Concentration ................................ ................................ .... 15 1.5 Cellular Content and Excreted Metabolites ................................ ................................ 18 1.6 Transients Controlled by the Transport Enzymes ................................ ........................ 18 1.7 Transient Dynamics Controlled by a Biosynthetic Limitation ................................ .... 20 1.8 Transient Response to Dilution Rate Shift Ups ................................ ........................... 25 2 MATERIALS AND METHODS ................................ ................................ ........................... 27 2.1 Growth Medium ................................ ................................ ................................ ........... 27 2.2 Bacterial Strain ................................ ................................ ................................ ............. 27 2.3 The Chemostat Setup ................................ ................................ ................................ ... 28 2.3.1 Wall Growth Limitation ................................ ................................ ..................... 28 2.3.2 Glucose Adaptation Limitation ................................ ................................ .......... 29 2.4 Cell Density Measurement and Growth Rate ................................ .............................. 30 2.5 Suga r Measurement, Yield Determination, and Uptake Rate ................................ ...... 31 2.6 Total Cell Protein Extraction and Measurement ................................ .......................... 32 2.6.1 Extraction ................................ ................................ ................................ ........... 32 2.6.2 Measurement ................................ ................................ ................................ ...... 32 2.7 Total Cell RNA Extraction and Measurement ................................ ............................. 33 2.7.1 Extraction ................................ ................................ ................................ ........... 33 2.7.2 Measurement ................................ ................................ ................................ ...... 33 2.8 Carbon Dioxide Evolution Measurement ................................ ................................ .... 33 2.9 Total Organic Carbon Measurement ................................ ................................ ............ 34 2.10 Elemental Content Measurement ................................ ................................ .......... 35 2.11 Cell Disruption Te chniques ................................ ................................ .................. 35 2.11.1 Chemical Disruption ................................ ................................ ...................... 36 2.11.1 Chemical Disruption Drawbacks ................................ ................................ ... 37 2.11.1 Mechanical Disruption ................................ ................................ ................... 38 2.11.2 Mechanical Disruption Advantages ................................ ............................... 38

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6 2.12 Enzymatic Assay of Glutamate D ehydrogenase ................................ ................... 39 2.13 Deactivation and Disposal of Microorganisms ................................ ..................... 40 3 PROTOCOL VALIDATION AND SYSTEM CHARACTERIZATION ............................. 41 3.1 Goals of Verification and Characterization ................................ ................................ 41 3.2 Steady State Biomass Yield ................................ ................................ ......................... 41 3.3 Carbon Dioxide Evolution Rate ................................ ................................ ................... 42 3.4 Protein and RNA Dry Weight Fractions ................................ ................................ ...... 43 3.5 Maximum Specific Growth Rate o n Minimal Media ................................ .................. 45 3.6 Initial Growth and Uptake Rate Response to Nutrient Excess ................................ .... 46 3.7 Concluding Remarks ................................ ................................ ................................ .... 48 4 STEADY STATE AND TRANSIENT CARBON FLUX ................................ ..................... 49 4.1 Introduction ................................ ................................ ................................ .................. 49 4.2 Materials and Methods ................................ ................................ ................................ 50 4.2.1 Organism and Cultivation Conditions ................................ ............................... 50 4.2.2 Carbon Measurement Methodology ................................ ................................ .. 51 4.2.3 The Continuous to Batch Shift Experiment ................................ ....................... 53 4.3 Results ................................ ................................ ................................ .......................... 54 4.4 Discussion ................................ ................................ ................................ .................... 58 5 ATTEMPT TO IDENTIFY THE BIOSYNTHETIC LIMITATION ................................ ..... 61 5.1 Introduction ................................ ................................ ................................ .................. 61 5.2 Materials and Methods ................................ ................................ ................................ 64 5.2.1 Organism and Cultivation Conditions ................................ ............................... 64 5.2.2 The Continuous to Batch Shift ................................ ................................ ........... 65 5.3 Results and Discussion ................................ ................................ ................................ 66 6 CONCLUDING REMARKS ................................ ................................ ................................ .. 69 APPENDIX A VISUAL BASIC PROG RAM FOR THE VAISALA GMT222 CO2 ANALYZER ............. 71 B RESULTS OF CARLO ERBA 1106 ELEMENTAL ANALYSIS OF E. COLI ML308 ...... 75 LIST OF REFERENCE S ................................ ................................ ................................ ............... 7 6 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 80

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7 LIST OF TABLES Table page 2 1 Minimal medium recipe used for all experimentation. ................................ ...................... 27 2 2. Volumes and concentrations of reagents used in the GDH assay. ................................ ..... 40 4 1 Frequency of data collection and approximate reactor volume taken during experiments ................................ ................................ ................................ ....................... 53 4 2 Growth rate, uptake rate, carbon dioxide evolution rate, and biomass yield before and during a batch mod e shift ................................ ................................ ................................ .. 57 5 1 Frequency of sample measurement / collection and approximate volume taken .......... 65

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8 LIST OF FIGURES Figure page 1 1 Example of a dilution rate shift up experiment .. ................................ ................................ 12 1 2 Conceptual model of a microb ial cell. ................................ ................................ ............... 14 1 3 D ilution rate dependence of biomass yield of microorganisms. ................................ ........ 16 1 4 D ilution rate dependence of the steady stat e glucose concentration. ................................ 16 1 5 D ilution rate dependence of the cellular RNA and protein content of microorganisms. ................................ ................................ ................................ ................. 17 1 6 D ilut ion rate dependence of the carbon dioxide e volution rate of microorganisms .......... 17 1 7 D ilution rate dependence of the glutamate dehydrogenase activity of microorganisms. ................................ ................................ ................................ ................. 18 1 8 Evidence for the transport enzyme limitation ................................ ................................ .... 19 1 9 Growth response of glucose limited cells exposed to glucose excess .............................. 21 1 10 Substrate uptake response of glucose limited cells exposed to glucose excess ................ 22 1 11 RNA content response of continuously grown Azobacter vineland ii to substrate excess. ................................ ................................ ................................ ................................ 24 1 12 The GDH activity response of continuously grown E. coli W to substrate excess. .......... 25 1 13 Response of a chemostat growing E. coli K 12 to a dilution rate shift up from D=0.2 to 0.6 hr 1 ................................ ................................ ................................ ........................... 26 2 1 Adaptation of E. coli ML308 to the glucose limited chemostat environment. .................. 30 2 2 Overall reaction mechanism of the enzyme glutamate dehydrogenase. ............................ 39 3 1 Steady state biomass yield of E. coli grown on glucose minimal media. .......................... 42 3 2 Steady state carbon dioxide evolution rate of E. coli grown on glucose minimal media ................................ ................................ ................................ ................................ 43 3 3 Steady State p rotein dry weig ht fraction of E. coli grown on glucose minimal media ..... 44 3 4 Steady state RNA dry weight fraction of E. coli grown on glucose minimal media. ........ 45 3 5 Initial growth rate response of E. coli grown on glucose minimal media ........................ 47

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9 3 6 Initial uptake rate response of E. coli grown on glucose minimal media. ......................... 47 4 1 Steady state c arbon mass fraction of dry weight measured in E. coli grown on glucose minimal media ................................ ................................ ................................ ..... 52 4 2 Results of a continuous to batch sh ift of a bioreactor growing E. coli continuously precultured at D=0.1 hr 1 ................................ ................................ ................................ ... 54 4 3 Results of a continuous to batch shift of a bioreactor growing E. coli continuously precultured at D=0. 3 hr 1 ................................ ................................ ................................ .. 55 4 4 Results of a continuous to batch shift of a bioreactor growing E. coli continuously precultured at D=0. 6 hr 1 ................................ ................................ ................................ .. 55 4 5 C omparison of the cell density evolution of the three preculture dilution rates. ............... 56 4 6 Comparison of the specific CO 2 evolution rate of the three preculture dilution rates ....... 56 4 7 A comparison of cellular carbon utilization patte rn during steady state and transient growth ................................ ................................ ................................ ............................... 58 5 1 D ilution rate shift up from the literatu re tracking growth rate and RNA level transient response ................................ ................................ ................................ ............................. 62 5 2 D ata from the literature showing that amino acids limit the growth of carbon limited cells exposed to excess glucose. ................................ ................................ ........................ 63 5 3 Data obtained from a continuous to batch shift of glucose limited E. coli precultured at D=0.3 hr 1 to excess glucose. ................................ ................................ ......................... 66 5 4 Data from Figure 5 3 converted to a per liter basis and scaled by their steady state value for the purpose of trend comparison. ................................ ................................ ....... 67 5 5 Oscillations present in growth rate of glucose limited cells exposed to excess glucose. ................................ ................................ ................................ .............................. 68

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10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DYNAMICS OF MICROBIAL GROWTH IN SINGLE SUBSTRATE CULTURE By Jason Noel August 2007 Chair: Spyros A. Svoronos Major: Chemical Engineering In this study the results of continuous to batch mode experiments conducted to characterize the initial transient response of carbon limited microbial cells growing in a chemostat to a pulse of the growth limiting substrate are presented. Changes in cellular rates of growth, substrate uptake, carbon dioxide evolution, and organic carbon excretion were recorded for three different preculture conditions. To ensure the validity of the da ta, ninety percent of added carbon was accounted for in generated biomass, evolved carbon dioxide, and in excreted organic products before and after the substrate pulse. The continuous shifts revealed that values of growth and respiration were proportional to the preculture dilution rate while the capacity to increase the respiration and growth rate was inversely proportional to the preculture dilution rate. Saturation of respiration and biosynthetic capacity led to a high amount of excretion at the interme diate and highest preculture dilution rates tested. Only the highest preculture dilution rate was able to utilize the excreted carbon before the specific carbon dioxide evolution rate fell considerably due to lack of substrate. The identity of the biosynth etic growth limitation was also explored in this study using similar continuous to batch shift experiments. Evidence was found to support the hypothesis that the biosynthetic limitation is an amino acid supply limitation and not a protein production

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11 capaci ty limitation. Continuous to batch mode shifts followed by a pulse of growth limiting carbon substrate show the transient response of the biosynthetic enzyme glutamate dehydrogenase qualitatively matched the transient response of the intracellular RNA conc entration suggesting the enzyme was more likely to be the cause of the biosynthetic growth limitation rather than the intracellular ribosome concentration.

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12 CHAPTER 1 GENERAL INTRODUCTION 1.1 Motivation for Research The chemostat is the best laboratory approximation to natural water bodies and industrial bioreactors. Naturally the transient response of a chemostat is a problem of significant biological and eng ineering interest. The simplest dynamics are obtained when a single microbial species is grown on a single growth limiting substrate. The dynamics are complex despite the simplicity of the system. The complexity arises from the ability of cells to adapt to changes in the environment around them. Certain intracellular components adapt to this change on a slow time scale of hours and even days. An example of the effects of this slow adaptation to environmental change can be seen in Figure 1 1. Figure 1 1. Dilution rate shift up from D=0.004 to 0.240 hr 1 of continuously grown K. aerogenes [Reprinted without permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of Aerobacter aerogenes at low dilution rates in a chemostat. Microbial Physiology an d Continuous Culture. Her Majesty's Stationary Office : 240 253. (Pages 360, Figure 2).] A least squares Monod Model (including death rate) fit of the cell density data and substrate concentration has been added. The error in the predicted cell density and a ctual cell density was minimized with non zero parameter value constraints and a upper limit of 0.8 for the yield. The Monod Model predicts the decrease in cell density but does so with unrealistic parameters values and a large amount of error in the react or substrate concentration. Clearly better models are needed to predict these kinds of transients.

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13 Upon an increase in the flow rate of feed in to the chemostat there is a pronounced transient decrease in the cell density and increase in the glucose conce ntration that ends fifteen hours after the dilution rate shift up. Transients such as these can lead to product deterioration in industrial bioreactors and regulatory violations in waste water treatment facilities as such systems can be disturbed with pert urbations to their feed flow rate. As seen in Figure 1 1 the estimated cell density time evolution based on the well known Monod Model does a poor job of predicting the transient response in microbial cells associated with an increase in the feed flow rate in to the chemostat. Better model based control of such systems would be necessary to lessen the impact of such transients in industrial systems. Based on a review of the experimental literature, conducted by Dr. Atul Narang, two slow variables were hypot hesized to control the dynamics of the chemostat: transport enzyme levels and biosynthetic capacity. Under certain operating conditions, the dynamics of the chemostat are controlled by only one of these slow variables. The goals of this research were two f old. The first was to characterize how microbial cells grown at a range of carbon limited growth rates respond to environmental conditions brought about by a feed rate increase perturbation. The second goal was to identify the intracellular cause of this b iosynthetic limitation. 1.2 Conceptual Model of the Cell Figure 1 2 is the conceptual model of growth used to design the experiments completed in this work. The dashed line corresponds to the boundary between the external and internal environments of a cel l. Carbonaceous substrate, denoted S enters the cell through transport enzymes, denoted E Typically microbial cells will modify sequestered substrate to an internalized form denoted by X There is possible positive feedback as internalized substrate can induce the synthesis of more of the transport enzyme responsible for the uptake of the substrate as seen with the lac operon. The internalized substrate is converted to a pool of precursor

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14 catabolites denoted by P There is negative feedback inhibition pos sible here as precursor catabolite excess could inhibit the uptake of more substrate. The precursor catabolites typically have three possible fates. These products of substrate catabolism can be either channeled into additional cell mass, fully oxidized to carbon dioxide, or excreted as a partially catabolized organic molecule. Storage of internalized carbohydrates such as glycogen is denoted by Ps and excretion of partially catabolized substrate is denoted by Px Synthesis of protein is denoted by C The designations GDH aa and RNA stand for glutamate dehydrogenase, amino acids, and RNA, respectively. Figure 1 2. Conceptual model of a microbial cell. 1.3 Steady state behavior of microbial cells For a given microbial species, growth limiting substrate, media compositio n, pH and temperature the steady states of a chemostat depend on two parameters: the substrate feed concentration and the residual substrate concentration of the reactor bulk. If the substrate feed concentration is increased at a fixed dilution rate, ther e is no change in the steady state substrate concentration. An increase in the feed concentration is compensated by a proportional increase in the cell density (41) Since the cellular steady state is completely determined by the state of the environment, the steady state values of the cellular variables must also be independent of the

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15 feed concentration. Variation of the steady states with respect to the dilution rate produces more complex results. Located in Figure 1 3, Figure 1 4, Figure 1 5, Figure 1 6, and Figure 1 7 are experimental data from the literature illustrating the dilution rate dependence of some common cellular variables in a carbon limited chemostat. 1.4 Cell density and Substrate Concentration Cells wash out at both small and large dilution rates. The lower washout dilution rate, called the minimum growth rate, has been argued to be caused by the specific uptake of glucose not being able to meet the minimum maintenance requirement of the cells. Tempest et al. found steady states of glycerol limited cultures of K. aerogenes at dilution rates as low as 0.004 hr 1 and found that the specific growth rate of the viable cells approached a minimum value of 0.009 hr 1 (33) The cell density passes through a maximum between the two washout dilution rates and since the steady state substrate concentration equals the substrate feed concentration at dilution rates above and below the two w ashout dilution rates the substrate concentration would pass through a minimum value. The data from Figure 1 4 shows the substrate concentration appears as an increasing function of the dilution rate.

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16 Figure 1 3. Example of dilution rate dependence of b iomass yield of microorganisms. The biomass yield data was from K. aerogenes grown on minimal media. [Reprinted without permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of Aerobacter aerogenes at low dilution rates in a chemostat. Microbia l Physiology and Continuous Culture. Her Majesty's Stationary Office : 240 253.1965 (Pages 361, Figure 3), Microbiological Research Establishment, Porton, Salisbury, Wilks, UK.] Figure 1 4. Example of dilution rate dependence of the steady state glucose co ncentration. The glucose data was from E. coli ML308 grown on minimal media. [Reprinted without permission. Senn, H., U. Lendenmann, M. Snozzi, G. Hamer, and T. Egli. 1994. The growth of Escherichia coli in glucose limited chemostat cultures: a re examina tion of the kinetics. Biochim Biophys Acta 1201: 424 36 (Page 428, Table 2). EAWAG, Dubendorf, Switzerland.]

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17 Figure 1 5. Examples of dilution rate dependence of the cellular RNA and protein content of microorganisms. Both sets of data were from A. aerog enes grown on minimal media. [Reprinted without permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of Aerobacter aerogenes at low dilution rates in a chemostat. Microbial Physiology and Continuous Culture. Her Majesty's Stationary Office : 240 253. (Pages 361, Figure 3).] Figure 1 6. Example of dilution rate dependence of the carbon dioxide evolution rate of microorganisms. The carbon dioxide evolution rate data was from E. coli K 12 grown on minimal media. [Reprinted without permission. Han K., H. C. Lim, and J. Hong. 1992. Acetic Acid Formation in Escherichia Coli Fermentation. Biotechnology and Bioengineering 39: 663 671. (Page 666, Figure 2).]

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18 Figure 1 7. Example of dilution rate dependence of the glutamate dehydrogenase activity of mic roorganisms. The glutamate dehydrogenase activity was from E. coli W grown on minimal media. [Reprinted without permission. Senior, P. J. 1975. Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous cult ure technique. J Bacteriol 123: 407 18 (Page 413, Figure 5).] 1.5 Cellular Content and Excreted Metabolites RNA and protein are the major cellular constituents comprising 80 85% of the dry weight of the cell. As the dilution rate increases, the RNA concent ration increases at the expense of the protein concentration. Carbohydrate concentration varies markedly with the identity of the microbial species and the carbon source. For a given microbial species and carbon source, the carbohydrate content is almost i ndependent of the dilution rate (29) Excreted metabolite concentrations also vary significantly depending on identify of the microbial species and the carbon source. Glycerol limited cultures of K. aerogenes show no measurable excretion of acetate at all dilution rates between 0.004 and 0.85 hr 1 (33) In contrast glucose and pyruvate limited cultures of E. coli show a high degree of excretion at high diluti on rates (11, 16, 17) 1.6 Transients Controlled by the Transport Enzymes Th e role of transport enzymes in chemostat dynamics is seen by switching the growth limiting substrate identity in the reactor feed. In this experiment a chemostat is allowed to reach

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19 a steady state at some dilution rate and substrate feed concentration. Aft er steady state is reached the growth limiting substrate is abruptly switched. If the transport enzymes for the new substrate are synthesized inducibly, their levels will be small when the switch occurs. Located in Figure 1 8 is an example of a transport e nzyme controlled transient taken from the literature. Figure 1 8. The results of glucose to n itrilotriacetate substrate switch experiment performed on a continuous culture of Chelatobacter heintzii grown on minimal media. [Reprinted without permissio n. Bally, M., and T. Egli. 1996. Dynamics of Substrate Consumption and Enzyme Synthesis in Chelatobacter heintzii during Growth in Carbon Limited Continuous Culture with Different Mixtures of Glucose and Nitrilotriacetate. Appl Environ Microbiol 62: 133 140 (Page 135, Figure 2 ).] A theoretical washout curve was added to illustrate the predicted cell density change assuming no cell growth. When the carbon source is switched from glucose to n itrilotriacetate (NTA) there is little to no growth for nearly twen ty hours as shown by the cell density matching the theoretical washout curve during this time. The cells simply cannot take up the NTA present in the environment as shown by the lack of NTA transport enzyme activity seen during the first twenty hours. This initial lack of transport enzyme causes the substrate NTA to accumulate in the reactor until the transport enzyme reaches a level that can support growth. The transport enzyme for NTA is produced at extremely low rates under glucose limited conditions hen ce the low rate of transport

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20 enzyme activity seen at the start of the experiment. The transport enzyme level is nearly zero for twenty hours and reaches fifty percent of its final value in the following twenty hours. The rapid buildup of transport enzyme after forty hours suggests the synthesis of the enzyme is autocatalytic. Evidence from molecular biology shows that enzyme induction is not only autocatalytic, but also cooperative, since inhibition of the repressor requires that two inducer molecules to b ind to it in the case of the lac operon repressor (46) Switching the growth limiting substrate from NTA to glucose produces a different transient behavior. Rapid consumption of glucose begins immediately after the switch occurs because glucose transport enzymes are constitutively produced keeping these transport enzymes at high levels even in the absence of glucose Still little to no growth was seen for the first two to three hours of the switch from NTA to glucose (2) An appropriate question might be what is prevent ing the specific growth rate from a djusting instantaneously as the cell is taking up substrate yet not growing ? Similar results were found when continuous cultures of E. coli (43) and K. aerogenes (36) were subjected to a substrate sw itch from glucose to xylose. For many hours there was a dramatic increase in the level of xylose and a pronounced decline in the cell density. These experiments imply that when the initial level of the inducible transport enzyme is low, the transient behav ior is controlled by the transport enzyme synthesis rate. There is little substrate uptake, hence little growth, until the enzyme level reaches a sufficiently high level. 1.7 Transient Dynamics Controlled by a Biosynthetic Limitation Continuous to batch sh ifts of glucose limited cells reveal the role of biosynthesis in limiting microbial growth. In the experiments shown in Figure 1 9 and Figure 1 10, a sample of glucose limited cells precultured in a chemostat are immediately exposed to supersaturating conc entrations of glucose in a batch reactor and the initial rates of various processes were measured. As seen in Figure 1 9 and 1 10, the growth rate and substrate uptake rate both increase

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21 upon exposure to excess glucose regardless of the preculture dilution rate at which the cells had been growing (22) A similar response has been seen in other studies as well (28, 32) This rapid incr ease occurs because the transport enzymes for glucose are constitutive so they are always present at high levels. The data from the figures shows the substrate uptake rate increases to a maximal level regardless of preculture dilution rate whereas the grow th rate increase is highly dependant on the preculture dilution rate. Appropriate questions here would be what is limiting the growth rate as the cell has excess substrate available and how is the cell utilizing excess substrate if it is not being channele d in to growth? Figure 1 9. Results of a continuous to batch shift where cells of E. coli ML308 grown in a glucose limited chemostat on minimal media were exposed to excess glucose. The transient growth rates were measured after thirty minutes of growth [Reprinted without permission. Lendenmann Phd dissertation (Page 72, Figure 8.1), EAWAG, Dubendorf, Switzerland.]

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22 Figure 1 10. Results of a continuous to batch shift where cells of E. coli ML308 grown in a glucose limited chemostat on minimal media we re exposed to excess glucose. The transient substrate uptake rates were measured during the first 5 7 minutes after the glucose pulse was administered. [Reprinted without permission. Lendenmann Phd dissertation (Page 72, Figure 8.1), EAWAG, Dubendorf, Swit zerland.] The specific biosynthesis rate (of RNA and protein) depends on the dilution rate at which the cells are growing before their withdrawal from the chemostat (18) This was shown in experiments whe re glycogenless mutants of E. coli B were exposed to saturating concentrations of glucose and the initial specific growth rate were measured. Since these mutants were unable to synthesize their major storage carbohydrate glycogen, the observed specific gro wth rate was roughly equal to the specific biosynthesis rate of only RNA and protein. Based on the measurements of the initial growth rates in these mutants it was concluded that at low dilution rates (D<0.3 hr 1 ) the specific biosynthesis rate of microbes increases rapidly but does not reach the maximum value for the strain and for high dilution rates the specific biosynthesis rate does not improve at all immediately. It stands to reason that in wild type cells there is an absence of rapid improvement in RNA and protein synthesis at the higher dilution rates. When wild type cells of E. coli drawn from a chemostat are exposed to saturating concentrations of glucose the specific growth rate,

PAGE 23

23 increases rapidly regardless of the dilution rate at which the cel ls have been growing implying that the increase is solely due to glycogen synthesis. The synthesis of glycogen in wild type cells is not surprising. The above experiments imply that when cells growing at large dilution rates are exposed to saturating con centrations of glucose, the substrate instantly enters the cells, but cellular metabolites derived from it cannot be completely channeled in to biosynthesis of RNA and protein. A large portion must therefore be respired, excreted, or stored. The specific c arbon dioxide evolution rate rapidly increases to maximal levels (levels seen at near washout dilution rates) (18, 30) The specific excretion rate (10, 12) and, if applicable, the specific glycogen synth esis rate (1 0, 19) increase to high levels when exposed to saturating glucose conditions. These experiments imply that if the initial level of transport enzyme is high, the transients are controlled by biosynthesis rather then by a transport limitation. There is a pr onounced substrate uptake, respiration, storage, and excretion but limited or no biosynthesis until more biosynthetic capacity has been synthesized. So what prevents the biosynthesis rate from instantly increasing to maximal levels upon exposure to exces s substrate? There are two possibilities: either one or more of the biosynthetic enzymes is saturated with their substrate(s) or the ribosomes are saturated with amino acids. Substrate saturation of a biosynthetic enzyme whose product(s) are required for g rowth would represent a rate limiting step. Biochemical pathways necessary for growth that require the products of the saturated biosynthetic enzyme will be forced to adjust their rate of reaction based on the rate of supply of products from the saturated biosynthetic enzyme. Similarly ribosomes saturated with their amino acid substrates would also limit growth as ribosomes generate the proteins that comprise the majority of cell mass. It is generally believed that the ribosome concentration is what limits biosynthesis (5, 31) ; however, there is evidence to suggest that

PAGE 24

24 saturation of biosynthetic enzymes, rath er than ribosome concentration, prevents the biosynthesis rate from instantly increasing to the maximum level. It has been seen that the addition of amino acids to a culture results in the rapid acceleration of protein synthesis (6, 20, 24) which implies that the ribosomes are in fact not saturated with amino acids. This would suggest the limitation lies with the supply of amino acids to the ribosomes and not the ribosomes themselves. A possible explanation for this response to increased protein synthesis upon exposure to excess amino acids could b e the substrate saturation of the biosynthetic enzyme glutamate dehydrogenase (GDH). Under carbon limited conditions this enzyme is the major pathway for incorporation of inorganic nitrogen in to the cell (26, 40) and could be a limiting step in the rate of amino acid synthesis expla ining why the ribosomes are not saturated. Figure 1 11 and Figure 1 12 show the transient response of RNA level and GDH activity, respectively in continuously grown microbial cells exposed to excess substrate. Figure 1 11. The RNA content response of con tinuously grown Azobacter vinelandii to substrate excess conditions. The bacteria were precultured on minimal media at three different dilution rates: 0.10, 0.15, and 0.20 hr 1 [Reprinted without permission. Nagai, S., Y., Nishizawa, I. Endo, and S. Aiba. 1968. Response of a chemostatic culture of Azobacter vinelandii to a delta type pul s e of glucose. J. Gen. Appl. Microbiol. 14: 121 134. ( Page 125, Figure 2). ]

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25 Figure 1 12. The GDH activity response of continuously grown E. coli W to substrate excess con ditions. The bacteria were precultured on minimal media. [Reprinte d without permission. Harvey, R. J. 1970. Metabolic regulation in glucose limited chemostat cultures of Escherichia coli. J Bacteriol 104: 698 706. (Page 704, Figure 10).] The slow accumulat ion of RNA (18, 27, 48) and GDH (18) after a continuous to batch shift follows sigmoidal kinetics. This suggests that the synthesis of GDH and RNA is autocatalytic. Such kinetics probably occur because an increase in the activity of GDH increases t he level of amino acid monomers. This stimulates the production of RNA and ribosomes resulting in the synthesis of even more GDH and RNA. 1.8 Transient Response to Dilution Rate Shift Ups In practice, chemostats regularly experience dilution rate and feed concentration shift ups. Laboratory studies have mos tly been concerned almost exclusively with dilution rate shift ups. In these experiments the chemostat is allowed to reach steady state at some dilution rate and then is abruptly increased. There is substantial literature on this topic (1, 4, 9, 13, 33, 42, 43, 48) but the cellular variables were measured in relatively few studies (4, 33, 48) The cellular studies suggest that when dilution rate shift ups are small, the transients are limited by the biosynthetic machinery, and when the shift ups are large the transients are limited by transport enzymes.

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26 Located in Figure 1 13 is th e response of a glucose limited culture growing inside a chemostat to a large dilution rate shift up. Herbert exposed a glycerol limited culture of K. aerogenes to a dilution rate shift up from D=0.004 to D=0.24 hr 1 which corresponds to a sixty fold incr ease in the substrate input rate. The transient response suggests that in the first few hours, there was a significant increase in the transport enzyme level. The initial rapid jump in the substrate uptake rate is due to saturation of the substrate uptake enzymes and the relatively slow increase after the initial jump is due to the gradual buildup of transport enzyme level because the specific substrate uptake rate is proportional to the transport enzyme level whenever the substrate concentration is saturat ing. The data suggests that the transport enzyme levels reach a maximum five hours after the shift up. During this time the RNA level is nearly constant. It increases significantly after the transport enzymes have peaked, and reaches the maximum more then ten hours after buildup (19) Figure 1 13. Response o f a chemostat growing E. coli K 12 to a dilution rate shift up from D=0.2 to 0.6 hr 1 Minimal media was used during the experiment. [Reprinted without permission. Yun, H. S., J. Hong, and H. C. Lim. 1996. Regulation of ribosome synthesis in Escherichia co li: Effects of temperature and dilution rate changes. Biotechnology and Bioengineering 52: 615 624. (Page 619, Figures 4 and 5).]

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27 CHAPTER 2 MATERIALS AND METHOD S 2.1 Growth Medium The growth medium was prepared with deionized water in 20 L polypropylene bottles. The medium used for these experiments was a modification of a recipe used by Lendenmann et al (22) The recipe is shown in Table 2 1. Before sterilizing the medium, it was supplemented with glucose and the pH was adjuste d to 3 with concentrated H 2 SO 4 The low pH prevents caramelization of the glucose during sterilization and back contamination of the feed line during operation of the chemostat Table 2 1 Minimal medium recipe used for all experimentation Component Conc (M) 1L (g) Potassium Phosphate (mono) 2.00E 02 2.7218 Ammonium Chloride 1.40E 02 0.7489 EDTA 2.20E 04 0.0819 Magnesium Sulfate Heptahydrate 2.30E 04 0.0567 Sodium Molybdate Dihydrate 1.00E 05 0.0024 Calcium Chloride Dihydrate ** 1.00E 04 0.0147 Manganese Chloride Tetrahydrate 2.50E 05 0.0049 Zinc Chloride 1.25E 05 0.0017 Cupric Chloride Dihydrate 5.00E 06 0.0009 Cobalt Chloride Hexahydrate 5.00E 06 0.0012 Ferric Chloride Hexahydrate 7.50E 06 0.0020 Note: Original source was Lendenmann 1995. PhD dissertation (Page 35, Table 4.1) Note: Adjusted reactor (not shake flask) media to pH 3 with reagent grade sulfuric acid Note: Replaced calcium carbonate with calcium chloride due to carbonate / acid reaction Note: Removed boric acid from me dia because borate adheres to the HPLC column 2.2 Bacterial Strain The microorganism used in this study was Escherichia coli ML308 (ATCC 15224) obtained from the American Type Culture collection. Stock cultures were prepared by freezing cells grown on co mplex media that had just entered stationary phase growth. The strain was preserved at 20C in 30 ml vials containing 50% of the culture, 50% glycerol cryoprotectant, and

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28 glass beads. The culture was resuscitated for experimental work by sterilely removing a glass bead from the stock culture and adding it to a shake flask containing the growth medium described below supplemented with 1 g/L glucose (35) 2.3 The Chemostat Setup The chemostat cultures were grown in a 1. 5 L Bioflow III f ermenter (New Brunswick Scientific Co.) with a working volume of 1 .2 L. The agitation speed was 1000 rpm and the aeration rate was 1.2 L/min. The bioreactor was equipped with automatic pH and temperature control. The pH was maintained at 7.0 + / 0.1 by add ition of 1M KOH / 1M NaOH solution and 10% H 3 PO 4 solutions. The temperature was maintained at 37 o C. The feed was pumped in by a Maste r flex L/S peristaltic pump (7523 70) equipped with a Masterflex EZ Load pump head (7534 04). 2.3.1 Wall Growth Limitation T he glucos e concentration in the reactor feed was kept at 200 mg/L or below Higher feed concentrations resulted in significant wall growth within a week of inoculating the reactor. Under higher feed conditions, the initial specific growth rates after conti nuous mode to batch mode shifts were observed to be significantly lower than the dilution rate at which the cells had been growing in the chemostat This probably reflects the fact that in the presence of wall growth, the steady state specific growth rate is lower than the dilution rate, since cells shearing off the wall become an additional source of cells inside the bioreactor (21) The reason for this lowerin g of the growth rate due to wall growth can easily be seen in the steady state cell mass balance, seen in Equation 2 cells. (2 1)

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29 The variables D, the growth rate of the cells in the bulk, and the rate of cell input in to the bulk from wall growth, zero then rg must be depressed to keep the chemostat operating at steady state. At feed concentrations of 100 mg/L of glucose, the reactor could be operated for up to month without significant wall growth. 2.3.2 Glucose Adaptation Limitation The glucose concentration is one of the last variables to achieve a steady value inside of a glucose limited chemostat. The time scale of this transient is on the order of days suggesting a genetic adaptation to low glucose levels. This makes sense from a natural selection point of view as those cel ls which possess a higher capacity for uptake for the growth limiting nutrient would grow faster then cells with a lower capacity for uptake in the chemostat environment. Eventually the better adapted mutant cells would out compete and take over the reacto r bulk given enough time. One such study found in the literature supported this idea (45) A deregulation mutation was fo und inside glucose limited E. coli cells grown in a chemostat that affected the transcription of a cell membrane glucose porin. Given enough time and selective pressure the cells would start producing additional glucose porin proteins that are normally not expressed under glucose limited conditions (45) These additional porins would facilitate glucose transport in to the cell and could confer a selective advantage over non adapted cells. A glucose adaptation experiment was conducted using the laboratory chemostat to glucose limited chemo stat post a shift to D=0.6 hr 1 The glucose concentration was measured and compared with a similar study found in the literature using the same strain of bacteria and a similar media composition (22) The results of this comparison are shown in Figure 2 1.

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30 Both glucose evoluion trend lines look extremely similar with a monotonic decrease in the conce ntration of glucose down to the same steady value. The time it took the lab system to achieve a steady glucose concentration was four days at D=0.6 hr 1 or 85 cell doublings. The experiments conducted in this research were sensitive to the glucose uptake c apacity thus this transient behavior introduced variation into collected data. This reproducibility problem was corrected by giving newly inoculated reactors four days time at D=0.6 hr 1 before any experiments were attempted to allow for microbial adaptati on to low glucose levels. This operational change increased the quality of the experimental data gathered immensely. Figure 2 1 Adaptation of E. coli ML308 to the glucose limited chemostat environment. The cultivation used minimal media and a glucose con centration of 100 mg/L. Substrate measurements were made daily post shifting the chemostat to D=0.6 hr 1 The comparison data was measured using E. coli ML308 grown on glucose minimal media. [Comparison data reprinted without permission. Lendenmann Phd dis sertation. (Page 44, Figure 5.1). EAWAG, Dubendorf, Switzerland.] 2.4 Cell Density Measurement and Growth Rate The concentration of cells inside the reactor at any time was determined by an absorbance measurement at 546 nm using a Spectronic Genesys 10UV s pectrophotometer. In order to ensure an accurate reading the 1 ml cuvette was given approximately 45 seconds to equilibrate before the value was recorded (15) Specific growth rates of cells growing exponentially were

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31 determined by fitting exponential curves over the time frame of inter est. The fitted equation takes the form seen in Equation 2 2. (2 2) The variables Co, C, and t are the initial cell density, final cell density, the growth rate, and time, respectively. 2.5 Sugar Measurement, Yield Determina tion, and Uptake Rate Sugar concentrations were measured with a Dionex 500 HPLC equipped with an anion exchange column (CarboPac PA10) and a pulsed electrochemical detector. In this method, the sugars are ionized by using a strong base as eluent (10 mM NaO H), separated based on their differential affinity for the anion exchange column, and detected by the pulsed amperometric detector. The manufacturer's protocol resulted in a rapid increase of retention times, presumably due to adsorption of bicarbonate on the column. The addition of 1 mM Ba(OAc ) 2 to the eluent, as recommended by (7, 8) precipitated the bicarbonate as Ba(CO 3 ) 2 The modified eluent dramatically improved the reproducibility and precision The sugar biomass yield of a culture is simply the amount of cells produced per amount of sugar consumed. The bio mass yield was determined for a time interval during exponential growth by plotting the cell density versus sugar concentration and using the slope of the linear fit. The final equation for yield determination during a given time interval presented as Equa tion 2 3. (2 3) The variables Y and (dC/dS) correspond to the biomass yield and the slope of the cell density versus sugar concentration plot. This method assumes that the biomass yield is constant over the time interval of inte rest. The slope of the linear fit trend line corresponds to the biomass yield

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32 during that time interval. The specific uptake rate of sugar during the time interval was determined by dividing the specific growth by the biomass yield. The final equation for the substrate uptake rate is presented as Equation 2 4. This equation assumes that the death or decay rate of cells is negligible for the purposes of calculating the specific substrate uptake rate. (2 4) The variables rs, rg, a nd Y are the specific uptake rate of sugar, the specific growth rate, and the biomass yield, respectively. 2.6 Total Cell Protein Extraction and Measurement 2.6.1 Extraction Total cell protein was extracted using a high temperature / low pH extraction meth od (15) These conditions denatu re cellular protein and therefore the protein product could not be used for enzymatic assays. Cells were harvested via low temperature centrifugation, washed with 0.15M sodium chloride sodium citrate solution (SSC), and digested with 0.5M sodium hydroxide (NaOH) at 100 o C. The harsh conditions of the digestion step solubilize the cellular protein. After centrifugation, the supernatant was retained and an equivalent volume and molar strength of trichloroacetic acid (TCA) was added to adjust the pH to a neutra l value. 2.6.2 Measurement Protein detection and quantification was preformed with colorimetric detection using a bicinchchoninic acid (BCA) kit available from Pierce Biotech (34) The color change reaction occurs from amino acid (cysteine, cystine, tryptophan, and tyrosine) catalyzed reduction of Cu 2+ to Cu 1+ The resulting BCA Cu complex exhibits a strong absorbance at 562 nm that shows a near linear response with protein concentr ation over a broad range. Bovine serum albumin was used as a standard to generate a calibration curve for cell protein quantification.

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33 2.7 Total Cell RNA Extraction and Measurement 2.7.1 Extraction Total cell RNA was extracted using a cold / hot TCA treat ment (15) Cells were harvested via low temperature centrifugation, washed with 0.15M SSC, and digested with 0.25 TCA at 0 o C. These digestion conditions lyse the cell and cause cellular RNA to precipitate as a solid. The insoluble fraction of the lysate was retained and resuspended in 0. 5M TCA and placed in a 70 o C water bath. The treatment solubilizes RNA but leaves other macromolecules such as proteins and lipids in a solid form. 2.7.2 Measurement RNA detection and quantification was performed with colorimetric detection using a slightl y modified acidified orcinol method (23) Solutions of ferric chloride dissolved in HCl and orcinol dissolved in ethanol are combined in a 50/50 ratio and added to the extracted RNA solution generated by the cold / hot TCA treatment. The RNA in the extract reacts with acidif ied orcinol producing a green chromogen that exhibits a strong absorbance at 665 nm that shows a near linear response with RNA concentration. Yeast RNA was used as a standard to generate a calibration curve for cell RNA quantification. 2.8 Carbon Dioxide E volution Measurement The carbon dioxide evolution of the chemostat contents was measured with a Vaisala GMT222 Infrared CO 2 analyzer. The device was connected to a computer for continuous data acquisition. This was especially useful for detecting any stead y state perturbations to ensure the initial conditions of the chemostat and bacteria growing within were acceptable before experiments were attempted. Special software for the CO 2 analyzer was written to allow for this continuous acquisition and recording. The code for this software appears in Appendix A.

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34 To ensure a rapid analyzer response time to the state of the chemostat all effluent air from the reactor system was passed through the CO 2 analyzer to ensure a rapid analyzer response time to changes in the chemostat condition. The air and liquid overflow outlets were the same overflow line in the chemostat setup so an air/liquid separator was created to isolate the two phases. The overflow line was connected to a 50 mL Erlenmeyer flask with two outlets. The air phase was forced up through an exit line at the top of the flask to the analyzer while the liquid phase fell to the bottom of the flask. The liquid phase was actively pumped out to a waste tank to prevent the flask from overflowing. The setup allow ed for detector response times on the order of seconds rather then minutes to changes in the chemostat carbon dioxide level. The detector output was recorded as a concentration of carbon dioxide in parts per million. The device came from the manufacturer p recalibrated but was checked periodically with specially made calibration gas. 2.9 Total Organic Carbon Measurement Total organic carbon measurements (TOC) were made with a Phoenix 8000 TOC Analyzer to quantify the amount of carbon excreted by cells in the form of organic acids or alcohols. Samples taken from the chemostat during an experiment contained excreted organic carbon as well as organic carbon from residual sugars and from the chelating agent EDTA present in the original media. The carbon contribut ion of the residual sugars was accounted for by first measuring the sugar concentration in the extracted sample via HPLC measurement and then contribution from EDTA was acc ounted for by measuring the TOC of the original media (before remaining TOC of a sample was modified forms of organic carbon because there were no other known sources of org anic carbon input in to the chemostat bulk.

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35 The Phoenix 8000 TOC Analyzer used UV initiated peroxydisulfate free radical chemistry to fully oxidize organic carbon to carbon dioxide (47) A nitrogen carrier gas transports the evolved carbon dioxide to an infrared probe fo r detection. TOC calibration curves were generated using a glucose standard. 2.10 Elemental Content Measurement Elemental analysis of dry cell mass was performed on a Carlo Erba 1106 Elemental Analyzer to determine the carbon content of cells for the purpo se of conducting carbon balances around the laboratory chemostat. Cell mass was collected and concentrated with low temperature (4 o C) centrifugation. The resulting cell pellet was washed with cold deionized water to remove excess salts from the cell mass t hat could skew the elemental analysis results. The washed cell pellet was incubated overnight at 80 o C to fully desiccate the cell biomass. The resulting dry mass was pulverized to a coarse powder prior to elemental analysis. The Carlo Erba 1106 Elemental Analyzer determined the H, N, and C content of the dry cell mass by using high temperature oxidation to convert organic H, N, and C to H 2 O, NO 2 and CO 2 respectively The evolved gases were quantified and compared with the initial dry cell mass to determi ne the percent content of the three elements. 2.11 Cell Disruption Techniques In order to test the enzymatic activity of intracellular proteins, cells must be disrupted in a way that does denature or inhibit the activity of cellular proteins. Two methods were used in this research to extract proteins with little deviation from the original media pH, non denaturing temperatures, and protease inhibitors to keep enzymatic activity as close to harvest conditions as possible.

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36 2.11.1 Chemical Disruption The Che mical Disruption method uses a surfactant and chicken egg white lysozyme to disrupt cells without the need for excessive mechanical force (15) First extracted cells were concentrated and washed using low temperature (4 o C) centrifugation and 0.15M SSC. The cell pellet was resuspended in 2 mL ice cold 50 mM Tris lysing buffer (pH=7.6) containing 100 mM NaCl, and 1 mM EDTA. This buffer has an osmolarity and pH at a level similar to chemostat conditions and more importantly will keep the pH constant during the extraction of prevent denaturin g of extracted proteins. The EDTA is present to scavenge any trace heavy metals that can inactivate enzymes by adhering to active or cofactor sites on the protein. The temperature of the disruption vial was kept at 0 o C for the duration of the disruption pr ocedure using an ice water 40) was then added to the suspension and vortexed vigorously (30 seconds) to permeabilize the outer membrane of the bacterial cells. The cell suspension was again centrifuged (4 o C) and resuspended in 0.11 ml of Tris lysing buffer. A vortexed and placed in the ice water bath for 20 minutes. After the sucrose equilibration a small volume ( disruption vial. The vial was vortexed and placed in the ice water bath for 20 minutes. The chicken egg white lysozyme weakens bacterial cells by degrading the peptidoglycan presen t in their cell walls. The sucrose added to the cell suspension prior to the lysozyme treatment helps force the lysozyme into the cell membrane holes made by the initial surfactant treatment. After the lysozyme digestion, cold deionized water (0.535 mL) wa s added to the cell suspension and vortexed. Next more of the surfactant, 10% NP 40 (0.179 mL), was then added to the disruption vial. Upon vortexing the suspension clears and the non soluble components of the cell are

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37 removed with low temperature (4 o C) ce ntrifugation. The supernatant was saved and contained the solubilized cell proteins in a non denatured state (38) 2.11.1 Chemical Disruption Drawbacks The supernatan t product from this technique is extremely viscous due to nucleic acid entanglement of liberated DNA. The viscosity of the supernatant made extracting accurate volumes with a pipette extremely difficult. Literature sources recommended sheering the crude ly sate by drawing it through a syringe but this proved to be a less then ideal solution (15) The enzyme DNAse was recommended as an alternate remedy for this problem but was never implemented as a solution as there was a larger problem with the chemical disruption technique. Of the dilut ion rates tested, significant cell lysis only occurred for dilution rates of 0.3 hr 1 or greater. When the chemical lysis technique was attempted with cells growing at D=0.1 hr 1 little to no cell lysis was observed. The reason for this resistance to chem ical lysing was thought to be due to the starvation response of E. coli (25) As the dilution rate of a carb on limited chemostat decreases the carbon substrate available to growing cells at steady state decreases as seen in Figure 1 4. Depending on the degree of starvation the cell may start activating stationary phase genes to help the organism persist in the n utrient limited environment in the hope that conditions conducive to growth will occur in the future. Formation of an extensive glycocalyx capsule around the starved cell is thought to be the source of resistance to chemical disruption (25, 29) It was hypothesiz ed that the extensive capsule created around a starved cell provides a barrier around the cell protecting the outer membrane and cell wall from surfactant and lysozyme attack effectively preventing chemical disruption. A more effective technique was found for the disruption of highly starved cells.

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38 2.11.1 Mechanical Disruption The Mechanical Disruption method uses sheer forces generated by vigorously vortexing cells with micron size glass beads (3, 39) The shear force generated by the rapidly moving glass beads leads to cell disruption in even the most robust of microorganisms (37) First extracted cells are concentrated and washed using low temperature (4 o C) centrifugation an d 0.15M SSC. Cells are resuspended in 0 .3 mL of ice cold 50 mM Tris disruption buffer (pH=7.6) containing 100 mM NaCl, and 1 mM EDTA. Ice cold glass beads (100 cell suspension on a w/v basis. The cell suspension was disrupted by vortexing it vigorously for six minutes at 20 o C. This was achieved by placing a table top vortex inside the lab freezer and modifying the device to hold disruption vials for the purpose of hands free vortexing. The subzero temperature ensured there was no increase in temperature due to mechanical energy input that might denature the cellular proteins. The cell suspension was diluted with 0.7 mL of ice cold disruption buffer after the vortexing step. The non soluble components of the cell were removed from the disrupted cell suspension w ith low temperature (4 o C) centrifugation. The supernatant was saved and contained the solubilized cell proteins in a non denatured state (44) 2.11.2 Mechanical Disruption Advantages The mechanical disruption technique solved many problems associated with the chemical disruption technique. Significant cell disruption was possible at all te sted dilution rates using the mechanical technique. The resulting crude lysate from the mechanical technique had a much lower viscosity then the crude lysate from the chemical technique due simply to the shearing action of the rapidly moving glass beads al lowing for more accurate volume extraction. Additionally the mechanical technique required the addition of nothing other then glass beads and a protease inhibitor solution to the cells being disrupted. The addition of surfactant, lysozyme enzyme, and other reagents in the chemical disruption technique affected the results of

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39 the protein assay and may have had an effect on cellular enzyme activity measurements. Overall the mechanical disruption technique was superior to the chemical disruption technique for the experiments being conducted in the laboratory. 2.12 Enzymatic Assay of Glutamate Dehydrogenase The enzyme glutamate dehydrogenase (GDH) is the protein responsible for the majority of inorganic nitrogen incorporation in E. coli under carbon limited cond itions (26) Specifically the ketoglutarate to the amino acid glutamate. The exact reaction can be seen in Figure 2 2. Figure 2 2 Overall reaction mechanism of the enzyme glutamate dehydrogenase. The reaction can procee d in the forward or reverse reaction depending on the concentrations of the reagents and products. The enzymatic activity of GDH in the crude lysate extracted with the chemical and mechanical disruption methods was measured by recording the consumption of NADPH (340 nm) over 15 minutes using a Beckman DU7500 spectrometer with all reagents in excess. The enzymatic assay was performed in 1 mL cuvettes at a temperature of 25 o C and a pH of 7.6. The contents of the 1 mL reaction volume are shown below in Table 2 2. All reagents were made in 50 mM Tris Buffer and adjusted to pH=7.6 with 1 M HCl and 1 M NaOH.

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40 Table 2 2 Volumes and concentrations of reagents used in the GDH assay. Vol. (mL) Reagent (pH=7.6) 0.6 50 mM Tris Buffer 0.1 50 mM alpha ketoglutarate 0.1 2.5 mM NADPH 0.1 40 mM NH 4 Cl 0.1 Lysate from Cell Disruption The crude lysate showed background NADPH activity that could not be attributed to the GDH activity of the sample. Control trials, where all reagents were present except NH 4 Cl, were run al ongside experimental trials to account for this excess NADPH consumption activity. The net rate of NADPH consumption was used to calculate the activity of the protein samples. The activity was expressed in micromoles of NADPH consumed per minute per millig ram of protein. 2.13 Deactivation and Disposal of Microorganisms By requirement of the Environmental Health and Safety Department of the University of Florida all used media and cellular material had to be deactivated before disposal. Reactor effluent was collected in a 50 gallon waste tank and subjected to overnight chlorination before being drained in to the building water disposal system. All other cellular material was autoclaved for 15 minutes at 121 o C before being disposed. Any accidental spills or c ontamination were first deactivated with a 50% v/v ethanol/water solution before cleaning (14)

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41 CHAPTER 3 PROTOCOL VALIDATION AND SYSTEM CHARACTER IZATION 3.1 Goals of Verification and Characterization Before novel experiments were attempted the laboratory model system and measurements were compared with similar literature data to give credibility to the laboratory results. The steady state of a microbial chemostat provides a reproducible initial condition for the cells growing inside the reactor bulk so data collected at different reactor dilution rates was used as the basis for comparison. Collecting this steady state data also was done to characterize the initial conditions of chemostat grown cells for the purposes of future experiments. The cellular variables quantified at a range of dilution rates were the biomass yield, the protein weight percent, the RNA weight percent, the carbon dioxide evolution rate, and the initial growth and substrate uptakes rates upon exposure of the cells to excess glucose. The maximum growth rate of E. coli ML308 on the laboratory medium was also measured during the char acterization experiments. Data from the literature that was used for comparison purposes was from systems with similar operating conditions to those of the laboratory. 3.2 Steady State Biomass Yield The biomass yield on glucose was measured at different d ilution rates and compared with data obtained using the same strain and similar media compositions (21) The two sets of data show comparable yields for matchi ng dilution rates. The biomass yield results and comparison data appear in Figure 3 1. For the lower dilution rates (0.03 0.1 hr 1 ) there is a sharp decrease in the yield as the dilution rate approaches zero. This trend makes sense assuming microbial cells have a minimum energy requirement for maintaining themselves. As the dilution rate decreases the energy input to the microbial cells will also decrease leaving less energy available to the cell for growth. If the energy maintenance requirement of the cell remains constant with decreasing

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42 dilution rate then the yield must decrease as a larger percentage of consumed substrate must be used for maintenance. At the extreme case of the low washout dilution rate the cells are utilizing all consumed substrate for maintenance energy causing the yield at this point to be zero as the cells at this condition are not generating cell mass. At intermediate dilution (0.1 0.6 hr 1 ) rates the biomass yield keeps a relatively steady value. The maximum biomass yield occurred a t a D=0.3 hr 1 Beyond D=0.6 hr 1 the biomass yield started decreasing markedly. This trend makes sense as a chemostat will eventually washout at sufficiently high dilution rates due to the inability of cells to grow at a rate matching or exceeding the di lution rate. Figure 3 1. Steady state biomass yield of E. coli ML308 grown on 100 mg/L glucose minimal media. The biomass yield was determined by dividing the cell density by the amount of glucose utilized at different dilution rates. Specific details ab out cultivation and measurement appear in the Materials and Methods section. [Comparison data reprinted without permission. Lendenmann Phd dissertation (Pages 158 160, Tables B1, B2, and B3). EAWAG, Dubendorf, Switzerland.] 3.3 Carbon Dioxide Evolution Rat e The specific carbon dioxide evolution rate of E. coli on glucose was measured at different dilution rates and compared with similar data found for the same species grown on glucose minimal media (16) The carbon dioxide evolution rate results and compariso n data used appear

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43 in Figure 3 2. The two sets of data show similar qualitative trends. There is a linear increase in the rate of carbon dioxide evolution with respect to the dilution rate from 0.1 to 0.4 hr 1 At dilution rates greater then 0.4 hr 1 the carbon dioxide evolution rate slows considerably. Figure 3 2. Steady state carbon dioxide evolution rate of E. coli ML308 grown on 100 mg/L glucose minimal media. Specific details about cultivation and measurement appear in the Materials and Methods sec tion. The effluent air carbon dioxide concentration, the chemostat bulk cell density, and the air flow rate were used to calculate a specific evolution rate. [Comparison data reprinted without permission. Han, K., H. C. Lim, and J. Hong. 1992. Acetic Acid Formation in Escherichia Coli Fermentation. Biotechnology and Bioengineering 39: 663 671. (Page 666, Figure 2).] 3.4 Protein and RNA Dry Weight Fractions The protein content of E. coli ML308 was measured at different dilution rates and compared with the res ults of a similar study done with a different strain of bacteria grown on glucose minimal media. Unfortunately no data was found for the dilution rate dependence of E. coli protein content for similar conditions. The results of the protein dry weight fract ion measurements and the comparison data used appear in Figure 3 3. The protein fraction was a decreasing function of the dilution rate ranging from 76% (at D=0.1 hr 1 ) to 61% (at D=0. 7). The data used for comparison matched the trend observed in the labo ratory extremely well despite being collected using a different species of bacteria.

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44 Figure 3 3. Protein dry weight fraction of E. coli ML308 grown on 100 mg/L glucose minimal media. Specific details about cultivation and measurement appear in the Materi als and Methods section. The comparison data was measured using A. aerogenes grown on glucose minimal media [Comparison data reprinted without permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of Aerobacter aerogenes at low dilution rates i n a chemostat. Microbial Physiology and Continuous Culture. Her Majesty's Stationary Office : 240 253. (Pages 361, Figure 3).] The RNA content of E. coli ML308 was measured at different dilution rates and compared with data from the same comparison data set The RNA fraction results appear in Figure 3 4. The RNA fraction was an increasing function of the dilution rate ranging from 12% (at D=0.1 hr 1 ) to 16% (at D=0. 7). The data used for comparison was a monotonic increasing trend as well but had a low RNA c ontent at lower dilution rates that increased at faster rate. At a dilution rate of 0.7 hr 1 the RNA level of both data sets had similar values. The difference in RNA trends could be a result of the fact the two trends collected were from different species of bacteria.

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45 Figure 3 4. RNA dry weight fraction of E. coli ML308 grown on 100 mg/L glucose minimal media. Specific details about cultivation and measurement appear in the Materials and Methods section. The comparison data was measured using A. aerogene s grown on glucose minimal media. [Comparison data reprinted without permission. Phipps, D. W. T. a. P. J. 1967. Studies on the growth of Aerobacter aerogenes at low dilution rates in a chemostat. Microbial Physiology and Continuous Culture. Her Majesty's Stationary Office : 240 253. (Pages 361, Figure 3), Microbiological Research Establishment, Porton, Salisbury, Wilks, UK.] 3.5 Maximum Specific Growth Rate on Minimal Media The maximum specific growth rate is the highest speed of cell division a microbial sp ecies can obtain on a given media at a specific pH, temperature, etc. All nutrients in the medium are at saturating concentrations with respect to the growing cells. The maximum growth rate on a particular medium is generally unique for different microbial species. Exposure of nutrient limited cells to excess nutrients will not result in an immediate adjustment of the cell specific growth rate to the maximal value. There is an adaptation period, on the order of days, for the cells to achieve their maximum s peed of cell division (21) The maximum specific growth rate of E. coli ML308 was determined and compared with a value published for the same strain and simil ar media composition (21) A population of E. coli was constantly exposed to a nutrient surplus (1 g/L glucose) for five days by sterile subculturing. The cell s were never allowed to enter stationary phase for the duration of the experiment. After

PAGE 46

46 five days the culture was growing at a growth rate of ~0.91 hr 1 which compared well with the value 0.92 hr 1 found in the literature. 3.6 Initial Growth and Uptake R ate Response to Nutrient Excess Chemostat grown glucose limited cells rapidly increase their growth rate and substrate uptake rate upon exposure to saturating glucose concentrations. The level of increase for the growth rates and uptake rates were dependan t on the preculture dilution of the chemostat grown cells. The initial growth rate and uptake rates of E. coli ML308 growing at different dilution rates were determined by measuring the cell density and glucose concentration of a culture exposed to excess glucose. Reactor volume (50 mL) from a glucose limited chemostat was extracted and transferred to a pre warmed shake flask (250 mL) and inoculated with a saturating concentration of glucose (50 mg/L). The shake flask was then placed in a tabletop shaker (2 000 RPM, 37 o C) and the cell density and glucose level were measured every five minutes for thirty minutes. The growth and uptake rates were calculated as described in the Materials and Methods section. The results of the initial rate experiments for three different dilution rates as well as comparison data from similar experiments taken from the literature (21) appear in Figure 3 5 and Figure 3 6. The arrows in dicate the increase in growth or uptake rate upon the shift from glucose limited continuous mode growth to glucose surplus batch mode growth. At all dilution rates tested both the substrate uptake rate and growth rate increased upon exposure to excess gluc ose. The growth rates compare well with the results of similar experiments done with the same species. The initial growth rates of cells growing at dilution rates less then 0.1 hr 1 have never been explored to the best of our knowledge. The increase in the growth rate upon exposure to excess glucose dropped considerably as the dilution rate approached zero. The highly starved cells were unable to maintain the capacity for increased growth upon exposure to nutrient excess.

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47 Figure 3 5. Initial growth rate increase of E. coli ML308 grown on 100 mg/L glucose minimal media upon exposure to excess glucose (50 mg/L). The growth rate under steady state conditions appears as the solid line. Specific details about cultivation and measurement appear in the Materials and Methods section. The comparison data was measured using E. coli ML308 grown on glucose minimal media. [Comparison data reprinted without permission. Lendenmann Phd dissertation (Page 72, Figure 8.1), EAWAG, Dubendorf, Switzerland.] Figure 3 6. Initi al uptake rate of E. coli ML308 grown on 100 mg/L glucose minimal media upon exposure to excess glucose (50 mg/L). The uptake rate under steady state conditions appears as the solid line. Specific details about cultivation and measurement appear in the Mat erials and Methods section. The comparison data was measured using E. coli ML308 grown on glucose minimal media. [Comparison data reprinted without permission. Lendenmann Phd dissertation (Page 72, Figure 8.1), EAWAG, Dubendorf, Switzerland.]

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48 The substrate uptake rates of the two different data sets had noticeable differences. The collected substrate uptake rates were lower then the comparison data but it has been hypothesized that the reason for this discrepancy is the timescale on which the substrate upta ke rates were measured. Lendenmann (1995) collected glucose samples over a seven minute period as opposed to over a thirty minute period. The reasoning for this discrepancy could be the specific growth rate oscillations discussed in Chapter 5 and shown in Figure 5 5. If the specific growth rate of the bacterial cells oscillates with time then the specific substrate uptake rate could be oscillating as well. 3.7 Concluding Remarks These characterization experiments were necessary to ensure the validity of t he new protocols and our model system. In all cases the results collected were comparable to those from similar experiments done by other researchers. In addition the data from these experiments provided a benchmark for the future experiments. The chemosta t steady state data collected characterized the initial state of cells of E. coli growing at different glucose limited growth rates. These benchmarks were checked routinely before conducting experiments to ensure the state of the chemostat grown cells were correct so reproducible data would be collected.

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49 CHAPTER 4 STEADY STATE AND TRA NSIENT CARBON FLUX 4.1 Introduction The transient response of a microbial chemostat is a challenge facing biology and engineering today. Continuously run bioreactors are inevitably disturbed with fluctuations in their feed flo w rate that can lead to lengthy transients involving massive cell loss and an overshoot in the limiting substrate concentration. These long transients can lead to product deterioration in industrial bioreactors and violations at wastewater treatment facili ties. Characterizing the initial carbon flux of these continuously cultivated microorganisms after one of these perturbations is the goal of this research. This knowledge could help in the development of a better model that could mitigate the effects of th ese reactor transients. A literature search yielded no systematic study of the transient carbon flux of continuously grown microorganisms at a range on carbon limited growth rates. Exposure of glucose limited cells to saturating concentrations of glucose will rapidly increase their specific growth rate and specific substrate uptake rate. As seen in Figure 3 5 and Figure 3 6 the substrate uptake rate increase appears to be independent of the preculture dilution rate at D=0.3 hr 1 and above but the increase in growth rate appears to change linearly with dilution rate. The cells are consuming increased amounts of substrate at all dilution rates tested yet grow at different rates. An appropriate question here would be how are cells utilizing this excess substra te if it is not being channeled into production of more cells? Microorganisms growing under aerobic conditions generally have three ways they can utilize consumed carbonaceous substrate. They can channel the carbon in to synthesis of cell biomass (biosynt hesis), fully oxidize the organic carbon to carbon dioxide for energy (respiration), or partially catabolize the substrate and discharge the product in to the environment

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50 around the cell (excretion). All three of these cellular carbon sinks for consumed ca rbonaceous substrate were measured during steady state growth and during transient growth immediately following removal of the substrate limitation. The measurements were conducted at D=0.1, 0.3, and 0.6 hr 1 to see the change in carbon flux response at a range of glucose limited preculture growth rates. 4.2 Materials and Methods 4.2.1 Organism and Cultivation Conditions The organism used in this work was E. coli ML308 (ATCC 15224) obtained from the American Type Culture collection. Cells were resuscitated from a frozen stock culture overnight on glucose (1 g/L) minimal media. The minimal media used in this work was described in the Materials and Methods Chapter and the components of which appear in Table 2 1. The chemostat cultures were grown in a 1. 5 L Bi oflow III fermenter (New Brunswick Scientific Co.) with a working volume of 1 .2 L. The agitation speed was 1000 rpm and the aeration rate was 1.2 L/min. The bioreactor was equipped with automatic pH and temperature control. The pH was maintained at 7.0 + / 0.1 by addition of 1M KOH / 1M NaOH solution and 10% H 3 PO 4 solutions. The temperature was maintained at 37 o C. The feed was pumped in by a Maste r flex L/S peristaltic pump (7523 70) equipped with a Masterflex EZ Load pump head (7534 04). The reactor was equ ipped with a Vaisala GMT222 CO 2 analyzer for continuous monitoring of the carbon dioxide output of the reactor. Chemostat cultures were precultured on glucose (100 mg/L) for four days at D=0.6 hr 1 before shifting to the trial dilution rate to ensure adap tation of the E. coli cells to low glucose conditions for reasons described in the Materials and Methods Chapter. The chemostat was then allowed to equilibrate for ten additional residence times to ensure the cells were adjusted to the new growth condition s (21) The cellular carbon dioxide evolution rate of the reactor was

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51 monitored during the adaptation and equilibration phases of preculturing to ensure the hi story and consequently the initial state of the chemostat grown cells were correct before attempting an experiment. 4.2.2 Carbon Measurement Methodology The carbon content of cell mass inside the reactor at any time was estimated using a carbon mass fracti on of cell dry weight conversion factor. The concentration of cells inside the reactor at any time was determined by an absorbance measurement at 546 nm using a Spectronic Genesys 10UV spectrophotometer. This carbon content conversion factor was found by m easuring the steady state carbon mass fraction using a Carlo Erba 1106 Elemental Analyzer at six different dilution rates (0.1 0.6 hr 1 ). Cell mass was collected, concentrated in a centrifuge (5,000 RPM, 4 o C), desiccated overnight in an 80 o C oven, and then pulverized to a fine powder prior to analysis. The carbon mass fraction of the cell dry weight was assayed three times with this method to ensure an accurate conversion factor. The steady state carbon fraction of the dry cell weight for the different stea dy states appears in Figure 4 1. This conversion factor was used to calculate the carbon content of cell biomass of the chemostat at any time as shown in Equation 4 1. (4 1) The variables Cb, CF, and C in Equation 4 1 correspond to the cell biomass carbon content of the chemostat (units mg C/L), the carbon weight fraction of cell mass of steady state cells at the preculture dilution rate (units g C/mg), and the cell density of the chemostat volume (units mg /L), respectively.

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52 Figure 4 1. Carbon mass fraction of dry weight E. coli ML308 grown at different glucose limited dilution rates. The analysis was performed on a Carlo Erba 1106 Elemental Analyzer. The carbon content of the effluent air was determined with output from t he Vaisala GMT 222 CO 2 analyzer and associated software as described in the Materials and Methods Chapter. The cell specific respiration rate calculated using Equation 2 1 was a five minute average of CO 2 values centered on the time a cell density measurem ent was taken. The rate of evolution was converted to milligrams of carbon respired per liter of reactor volume by the conversion shown in Equation 4 2. (4 2) The variables Cr, qCO2, C, and dt in Equation 4 2 correspond to the r espired carbon evolved over the dt time period (units mg C/L), the cell specific CO 2 evolution rate (units mmoles C/mg hr), the cell density of the chemostat (units mg/L), and the time interval (unit hr), respectively. The carbon content glucose and excre ted products in the chemostat bulk were determined with a Dionex DX 500 HPLC and a Phoenix 8000 TOC Analyzer. The details of how these measurements were performed appear in the Materials and Methods Chapter. Little manipulation

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53 was needed to convert the gl ucose concentration and excreted carbon concentration to the correct form (units mg C/L) necessary for analysis. 4.2.3 The Continuous to Batch Shift Experiment The continuous to batch shift of a chemostat was achieved by simple switching off the feed pump To simulate the environment a microbial cell experiences during a dilution rate shift up, a 50 mg/L pulse of glucose was injected in to the reactor bulk immediately following the shift from continuous to batch mode. Shifting the chemostat to a batch reac tor was necessary for data collection as extraction of large sample volumes were necessary for measuring the total organic content of the reactor bulk as kinetics of batch growth are independent of the active volume of the reactor. Frequency of variable me asurement, sample collection, and approximate volumes taken for measurement appear in Table 4 1. Table 4 1. Frequency of data collection and approximate reactor volume taken for each measurement during the continuous to batch shift experiments. Sample ext raction for the total organic carbon measurement only applied during batch mode growth as significant reactor volume was required. Steady state TOC samples were only taken immediately before the shift from continuous to batch mode. Variable Vol. (ml) Freq. (min) Cell Density 6 5 CO2 Conc. 1 Glucose 2 10 TOC 20 10 To observe the change in carbon utilization pattern during a pseudo dilution rate shift up, most of the steady state variables were monitored for an hour prior to the continuous mode to batch mode shift. After the 50 mg/L pulse of glucose was added to the reactor the indicated variables were monitored until exhaustion of glucose denoted by a sudden, rapid decrease in the carbon dioxide concentration and an unchanging reactor cell dens ity.

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54 To ensure the validity of collected data at least 90% of the added glucose carbon was accounted for in either the generated cell biomass, respired carbon dioxide, and in excreted organic carbon both during continuous mode growth and at the end of ba tch mode growth. Each dilution rate was tested at least twice to ensure a reproducible trend. 4.3 Results Figure 4 2, Figure 4 3, and Figure 4 4 show example data from each of the three preculture dilution rates tested. Figure 4 5 shows a comparison in the cell density trends among the three different preculture dilution rates and Figure 4 6 shows a comparison in the specific CO 2 evolution rate trends among the three dilution preculture dilution rates. Table 4 2 shows numerical values for growth rate, subst rate uptake rate, CO 2 evolution rate, and biomass yield before and during batch mode growth. Figure 4 2. Results of a continuous to batch shift of a bioreactor growing E. coli ML308 continuously at D=0.1 hr 1 on 100 mg/L glucose minimal media. Batch mode shift and injection of ~50 mg/L glucose occurred at 1 hour.

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55 Figure 4 3. Results of a continuous to batch shift of a bioreactor growing E. coli ML308 continuously at D=0.3 hr 1 on 100 mg/L glucose minimal media. Batch mode shift and injection of ~50 mg/L glucose occurred at 1 hour. Figure 4 4. Results of a continuous to batch shift of a bioreactor growing E. coli ML308 continuously at D=0.6 hr 1 on 100 mg/L glucose minimal media. Batch mode shift and injection of ~50 mg/L glucose occurred at 1 hour. A t every dilution rate tested there was a seemingly immediate increase in substrate uptake rate and growth rate. The dilution rate D=0.6 hr 1 attained higher overall growth rates and carbon dioxide evolution rates as seen in Figure 4 5 and Figure 4 6. The g reatest increase in growth rate,

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56 substrate uptake rate, and carbon dioxide evolution rate were found at the dilution rate D=0.1 hr 1 as seen in Table 4 2. Figure 4 5. Comparison of the cell density evolution of the three preculture dilution rates before during, and after the continuous to batch shift. Figure 4 6. Comparison of the cell density evolution of the three preculture dilution rates before, during, and after the continuous to batch shift.

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57 Table 4 2. Growth rate, uptake rate, carbon dioxi de evolution rate, and biomass yield before and during a batch mode shift for the six different trials. Cont. Batch Cont. Batch Cont. Batch Cont. Batch D (1/hr) rg (1/hr) rg (1/hr) rs (1/hr) rs (1/hr) Y (gdw/g) Y (gdw/g) qCO2 (mm/g hr) qCO2 (mm/g hr) 0 .10 0.10 0.38 0.26 0.61 0.40 0.54 4.85 8.06 0.10 0.10 0.33 0.25 0.70 0.39 0.54 4.42 7.73 0.30 0.30 0.52 0.60 1.17 0.54 0.45 6.93 7.53 0.30 0.30 0.59 0.55 1.11 0.54 0.53 7.70 11.44 0.60 0.60 0.81 1.08 1.38 0.55 0.59 10.93 11.45 0.60 0.60 0.81 1.13 1.58 0.53 0.52 11.96 10.20 It is worth mentioning that at D=0.1 hr 1 there is an increase in yield upon exposure to excess glucose while at D=0.3 and 0.6 hr 1 the batch mode yield stays relatively constant. This was unexpected as other studies looking into t ransient yields after a dilution rate shift up show a marked drop in the yield (13) The experiments in question had transients that operated on a much longer time scale then the transients in this study and was thought to be the cause of the yield discrepancy. Simple shake flask experiments done at the three different dilution rates using 1 g/L glucose confirmed that the yield does decrease at all dilution rates on a longer time scale. As stated earlier at least 90% of the injected glucose was accounted for in carbon balances done at steady state and upon the exhaustion of glucose during batch mode growth. Carbon utilization by cells does change under substrate excess conditions and varies with preculture dilution rate. A comparison of carbon utilization between steady state growth and transient growth appears in Figure 4 7. During steady state growth at D=0.1 hr 1 about 50% of the consumed glucose went to respiration and 50% is going to biosynthesis. During excess glucose conditions most of the consumed carbon is channeled towards biosynthesis resulting in th e much higher yield seen during batch growth. Little excretion is seen during steady state and transient growth at D=0.1 hr 1 At D=0.3 hr 1 the split between biosynthesis and respiration was 64% and 39%, respectively. During substrate excess conditions th e fraction of consumed carbon being used for growth stays

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58 relatively constant while the fraction being excreted increases immensely. At D=0.6 hr 1 the trend is different still. This dilution rate shows the least amount of change upon exposure to excess glu cose. The fraction of carbon being utilized during steady state and transient growth stays roughly the same before and after the shift at 66% being used for biosynthesis and 27% being respired to carbon dioxide. While the cells did excrete organic compound s during batch growth, they were completely consumed by the end of the transient (including some leftover excretory products from steady state growth). Figure 4 7. A comparison of cellular carbon utilization pattern during continuous mode growth and during batch mode growth for D=0.1, D=0.3, and D=0.6. 4.4 Discussion The most glucose starved dilution rate, D=0.1 hr 1 was extremely efficient at incorporating consumed substrate into biomass. A sizable increase in the amount of carbon going toward bio synthesis is observed upon exposure to excess glucose leading to a higher yield. Little to no excretion was seen during batch growth. For the intermediate dilution rate, D=0.3 hr 1 the bacteria were less starved during the preculturing phase which proved to make them more wasteful in terms of carbon utilization. Cells growing at this dilution rate utilized the same

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59 percentage of consumed substrate in biosynthesis during glucose limited growth and glucose excess growth. There was an increase in the carbon d ioxide evolution rate, but a large percentage of the consumed substrate during transient growth was excreted back in to the medium. This suggests saturation of respiration and biosynthetic capacity forcing the cell to excrete the excess consumed substrate as partially catabolized organic molecules. The cells precultured at D=0.3 hr 1 did not utilize the excreted organic products before exponential growth ended. Cells growing at the highest dilution rate tested, D=0.6 hr 1 were least affected by the excess glucose conditions. Carbon accounting at the end of the exponential growth phase showed the excess carbon was utilized by the cell in percentages proportional to those seen at steady state. At D=0.6 hr 1 there was little or no increase in the carbon dioxid e evolution rate leading to the same trend seen at D=0.3 hr 1 where the cells tend to excrete a large fraction of the consumed substrate. The excreted organic products were completely consumed by the end of the exponential growth phase signifying that cell s grown at D=0.6 hr 1 have a higher capacity to utilize their excretory products than the intermediate dilution rate of D=0.3 hr 1 In conclusion as the preculture growth rate (and consequently degree of glucose availability) increases there tends to be le ss excess capacity for respiration and growth leading to more excretion of excess consumed carbon. Only at the highest dilution rate of D=0.6 hr 1 were the bacteria able to utilize the excreted compounds before the transient ended. The steady state and transient yields support the idea that as preculture dilution rate increases the capacity for excess growth and respiration decreases. At D=0.1 hr 1 the steady state yield is significantly lower then those seen at D=0.3 hr 1 and D=0.6 hr 1 The higher dilu tion rates show an equivalent and higher yield. This suggests that cells growing at D=0.1 hr 1 inside a chemostat have to use more of their consumed substrate for the production of energy

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60 (respiration) than in biosynthesis unlike the higher dilution rates. This idea is also supported by the fact that little to no excretion is seen during steady state growth at D=0.1 hr 1 as the cell is either fully oxidizing carbon for energy or incorporating it in to cell biomass. Upon exposure of cells growing at D=0.1 hr 1 to excess glucose, the cells increase the fraction of consumed carbon going to biosynthesis to levels seen at D=0.3 hr 1 and D=0.6 hr 1 This suggests that cells keep an excess capacity to grow at their desired yield under starved conditions. This trend probably does not hold at dilution rates less then D=0.1 hr 1 There is a drastic decrease in the glucose excess growth rate seen at extremely low dilution rates, as seen in Figure 4 5, as more substrate is needed for energy production just to maintain th e cell. The capacity for excess growth starts decreasing markedly below D=0.1 hr 1 as the microbial cells become increasingly starved. In all cases the initial transient yield on the time scale measured increased or remained constant upon exposure to exce ss glucose. This is in contrast to what has been seen in the literature. Others have seen that exposure of substrate limited microorganisms to excess substrate leads to an overall decrease in the yield during transient growth. Shake flask experiments done with higher concentrations of inoculated glucose revealed that the yield does indeed decrease on a much longer time scale. So there appears to be an initial yield increase on a fast time scale and a decrease on a longer time scale. The cause of this yield variation with time is yet to be determined

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61 CHAPTER 5 ATTEMPT TO IDENTIFY THE BIOSYNTHETIC LIM ITATION 5.1 Introduction The inability of microbial cells, growing under carbon limited conditions, to instantly adjust their growth rates to maximal levels has been seen in the literature and from data col lected in previous experiments. Currently the concentration of ribosomes inside microbial cells is thought to be the cause of the intracellular growth limitation and is used as such for modeling purposes (5) At different p reculture dilution rates the initial increase in the growth rate upon exposure to excess substrate has been found to be a linearly increasing function of the dilution rate much like the ribosome level. Since ribosomes are necessary for the production of pr otein; which constitutes the majority of cell biomass, one can easily draw the conclusion that ribosome levels control the growth rate during a dilution rate shift up. Experimental data collected where the growth rate and ribosome level have been monitored simultaneously certainly suggest this is the case. Figure 5 1 is a dilution rate shift up experiment conducted where the cell specific growth rate and the RNA level of the cell were recorded during transient growth. The majority of cellular RNA (97%) is ribosomal RNA making total RNA content a good indirect measure of ribosome content (29) Clearly the cell specific gro wth rate and RNA level are a good qualitative match; however, there are lines of evidence to suggest that the ribosome content of a cell is not the true intracellular biosynthetic limitation.

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62 Figure 5 1 Example dilution rate shift up (0.075 to 0.409 hr 1 ) from the literature where the growth rate and RNA level were measured versus time. The microorganism used was Lactococcus cremoris grown on glucose minimal media. [Reprinted without permission. Benthin, S., and J. Villadsen. 1991. Growth energetics of L actococcus cermoris FD1 during energy carbon and nitrogen limitation in steady state and transient cultures. Chem. Eng. Sc. 49: 589 609. (Page 4237, Figure 5).] The well known fact that microbial cells grow much faster on complex media than on minimal medi a should already be enough evidence to show that the ribosome content of a cell cannot be the true biosynthetic growth limitation seen in carbon limited microbial cells. Analysis of data from the literature seems to suggest that the biosynthetic limitation is actually a result of a lack of amino acids. Figure 5 2 is a comparison of transient growth rates among cultures of E. coli grown under different preculture conditions and transient conditions. Cells extracted from a carbon limited chemostat and exposed to excess glucose and excess amino acids produced growth rates that exceeded even the maximum growth rate attainable on minimal media alone. The ribosomes of microbial cells exposed to excess glucose are most likely unsaturated with their amino acid subst rates making the supply of amino acids to the ribosomes and not the ribosome level itself the cause of the biosynthetic limitation.

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63 Figure 5 2. Example data from the literature showing that amino acids limit the growth of carbon limited cells exposed to excess glucose. The microorganism used was E. coli B precultured on glucose limited minimal media. The open box trend refers to cells growing at D=0.37 that would show no improvement in the growth rate if exposed to excess glucose. The open circle trend refers to the growth rate trend observed when cells grown at D=0.37 were exposed to excess glucose. The open triangle trend refers to cells growing at their maximum growth rate on glucose. The open diamond trend refers to cells growing at D=0.34 exposed to excess glucose and excess amino acids. All data was scaled by their initial cell densities for trend comparison. [Reprinted without permission. Harvey, R. J. 1970. Metabolic regulation in glucose limited chemostat cultures of Escherichia coli. J Bacteriol 104: 698 706. (Pages 669 and 702, Figures 1 and 6).] Since amino acids are clearly a limiting factor in the growth rate of microbial cells the enzyme glutamate dehydrogenase (GDH) was thought to be a potential cause of the biosynthetic growth limitation. T his enzyme is responsible for the majority of inorganic nitrogen assimilation in to cell biomass under carbon limited conditions and its amino acid product glutamate is required for the production of many other amino acids (26) For these reasons the enzyme was thought to be a better candidate for the intracellular growth limitation of microbial cells growing with substrate excess. To test this hypothesis cells precultured un der glucose limited conditions were exposed to excess glucose and the cell density, RNA level, and GDH activity were all monitored during the transient. If the GDH enzyme was the cause of the intracellular limitation y during transient conditions would mirror the change

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64 seen in the RNA level as the production rate of ribosomes would be indirectly limited by the 5.2 Materials and Methods 5.2.1 Organism and Cultivation Conditions The organism used in this work was E. coli ML308 (ATCC 15224) obtained from the American Type Culture collection. Cells were resuscitated from a frozen stock culture overnight on glucose (1 g/L) minimal media. The minimal media used in this work was described in the Materials and Methods Chapter and the components of which appear in Table 2 1. The chemostat cultures were grown in a 1. 5 L Bioflow III fermenter (New Brunswick Scientific Co.) with a working volume of 1 .2 L. The agitation speed was 1000 rpm and th e aeration rate was 1.2 L/min. The bioreactor was equipped with automatic pH and temperature control. The pH was maintained at 7.0 + / 0.1 by addition of 1M KOH / 1M NaOH solution and 10% H 3 PO 4 solutions. The temperature was maintained at 37 o C. The feed wa s pumped in by a Maste r flex L/S peristaltic pump (7523 70) equipped with a Masterflex EZ Load pump head (7534 04). The reactor was equipped with a Vaisala GMT222 CO 2 analyzer for continuous monitoring of the carbon dioxide output of the reactor. Chemostat cultures were precultured on glucose (200 mg/L) for four days at D=0.6 hr 1 before shifting to the trial dilution rate to ensure adaptation of the E. coli cells to low glucose conditions for reasons described in the Materials and Methods Chapter. The chem ostat was then allowed to equilibrate for ten additional residence times to ensure the cells were adjusted to the new growth conditions (21) The cellular carb on dioxide evolution rate of the reactor was monitored during the adaptation and equilibration phases of preculturing to ensure the history and consequently the initial state of the chemostat grown cells were correct before attempting an experiment.

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65 The c ell density, RNA concentration, and GDH activity were measured by the methods covered in the Materials and Methods Chapter. 5.2.2 The Continuous to Batch Shift Continuous to batch shifts with a subsequent glucose pulse were again employed to simulate the environment cells experience during a dilution rate shift up. The reactor media was pulsed with 1 g/L of glucose to ensure substrate level never became limiting during for length of the experiment. Towards the end of the experiment the cell density is rath er high compared to the starting cell density and consequently the rate of substrate consumption would be high at this point as well. The duration of the experiment was decided to be two and a half hours long after finding an example GDH transient in the l iterature (18) The frequency of variable measurement and sample collection can be found in Table 5 1. Table 5 1. Frequency of sample measurement / collection and approximate volume taken. Sample Vol. ( mL) Freq. (min) Cell Density 6 5 RNA Level 2 10 GDH Activity 25 10 Note: Samples taken for GDH activity ranged from 5 25 mL depending on the cell density. For qualitative comparison of the time evolution of all three variables the measured va lues were converted to a per liter of reactor volume basis and then scaled by their steady state value. The purpose of this analysis was to visualize the improvement in the ribosomal capacity and GDH biosynthetic capacity as it compared to the increase in cell density. Unfortunately the experiment was conducted only once at the intermediate dilution rate D=0.3 hr 1 due to time constraints.

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66 5.3 Results and Discussion The results of the measured trial conducted are displayed in Figure 5 3 and the scaled comp arison of the three different variables appears in Figure 5 4. As hypothesized the scaled GDH activity level and RNA level of the reactor volume are qualitatively similar initially upon exposure of the culture to excess glucose. This data coupled with the fact that amino acids instantly increase the growth rate of E. coli beyond the maximum attainable growth rate supports the idea that GDH and not ribosome level is the better candidate for the biosynthetic growth limitation. Unfortunately one trial at one p reculture dilution rate is not sufficient to make this a substantial conclusion. Figure 5 3. Data obtained from a continuous to batch shift of glucose limited E. coli ML308 precultured at D=0.3 hr 1 to excess glucose. The feed concentration of glucose wa s 200 mg/L and the glucose pulse at half an hour was to 1 g/L glucose.

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67 Figure 5 4. Data from Figure 5 3 converted to a per liter basis and scaled by their steady state value for the purpose of trend comparison. Another interesting discovery was found w hen trying to find a way to analyze the data from this experiment was the presence of major oscillations in the growth rate with time upon exposure of carbon limited microbial cells to excess carbon. Fifteen minute growth rate fits were calculated for each cell density measurement collected using the cell density measurements five minutes before and five minutes after. The seemingly large amount of scatter seen in the collected data was first attributed to spectrometer noise, operator error, or a mechanical forcing influence brought about by the reactor itself. All three of these sources of oscillation were eliminated by a simple shake flask experiment where nothing but the cell density was measured. The magnitude and time interval of the oscillations from t he reactor trial and the shake flask trial were similar. The properties of the mechanical shaker were much different then those of the reactor setup supporting the idea that the observed oscillations were a cell driven phenomenon. The results of the reacto r and shake flask growth rate trials appear in Figure 5 5. The cause of these oscillations is still an unknown up to this point.

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68 Figure 5 5. Oscillations present in growth rate of glucose limited cells exposed to excess glucose. Cells were precultured at D=0.3 hr 1 on 200 mg/L glucose minimal media. Upon extraction or a switch to batch mode the reactor volume was pulsed with glucose to a concentration of 1 g/L. The experiment was done in a shake flask as well as in a reactor to ensure mechanical oscillati ons were not forcing the cell growth rate oscillations.

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69 CHAPTER 6 CONCLUDING REMARKS The study was a success from the point of view that new knowledge is available for understanding how microorganisms respond to the environment around them. Even the time spent on protocol validation and system characterization provided information on E. coli not published in the literature: protein and RNA content versus glucose limited dilution rate and low dilution rate growth response of glucose limited cells exposed to excess glucose. The experiments completed provide furthe r insight in to how microorganisms respond to removal of the nutrient growth limitation. The carbon flux experiments provided a lot of insight in to how the cell utilizes substrate before and after exposure to excess glucose at different glucose limited gr owth rates. Cells grown at all dilution rates tested were able to instantly improve their growth rate and substrate uptake rate upon removal of the glucose growth limitation. How the cells utilize this excess glucose is highly dependent on the preculture g lucose limited growth rate. Unfortunately the biosynthetic limitation experiments remain incomplete due to time constraints but it still remains that ribosomes are not the key cause of biosynthetic limitation as excess amino acids will greatly speed the g rowth rate of carbon limited microbial cells. The data that was collected did support the hypothesis that the enzyme glutamate dehydrogenase mirrors the RNA concentration well and could be the cause of biosynthetic limitation rather then the ribosome level The detection of oscillations in growth rate of glucose limited cells exposed to excess glucose was not the purpose of the experiments here but was found regardless. This is a prime example of how research can answer questions but in the end can pose mor e. Finding the source of this specific growth rate oscillation would be the potential next step in this research.

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70

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71 APPENDIX A VISUAL BASIC PROGRAM FOR THE VAISALA GMT2 22 CO2 ANALYZER CODE Public CO2, X As Single Public CollectingData As Boolean Dim CO2Array(1 To 720, 1 To 2) As Single Public ClockSeconds, ClockMinutes, ClockHours, TotalCollectionTime, MinuteTimer As I nteger Sub GetCO2() CO2 = MSComm1.Input CO2 = Round(Val(CO2), 0) End Sub Private Sub Form_Load() 'Initialization subroutine. Setup for COM Port, array for data storage, and main ch art. MSComm1.InBufferSize = 6 MSComm1.CommPort = 6 MSComm1.PortOpen = True For X = 0 To 39 RealTime.List(X) = "" Next X For X = 1 To 720 CO2Array(X, 1) = X CO2Array(X, 2) = 0 Next X With CO2Chart .chartType = VtChChartType2dXY .Plot.UniformAxis = False .Plot.SeriesCollection(1).ShowLine = False .Plot.SeriesCollection(1).SeriesMarker.Auto = False .Plot.SeriesCollection(1).DataPoints( 1).Marker.Style = VtMarkerStyleFilledCircle .Plot.SeriesCollection(1).DataPoints( 1).Marker.Size = 60 .Plot.SeriesCollection(1).DataPoints( 1).Marker.Visible = True .Plot.AutoLayout = False .Plot.Axis(VtChAxisIdY).ValueScale.Auto = False .Plot.Axis(VtChAxisIdY).ValueScale.Maximum = 1000 .Plot.Axis(VtChAxisIdY).Value Scale.Minimum = 0 .Plot.Axis(VtChAxisIdY).ValueScale.MajorDivision = 10 .Plot.Axis(VtChAxisIdX).ValueScale.Auto = False .Plot.Axis(VtChAxisIdX).ValueScale.Maximum = 720 .Plot.Axis(VtChAxisIdX).ValueScale.Minimum = 0

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72 .Plot.Axis(VtChAxisI dX).ValueScale.MajorDivision = 12 .Plot.Axis(VtChAxisIdY).AxisTitle = "CO2 Concentration (ppm)" .Plot.Axis(VtChAxisIdX).AxisTitle = "Data Point Taken Every Minute" .ChartData = CO2Array End With End Sub Private Sub Updategraph() data array and adjusts the chart accordingly. If MinuteTimer = 60 Then MinuteTimer = 0 MinuteTimer = MinuteTimer + 1 If MinuteTimer Mod 60 = 0 Then For X = 1 To 719 CO2Array(721 X, 2) = CO2Array(720 X, 2) Next X CO2Array(1, 2) = CO 2 CO2Chart.ChartData = CO2Array End If End Sub Private Sub SendCommandButton_Click() MSComm1.Output = Command.Text End Sub Private Sub StartCollectionButton_Click() h the entered name and starts recording timestamped data to the file. If Interval.Text = "" Or Filename.Text = "" Then Else Open Filename.Text For Output As #1 Print #1, "Time (sec),CO2 (ppm)" CollectingD ata = True Interval.Locked = True Filename.Locked = True StartCollectionButton.Enabled = False SendCommandButton.Enabled = False End If End Sub Private Sub StopCollectionButton_Click() llection clock off. Close #1 CollectingData = False Interval.Locked = False

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73 Filename.Locked = False StartCollectionButton.Enabled = True SendCommandButton.Enabled = True Hours.Caption = 0 Minutes.Caption = 0 Seconds.Caption = 0 End Sub Private Sub Timer1_ Timer() Call RealTimeCO2 Call DataCollectionClock Call Updategraph Call DataCollection End Sub Private Sub RealTimeCO2() ues shown every second. Call GetCO2 For X = 0 To 39 RealTime.List(39 X) = RealTime.List(38 X) Next X RealTime.List(0) = CO2 End Sub Private Sub DataCollectionClock() If Coll ectingData = True Then ClockHours = Val(Hours.Caption) ClockMinutes = Val(Minutes.Caption) ClockSeconds = Val(Seconds.Caption) ClockSeconds = ClockSeconds + 1 If ClockSeconds = 60 Then ClockMinutes = ClockMinutes + 1 Clo ckSeconds = 0 End If If ClockMinutes = 60 Then ClockHours = ClockHours + 1 ClockMinutes = 0 End If Hours.Caption = ClockHours Minutes.Caption = ClockMinutes Seconds.Caption = ClockSeconds TotalCollectionTime = Cl ockHours 3600 + ClockMinutes 60 + ClockSeconds End If End Sub

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74 Private Sub DataCollection() If CollectingData = True Then If TotalCollectionTime Mod Val(Interva l.Text) = 0 Then Print #1, Str(TotalCollectionTime) + "," + Str(CO2) End If End If End Sub FORM SETUP USER INTERFACE

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75 APPENDIX B RESULTS OF CARLO ERB A 1106 ELEMENTAL ANALY SIS OF E. COLI ML308

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76 LIST OF REFERENCES 1. Aiba, S., N. Y. Nishizawa, and M. Onodera. 1967. Nucleic approach to some response of chemostatic culture of Azobacter vinelandii. Gen. Appl. Microbiol. 35: 85 101. 2. Bally, M., and T. Egli. 1996. Dynamics of Su bstrate Consumption and Enzyme Synthesis in Chelatobacter heintzii during Growth in Carbon Limited Continuous Culture with Different Mixtures of Glucose and Nitrilotriacetate. Appl Environ Microbiol 62: 133 140. 3. Benov, L., and J. Al Ibraheem. 2002. Disr upting Escherichia coli: a comparison of methods. J Biochem Mol Biol 35: 428 31. 4. Benthin, S., and J. Villadsen. 1991. Growth energetics of Lactococcus cermoris FD1 during energy carbon and nitrogen limitation in steady state and transient cultures. Chem Eng. Sc. 49: 589 609. 5. Brunschede, H., T. L. Dove, and H. Bremer. 1975. Establishment of exponential growth after a nutritional shift up in Escherichia coli B/r: Accumulation of deoxyribonucleic acid, ribonucleic acid, and protein. J Bacteriol 129: 1020 1033. 6. Brunschede, H., T. L. Dove, and H. Bremer. 1975. Establishment of exponential growth after a nutritional shift up in Escherichia coli B/r: Accumulation of deoxyribonucleic acid, ribonucleic acid, and protein. J Bacteriol 129: 355 358. 7. Cataldi T. R., C. Campa, M. Angelotti, and S. A. Bufo. 1999. Isocratic separations of closely related mono and disaccharides by high performance anion exchange chromatography with pulsed amperometric detection using dilute alkaline spiked with barium acetate. J Chromatogr A 855: 539 50. 8. Cataldi, T. R. I., C. Campa, and G. E. De Benedetto. 2000. Carbohydrate analysis by high performance anion exchange chromatography with pulsed amperometric detection: The potential is still growing. Fresenius Journal of Analyt ical Chemistry 368: 739 758. 9. Chi, C. T., and J. A. Howell. 1976. Transient behavior of a continuous stirred tank biological reactor utilizing phenol as an inhibitory substrate. Biotechnol Bioeng 18: 63 80. 10. Cooney, C. L., and D. I. Wang. 1976. Transi ent response of Enterobacter aerogenes under a dual nutrient limitation in a chemostat. Biotechnol Bioeng 18: 189 98. 11. Cooney, C. L., D. I. Wang, and R. I. Mateles. 1976. Growth of Enterobacter aerogenes in a chemostat with double nutrient limitations. Appl Environ Microbiol 31: 91 8. 12. Daniels, L., R. S. Hanson, and J. A. Phillips. 1994. Chemical Analysis. American Society of Microbiology.

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77 13. Duboc, P., U. von Stockar, and J. Villadsen. 1998. Simple generic model for dynamic experiments with Saccharo myces cerevisiae in continuous culture: decoupling between anabolism and catabolism. Biotechnol Bioeng 60: 180 9. 14. EH&S. 1998. Biological Safety Manual. University of Florida, Gainesville, FL. 15. Gerhardt, P., R. G. E. Murray, W. A. Wood, and N. R. Kr ieg. 1994. Methods for General and Molecular Bacteriology. American Society of Microbiology, Washington, D. C. 16. Han, K., H. C. Lim, and J. Hong. 1992. Acetic Acid Formation in Escherichia Coli Fermentation. Biotechnology and Bioengineering 39: 663 671. 17. Hans Peter Meyer, e. a. 1984. Acetate formation in continuous culture of E. coli K12 D1 on defined and complex media. Journal of Biotechnology 1: 355 358. 18. Harvey, R. J. 1970. Metabolic regulation in glucose limited chemostat cultures of Escherichi a coli. J Bacteriol 104: 698 706. 19. Herbert, D., R. Elsworth, and R. C. Telling. 1956. The continuous culture of bacteria; a theoretical and experimental study. J Gen Microbiol 14: 601 22. 20. Koch, A. L., and C. S. Deppe. 1971. In vivo assay of protein synthesizing capacity of Escherichia coli from slowly growing chemostat cultures. J Mol Biol 55: 549 62. 21. Lendenmann, U. 1994. Growth Kinetics of Escherichia coli with mixtures of sugars. 22. Lendenmann, U., and T. Egli. 1995. Is Escherichia coli growi ng in glucose limited chemostat culture able to utilize other sugars without lag? Microbiology 141 ( Pt 1): 71 8. 23. Lin, R. I. S., and O. A. Schjeide. 1969. Micro Estimation of Rna by Cupric Ion Catalyzed Orcinol Reaction. Analytical Biochemistry 27: 473 &. 24. Maaloe, O. a. N. O. K. 1966. A study of DNA, RNA, and protein synthesis. W. A. Benjamin, New York. 25. Maier, R. M., I. L. Pepper, and C. P. Gerba. 2000. Envrionmental Microbiology. Academic Press, San Diego, CA. 26. Meers, J. L., D. W. Tempest, and C. M. Brown. 1970. 'Glutamine(amide):2 oxoglutarate amino transferase oxido reductase (NADP); an enzyme involved in the synthesis of glutamate by some bacteria. J Gen Microbiol 64: 187 94. 27. Nagai, S., Y., Nishizawa, I. Endo, and S. Aiba. 1968. Respo nse of a chemostatic culture of Azobacter vinelandii to a delta type pulse of glucose. J. Gen. Appl. Microbiol. 14: 121 134.

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78 28. Natarajan, A., and F. Srienc. 2000. Glucose uptake rates of single E. coli cells grown in glucose limited chemostat cultures. J Microbiol Methods 42: 87 96. 29. Neidhardt, F. C. e. a. 1990. Physiology of the Bacterial Cell: A Molecular Approach. Frank Sinauer Associates, Sunderland. 30. Neijssel, M., S. Hueting, and D. W. Tempest. 1977. Glucose transport capacity is not rate limit ing in the growth of some wild type strains of Escherichia coli and Klebsiella aerogen in chemostat culture. FEMS Microbiol Lett 2: 1 3. 31. Nielsen, J., and J. Villadsen. 1992. Modelling of Microbial Kinetics. Chem. Eng. Sc. 47: 4225 4270. 32. Obrien, R. W., O. M. Neijssel, and D. W. Tempest. 1980. Glucose Phosphoenolpyruvate Phosphotransferase Activity and Glucose Uptake Rate of Klebsiella Aerogenes Growing in Chemostat Culture. Journal of General Microbiology 116: 305 314. 33. Phipps, D. W. T. a. P. J. 1 967. Studies on the growth of Aerobacter aerogenes at low dilution rates in a chemostat. Microbial Physiology and Continuous Culture. Her Majesty's Stationary Office : 240 253. 34. Pierce Biotechnology, I. 2003. BCA Protein Assay Kit, Rockford, IL. 35. Pri mrose, S. B., and A. C. Wardlaw. 1982. Sourcebook of Experiments for the Teaching of Microbiology. Academic Press, St. Louis, MO. 36. Ramkrishna, B. a. 1990. Metabolic regulation in bacterial continuous cultures. Biotechnol Bioeng 29: 940 943. 37. Schulze U., M. E. Larsen, and J. Villadsen. 1995. Determination of intracellular trehalose and glycogen in Saccharomyces cerevisiae. Anal Biochem 228: 143 9. 38. Schwinghamer, E. A. 1980. Method for Improved Lysis of Some Gram Negative Bacteria. Fems Microbiolog y Letters 7: 157 162. 39. Scopes, R. K. 1994. Protein Purification, 3rd ed. Springer+Business Media, Inc., New York, NY. 40. Senior, P. J. 1975. Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous cu lture technique. J Bacteriol 123: 407 18. 41. Senn, H., U. Lendenmann, M. Snozzi, G. Hamer, and T. Egli. 1994. The growth of Escherichia coli in glucose limited chemostat cultures: a re examination of the kinetics. Biochim Biophys Acta 1201: 424 36.

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79 42. So nnleitner, B., S. A. Rothen, and H. Kuriyama. 1997. Dynamics of glucose consumption in yeast. Biotechnol Prog 13: 8 13. 43. Standing, C. N., A. G. Fredrickson, and H. M. Tsuchiya. 1972. Batch and continuous culture transients for two substrate systems. Ap pl Microbiol 23: 354 9. 44. Teplitksi, M. 2007. Method for Bead Disruption of Starved Microorganisms. In J. Noel (ed.), Gainesville, FL. 45. Wick, L. M., M. Quadroni, and T. Egli. 2001. Short and long term changes in proteome composition and kinetic prop erties in a culture of Escherichia coli during transition from glucose excess to glucose limited growth conditions in continuous culture and vice versa. Environ Microbiol 3: 588 99. 46. Yagil, G., and E. Yagil. 1971. Relation between Effector Concentration and Rate of Induced Enzyme Synthesis. Biophysical Journal 11: 11 &. 47. Yang, S. S., M. Miller, J. Martin, and J. Harris. 2003. Evaluation and Application of a New Total Organic Carbon Analyzer, Teledyne Instruments Application Note, Mason, OH. 48. Yun, H. S., J. Hong, and H. C. Lim. 1996. Regulation of ribosome synthesis in Escherichia coli: Effects of temperature and dilution rate changes. Biotechnology and Bioengineering 52: 615 624.

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80 BIOGRAPHICAL SKETCH Jason Noel received his B.S. from Virginia Commonwealth University in Richmond, Virginia in August of 2003. Thereafter he has worked as a graduate student in the Chemical Engineering Department of the University of Florida studying the growth kinetics of bacteria and received his doctorial degree in August of 2007.