METABOLIC ENGINEERING FOR ENHANCED PROPIONIC ACID FERMENTATION BY PROPIONIBACTERIUM ACIDIPROPIONICI

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Supaporn Suwannakham, B.Eng.

*****

The Ohio State University

2005

Dissertation Committee: Approved by Professor Shang-Tian Yang, Adviser Professor Jeffrey J. Chalmers __________________________________ Professor Hua Wang Adviser Graduate Program in Chemical Engineering

ii

ABSTRACT

Propionic acid is widely used in food and dairy industries. As a result of its

antimicrobial activity, propionic acid and its salts are widely used as preservatives in

foods and grains. Currently, the market of propionic acid is mainly supplied by

production via petrochemical routes. Fermentation by propionibacteria produces mainly

propionic and acetic acids from sugars; however, the fermentation suffers from low

propionic acid production due to by-product formation and strong propionic acid

inhibition on cell growth and the fermentation. The high demand of propionic acid for

use as a natural preservative in foods and grains has stimulated developments of new

fermentation processes to achieve improved propionic acid production from low-cost

biomass and food processing wastes. In this research, novel approaches, at process

engineering, metabolic engineering, and genetic engineering levels, were developed for

enhanced propionic acid production by Propionibacterium acidipropionici.

Fed-batch fermentation of glucose by P. acidipropionici immobilized in a fibrous-

bed bioreactor (FBB) with a high cell density (>45 g/L) produced a high final propionic

acid concentration of 72 g/L and a high propionate yield of up to 0.65 g/g. A mutant with

improved propionate tolerance was obtained by adaptation in the FBB, which resulted in

significant physiological and morphological changes. The mutant cells were less sensitive

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to propionate inhibition and had a higher saturated fatty acid content in the cell

membrane and a slimmer shape with an increased specific surface area.

Metabolic stoichiometric analysis was applied to quantitatively describe the

global cellular mechanism in propionic acid fermentation. By feeding carbon sources

with different oxidation states, different fermentation end-product compositions were

obtained, indicating different controlling mechanisms involving various acid-forming

enzymes with significant changes in their activities and overall protein expression

pattern. In general, the metabolic pathway shifted toward more propionate formation with

a more-reduced substrate.

Gene inactivation via gene disruption and integrational mutagenesis was used to

knock out the acetate kinase (ack) gene with the goal of eliminating acetate formation and

further enhancing propionic acid production by P. acidipropionici. Mutants were

obtained by transforming the cells with a partial ack gene fragment, which was

introduced either as a linear DNA fragment with a tetracycline resistance cassette within

the partial ack gene or in a non-replicative integrational plasmid containing the

tetracycline resistance cassette. The ack inactivation in the mutants showed a profound

impact on cell growth rate. Compared to the wild type, the ack-deleted mutants achieved

~10% increase in propionate yield and ~10% decrease in acetate yield.

iv

The FBB, the knowledge of the underlying mechanism in controlling propionic

acid fermentation, and the mutants obtained in this research should allow us to develop

an economical bioprocess for the production of propionic acid from sugars.

v

Dedicated to my mother

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ACKNOWLEDGMENTS

My great appreciation goes to my adviser, Dr. Shang-Tian Yang, for intellectual

and financial support as well as for his inspiring advice, encouragement, enthusiasm, and

flexibility throughout my study. I have gained a lot from his insights and I have greatly

enjoyed my staying here as his student.

I would like to acknowledge with sincere gratitude to the members of my

dissertation committee, Dr. Jeffrey J. Chalmers and Dr. Hua Wang. I am grateful for their

helpful advices on a variety of topics.

I wish to thank Dr. Yan Huang for her help in the work reported in Chapter 5. I

am greatly indebted to Dr. Ying Zhu for teaching me the fundamental laboratory skills in

conducting the fermentation experiments and the basic molecular biology skills in

performing the genetic engineering experiments.

I also appreciate Mr. Carl Scott, Mr. Leigh Evrard, and Mr. Paul Green for their

technical assistance.

My colleagues in my research group, especially Dr. Nuttha Thongchul and Ms.

Suwattana Pruksasri, offered many kinds of support and help over the last four years. I

benefited a lot from discussions with them and sharing the knowledge on my research.

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I would like to thank Dr. Daniel R. Zeigler (Bacillus Genetic Stock Center, The

Ohio State University, Columbus, OH) for supplying pBEST309 and pDG1515, Dr. Desh

Pal Verma (Department of Molecular Genetics, The Ohio State University, Columbus,

OH) for supplying electroporation device, and Dr. Mitsuo Yamashita (Department of

Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan) for his

suggestions on genetic engineering experiments.

Financial supports from the U.S. Department of Agriculture (CSREES 99-35504-

7800) and the Consortium for Plant Biotechnology Research, Inc. to various phases of

this work are also acknowledged.

I appreciate Mrs. Panitee Panjakup for her kindly help and encouragement on

pursuing my Ph.D. study.

I wish to thank best friends of mine, Mr. Chanin Hunsakunathai, Mr. Vichian

Suksoir, and Ms. Ratchat Chantawongvuti, for their warm support and understanding

during the last four years.

Special thank goes to my family for their love and warm support. With their love

and support, I could be through ups and downs during my study.

Finally, my heartfelt gratitude goes to my mother, Ms. Suwannee, my

grandmother, Mrs. Sunee, and my aunt, Ms. Sumalee Peerapongsathorn, for their love

and inspiration. My graduation could only be achievable with their warmest support and

understanding.

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VITA August 5, 1978…………………………...............................Born – Bangkok, Thailand March, 1999……………………………...............................B.Eng. Chemical Engineering Chulalongkorn University Bangkok, Thailand September, 2000 – March 2005…………………………….Graduate Research Associate Chemical Engineering The Ohio State University

PUBLICATION Suwannakham S, Yang S-T. 2005. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol Bioeng, in press.

FIELD OF STUDY Major Field: Chemical Engineering Specialty: Biochemical Engineering

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TABLE OF CONTENTS PageAbstract……………………………………………………………………...............……iiDedication………………………………………………………………………........……vAcknowledgments………………………………………………………………….....….viVita…………………………………………………………………………………..…viiiList of Tables……………………………………………………………...................….xivList of Figures……………………………………………………………………….….xvi Chapters: 1. Introduction……………………………………………………………………..

……..1

2. Literature Review………………………………………………………………. 2.1 Propionic Acid Fermentation…....................................................................... 2.1.1 Propionic Acid………………………………………………………... 2.1.2 Microorganisms………………………………………………………. 2.1.3 Metabolic Pathway…………………………………………………… 2.1.4 Fermentation Processes………………………………………………. 2.2 Cell Immobilization and Fibrous-Bed Bioreactor…………………………… 2.2.1 Cell Immobilization…………………………………………………... 2.2.2 Immobilized-Cell Bioreactor…………………………………………. 2.2.3 Fibrous-Bed Bioreactor………………………………………………. 2.3 Immobilized-Cell Fermentation……………………………………………... 2.4 Metabolic Engineering………………………………………………………. 2.4.1 Metabolic Flux Analysis……………………………………………… 2.4.2 Applications of Other Metabolic Engineering Techniques…………... 2.5 Genetic Engineering of Propionibacteria……………………………………. 2.5.1 Acetic Acid Formation in Propionic Acid Fermentation…………….. 2.5.2 Acetic Acid Formation Pathway Genes and Enzymes……………….. 2.5.3 Genetics and Molecular Biology of Propionibacteria………………... 2.6 References……………………………………………………………………

….….9

……..9…….9……12……14……17.….30…..30

……31……32……33……36……36……39……40……40……42……43……50

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3. Enhanced Propionic Acid Fermentation by Propionibacterium acidipropionici Mutant Obtained by Adaptation in a Fibrous-Bed Bioreactor……………......... Summary……………………………………………………………………….... 3.1 Introduction………………………………………………………………….. 3.2 Materials and Methods………………………………………………………. 3.2.1 Culture and Media……………………………………………………. 3.2.2 Free-Cell Fermentation……………………………………………..... 3.2.3 Immobilized-Cell Fermentation……………………………………… 3.2.4 Effect of Propionic Acid on Cell Growth…………………………..... 3.2.5 Enzyme Assays……………………………………………………..... 3.2.6 Membrane-Bound ATPase Assay……………………………………. 3.2.7 Cell Membrane Fatty Acid Analysis…………………………………. 3.2.8 Cell Viability Assay.…………………………………………………. 3.2.9 Scanning Electron Microscopy……………………………………….. 3.2.10 Analytical Methods…………………………………………………... 3.3 Results and Discussion………………………………………………………. 3.3.1 Fermentation Kinetics………………………………………………... 3.3.2 Propionic Acid Inhibition…………………………………………….. 3.3.3 Acid-Forming Enzyme Activities…………………………………….. 3.3.4 Membrane-Bound ATPase…………………………………………… 3.3.5 Membrane Fatty Acid Composition………………………………….. 3.3.6 Morphological Change in Mutant……………………………………. 3.3.7 Effects of Cell Immobilization in FBB………………………………. 3.4 Conclusion…………………………………………………………………… 3.5 References……………………………………………………………………

……64

……64……66……68……68……68……69……70……70……71……72……72……72……73……73……73……76……78……81……83……83……84……87……88

4. Effect of Carbon Sources on Propionic Acid Fermentation by Propionibacterium acidipropionici…………………………………………….. Summary……………………………………………………………………...….. 4.1 Introduction………………………………………………………………….. 4.2 Materials and Methods..................................................................................... 4.2.1 Culture and Media…………………………………………………..... 4.2.2 Batch Fermentation………………………………………………...…. 4.2.3 Analytical Methods………………………………………………...…. 4.2.4 Metabolic Stoichiometric Analysis…………………………………… 4.2.5 Preparation of Cell Extract…………………………………………… 4.2.6 Enzyme Assays……………………………………………………......

….105

…105…107.…110.…110…110.…111…111….112….112

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4.2.7 Protein Expression: SDS-PAGE……………….…………………….. 4.3 Results……………………………………………………………………...... 4.3.1 Effect of Carbon Sources on Fermentation Kinetics……...………….. 4.3.2 Comparison of Fermentation Kinetics between Wild Type and Adapted Mutant……………………………………… 4.3.3 Metabolic Pathway Analysis…………………………………………. 4.3.4 Metabolic Stoichiometric Analysis (MSA)…………………………… 4.3.5 Effect of Carbon Sources on MSA……………………………….…... 4.3.6 Comparison of MSA between Wild Type and Adapted Mutant…...… 4.3.7 Effect of Carbon Sources on Activity of Acid-Forming Enzymes...…. 4.3.8 Effect of Carbon Sources on Protein Expression Pattern…………...... 4.4 Discussion…………………………………………………………………..... 4.4.1 Effect of Carbon Sources on Fermentation by P. acidipropionici...… 4.4.2 Effect of Carbon Sources on Protein Expression Pattern…………….. 4.5 Conclusion…………………………………………………………………… 4.6 References…………………………………………………………………....

…..113…113….113

.…114…115…119…122…123…123…124…125.…125…133.…134.…136

5. Construction and Characterization of ack Gene Deleted Mutants of Propionibacterium acidipropionici for Propionic Acid Fermentation………..... Summary…………………………………………………………………………. 5.1 Introduction………………………………………………………………….. 5.2 Materials and Methods………………………………………………………. 5.2.1 Bacterial Strains and Plasmids……………………………………….. 5.2.2 Medium Preparation and Growth Conditions………………………… 5.2.3 DNA Preparation……………………………………………………… 5.2.4 PCR Amplification…………………………………………………… 5.2.5 Gene Disruption: Construction of Disrupted ack…………………….. 5.2.6 Integrational Mutagenesis: Construction of Integrational Plasmid of ack........................................ 5.2.7 Transformation of P. acidipropionici................................................... 5.2.8 Southern Hybridization…………………………………………….…. 5.2.9 Mutant Characterization……………………………………………… 5.3 Results……………………………………………………………………….. 5.3.1 PCR Amplification and Sequence Analysis……………………….…. 5.3.2 Transformation of P. acidipropionici................................................... 5.3.3 Disruption of ack……………………………………………………… 5.3.4 Integrational Mutagenesis (pTAT)…………………………………… 5.3.5 Southern Hybridization………………………………………………..

…154

…154…156.…159…159…159…160…160…161

…162…162.…163.…164.…166…166….167…167…168…168

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5.3.6 Overall Protein Expression Pattern…………...………………….….... 5.3.7 Acid-Forming Enzyme Activities………………………………….…. 5.3.8 Fermentation Kinetics……………………………………………....… 5.4 Discussion………………………………………………………………….… 5.4.1 Genetic Manipulation of P. acidipropionici………………………..... 5.4.2 Overall Protein Expression Pattern…...…………………………….… 5.4.3 Effect of ack Gene Inactivation………………………………………. 5.5 Conclusion…………………………………………………………………… 5.6 References………………………………………………………………….…

…169…169…170…170…170…172…173…175…176

6. Conclusions and Recommendations………………………………………….… 6.1 Conclusions………………………………………………………………….. 6.1.1 Fermentation Kinetics………………………………………………… 6.1.2 Fibrous-Bed Bioreactor………………………………………………. 6.1.3 Metabolic Engineering……………………………………………….. 6.2 Recommendations………………………………………………………….… 6.2.1 Propionic Acid Fermentation by P. acidipropionici………………… 6.2.2 Metabolic Engineering and Genetic Engineering of P. acidipropionici…………………………………………………….

…192…192…192…193…194…196…196

…197

Bibliography………………………………………………………………………...…199

Appendices…………………………………………………………………………… Appendix A Medium Compositions……………………………………………... A.1 Medium Compositions for Propionibacterium acidipropionici……..... A.2 Medium Compositions for Escherichia coli…………………………... Appendix B Analytical Methods……………………………………………….… B.1 Cell Concentration……………………………………………...….….. B.2 Cell Viability…………………………………………………………... B.3 High-Performance Liquid Chromatography……...……...………….… B.4 Preparation of Cell Extracts and Protein Assay…………...…………... B.5 Enzyme Assays………………………………………………...….…..… B.6 SDS-PAGE………………..…………………………...…………….… B.7 Membrane Fatty Acid Composition……………...………………….… B.8 Scanning Electron Microscopy…………………………………..……. Appendix C Bioreactor Construction and Operation………………………….… C.1 Construction of Immobilized-Cell Bioreactor…………..……….………

….213

…213…214…214

…..215…216…216…217…220….221…225…227…234…235…236

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C.2 Bioreactor Start-Up and Operation……………………………………. Appendix D Genetic Engineering Protocols…………………………………….. D.1 Preparation of Genomic DNA from P. acidipropionici with QIAGEN Genomic DNA Kit…………..………………………... D.2 Preparation of Plasmid DNA by QIAprep Spin Miniprep Kit…...…… D.3 Preparation of Plasmid DNA by HiSpeed Plasmid Maxi Kit……...….. D.4 DNA Purification by QIAquick Spin Gel Extraction Kit………...…… D.5 DNA Electrophoresis…………………………...……………………... D.6 PCR Amplification of ack Gene from P. acidipropionici……………. D.7 TOPO TA Cloning Protocol (Invitrogen TOPO TA Cloning Kit)……. D.8 Construction of TOPOACK1………………………..…………….….. D.9 DNA Digestion and Ligation…………………………...………….….. D.10 DNA Transformation in P. acidipropionici…………………...……… D.11 Construction of pSSF1………………...…………………………….… Appendix E Reagents and Buffers………………………………………………..

…237…239

…240…241…242…244…245…246…247…249…251…252…253…256

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LIST OF TABLES Table Page 2.1 Development of fermentation processes for improved propionic acid production…………………………………………... 2.2 Genetic manipulation in propionibacteria…………………………………….. 3.1 Comparison of product yields and maximum propionic acid concentrations from fed-batch fermentations with free cells and immobilized cells in the FBB…………………………………………….. 3.2 Comparison of rate constants for specific growth rate and several key enzymes in P. acidipropionici wild type and mutant from the FBB……………………………………………………… 3.3 Comparison of membrane fatty acid compositions of P. acidipropionici wild type and mutant from the FBB………………………. 4.1 Kinetics of fermentations by P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources……..…………. 4.2 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using glucose…………………………………………….… 4.3 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using sorbitol……………………………………………… 4.4 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using gluconate……………………………………………. 4.5 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using xylose………………………………………………..

.….60

……61

……92

……93

……93

…..139

….140

…..141

….142

…..143

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4.6 Metabolic stoichiometric analysis of fermentations by P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources………………………………………………… 4.7 Specific activity of major enzymes involving in the dicarboxylic acid pathway of P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources…..……………. 5.1 Strains and plasmids……………………………………………………….….. 5.2 Comparison of amino acid sequences from ack gene of P. acidipropionici to the corresponding sequences from other microorganisms……………….… 5.3 Comparison of fermentation kinetics from batch fermentations by P. acidipropionici wild type, ACK-Tet mutant, and pTAT-ACK-Tet mutant…………………………….…. B.1 Gel compositions for SDS-PAGE electrophoresis………...………………….

….144

….145

….180

….181

….182

…227

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LIST OF FIGURES Figure Page 1.1 Research objectives and the scope of this study………………………………. 2.1 The dicarboxylic acid pathway of P. acidipropionici………………………… 2.2 Integrational mutagenesis……………………………………………………... 3.1 Fed-batch fermentations of glucose by P. acidipropionici at pH 6.5, 32°C. A. Free-cell fermentation; B. Immobilized-cell fermentation in the FBB….… 3.2 Effect of propionic acid on the volumetric productivity of propionic acid in fed-batch fermentations with free cells and immobilized cells…………….. 3.3 Comparison of product yields from glucose in fed-batch fermentations with free cells and immobilized cells of P. acidipropionici. A. Cumulative propionic acid production vs. glucose consumption; B. Cumulative acetic acid production vs. glucose consumption; C. Cumulative succinic acid production vs. glucose consumption…………… 3.4 Effects of propionic acid on specific growth rates of P. acidipropionici wild type and mutant from the FBB…………………………………………… 3.5 The dicarboxylic acid pathway for the conversion of glucose to propionic, acetic, and succinic acids……………………………………….. 3.6 Effect of propionic acid on oxaloacetate transcarboxylase (A) and propionyl CoA: succinyl CoA transferase (B) activities of P. acidipropionici wild type and mutant from the FBB ……………………… 3.7 Effect of propionic acid on phosphotransacetylase (A) and acetate kinase (B) activities of P. acidipropionici wild type and mutant from the FBB……………………………………………………….….

…….8

……62

……63

……94

……95

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.….98

…..99

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…101

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3.8 Effect of propionic acid on PEP carboxylase activity of P. acidipropionici wild type and mutant from the FBB………………………. 3.9 Effect of propionic acid on membrane-bound ATPase activity of P. acidipropionici wild type and mutant from the FBB. A. Cells in the exponential phase; B. Cells in the stationary phase…………… 3.10 Scanning electron micrographs of P. acidipropionici showing morphological difference between the wild type and the mutant from the FBB. A. Wild type cells with a short rod shape; B. Mutant cells with an elongated slimmer rod shape………………………… 4.1 Fermentations by P. acidipropionici using glucose. A. Wild type; B. Adapted mutant from the FBB…………..……………………………….… 4.2 Fermentations by P. acidipropionici using sorbitol. A. Wild type; B. Adapted mutant from the FBB……………..…………………………….… 4.3 Fermentations by P. acidipropionici using gluconate. A. Wild type; B. Adapted mutant from the FBB………………………..………………….… 4.4 Fermentations by P. acidipropionici using xylose. A. Wild type; B. Adapted mutant from the FBB……………………..………………….…… 4.5 The dicarboxylic acid pathway for the formations of propionic, acetic, and succinic acids……………………………………….…. 4.6 Proposed pathways of sugar utilization in P. acidipropionici………………… 4.7 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici wild type using different carbon sources……………………………………… 4.8 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici adapted mutant from the FBB using different carbon sources…………..……. 5.1 Construction of an integrational plasmid, pTAT, with a fragment of 0.5-kb ack gene cloned from P. acidipropionici…………………………….…

…102

…103

….104

…146

…147

…148

…149

…150

…151

…152

…153

…183

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5.2 PCR product of partial ack gene…………………………………….………… 5.3 DNA and amino acid sequences of partial ack gene from P. acidipropionici... 5.4 Restriction enzyme map of partial ack gene from P. acidipropionici………… 5.5 Alignment of amino acid sequences of AK from E. coli, M. thermophila, B. subtilis, and P. acidipropionici………………....... 5.6 Southern blots of mutant ACK-Tet. A: 0.5-kb ack fragment as a probe; B: 1.9-kb Tetr as a probe……………………………………………………..... 5.7 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici……...….. 5.8 Relative activities of AK and PTA in ACK-Tet and pTAT-ACK-Tet mutants as compared to the wild type……………………………………………….…. 5.9 Fermentations by P. acidipropionici. A: Wild type; B: ACK-Tet mutant; C: pTAT-ACK-Tet mutant……………………………………………….…… B.1 The HPLC chromatogram for standard containing glucose, succinic acid, lactic acid, acetic acid, and propionic acid..…………………… B.2 The sample HPLC chromatogram of propionic acid fermentation by P. acidipropionici using glucose as the substrate…...…..………………… B.3 The standard curve of protein assay using bovine serum albumin…………..… B.4 An inorganic phosphate standard curve…………………...………………….. B.5 The analysis report of membrane fatty acid content of P. acidipropionici wild type…………………………………..……………..… B.6 The analysis report of membrane fatty acid content of P. acidipropionici adapted mutant from the FBB………………...……….…. C.1 A fibrous-bed bioreactor construction……………...……………………….…

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C.2 The system of propionic acid fermentation with a fibrous-bed bioreactor….... D.1 The genomic DNA isolated from P. acidipropionici……..………….….….… D.2 EcoRI digestion of TOPOACK1……………..………………….………….… D.3 Restriction enzyme digestion of pSSF1…………...………………………......

…237

…241

…249

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1

CHAPTER 1

INTRODUCTION

Propionic acid has numerous applications in various food and chemical industries.

It and its salts are mainly used as a mold inhibitor for animal feeds and as a preservative

for foods such as cheeses and baked goods. It plays an important role as an intermediate

in production of herbicides and cellulose plastics. Its derivatives are applied in

pharmaceuticals, plasticizers, and perfumes. In animal therapy, it is used in dermatoses,

wound infections, anti-arthritic drugs, and conjunctivitis (Boyaval and Corre, 1995).

Propionate ester is widely used as an additive for artificial fruit flavors. Currently,

propionic acid is industrially synthesized from petroleum feedstock. The current

production is around 440 million pounds with an annual growth rate of 1.8% through

2006. The current US market price is $0.51-$0.54 per pound (Chemical Market Reporter,

2003). More than 65% of total propionic acid demand is for animal feed and grain

preservatives (45%) and for human food preservatives (calcium and sodium salts) (21%).

The market demand of feed and grain preservatives has an average growth of ~2.5% per

year through 2006 and the future growth of human food preservatives, calcium and

sodium salts, is projected to increase with population growth (Chemical Market Reporter,

2003). Since propionic acid is mainly synthesized via the oxo-process or via the

2

liquid-phase oxidation of propane, propionaldehyde, or propanol, its current production is

vulnerable to sudden price fluctuations of propane and natural gas (Gu et al., 1999).

Currently, the consumer’s demand for fermentation-produced propionic acid as a natural

food preservative is high (Boyaval and Corre, 1995; Huang et al., 2002; Yang et al.,

1994). Propionibacteria have been granted GRAS (generally recognized as safe) status by

the United States Food and Drug Administration (Salminen et al., 1998) and are widely

used in the cheese industry. Therefore, fermentation by propionibacteria has a good

potential for the production of natural propionic acid to satisfy the market demand.

However, current propionic acid fermentation processes suffer from low

propionic acid yield, final propionic acid concentration, and reactor productivity caused

by a strong inhibition of propionic acid. Because of the low propionic acid concentration

and purity in the fermentation broth, the product recovery and purification cost is high. It

is thus important to develop an effective fermentation technology for economical

production of propionic acid.

The interest to improve propionic acid fermentation has been high. New

bioprocesses and mutant strains have been developed to improve propionic acid

production in terms of its yield, final product concentration, and productivity, but with

limited success (Emde and Schink, 1990; Huang et al., 1998; Jin and Yang, 1998; Lewis

and Yang, 1992; Paik and Glatz, 1994; Rickert et al., 1998; Solichien et al., 1995;

Woskow and Glatz, 1991). One significant obstacle in propionic acid fermentation is the

strong end-product inhibition caused by propionic acid even at a very low concentration

of 10 g/L (Gu et al., 1998; Hsu and Yang, 1991). A higher final product concentration in

3

the fermentation broth would facilitate product separation and recovery, and significantly

reduce the production costs (Van Hoek et al., 2003). A fibrous-bed bioreactor (FBB) has

been developed to improve final product concentration, product yield, and volumetric

productivity in several organic acid fermentations (Huang and Yang, 1998; Lewis and

Yang, 1992; Silva and Yang, 1995; Yang et al., 1995; 1994; Zhu et al., 2002; Zhu and

Yang, 2003). Cells immobilized in the FBB experienced rapid adaptation that enabled

them to tolerate higher concentrations of inhibitory fermentation products (Huang et al.,

1998; Huang and Yang, 1998; Zhu and Yang, 2003). In this work, fed-batch fermentation

using the FBB was performed to evaluate the potential to further enhance the final

propionic acid concentration and yield and to obtain mutants with high tolerance to

propionic acid.

Different carbon sources have been used to enhance propionic acid production;

however, little is known about the underlying mechanism of propionic acid fermentation.

Elucidation of metabolic patterns in propionic acid fermentation and a quantitative

understanding of flux distributions among various metabolic pathways are useful in

investigating and controlling a complex metabolism, which could lead to a potential

means of improving propionic acid production. Metabolic stoichiometric analysis has

been extensively used as a metabolic engineering tool to quantitatively evaluate alteration

of carbon flux distributions in the metabolic network influenced by different

physiological conditions. In this work, the controlling mechanism in propionic acid

fermentation affected by feeding various carbon sources with different oxidation states

was determined via metabolic stoichiometric analysis.

4

A quantitative understanding of flux distributions among various metabolic

pathways can provide a guideline for improving end-product production via directing

carbon flux toward a desired product (i.e., propionic acid) and eliminating or reducing the

formation of an undesired byproduct (e.g., acetic acid). Via genetic engineering

techniques, certain undesired genes can be knocked out in order to redistribute carbon

flux in the network toward the desirable products. Although genetic manipulations of

propionibacteria have been limited to the improvement of vitamin B12 production, these

techniques would be useful and helpful for genetic manipulation for enhanced propionic

acid production. In this work, the inactivation of an undesired gene was conducted to

evaluate the potential of using genetic engineering for enhanced propionic acid

production.

5

Objectives

The overall goal of this study was to develop a novel fermentation technology for

improved production of propionic acid by P. acidipropionici. To achieve this goal, the

following three specific objectives were proposed:

Objective 1. To improve propionic acid fermentation at process engineering level

A fibrous-bed bioreactor (FBB) was used to enhance propionic acid fermentation

by P. acidipropionici. Kinetics of fed-batch free-cell fermentation and immobilized-cell

fermentation using the FBB were compared to investigate the advantages of the FBB

over the conventional free-cell fermentation. Propionate inhibition on cell growth and

activities of major enzymes as well as the activity of membrane-bound ATPase and the

composition of membrane fatty acids were studied. Significant differences in cell

physiology and morphology between the wild type and the adapted mutant from the FBB

were observed. Not only did the immobilization of P. acidipropionici in the FBB enhance

propionic acid production with reduced by-product formation, but it also provided an

effective means to obtain a metabolically advantageous mutant with improved propionate

tolerance.

6

Objective 2. To elucidate the metabolic pathway of P. acidipropionici using different

carbon sources

Understanding the metabolism of different carbon sources in P. acidipropionici is

critical to metabolic engineering of the bacterium for enhancing propionic acid

production. In this work, the underlying mechanism in propionic acid fermentation was

investigated using various carbon sources (glucose, xylose, sorbitol, and gluconate) with

different oxidation states. Batch free-cell fermentations by both wild type and adapted

mutant from the FBB using these carbon sources were carried out. Metabolic

stoichiometric analysis was used to quantitatively evaluate metabolic flux distributions

and to better understand the fermentation pathway and flux control mechanism. The

activities of major enzymes in the metabolic pathway and overall protein expression

patterns were studied. The results indicated that propionic acid production could be

improved with reduced by-product formation by using a more-reduced carbon source

such as sorbitol. This finding can provide a guideline for genetic engineering to eliminate

by-product (acetate) formation in propionic acid fermentation.

7

Objective 3. To enhance propionic acid production by genetic engineering

Propionic acid production with reduced by-product formation would be more

feasible for commercial processes due to increased efficiency in substrate utilization and

decreased separation costs. Aiming to eliminate acetate formation, inactivation of the ack

gene, encoding acetate kinase that is one of key acetate-forming enzymes, was conducted

via gene disruption and integrational mutagenesis. Fermentation kinetics, the activities of

acetate-forming enzymes (AK and PTA), and the overall protein expression patterns of

the wild type and the ack-deleted mutants were studied to evaluate the effects of the ack

gene deletion on propionic acid fermentation. The results indicated that the ack-deleted

mutants improved propionic acid production by ~10% with a corresponding reduction in

acetate formation.

Figure 1.1 provides the overview of research objectives and the scope of this

study. The conclusions and recommendations are discussed in Chapter 6.

8

Figure 1.1 Research objectives and the scope of this study.

Overall Objective

To develop a fermentation process for economical production of propionic acid

Genetic Engineering * Gene Inactivation • Gene Disruption • Integrational Mutagenesis

- Fermentation kinetics - Acetate-forming enzyme activity - Protein expression pattern

(Chapter 5)

Process Engineering * Fibrous-Bed Bioreactor * Adaptation of P. acidipropionici

- Fermentation kinetics - Propionate tolerance (cell growth and enzymes) - Membrane ATPase - Membrane fatty acids - Morphology

(Chapter 3)

Metabolic Pathway Analysis * Effect of carbon sources with different oxidation states

- Fermentation kinetics - Metabolic stoichiometric analysis - Enzyme activity - Protein expression pattern

(Chapter 4)

9

CHAPTER 2

LITERATURE REVIEW 2.1 Propionic Acid Fermentation

2.1.1 Propionic Acid

Propionic acid, CH3CH2COOH, is one of important organic acids. It has been

applied in various food and chemical industries. The current production of propionic acid

is around 440 million pounds with the increasing growth of 1.8% annually through 2006.

The current price of propionic acid in the US market is $0.51-$0.54 per pound (Chemical

Market Reporter, 2003). Currently, in the US the major uses of propionic acid are for

animal feed and grain preservatives (45%), human food preservatives (21%), herbicides

(19%), cellulose acetate propionate (CAP) (11%), and miscellaneous uses (4%)

(Chemical Market Reporter, 2003). As a result of an antimicrobial activity, especially as

a mold inhibitor, salts of propionic acid are mainly used as food and feed preservatives.

Ammonium propionate is utilized in animal feeds and grains whereas calcium and

sodium propionates are used in human foods such as breads and cheeses. Moreover,

propionic acid is an important intermediate used in production of herbicides, cellulose

acetate propionate, and perfume. Propionate esters, classified as non-hazardous

10

air pollutant (non-HAPs) solvents, are substitutes for HAP solvents, mainly xylenes and

certain ketones (Chemical Market Reporter, 2003). Furthermore, propionic acid and its

derivatives are applied in production of pharmaceuticals, artificial fruit flavors (e.g.,

citronellyl and geranyl propionate), and plasticizers (e.g., glycerol tripropionate and

phenyl propionate) (Boyaval and Corre, 1995). In addition to the application in food

preservation, sodium propionate is also used in animal therapy: dermatoses, wound

infections, anti-arthritic drugs, and conjunctivitis (Boyaval and Corre, 1995).

At present, commercial production of propionic acid is solely via petrochemical

routes. Most propionic acid is synthesized by the oxo-process or by the liquid-phase

oxidation of propane. The oxo-process involves a reaction of ethylene and carbon

monoxide to obtain propionaldehyde, an intermediate, which is further oxidized to finally

obtain propionic acid (Chemical Market Reporter, 2003). Although the chemical

synthesis route is economical, a utilization of non-renewable petroleum feedstock, an

increase of propionic acid market price due to the increasing cost of petroleum feedstock,

and a cause of environmental and safety hazards are major concerns of most

manufacturers. Production via fermentation by propionibacteria is another route to supply

propionic acid. In general, fermentation is environmentally friendly and it can utilize

inexpensive substrates such as renewable resources, biomass, and food and dairy wastes

such as whey permeate and corn steep liquor (Begin et al., 1991; Huang et al., 1998;

2002; Yang et al., 1992), leading to a reduction of waste disposals from food and dairy

industries. Propionic acid obtained from the fermentation route is considered as a natural

acid preferred to be used in food and feed preservations.

11

Propionic acid production via fermentation can be a potentially competitive with

the production via petrochemical routes due to 1) the growing market of preservatives:

the average growth of 2.5% per year through 2006 of feed and grain preservatives and the

consumption of propionate salts as human food preservatives, which is relatively

increasing with the population growth (Chemical Market Reporter, 2003), 2) the increase

of consumer’s demand for natural propionic acid, 3) propionic acid produced by

propionibacteria, which have been granted GRAS (generally recognized as safe) status by

the United States Food and Drug Administration (Salminen et al., 1998), 4) the lower

cost of raw material feedstock for fermentation from waste biomass, and 5) the

fluctuation of cost of petroleum stock, which is a raw material for production via

petrochemical routes. However, a conventional fermentation process is economically

uncompetitive since the fermentation suffers from low propionic acid production: low

product concentration (<40 g/L), low product yield (<0.5 g/g), and low productivity (<1

g/L/h) (Jin and Yang, 1998). In order to make propionic acid via microbial production

economically attractive, the development of new fermentation processes is required.

12

2.1.2 Microorganisms

Propionibacterium and several other genera of anaerobic bacteria such as

Veillonella, Selenomonas, and Clostridium, especially C. propionicum, produce propionic

acid as a main fermentation product (Boyaval et al., 1994; Playne, 1985; Seshadri and

Mukhopadhyay, 1993). Other genera except for Propionibacterium can utilize more

limited range of sugar types as substrate; for example, Veillonella parvula can use only

lactate, pyruvate, and succinate but it cannot use carbohydrates as substrates (Playne,

1985). Therefore, Propionibacterium species have been most extensively applied and

studied for propionic acid production and granted GRAS (generally recognized as safe)

status by the United States Food and Drug Administration. Propionibacteria have been

used in production of Swiss cheeses (Hettinga and Reinbold, 1972b; Langsrud and

Reinbold, 1973), vitamin B12 (Hatanaka et al., 1988; Playne, 1985; Ye et al., 1999),

probiotics (Jan et al., 2001; Mantere-Alhonen, 1987; Mantere-Alhonen, 1983), and

propionic acid (Hsu and Yang, 1991; Lewis and Yang, 1992a;b;c; Paik and Glatz, 1994;

Woskow and Glatz, 1991). In swiss-type cheeses, propionibacteria consume lactate and

produce propionic acid, acetic acid, and CO2. The produced acids, accumulated proline

(Quelen et al., 1995), and metabolites from amino acid catabolism are responsible for

development of cheese flavor (Hettinga and Reinbold, 1972c). Moreover, the ‘eyes’ or

holes in the swiss-cheese body are formed as a result of CO2 produced from

propionibacteria (Langsrud and Reinbold, 1973). Propionibacteria such as P.

acidipropionici (Goswami and Srivastava, 2000; Himmi et al., 2000; Huang et al., 2002;

13

Lewis and Yang, 1992a;b;c; Paik and Glatz, 1994; Yang et al., 1994), P. shermanii

(Begin et al., 1991; Nanba et al., 1983; Neronova et al., 1967), P. freudenreichii

(Balamurugan et al., 1999), P. freudenreichii subsp. shermanii (Himmi et al., 2000), and

P. freudenreichii subsp. freudenreichii (Emde and Schink, 1990) are commonly used for

propionic acid production. However, P. acidipropionici has been the most used species

for developments of industrial propionic acid production (Colomban et al., 1993).

Propionibacteria are Gram-positive, non-motile, non-sporulating, short-rod-

shaped, mesophilic anaerobes. Since propionibacteria play an important role in a variety

of industrial applications, the nutrient requirements, growth conditions, and properties of

their metabolism have been extensively studied (Hettinga and Reinbold, 1972a;b;c;

Playne, 1985). All essential nutrients including carbon, nitrogen, and trace elements for

cell growth, product formation, and cellular maintenance are required in the fermentation

medium (Balamurugan et al., 1999). Mg2+ and Mn2+ in the medium have a major role in

the conversion efficiency of cell metabolism (Balamurugan et al., 1999). In P.

arabinosum, the decarboxylation of succinate, the first half of the last couple reactions in

the propionate formation pathway catalyzed by CoA transferase finally leading to

propionic acid formation, was accelerated by the presence of metallic cations like Mg2+

and Mn2+ in the medium (Katagiri and Ichikawa, 1953). The optimal growth conditions

are a pH range of 6 and 7 (Hsu and Yang, 1991) and a temperature range of 30 to 32°C

under anaerobic condition with N2. If the pH is below 4.5, there is practically no growth

and cell activity observed (Hsu and Yang, 1991; Jin and Yang, 1998; Playne, 1985).

14

2.1.3 Metabolic Pathway

Propionibacteria mainly utilize carbon sources to produce propionic acid as a

main product via the dicarboxylic acid pathway, as shown in Figure 2.1. The following

equation (solely EMP pathway was taken into consideration) represents a theoretical

formulation of propionic acid fermentation from glucose (Allen et al., 1964; Boyaval and

Corre, 1995):

1.5 glucose 2 propionate + acetate + CO2 + H2O + 6 ATP (2.1)

Glucose is first transported into the cellular cytoplasm toward the glycolytic

pathway. Glycolysis is responsible for catabolizing glucose into PEP, a final intermediate

of the glycolytic pathway. There are two alternative pathways: Embden-Meyerhorf-

Parnaz (EMP) pathway and Hexose Monophosphate (HMP) pathway in glycolysis. The

HMP pathway was proven for the existence in propionibacteria (Papoutsakis and Meyer,

1985b). Via the EMP pathway, 1 mole of glucose is converted into 2 moles of PEP and 2

moles of NADH, while the HMP pathway provides 5 moles of PEP and 11 moles of

NADH from 3 moles of glucose.

After glycolysis, at a PEP node, PEP, an energy-rich intermediate, is converted

into two intermediates, pyruvate and oxaloacetate. Pyruvate, a main branch-point

intermediate, is obtained from a conversion of a majority of PEP whereas the remaining

PEP is converted into oxaloacetate. Oxaloacetate is further utilized in succinate formation

pathway and finally succinate is formed as a byproduct. For pyruvate production, 1 mole

of PEP is converted into 1 mole of pyruvate and 1 mole of ATP obtained from a transfer

15

of one phosphoryl group from the high-energy intermediate, PEP, to the low-energy

compound, ADP. The total ATP obtained from the EMP and HMP pathways per mole of

glucose is 2 and 5/3 moles, respectively. It can be noted that when 1 mole of glucose is

consumed, glycolysis via the EMP pathway provides a lower amount of NADH (EMP:

HMP = 2: 11/3) but a higher amount of ATP (EMP: HMP = 2: 5/3). The ratio of EMP to

HMP utilization in glycolysis is dependent on propionibacterium species, substrates of

fermentation such as hexose and pentose sugars, and fermentation conditions

(Papoutsakis and Meyer, 1985b).

At a pyruvate branch point, pyruvate is directed toward three major pathways.

Most of pyruvate is converted into propionic acid via the Wood-Werkman cycle, a

propionate formation pathway. Some of pyruvate flows toward acetate formation

pathway while some is incorporated into biomass.

In the propionate formation pathway, after pyruvate enters the Wood-Werkman

cycle, oxaloacetate transcarboxylase catalyzes a couple reaction of pyruvate to

oxaloacetate and methylmalonyl CoA to propionyl CoA. In this couple reaction, the

carboxyl group transferred from methylmalonyl CoA to pyruvate to form propionyl CoA

and oxaloacetate is never released from the reaction or no exchange between this

carboxyl group with the dissolved CO2 in the fermentation broth is observed (Wood,

1981). Next, oxaloacetate is converted into malate by malic dehydrogenase and then

malate is channeled toward fumarate by fumarase. Then, a reaction of fumarate to

succinate is catalyzed by succinate dehydrogenase. After that succinate is converted into

succinyl CoA, which is then converted into methylmalonyl CoA. As previously

16

mentioned, methylmalonyl CoA is converted into propionyl CoA by oxaloacetate

transcarboxylase. Finally, propionyl CoA is converted into propionate along with a

reaction of succinate to succinyl CoA. This coupled reaction is catalyzed by propionyl

CoA: succinyl CoA transferase. After 1 mole of pyruvate enters the Wood-Werkman

cycle, 1 mole of propionate, 2 moles of NAD+, and 1 mole of ATP are generated. Not

only is propionic acid, a main fermentation product, produced in the Wood-Werkman

cycle, but also there is the NAD+ regeneration for glycolysis occurring in this cycle. The

regeneration is necessary for glycolysis and the Wood-Werkman cycle is a sole source of

NAD+ produced in cell metabolism.

In acetate formation pathway, a conversion of pyruvate to acetyl CoA and CO2 is

catalyzed by pyruvate dehydrogenase complex. Acetyl CoA, a high-energy thioester, is

converted into acetyl phosphate by phosphotransacetylase. Eventually, a conversion of

acetyl phosphate to acetate is catalyzed by acetate kinase. In acetate formation pathway, 1

mole of acetate, CO2, NADH, and ATP are obtained from 1 mole of pyruvate. The

acetate-formation pathway is an ATP-generating source of propionibacteria. Therefore,

propionic acid production is usually accompanied by the acetate formation as the major

ATP production pathway supplying energy for cellular metabolism (Goswami and

Srivastava, 2000; Rickert et al., 1998).

17

Some pyruvate is incorporated into cell biomass. Most of NADH and ATP

produced in cellular metabolism are utilized for biomass formation. As previously

mentioned, the remaining PEP flows toward oxaloacetate. The conversion of PEP and

CO2 to oxaloacetate is catalyzed by PEP carboxylase. Oxaloacetate is converted into

malate, then malate into fumarate, and finally fumarate into succinate. These steps are

similar to the succinate formation steps, which are parts of propionate formation.

2.1.4 Fermentation Processes

In general, fermentation by propionibacteria produces propionic acid as the main

product with acetic acid, succinic acid, and CO2 as byproducts. Depending on the

fermentation conditions, the product mole ratio between propionate and acetate with

glucose as the carbon source can vary between 2.1 and 14.7 (Wood, 1981; Wood and

Werkman, 1936). The major bottlenecks in biotechnological production of propionic acid

via fermentation are the notoriously slow growth of propionibacteria, a strong inhibition

of end products, especially propionic acid (Blanc and Goma, 1987a; Nanba et al., 1983;

Neronova et al., 1967), and difficulty in product purification processes due to the low

selectivity for propionic acid production and the low concentration of propionic acid in

the fermentation broth (Yang et al., 1994). Due to the strong inhibition of propionic acid,

the low selectivity for propionic acid production, and the slow growth of

propionibacteria, conventional fermentation suffers from low final propionic acid

concentration (<40 g/L), low propionic acid yield (<0.5 g/g), and low fermentation rate

18

(<1 g/L/h) (Jin and Yang, 1998). Due to the low propionic acid production and a costly

product recovery process, the production of propionic acid via fermentation has not been

economically competitive with the production via petrochemical routes. It was reported

that in the presence of propionic and acetic acids, the effects of the two acids on

fermentation are not additive. The acid with a relatively higher concentration in the

fermentation broth will play an important role in inhibiting cell growth and fermentation

(Neronova et al., 1967). In general, acetic acid had a weaker inhibitory effect on the

growth of P. shermanii (Neronova et al., 1967) and propionic acid is the strongest

inhibitor suppressing further propionic acid production (Obaya et al., 1994).

2.1.4.1 Inhibitory Effects of Propionic Acid on Cell Growth and Propionic Acid

Production

The major problem of propionic acid fermentation is a strong inhibitory effect of

propionic acid on further growth of propionibacteria and propionic acid production

(Blanc and Goma, 1987a; Gu et al., 1998; Herrero, 1983; Ibragimova et al., 1969; Lewis

and Yang, 1992a; Lueck, 1980; Neronova et al., 1967; Ozadali et al., 1996; Paik and

Glatz, 1994). It was reported that in the presence of propionic acid concentration more

than 2 g/L, cell growth and acid production were inhibited (Ramsay et al., 1998). The

increase of propionic acid concentration in the fermentation broth from 2.77 to 30.41 g/L

resulted in a two-third decrease in cell growth and a decrease in the specific propionic

acid production from 0.059 to 0.015 g/g cell/h (Gu et al., 1998). The decrease of

19

productivity with the increase of propionic acid concentration may be explained by a

combination of suppressed cellular metabolism and less selective production of propionic

acid (Gu et al., 1998). Moreover, it was found that more byproducts such as acetic, lactic,

and succinic acids were produced when there was an excessive amount of propionic acid

present in the medium. This alteration of bacterial metabolism resulted in the decreased

propionic acid yield from 0.52 to 0.41 g/g glucose (Gu et al., 1998).

Like other weak organic acids, propionic acid suppresses cellular metabolic

process in both cell growth and propionic acid production. The inhibition of propionic

acid was caused by its disruptive effect on the pH gradient, an essential motive force for

propionibacteria to transport nutrients and metabolites in and out of bacterial cells.

Maintaining a constant pH gradient is necessary for cells in a normal condition. The

presence of propionic acid in the medium disturbs the pH gradient across the cell

membrane. The cytoplasmic membrane prevents ionized compounds from diffusing into

the bacterial cells (Obaya et al., 1994). Thus, only the undissociated propionic acid is able

to diffuse through the bacterial membrane into the cytoplasm. At an alkaline pH inside

the cytoplasm, the undissociated acid then dissociates into a proton and a propionate

anion, creating the excess of protons inside the cytoplasm or the inward “leak” of protons

(Gu et al., 1998; Pèrez Chaia et al., 1994). The maintenance of a functional proton

gradient across the membrane is required for cell survival; therefore, H+-ATPase utilizes

extra ATP to extrude the excess protons to the outside. As a result, less ATP is provided

for cell metabolism, which inhibits cell growth and propionic acid production (Gu et al.,

1998; Pèrez Chaia et al., 1994).

20

2.1.4.2 Development of Fermentation Processes

Many efforts have been made through improvements of fermentation processes to

enhance propionic acid production. As seen in Table 2.1, fermentations with cell recycle

and cell immobilization systems have been extensively used to improve propionic acid

production, including productivity, yield, and final concentration.

Fermentation with cell recycle has been developed to improve propionic acid

production, especially reactor productivity, as a result of the high cell density maintained

in the reactor unit. Fermentation with cell recycle by ultrafiltration unit using whey

permeate obtained a high cell density of 100 g/L and achieved significantly high

productivity of 14.3 g/L/h (Boyaval and Corre, 1987).

Fibrous-bed bioreactors (FBB) and immobilization of cells in calcium alginate

beads, two of the most widely used cell immobilization systems, have also successfully

improved propionic acid production. High density of viable cells obtained in the system

led to a significantly enhanced acid production. Not only were significantly increased

final propionic acid concentration and propionate yield achieved by the immobilized-cell

fermentation as compared to the conventional free-cell fermentation, but improved

volumetric productivity was also obtained. Recycle-batch immobilized-cell fermentation

in the FBB using de-lactose whey permeate achieved a maximum propionic acid

concentration of 65 g/L, ~0.5 g/g propionic acid yield, and 0.22-0.47 g/L/h productivity

(Yang et al., 1995). Fed-batch fermentation with cells immobilized in calcium alginate

beads obtained a high final concentration of 57 g/L with 0.3 g/L/h productivity (Paik and

21

Glatz, 1994). The success of using cell immobilization system to enhance propionic acid

production indicates a potential of developed fermentation processes to supply

economical propionic acid to the growing market.

In addition, integration of fermentation with separation has been of interest.

Extractive fermentation using a hollow-fiber membrane extractor was developed. High

propionic acid concentration of 75 g/L with 0.66 g/g propionic acid yield and 1 g/L/h

productivity was achieved (Jin and Yang, 1998).

A fermentation process equipped with a three-electrode amperometric culture

system achieved a very high propionic acid yield of 0.973 g/g (Emde and Schink, 1990).

The developed fermentation processes showed their ability to achieve the high

performance in propionic acid production. This indicates propionic acid production via

fermentation could be potentially competitive with production via petrochemical routes.

2.1.4.2.1 Fermentation Processes for An Improvement of Final Propionic Acid

Concentration

As a consequence of the strong end-product inhibition, the conventional batch

fermentation hardly obtains the final concentration of >4% (w/v) (Herrero, 1983; Obaya

et al., 1992). Playne reported that most propionibacteria provided a final propionic acid

concentration ranging from 20 to 30 g/L (Playne, 1985). With 1% (w/v) propionic acid in

the medium, the specific growth rate of suspended cells in conventional fermentation

would decrease by more than 50% (Blanc and Goma, 1987b; Jin and Yang, 1998). When

22

the propionate concentration reached ~2% (w/v), the declination of cell concentration in

the fermentation broth was observed (Yang et al., 1995) and further propionic acid

production was suppressed or stopped due to the strong inhibitory effect of propionic acid

on cell growth and acid production. Sequential-batch fermentation with cell recycle using

ultrafiltration was applied to achieve propionic acid concentration between 30 and 40 g/L

(Colomban et al., 1993). A maximum propionic acid concentration of 47 g/L was

obtained from the fed-batch fermentation with propionate- tolerant P. acidipropionici

(Woskow and Glatz, 1991). At the acidic pH (pH 4.5-5.5) fermentation rates were much

slower than those at the optimal pH (pH 6-7) because of the strong acid inhibition at the

acidic pH (Hsu and Yang, 1991). Batch fermentation with P. acidipropionici using

lactose operated at the acidic pH (pH 5.5) achieved the final propionic acid concentration

of 2.70% (w/v) (Hsu and Yang, 1991).

2.1.4.2.2 Fermentation Processes for An Enhancement of Propionic Acid Yield

Propionic acid fermentation is usually accompanied by a formation of acetate to

maintain the hydrogen and redox balances in cellular metabolism and for stoichiometric

reasons (Lewis and Yang, 1992b; Martínez-Campos and de la Torre, 2002). Due to the

heterofermentative nature of fermentation, some of the carbon flux is directed toward

byproducts such as acetic acid, succinic acid, and CO2, and some is incorporated into

biomass, resulting in low propionic acid yield. Via the EMP pathway of glycolysis and

with no glucose used for the formation of cell biomass and succinic acid, theoretical

23

yields of propionic acid, acetic acid, and CO2 are 0.548, 0.22 and 0.17 g/g, respectively,

and the theoretical molar ratio of propionate: acetate (P/A) is 2:1. In fact, some of carbon

source must be consumed for biomass formation; therefore, the actual propionic acid

yield obtained from the conventional fermentation is less than 0.5 g/g glucose, usually in

the range of 0.25-0.4 g/g (Lewis and Yang, 1992b; Obaya et al., 1994). Product ratios

such as the P/A ratio are controlled for thermodynamic reasons and for ATP production

and entropy generation (Himmi et al., 2000; Lewis and Yang, 1992b). Propionic acid

fermentation is sensitive to pH (Hsu and Yang, 1991). It was found that the actual

fermentation product yield and P/A ratio were significantly dependent on pH and growth

conditions (Hsu and Yang, 1991; Lewis and Yang, 1992b; Seshadri and Mukhopadhyay,

1993). At lower pH values, higher propionic acid yield and P/A ratio were obtained but

cell growth and productivity were significantly sacrificed (Hsu and Yang, 1991).

Under two atmospheres of hydrogen, nearly sole propionic acid production was

obtained from Propionispira arboris as a result of the hydrogenase activity in this

bacterium (Thompson et al., 1984). It was reported that the significantly improved

propionic acid yields of 0.973 and 0.900 g/g were achieved when the fermentation of P.

freudenreichii subsp. freudenreichii using glucose was operated with a three-electrode

amperometric culture system and the fermentation medium contained 0.4 mM cobalt

sepulchrate and 0.5 mM anthraquinone 2,6 disulfonic acid, respectively (Emde and

Schink, 1990).

24

2.1.4.2.3 Fermentation Processes for An Improvement of Propionate: Acetate (P/A)

Ratio

Theoretically, the P/A molar ratio is 2; however, it was reported that the wide

variation of the P/A ratio from 2.1 to 14.7 has been observed for glucose as a carbon

source (Wood, 1981; Wood and Werkman, 1936). The P/A ratio changes greatly with

growth conditions (Seshadri and Mukhopadhyay, 1993). The P/A ratio decreases with an

increase in operating temperature (Seshadri and Mukhopadhyay, 1993). The decrease in

P/A ratio was reported when the temperature of fermentation by P. shermanii using

molasses as a carbon source was increased (Virkar et al., 1985). Temperature probably

influenced the specificity of enzymatic reactions, cell membrane permeability, and

metabolic regulatory mechanism of cells (Forage et al., 1985). pH is also a parameter

affecting the ratio of P/A. When pH decreased, an increase in P/A ratio was observed.

Moreover, the concentration of carbon source influences the P/A ratio as well. When the

initial glucose concentration of >100 g/L (105-115 g/L) was used, the P/A ratio of 9 g/g

was attained (Rickert et al., 1998). In addition, the type of substrates is another factor

affecting the P/A ratio. Fermentation of P. acidipropionici using mixed substrates of

lactate and glucose at the molar ratio of 4:1 provided the P/A ratio of 7.63 whereas that

using glucose as a pure substrate provided the ratio of 1.85 (Martínez-Campos and de la

Torre, 2002).

25

2.1.4.2.4 Fermentation Processes for An Enhancement of Propionic Acid

Productivity

It was found that the specific growth rate of propionibacteria is dependent on

propionic acid concentration (Balamurugan et al., 1999; Neronova et al., 1967).

Conventional batch fermentation by propionibacteria normally takes several days to

produce 1 to 3% (w/v) propionic acid concentration and to reach a batch completion

because of the very slow growth of propionibacteria (Blanc and Goma, 1989; Carrondo et

al., 1988; Paik and Glatz, 1994), which results in a low propionic acid productivity.

Fermentation with high cell density in the reactor has been developed in order to increase

propionic acid productivity. Higher productivities and acid concentrations can be

achieved when the higher concentration of living cells is maintained in the reactor (Paik

and Glatz, 1994) and the inhibitory effect of propionic acid on the cells is reduced

(Boyaval and Corre, 1987). Continuous fermentation using glycerol with a membrane

bioreactor containing high density of cells provided a maximum volumetric productivity

of 1 g/L/h (Boyaval et al., 1994). The fermentation with high cell density of 50 g/L

maintained in the reactor by cell recycling operated with sequential-batch mode attained

1.2 g/L/h propionic acid productivity (Colomban et al., 1993). Fermentation using xylose

coupled to membrane unit operated with a continuous stirred-tank reactor obtained a 2.2

g/L/h maximum volumetric productivity (Carrondo et al., 1988). Operated with a

continuous mode, fermentation by P. acidipropionici with a maximum cell biomass

concentration of 130 g/L obtained in the reactor by cell recycle using ultrafiltration

26

successfully obtained a maximum volumetric productivity of 5 g/L/h from whey as the

substrate (Blanc and Goma, 1989). Compared to the conventional fermentation providing

only 0.03 g/L/h productivity, the 14.3 g/L/h volumetric productivity was achieved from

P. acidipropionici fermentation using lactose from whey permeate in a continuous

stirred-tank reactor with recycling cells using an ultrafiltration unit for maintaining the

high cell density in the reactor (Boyaval and Corre, 1987). In conclusion, the method of

high cell density maintained in the reactor successfully attained the improved

productivity for propionic acid production.

2.1.4.2.5 Integration of Fermentation and Separation Processes for Improved

Propionic Acid Production

Another way to improve fermentation productivity is to overcome or eliminate

end-product, especially propionic acid, inhibition. Fermentations integrated with various

separation processes have been developed in order to reduce the inhibitory effect of

propionic acid on further cell growth and propionic acid production, leading to enhanced

reactor productivity for organic acid fermentation (Bar and Gainer, 1987; Daugulis, 1988;

Lewis and Yang, 1992c; Roffler et al., 1984; Yabannavar and Wang, 1991). Among

various separation processes, liquid-liquid extraction is the most widely studied (Gu et

al., 1999). There are a variety of extraction systems applied such as a system of non-toxic

extractant with a hollow-fiber membrane extractor (Gu et al., 1999; Jin and Yang, 1998)

and a flat-sheet-supported liquid membrane system (Ozadali et al., 1996) to continuously

27

remove propionic acid from the fermentation broth. Fed-batch fermentation of P.

acidipropionici using lactose was integrated with the extractive system of an amine

extractant and a hollow-fiber membrane extractor. Compared to the conventional

fermentation, a five-fold increase in propionic acid productivity (~1 g/L/h), ~20%

increase in propionic acid yield (up to 0.66 g/g), and a higher final concentration of

propionic acid (up to 75 g/L) with ~90% product purity were obtained from the integrated

process (Jin and Yang, 1998).

In order to maintain pH at a certain value during the fermentation to increase

fermentation productivity, propionate salts such as sodium and calcium propionate are

obtained after an addition of base such as NaOH. Electrodialysis with bipolar membranes

and electro-electrodialysis were developed to convert propionate salt, especially sodium

salt, to concentrated propionic acid with the current efficiency of >85% (Boyaval et al.,

1993). Moreover, separation of propionic and acetic acids by pertraction in a

multimembrane hybrid system was also reported (Wόdzki et al., 2000).

28

2.1.4.2.6 Fermentation Processes Using Waste Biomass

Propionibacteria are capable of utilizing a broad range of carbon sources. A

variety of carbon sources such as glucose (Himmi et al., 2000; Lewis and Yang, 1992b;

Rickert et al., 1998), lactose (Goswami and Srivastava, 2000; Hsu and Yang, 1991; Jin

and Yang, 1998), xylose (Carrondo et al., 1988), sucrose (Quesada-Chanto et al., 1994),

and lactate (Lewis and Yang, 1992a; Rickert et al., 1998). In addition to these hexose and

pentose sugars as well as lactate, numerous efforts of using low-cost substrates and

substrates without supplementary nutrients, as yeast extract and trypticase, have been

made in order to reduce the raw material cost.

There are a variety of waste biomass applied for propionic acid fermentation such

as whey permeate, a disposed byproduct of whey protein and whey lactose recovery

process (Boyaval and Corre, 1987; Yang et al., 1995; 1994), and corn steep liquor, a

byproduct of the corn wet milling process (Paik and Glatz, 1994). Moreover, plant

biomass, as hemicellulose (Ramsay et al., 1998), corn meal (Huang et al., 2002), and corn

(www.ag.iastate.edu/center/ccur/developingsubs.2.html), has been another alternative

carbon source for propionic acid fermentation. In addition to the use of carbohydrates as

carbon sources, the utilization of glycerol, a byproduct from fat industries such as

concentrated glycerine and glyceric wastewaters, (Barbirato et al., 1997; Himmi et al.,

2000) has been an interesting choice of substrate. It was found that batch fermentation of

P. acidipropionici using glycerol provided two-fold lower final acetic acid concentration

as compared to that using glucose (Himmi et al., 2000). The P/A ratio of shake-flask

29

fermentation using glycerol, glucose, and lactose were 37, 4, and 2.5, respectively.

Fermentation using glycerol could provide propionic acid production with low acetic acid

formation; however, succinic and formic acids as well as n-propanol are other byproducts

of fermentation.

Utilization of low-cost raw material for fermentation will allow propionic acid

production via biotechnological fermentation to supply propionic acid with low market

price; thus making propionic acid production via fermentation economically competitive

with the production via chemical synthesis.

In conclusion, a variety of fermentation processes, fermentation substrates,

integrated fermentation/separation processes have been developed to enhance propionic

acid production. Up till now, the highest propionic acid concentration obtained from free-

cell semicontinuous fermentation of propionate-tolerant P. acidipropionici using glucose

was 47 g/L with the propionic acid yield of 0.55 g/g and the volumetric productivity of

0.37 g/L/h (Woskow and Glatz, 1991). A maximum volumetric productivity of 14.3 g/L/h

was attained by P. acidipropionici fermentation in continuous stirred-tank reactor with

recycles using an ultrafiltration (Boyaval and Corre, 1987). The highest propionic acid

yield of 0.973 g/g was achieved in fermentation by P. freudenreichii operated with a

three-electrode amperometric culture system with fermentation medium containing 0.4

mM cobalt sepulchrate (Emde and Schink, 1990).

30

2.2 Cell Immobilization and Fibrous-Bed Bioreactor

2.2.1 Cell Immobilization

Immobilization of cells as biocatalysts is a powerful tool for utilizing enzymes or

enzyme systems from microorganisms in fermentation (Rickert et al., 1998). Cell

immobilization is defined as “The restriction of cell mobility within a defined space”

(Shuler and Kargi, 1992).

Cell immobilization has been widely used to effectively and economically

improve performance and production of fermentation processes. The immobilized-cell

fermentation offers several potential advantages over the free-cell fermentation; 1) the

high cell density obtained in the immobilized-cell bioreactor improves the reactor

volumetric productivity, 2) the expensive cell separation is eliminated due to cell

immobilization facilitates the separation of cells from products in the fermentation broth,

3) the microenvironment in the matrix of the system offers a protection to immobilized

cells against unfavorable physiological conditions such as extreme pH or temperature, 4)

there are no problems of cell washout when it is operated with continuous mode with a

high dilution rate, 5) a better performance leads to improvements of production of desired

fermentation products, and finally it was found that immobilized cells were more tolerant

to inhibitory fermentation products present in the medium as compared to the free cells

(Keweloh et al., 1989).

31

Entrapment and binding are two major methods of active cell immobilization.

There are several methods of entrapment such as physical entrapment, encapsulation, and

macroscopic membrane-based reactors. The most extensively used technique of cell

immobilization is physical entrapment within porous matrices (Shuler and Kargi, 1992).

For instance, P. thoenii immobilized in calcium alginate beads has been developed to

improve propionic acid production (Rickert et al., 1998). Binding, the other method, is

divided into 2 groups: physical adsorption and covalent binding. Physical adsorption on

inert support surfaces has been the most widely used technique for cell immobilization

while covalent binding has not been mainly used in cell immobilization but widely used

for enzyme immobilization (Shuler and Kargi, 1992).

2.2.2 Immobilized-Cell Bioreactor

Several types of immobilized-cell bioreactors such as a packed-bed bioreactor, a

hollow-fiber membrane, and a fluidized-bed bioreactor have been developed. The high

cell density obtained in the bioreactor offers the advantage of improving propionic acid

production in both final concentration and volumetric productivity over the conventional

free-cell fermentation. However, these conventional packed-bed and membrane

bioreactors suffer from 1) unstable long-term production due to the loss of cell viability

over the period of operating time, 2) clogging by cell biomass and dead cells, 3) high

pressure drop and gas entrapment inside the bed of reactor, reducing the reactor working

32

volume, and 4) the loss of reactor productivity over time. Fluidized-bed bioreactors also

struggled with a problem of unstable bed expansion due to a formation of biofilm.

2.2.3 Fibrous-Bed Bioreactor

The significantly efficient fibrous-bed bioreactor (FBB) has been developed and

extensively used for improving production of numerous carboxylic acids such as

propionic acid (Huang et al., 2002; Yang et al., 1995; 1994), butyric acid (Zhu et al.,

2002; Zhu and Yang, 2003), lactic acid (Silva and Yang, 1995), and acetic acid (Huang et

al., 1998; 2002).

The FBB has overcome problems the conventional immobilized-cell bioreactors

encountered. A spiral wound fibrous matrix was packed in the bioreactor. The matrix is

highly porous providing large void volume for cell entrapment and it has a large surface

area for cell attachment. A proper thickness and a high porosity of the matrix layer

efficiently maintain high mass transfer rates in the reactor. The built-in vertical gaps

among the matrix layers allow gas such as CO2 to escape from the top of the reactor,

eliminating gas entrapment, and permit sloughed-off non-active cells to fall off to the

bottom of the reactor, overcoming a problem of high pressure drop. The FBB also has a

regenerative ability with continuously replacing old and non-active cells with new and

active cells, allowing the FBB to maintain a stable long-term productivity. It has been

proven to provide many advantages over the conventional bioreactors. Material and

construction of the FBB are inexpensive and it is easy to scale up. The FBB contains high

33

viable-cell concentration, resulting in improved organic acid production with a stable

long-term productivity. No contamination is observed when the FBB is operated.

2.3 Immobilized-Cell Fermentation

The maintenance of high concentration of cells in the reactor, either by

immobilizing cells in or on a solid support or by recycling free cells back to the reactor, is

able to compensate for the slow growth of propionibacteria (Gu et al., 1998) and to allow

the improved propionic acid concentration and volumetric productivity to be achieved

(Lewis and Yang, 1992a; Paik and Glatz, 1994). It was reported that growth condition

was a factor controlling propionic acid production to be either growth-associated or non-

growth-associated (Hsu and Yang, 1991). It was found that for propionate-tolerant strain

of P. acidipropionici immobilized in calcium alginate beads further propionic acid

production still continued even if the further growth had ceased (Paik and Glatz, 1994).

This could be considered that these immobilized cells are non-growing but in a

metabolically active state (Paik and Glatz, 1994; Woskow and Galtz, 1991). Propionate-

tolerant strain of P. acidipropionici (P200910) showed the pattern of non-growth-

associated product formation (Woskow and Glatz, 1991). Similar to Paik and Glatz’s

conclusion, the growth phase of P. acidipropionici does not normally affect the propionic

acid production rate (Hsu and Yang, 1991). Repeated-batch fermentation of P. thoenii

immobilized in calcium alginate beads using 75 g/L glucose achieved the final propionic

acid concentration of 34 g/L, the propionate yield of 0.45 g/g, and the P/A ratio of 4.8

34

(Rickert et al., 1998). A maximum propionic acid concentration of 57 g/L and 45.6 g/L

were obtained from P. acidipropionici fed-batch fermentations with cells immobilized in

calcium alginate beads using semidefined medium and corn steep liquor, respectively

(Paik and Glatz, 1994). Moreover, the volumetric productivity increased from 0.3 g/L/h

to 0.96 g/L/h when the fermentation mode was changed from fed-batch to continuous

modes (Paik and Glatz, 1994).

The FBB has been widely used with various substrates and fermentation modes to

achieve improved propionic acid production. Continuous fermentation of P.

acidipropionici immobilized in the FBB using lactate was developed and a high cell

density of ~37 g/L was obtained in the FBB. It was reported that the reactor productivity

obtained from the immobilized-cell fermentation was four-time higher than that from the

conventional free-cell fermentation (Lewis and Yang, 1992a). The FBB could be

operated with low nutrient and low pH without a large expense of reactor productivity

(Lewis and Yang, 1992a). The highest propionic acid concentration of 65 g/L was

achieved in the fermentation of immobilized P. acidipropionici in the FBB with a

recycle-batch mode using de-lactose whey permeate (Yang et al., 1995). The volumetric

productivity increased from 0.22-0.47 g/L/h, which has already been higher than the

conventional batch fermentation with unsupplemented whey permeate (~0.045 g/L/h)

(Woskow and Glatz, 1991), to 0.675 g/L/h when the whey permeate was supplemented

with yeast extract (Yang et al., 1995). With no nutrient supplementation, the

immobilized-cell fermentation in the FBB using whey permeate operated with a

continuous mode provided the higher volumetric productivity (0.35-0.78 g/L/h) (Yang et

35

al., 1994) than that with a recycle-batch mode (0.22-0.47 g/L/h) (Yang et al., 1995).

Application of whey permeate with no nutrient supplementation as substrate for the

fermentation would lower the raw material cost of the fermentation and probably make

the fermentation be more feasible. Due to its capability of providing improved propionic

acid production, plain whey permeate with no nutrient supplementation would be a

promising feed substrate for propionic acid production via the long-term continuous

fermentation with cells immobilized in the FBB (Yang et al., 1994). Corn meal is another

substrate used for propionic acid production via the immobilized-cell fermentation in the

FBB. The immobilized P. acidipropionici fermentation in the FBB using corn meal

hydrolyzate achieved 0.58 g/g propionic acid yield and 2.12 g/L/h volumetric

productivity (Huang et al., 2002). The yield was higher than the theoretical yield of 0.548

g/g. This could be explained that other nutrients present in the hydrolyzate may

contribute to the enhanced end-product formation (Huang et al., 2002).

So far, the highest propionic acid concentration of 65 g/L was obtained from the

immobilized-cell fermentation in the FBB using whey permeate operated with a recycle-

batch mode. A maximum volumetric productivity of 2.12 g/L/h and a yield of ~0.58 g/g

were achieved from the immobilized-cell fermentation in the FBB using corn meal

hydrolyzate. The immobilized-cell fermentation in the FBB using waste biomass, as

whey permeate, would be a promising fermentation process that allows propionic acid

production via fermentation to be economically competitive with production via

petrochemical routes.

36

2.4 Metabolic Engineering

Metabolic engineering is defined as the manipulation of the cellular metabolism

to achieve a desired goal of process optimization (Bailey, 1991; Desai et al., 1999a;b;

Farmer and Liao, 1996; Koffas et al., 1999; Lee and Papoutsakis, 1999). Metabolic

engineering integrates the methodology of synthesis and analysis to improve the flux

distribution of cellular metabolic pathways (Shimizu, 2002). In metabolic engineering

field, the combination of macroscopic analysis of whole bioprocesses such as

fermentation and microscopic analysis of intracellular networks contributed to

biotechnology (Shimizu, 2002). The metabolic pathway of a particular organism has been

focused in order to maximize the yield of desired metabolites (Venkatesh, 1997).

Metabolic engineering has many significant applications such as the improvement of

production of metabolites produced by host organisms and the modification of cell

properties facilitating biotechnological processes such as fermentation and/or product

purification (Lee and Papoutsakis, 1999). These applications can greatly improve organic

acid production via fermentation and make the process economically competitive as

compared with the current chemical synthesis process.

2.4.1 Metabolic Flux Analysis

Metabolic engineering has been applied through several effective methodologies

such as metabolic flux analysis (MFA), metabolic control analysis (MCA), 13C-NMR and

37

GCMS measurements as well as kinetic modeling. Metabolic flux analysis (MFA) is a

systematic approach to assess the role of individual metabolic reactions in the metabolic

pathway by the calculation of the fluxes through various pathways with the analysis of

factors affecting the flux distribution. MFA is used to quantify various pathway fluxes, to

determine intracellular carbon flows, to identify possible branch points in the metabolic

pathways, to calculate non-measured extracellular and intracellular fluxes, and to

estimate the maximum theoretical yield based on a stoichiometric model of the particular

microorganism (Granström et al, 2002; Nielsen, 1998; Stephanopoulos et al., 1998). The

data of substrate uptake rates, metabolite secretion rates, metabolic stoichiometry, and

quasi-steady state mass balances on intracellular metabolic intermediates are combined to

determine intracellular metabolic fluxes under different environmental conditions

(Stephanopoulos, 1999). The number of biochemical reactions selected to represent the

metabolic pathway is mainly dependent on the culture conditions and the information

required for the desired goal (Venkatesh, 1997). The set of mass balance equations for the

intracellular and extracellular metabolites included in the stoichiometric relationships of

reactions can be represented as

A⋅ X(t) = Y (2.2)

where A is a matrix of stoichiometric coefficients, X(t) is a matrix containing vector of

the individual branch flux, and Y is a matrix of accumulation rates of intracellular and

extracellular metabolites. A pseudo-steady state approximation, a zero net accumulation

rate, on the accumulation of the intracellular metabolic intermediates is based on the fact

that the accumulation of intermediates is very small as compared to the cell growth rate

38

or production and consumption of these intermediates. The reaction stoichiometry was

developed based on the fermentation biochemistry, the assumption of ATP yield, which

is dependent on types of carbon source used for cell growth, and two well-accepted

biological regularities, the carbon weight fraction in biomass and the composition

reductance degree of biomass (Papoutsakis and Meyer, 1985a). The stoichiometric

equations for propionibacteria were derived (Papoutsakis and Meyer, 1985b). These

equations can be used to calculate the maximal product yield and the selectivity for

various fermentation products and to check the consistency of experimental data

(Papoutsakis and Meyer, 1985a). It was found that Hexose Monophosphate (HMP)

pathway exists in propionibacteria and the glycolytic pathway of propionibacteria is via a

combination of Embden-Meyerhof-Parnas (EMP) and HMP pathways (Papoutsakis and

Meyer, 1985b). In addition, fermentation equations are useful for calculating the extent of

utilization of the EMP and HMP pathways in glycolysis (Papoutsakis and Meyer, 1985a).

The information on metabolic flux distributions obtained from MFA has been used to

understand the phenomenon in many fermentation processes (Hua et al., 2001; Jorgensen

et al., 1995; Kiss and Stephanopoulos, 1991; Papoutsakis and Meyer, 1985b;

Vanrolleghem et al., 1996).

MFA has been applied to elucidate metabolic pathways of several

microorganisms. MFA was first developed to elucidate the metabolic pathway of

Corynebacterium glutamicum during growth and lysine synthesis in a defined medium

(Vallino and Stephanopoulos, 1993). MFA was applied to reveal the roles of the acid-

forming enzymes in the complex primary metabolism of C. acetobutylicum (Desai et al.,

39

1991a). Effects of pH on the uptake rates of lactose and influences of lactate ion

concentration on the flux distribution in the metabolic network of Streptococcus lactis

were determined by using MFA (Venkatesh, 1997). It was found that based on MFA

changes in environmental conditions as pH and culture redox potential or the introduction

of either a hydrogen atmosphere or nicotinic acid to affect the intracellular NADH:

NAD+ ratio substantially influenced the intracellular carbon flux in Clostridium

thermosuccinogenes (Sridhar and Eiteman, 2001). Moreover, in vitro enzyme assay was

introduced and combined with MFA for the study of Candida tropicalis growing on

xylose in an oxygen-limited chemostat (Granström et al., 2002). The in vitro enzyme

assay allowed the effects of different metabolic pathways on ATP yield in xylose and

xylose-formate cultivations to be implicated (Granström et al., 2002). In conclusion,

metabolic flux analysis has been proven to be a powerful technique to quantify the

metabolic flux distributions and elucidate an alteration of the metabolic pathway under

particular physiological conditions.

2.4.2 Applications of Other Metabolic Engineering Techniques

In vivo NMR studies have been applied to elucidate metabolic pathways, to

extract additional information about metabolic pathway fluxes, and to confirm flux

estimations obtained by material balancing (Stephanopoulos, 1999). In vivo 13C-NMR

was used to elucidate the role of carboxylation reactions in propionate metabolism

(Houwen et al., 1991). 13C-NMR of chloroform-methanol extracts was applied to

40

establish the intracellular composition of dairy propionibacteria (Rolin et al., 1995).

Confirmed with the 13C-NMR spectral analysis, propionate is oxidized to pyruvate via a

reversed Wood-Werkman cycle when P. freudenreichii was grown under aerobic

condition (Ye et al., 1999). The bi-directional reaction of the Wood-Werkman cycle was

studied by using the in vivo 13C-NMR and it was found that these reactions involved in

central carbon metabolic pathways of propionibacteria during pyruvate catabolism

(Deborde et al., 1999).

2.5 Genetic Engineering of Propionibacteria

2.5.1 Acetic Acid Formation in Propionic Acid Fermentation

In general, fermentation by P. acidipropionici produces propionic acid as a main

product accompanied by acetic acid as a key byproduct. Based on the theoretical

formulation of propionic acid fermentation from glucose, the maximum propionic acid

yield would be 2 moles of propionic acid per mole of glucose (0.822 g/g) if glucose was

completely utilized via the EMP pathway of glycolysis and formations of byproducts,

acetate and CO2, and biomass could be completely eliminated. Reduced acetate formation

in propionic acid production could dramatically lower the separation cost, resulting in

reduced cost of fermentation products.

Several studies have focused on eliminating acetate formation in propionic acid

fermentation. The improvement of propionic acid yield (>0.9 g/g) has been achieved via

41

the use of sophisticated processes operated either under two atmospheres of hydrogen

(Thompson et al., 1984) or with a three-electrode amperometric culture system (Emde

and Schink, 1990). The molar ratio of propionate: acetate increased from 2 to 14 when

the fermentation was operated under a low pH condition (Hsu and Yang, 1991; Seshadri

and Mukhopadhyay, 1993). Unfortunately, these processes are impractical for

commercial production due to extremely low productivity, high capital input required for

process setup, and difficulty in scale up.

The use of a sole carbon source or a mixture of carbon sources has achieved the

improved propionate yield with the decreased acetate yield, leading to the enhanced P/A

molar ratio. The P/A molar ratio of 7.63 was obtained from P. acidipropionici using a

mixture of lactate: glucose at a molar ratio of 4 (Martínes-Campos and de la Torre, 2002).

Glycerol as the substrate gave a high propionate yield (0.844 mol/mol) with low acetate

formation (0.023 mol/mol) (Barbirato et al., 1997). A high propionate yield of 0.79

mol/mol with low acetate yield of 0.17 mol/mol was also reported (Himmi et al., 2000).

However, the formation of other unconventional byproducts, such as formic acid and n-

propanol, was accompanied when glycerol was used as the substrate. Genetic

manipulation of the acetic acid formation pathway can be one effective means to enhance

propionic acid production with reduced acetate formation by directing carbon flow from

the acetate formation pathway toward the propionate formation pathway.

42

2.5.2 Acetic Acid Formation Pathway Genes and Enzymes

The acetate formation pathway, one of energy (ATP) sources besides the sugar

utilization pathway (i.e., glycolysis), consists of the conversion of pyruvate to acetyl CoA

by pyruvate dehydrogenase complex, acetyl CoA to acetyl phosphate by

phosphotransacetylase (PTA, EC 2.3.1.8), and acetyl phosphate to acetate by acetate

kinase (AK, EC 2.7.2.1). The PTA - ACK pathway may play a more important role in

regulating the intracellular concentration of acetyl phosphate, a global regulator that

could regulate the cell responses to the environmental stimulations and the cell mobility

in a metabolic network, than in the energy conversion (McCleary et al., 1993; Wanner

and Willmes-Reisenberg, 1992).

To date, ack and pta genes have been cloned, sequenced, and characterized from

E. coli (Kakuda et al., 1994a; Matsuyama et al., 1989), Methanosarcina thermophila

(Latimer and Ferry, 1993), Bacillus subtilis (Grundy et al., 1993), and Clostridium

acetobutylicum (Boynton et al., 1996). The DNA sequence of the ack from Mycoplasma

genitalium and Haemophilus influenzae are also available in the genome database but

only defined by sequence homology (Boynton et al., 1996). AKs from E. coli and

Salmonella typhimurium had a homodimer with 40 kDa molecular mass (Fox and

Roseman, 1986) while those from M. thermophila and C. thermoaceticum had ones with

94 kDa (Aceti and Ferry, 1988) and 60 kDa (Schaupp and Ljungdahl, 1974), respectively.

The ack deletion generally affected cell growth as the mutant had lower specific

growth rate than the wild type. However, similar or indistinguishable kinetics of

43

fermentation was observed in the ack-deleted mutant as compared to the wild type

(Kakuda et al., 1994b). On the other hand, significant decrease in acetate formation was

observed in pta- or pta-ack-deleted mutants (Diaz-Ricci et al., 1991; Kakuda et al.,

1994b).

2.5.3 Genetics and Molecular Biology of Propionibacteria

2.5.3.1 Introduction

Development of genetic manipulation system in propionibacteria can enable

production of heterologous proteins for use in the food industry (Kiatpapan and Murooka,

2002). However, there have been few genetic manipulation studies in propionibacteria

because of 1) the high GC content of propionibacteria, 2) the lack of information on

plasmid sequence determination, 3) the lack of available vectors for gene transfer, 4) the

presence of a strong restriction-modification system in propionibacteria, and 5) the lack

of an appropriate selective marker (Kiatpapan and Murooka, 2002).

Recently, research in genetics and molecular biology of propionibacteria has

made some progress (van Luijk et al., 2002). Table 2.2 shows an overview of genetic

manipultation in propionibacteria. The high restriction-modification system in

propionibacteria; for example, has been overcome by the use of the shuttle vector

prepared from propionibacteria (Kiatpapan and Murooka, 2002; van Luijk et al., 2002).

Moreover, the efficient transformation (Kiatpapan and Murooka, 2002; van Luijk et al.,

44

2002) and the development of expression vectors with native promoters from

propionibacteria (Kiatpapan and Murooka, 2001) were achieved. To date, approximately

30 gene sequences with attributed coding functions from propionibacteria are available

on databases (van Luijk et al., 2002). In addition, the complete 2.6-megabase genome of

P. freudenreichii subsp. freudenreichii (ATCC 6207) was successfully sequenced by

DSM Food Specialties and Friesland Coberco Dairy Foods as announced at the 3rd

International Symposium on Propionibacteria in Zurich (2001) (van Luijk et al., 2002).

2.5.3.2 Plasmids in Propionibacteria

Rehberger and Glatz, 1990 first screened and characterized endogenous plasmids

in propionibacteria. Plasmids ranged in size from 4.4 MDa to 119 MDa or higher. P.

acidipropionici strain contained only the pRG01 of 4.4 MDa while P. freudenreichii

strain contained the most diverse profile of plasmids (Rehberger and Glatz, 1990). RepA,

encoded by orf1 of pRG01, had homology to the theta replicase found in several Gram-

positive bacteria with the high GC content, indicating that the pRG01 may replicate via

the theta-type replication. Moreover, RepB, encoded by orf2 of pRG01, may play a role

in the initiation of plasmid replication (Kiatpapan and Murooka, 2002).

45

2.5.3.3 Construction of Shuttle Vectors

Development of shuttle vectors has recently been achieved by using a replicon

from an endogenous plasmid of propionibacteria and appropriate selection markers (Jore

et al., 2001; Kiatpapan et al., 2000). A pPK705 was constructed using the pRG01

replicon, pUC18, and the Streptomyces hygBr gene as a selection marker (Kiatpapan et

al., 2000). Construction of a pBRESP36A by using the p545 replicon, pBR322, and the

erythromycin resistance gene from Saccharopolyspora erythraea has also been done

(Jore et al., 2001).

2.5.3.4 Development of Expression Vectors

pKHEM01 and pKHEM04 were constructed by subcloning native promoters,

p138 and p4, respectively, from propionibacteria into the shuttle vector pPK705. These

expression vectors have been successfully used in the production of 5-aminolevulinic

acid (ALA) (Kiatpapan and Murooka, 2001). This indicates that the efficient expression

vectors would facilitate genetic studies of propionibacteria for production of both vitamin

B12 and propionic acid.

46

2.5.3.5 Molecular Analysis of Promoter Elements

Strong promoters are essential for efficient foreign gene expressions in

propionibacteria (Piao et al., 2004a). A consensus sequence of promoters in P.

freudenreichii was proposed, facilitating the selection or development of promoter

sequences for achieving high-level heterologous gene expression in medically and

industrially important organisms (Piao et al., 2004a).

2.5.3.6 Transformation of Propionibacteria

The transformation can be performed by either protoplast transformation or

electroporation (Kiatpapan and Murooka, 2002; Luchansky et al., 1988). Electroporation

is considered as a non-specific method for gene transfers in a variety of microorganisms.

Several treatments have been tried in order to improve the transformation efficiency.

Johnson and Cummins (1972) found that lysozyme treatment was not effective for

propionibacteria because of lysozyme resistance in many strains of propionibacteria. The

high efficiency of electro-transfection of 105 cfu/µg of DNA was successfully obtained

by treating cells in the medium containing glycin and using Propionibacterium

bacteriophage DNA (Gautier et al., 1995). Since glycin was incorporated into the cell

wall components, which resulted in a less cross-linked cell wall, the barrier by the thick

cell wall of propionibacteria was overcome. However, the DNA used in this study was

47

isolated from the Propionibacterium phage, which would not provide a general method

for obtaining stable genetic transformants (van Luijk et al., 2002).

Recently, efficient electro-transformation systems for propionibacteria have been

reported (Jore et al., 2001; Kiatpapan et al., 2000). Kiatpapan et al. (2000) prepared

competent cells in 10% glycerol and used the pPK705 as the transferred DNA. The

electroporation condition was set at the electric field strength of 6.0 kV/cm, the

capacitance of 25 µF, and the resistance of 129 Ω. As a result, high transformation

efficiency of 106-107 cfu/µg of DNA was obtained in P. pentosaceum and P.

freudenreichii (Kiatpapan et al., 2000). In the study by Jore et al. (2001), cells were

prepared in 0.5 M sucrose buffered with 1 mM potassium acetate (pH 5.5) and

pBRESP36A was used for transformation at 20 kV/cm, 25 µF, and 200 Ω to obtain a high

transformation efficiency of ≥108 cfu/µg of DNA. The success in development of

efficient transformation systems in propionibacteria may be due to the use of the replicon

from the endogenous plasmid and the appropriate selection marker (Jore et al., 2001;

Kiatpapan et al., 2000). The existence of the strong system of restriction-modification in

propionibacteria was overcome by the use of vectors prepared from propionibacterium

cells (Kiatpapan et al., 2002).

48

2.5.3.7 Strategies for Genetic Manipulation in P. acidipropionici

Both gene inactivation and gene overexpression, two useful metabolic

engineering tools, have been extensively applied for genetic modifications of many

microorganisms such as C. acetobutylicum (Boynton et al., 1996; Green et al., 1996), E.

coli (Kakuda et al., 1994b), M. thermophila (Latimer and Ferry, 1993), and

Thermococcus kodakaraensis KOD1 (Sato et al., 2003). Two methods have been mainly

used for the inactivation of gene on the host chromosome. Gene disruption is the first

method whose strategy is to insert an antibiotic resistance cassette in the middle of the

cloned gene of interest. The linear fragment of the disrupted gene would be used to

transform host cells and the original copy of the target gene in the genome would be

replaced with the linear fragment via homologous recombination. The linear fragment

containing two trp regions at both ends of the pyrF, a marker cassette, was used to obtain

gene disruption by homologous recombination in T. kodakaraensis KOD1 (Sato et al.,

2003). The second is integrational mutagenesis by using a non-replicative integrational

plasmid containing a fragment of the target gene and an antibiotic resistance cassette, the

selection marker, to transform the host cells. The partial gene in the non-replicative

plasmid can recombine with the internal region of the original target gene in the parental

chromosome, resulting in the insertional inactivation of the target gene. The insertion

occurs in a Campbell-like fashion (Campbell, 1962), which results in duplicated

homologous regions flanking the plasmid DNA. Integrational mutagenesis could occur if

the homologous DNA fragments are internal to the transcription unit, resulting in the

49

disruption of the unit and the loss of unit function and producing a mutant phenotype

(Figure 2.2). The non-replicative plasmid (pJC4) with the partial pta gene was

constructed and integrated into the homologous region of original pta gene on the

chromosome of C. acetobutylicum ATCC 824, resulting in a reduction of PTA and AK

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(Wilkinson and Young, 1994).

50

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Table 2.1 Development of fermentation processes for improved propionic acid production.

Propionic Acid Production

Fermentation Process/ Mode Substrate Cell Density (g/L)

Final Conc. (g/L)

Yield (g/g)

Productivity (g/L/h)

References

Cell Recycle (Ultrafiltration) continuous whey permeate 100 25 - 14.3 Boyaval and Corre, 1987 continuous whey permeate 130 17 - 5 Blanc and Goma, 1989 sequential-batch whey permeate 50 30-40 - 1.2 Colomban et al., 1993 Cell Immobilization Fibrous-Bed Bioreactor

batch corn meal hydrolyzate - - 0.58 2.12 Huang et al., 2002

recycle-batch de-lactose whey permeate 34 65 0.5 0.22-0.47

(0.68 with yeast extract) Yang et al., 1995

Calcium- Alginate Beads

fed-batch semidefined medium

9.8x109 cells/g beads 0.1 g beads/ mL medium 57 - 0.3

(0.96: continuous mode) Paik and Glatz, 1994

repeated-batch glucose 2x1011 cells/g beads

beads (g)= 40% of broth volume 34 0.45 - Rickert et al., 1998 Extractive Fermentation (Hollow-fiber membrane)

fed-batch lactose - 75 0.66 1 Jin and Yang, 1998 Three-Electrode Amperometric Culture System

batch glucose - - 0.973 - Emde and Schink, 1990

60

58

Genetic Manipulation Performance References

Improved Transformation Efficiency

Construction of shuttle vector, pPK705 106-107 cfu/µg DNA Kiatpapan et al., 2000

Construction of shuttle vector, pBRESP36A ≥108 cfu/µg DNA Jore et al., 2001

Improved Production of Desired Products

Cholesterol oxidase

(Expression vector harboring Streptomyces choA ) - Kiatpapan et al., 2001

5-Aminolevulinic acid (ALA)

(Expression vector harboring hemA from

Rhodobacter spheroides)

8.3 mM ALA (comparable with that by recombinant E. coli)

Kiatpapan and Murooka, 2001

Vitamin B12

(Expression vector harboring hemA from

R. spheroides and endogenous hemB and cobA)

1.7 mg/L Piao et al., 2004b

Table 2.2 Genetic manipulation in propionibacteria.

61

62

Figure 2.1 The dicarboxylic acid pathway of P. acidipropionici. The numbers indicate enzymes as follows: (1) Pyruvate kinase; (2) Pyruvate dehydrogenase complex; (3) Phosphotransacetylase; (4) Acetate kinase; (5) PEP carboxylase; (6) Oxaloacetate transcarboxylase; (7) Malic dehydrogenase; (8) Fumarase; (9) Succinate dehydrogenase; (10) Propionyl CoA: succinyl CoA transferase; (11) Methylmalonyl isomerase.

HMP

NAD+

NADH + CO2

Glucose

EMP

NAD+

NADH

(11)

(10) (9)

(8)

(7)

(6)

(5)

(4)

(3)

(2)

(1)

NADH CoA

NAD+

CO2

Acetate

ADP

ATP

Acetyl CoA

Acetyl phosphate

Pi

CoA

Propionyl CoA

Pyruvate

Phosphoenolpyruvate ADP

ATP

Succinate

Methylmalonyl CoA

Succinyl CoA

Propionate

GDP

GTP

CO2

Oxaloacetate

NADH

Malate

Fumarate

NADH + ADP

FPH2

FP

FPH2

FP

NAD+

NAD+ + ATP

63

Figure 2.2 Integrational mutagenesis.

P T

CAMoriE

CAM oriE P T

64

CHAPTER 3

ENHANCED PROPIONIC ACID FERMENTATION BY PROPIONIBACTERIUM

ACIDIPROPIONICI MUTANT OBTAINED BY ADAPTATION IN A FIBROUS-

BED BIOREACTOR

Summary

Fed-batch fermentations of glucose by P. acidipropionici ATCC 4875 in free-cell

suspension culture and immobilized in a fibrous-bed bioreactor (FBB) were studied. The

latter produced a much higher propionic acid concentration (71.8 ± 0.8 vs. 52.2 ± 1.1

g/L), indicating enhanced tolerance to propionic acid inhibition by cells adapted in the

FBB. Compared to the free-cell fermentation, the FBB culture produced 20-59% more

propionic acid (0.40–0.65 ± 0.02 vs. 0.41 ± 0.02 g/g), 17% less acetic acid (0.10 ± 0.01

vs. 0.12 ± 0.02 g/g), and 50% less succinic acid (0.09 ± 0.02 vs. 0.18 ± 0.03 g/g) from

glucose. The higher propionate production in the FBB was attributed to mutations in two

key enzymes, oxaloacetate transcarboxylase and propionyl CoA: succinyl CoA

transferase, leading to the production of propionic acid from pyruvate. Both showed

higher specific activity and lower sensitivity to propionic acid inhibition in the mutant

than in the wild type. In contrast, the activity of PEP carboxylase, which converts PEP

65

directly to oxaloacetate and leads to the production of succinate from glucose, was

generally lower in the mutant than in the wild type. For phosphotransacetylase and

acetate kinase in the acetate formation pathway; however, there was no significant

difference between the mutant and the wild type. In addition, the mutant had a striking

change in its morphology. With a three-fold increase in its length and ~24% decrease in

its diameter, the mutant cell had a ~10% higher specific surface area that should have

made the mutant more efficient in transporting substrates and metabolites across the cell

membrane. A slightly lower membrane-bound ATPase activity found in the mutant also

indicated that the mutant might have a more efficient proton pump to allow it to better

tolerate propionic acid. In addition, the mutant had more longer-chain saturated fatty

acids (C17:0) and less unsaturated fatty acids (C18:1), both of which could decrease

membrane fluidity and thus might have also contributed to the increased propionate

tolerance. The enhanced propionic acid production from glucose by P. acidipropionici

was thus attributed to both a high viable cell density maintained in the reactor and

favorable mutations resulted from adaptation by cell immobilization in the FBB.

66

3.1 Introduction

Propionibacteria are Gram-positive, nonspore-forming, nonmotile, anaerobic, rod-

shaped bacteria. They are widely used in probiotic and cheese industries (Playne, 1985).

They also can be used to produce vitamin B12, tetrapyrrole compounds, and propionic

acid. The latter is an important chemical used in production of cellulose plastics,

herbicides, and perfumes. As a strong mold inhibitor, calcium, sodium, and potassium

salts of propionate are also widely used as food and feed preservatives. Currently, almost

all propionic acid is produced by petrochemical processes, at an annual production rate of

~400 million lbs in the U.S. Although there has been a high interest to produce propionic

acid from biomass via fermentation with propionibacteria, the relatively low propionic

acid concentration, yield, and production rate from the fermentation have been the major

barriers for economical production of propionic acid from glucose and other fermentable

sugars.

The problems associated with conventional propionic acid fermentation largely

stem from the strong end-product inhibition caused by propionic acid even at a very low

concentration of 10 g/L (Gu et al., 1998; Hsu and Yang, 1991). The conventional batch

propionic acid fermentation usually takes more than 3 days to reach ~20 g/L propionic

acid, with a product yield usually less than 0.45 g/g sugar fermented. Attempts to

improve propionic acid fermentation in terms of its yield, final product concentration, and

production rate have resulted in the development of new bioprocesses and mutant strains

but with limited success (Emde and Schink, 1990; Huang et al., 1998; Jin and Yang,

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1998; Lewis and Yang, 1992; Paik and Glatz, 1994; Rickert et al., 1998; Solichien et al.,

1995; Woskow and Glatz, 1991). Among all these previous efforts, a fibrous-bed

immobilized-cell bioreactor has shown to be a promising technology that can

significantly improve volumetric productivity, product yield, and final product

concentration in several organic acid fermentations (Huang and Yang, 1998; Lewis and

Yang, 1992; Silva and Yang, 1995; Yang et al., 1995; 1994; Zhu et al., 2002; Zhu and

Yang, 2003).

The potential of using the fibrous-bed bioreactor to produce high-concentration

propionic acid from glucose was evaluated in this study. We found that a final

concentration of more than 70 g/L of propionic acid could be produced from glucose in a

fed-batch fermentation. This propionic acid concentration was not only much higher than

that from a similar fed-batch fermentation with free cells grown in a conventional stirred-

tank bioreactor, but also higher than the highest concentration (57 g/L) previously

reported for an acid-tolerant strain immobilized in calcium alginate beads (Paik and

Glatz, 1994).

In order to understand the underlying mechanisms contributing to the improved

propionic acid production and tolerance by cells immobilized in the FBB, the effects of

propionic acid on both the original (wild type) and adapted (from the FBB) cultures were

also studied and compared, with respect to their specific growth rates, cellular activities

of several key enzymes in the dicarboxylic acid pathway, and membrane-bound ATPase.

In addition to significant differences in some of these factors studied, it was also found

that there were significant changes in the membrane fatty acid composition and cell

68

morphology. Based on these results, how the adapted (mutant) cells from the FBB

acquired the ability to tolerate and produce more propionic acid is also discussed in this

paper.

3.2 Materials and Methods

3.2.1 Culture and Media

P. acidipropionici ATCC 4875 was grown in a synthetic medium, described in

Appendix A.1, with 50-100 g/L glucose as the substrate. The medium was sterilized by

autoclaving at 121°C, 15 psig for 30 min. The stock culture was kept in serum bottles

under anaerobic conditions at 4°C.

3.2.2 Free-Cell Fermentation

Unless otherwise noted, the fermentation was carried out in a 5-L fermentor

(Marubishi MD-300) containing 2 L of the synthetic medium. The operating condition

was maintained at 32°C, pH 6.5 by the addition of 6 M NaOH, and 100 rpm for agitation.

Anaerobiosis was established by sparging the medium with N2 for ~30 min, and thereafter

the fermentor headspace was maintained under 5 psig N2. The fermentor was inoculated

with ~100 mL of a fresh culture grown in a serum bottle (OD600 ~ 2.0), and liquid

samples were withdrawn at regular time intervals. The glucose level in the fermentation

69

broth was replenished by pulse feeding a concentrated glucose solution to allow the

fermentation to continue until reaching the maximum propionic acid concentration.

3.2.3 Immobilized-Cell Fermentation

The fermentation was carried out with cells immobilized in a fibrous-bed

bioreactor (FBB) connected to a 5-L fermentor (Marubishi MD-300) through a

recirculation loop (~1 m long, tubing ID: 3.1 mm; Microflex Norprene 06402-16, Cole

Palmer, Chicago, IL) and operated under well-mixed conditions with pH and temperature

controls. The FBB was constructed by packing a spiral wound cotton towel into a glass

column bioreactor and had a working volume of ~690 mL. Detailed description of the

reactor construction is given in Appendix C.1. After inoculation with ~100 mL of cell

suspension (OD600 ~ 2.0) into the fermentor, cells were grown for 3-4 days to reach an

optical density (OD600) of ~3.5. The fermentation broth was then circulated at a flow rate

of ~30 mL/min through the FBB to allow cells to attach and be immobilized in the

fibrous matrix. The process continued for 60-72 h until most cells had been immobilized

in the FBB. The medium circulation rate was then increased to ~80 mL/min. In the

bioreactor start-up (Appendix C.2), the FBB was operated at a repeated-batch mode to

obtain a high cell density in the fibrous bed. Fed-batch fermentation with pulse additions

of concentrated glucose solution was then performed to study the fermentation kinetics

and to evaluate the achievable maximum propionic acid concentration. Samples were

taken at regular time intervals throughout the fermentation for analyses. At the end of the

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fed-batch fermentation, adapted cells (mutants) in the FBB were removed from the

fibrous matrix by vortexing the matrix in sterile distilled water. The cells were collected

for viability assay and subcultured in serum bottles for further analyses.

3.2.4 Effect of Propionic Acid on Cell Growth

To determine the inhibition effect of propionic acid on cell growth, cells were

grown under anaerobic condition in serum tubes containing 10 mL of the synthetic

medium with 20 g/L glucose and varying amounts of propionic acid (0-100 g/L). Cell

growth was followed by measuring the optical density (OD) at 600 nm. The experiment

was done in duplicate. The specific growth rates at various initial propionic acid

concentrations were then estimated from the semilogarithmic plots of the OD versus

time.

3.2.5 Enzyme Assays

Cells grown in the synthetic medium (50 mL) at 32°C to the exponential phase

(OD600 ~ 1.8) were harvested, washed three times, and resuspended in 15 mL of 25 mM

Tris/HCl (pH 7.4). The cell suspension was then sonicated to break cell walls for 12 min

and centrifuged at 10,000 rpm, 4°C for 1 h to remove cell debris. The cell extracts were

kept cold on ice before they were used in the enzyme activity assays. The protein content

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of the extracts was determined in triplicate by Bradford protein assay (Bio-Rad) with

bovine serum albumin as the standard protein (see Appendix B.4).

The activities of oxaloacetate transcarboxylase, propionyl CoA: succinyl CoA

transferase (CoA transferase), phosphotransacetylase (PTA), acetate kinase (AK), and

phosphoenolpyruvate carboxylase (PEP carboxylase) were assayed in duplicate as

described in Appendix B.5.

In studies of the propionate tolerance of oxaloacetate transcarboxylase, CoA

transferase, PTA, and AK, sodium propionate (0-100 g/L) was added into the enzyme

assay reaction mixtures. Non-competitive inhibition kinetics was evaluated by plotting

1/activity versus the concentration of propionate in the reaction medium.

3.2.6 Membrane-Bound ATPase Assay

ATPase activity was determined based on the method described in Appendix B.5.

One unit of activity is defined as the amount of enzyme that releases 1 µmole of Pi per

minute, and the specific activity of ATPase is defined as the unit of activity per mg

biomass. Sodium propionate was initially added into the medium to obtain final propionic

acid concentration of 10 g/L for the activity study. The results were compared with those

from the culture without the addition of propionate.

72

3.2.7 Cell Membrane Fatty Acid Analysis

Fatty acid methyl ester (FAME) analysis was used to analyze cellular membrane

fatty acid compositions. The membrane fatty acids of cells, harvested at the exponential

phase, were extracted with solvents and then methylated before analysis with gas liquid

chromatography as done by Microcheck, Inc. (Northfield, VT) (Appendix B.7).

3.2.8 Cell Viability Assay

Cell viability was determined as described in Appendix B.2. The cell viability (%)

is reported with cells cultured in serum bottles and harvested in the exponential phase as

the control with 100% viability.

3.2.9 Scanning Electron Microscopy

Samples, initially treated through fixing and dehydration processes (Appendix

B.8), were scanned and photographed with a Philips XL 30 scanning electron microscope

at 15 kV.

73

3.2.10 Analytical Methods

Fermentation samples were analyzed for the optical density at 600 nm (OD600)

using a spectrophotometer (Sequoia-turner, Model 340). One unit of OD600 was

equivalent to 0.435 g/L of cell dry weight (Appendix B.1). The concentrations of glucose

and acid products (mainly, propionic, acetic, and succinic acids) were analyzed by high-

performance liquid chromatography (HPLC) as described in Appendix B.3.

3.3 Results and Discussion

3.3.1 Fermentation Kinetics

Figure 3.1 shows typical kinetics for fed-batch fermentations with free cells in a

stirred-tank fermentor and immobilized cells in the fibrous-bed bioreactor. These fed-

batch fermentations were allowed to continue until cells ceased to consume glucose or

produce propionic acid. The purpose of the experiment was to allow cells to gradually

adapt to the high-propionate concentration environment so as to test the maximum

propionic acid concentration that can be produced by the bacteria. As seen in Figure 3.1,

the immobilized-cell fermentation in the FBB produced more propionate from glucose

and reached a higher final propionic acid concentration of 71.8 g/L, which was ~40%

higher than that from the free-cell fermentation (52.2 g/L). It should be noted that 71.8

g/L is the highest propionic acid concentration ever produced in propionic acid

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fermentation. Previously, the maximum propionate concentration produced from glucose

was 57 g/L by a propionate-tolerant strain P. acidipropionici P200910 immobilized in

calcium alginate beads (Paik and Glatz, 1994). A propionic acid concentration of 65 g/L

was also attained from the fermentation with cells immobilized in the FBB using de-

lactose whey permeate as the substrate (Yang et al., 1995). The higher final product

concentration obtained in the fermentation would facilitate product separation and

recovery, and significantly reduce the production costs (Van Hoek et al., 2003).

The higher propionic acid concentration produced in the immobilized-cell

fermentation indicated that cells in the FBB were possibly less sensitive to propionic acid

inhibition. As shown in Figure 3.2, at comparable propionic acid concentrations, the

volumetric productivity for propionic acid was much higher in the immobilized-cell

fermentation than in the free-cell fermentation. The higher volumetric productivity in the

immobilized-cell fermentation could also be attributed to the high cell density (>45 g/L

reactor volume) and cell viability in the FBB. At the end of the fed-batch fermentation,

the total amount of cells in the FBB was ~32 grams, of which more than 95% was

immobilized in the fibrous matrix, with an average cell viability of greater than 70%. The

volumetric productivities shown in Figure 3.2 were calculated based on the total volume

of the liquid medium (2 L) in the fermentation, instead of the actual working volume of

the FBB (0.69 L), which was about one third of the total liquid volume. It should be

noted that the specific productivity in the immobilized-cell fermentation was approx.

80% of that in the free-cell fermentation because of the much higher cell density in the

immobilized-cell system (11.5 g viable cell/L vs. 2.4 g viable cell/L for the free-cell

75

fermentation). A lower specific productivity was often found in immobilized-cell

fermentations (Goncalves et al., 1992; Krischke et al., 1991; Norton et al., 1994;

Senthuran et al., 1997) because of nutrient limitation and reduced cell growth.

Compared to the free-cell fermentation, the immobilized-cell fermentation in the

FBB produced more propionic acid and less succinic and acetic acids from glucose.

Figure 3.3 shows cumulative consumption of glucose and production of propionate,

acetate, and succinate in the fed-batch fermentations. It is noted that the overall propionic

acid yield from the immobilized-cell fermentation was high, ~0.65 g/g, for propionic acid

concentrations up to 45 g/L, but then decreased to 0.4 g/g as the propionic acid

concentration increased to 71 g/L. In contrast, the average propionic acid yield from the

free-cell fermentation was 0.41 g/g. The lower propionic acid yield in the free-cell

fermentation can be partially attributed to the fact that more carbon sources are used for

cell growth and energy production. In the FBB with high cell density, cell growth was

limited (Huang et al., 2002; Yang et al., 1994; Zhu and Yang, 2003), as indicated by the

relatively low OD value throughout the fermentation (see Fig. 3.1). Furthermore, more

acetate and succinate were produced in the free-cell fermentation than in the

immobilized-cell fermentation. As can be seen in Table 3.1, the free-cell fermentation

produced 20% more acetate (0.12 vs. 0.10 g/g) and 100% more succinate (0.18 vs. 0.09

g/g) than the immobilized-cell fermentation. The reduced cell growth in the FBB would

lower the ATP demand for biomass formation and thus result in decreased acetate

production as the acetate formation pathway is the major pathway for ATP production in

propioinibacteria (Goswami and Srivastava, 2000; Rickert et al., 1998). The lower

76

succinate production in the immobilized-cell fermentation can be attributed to changes in

the activities of several key enzymes in the dicarboxylic acid pathway, which will be

discussed later in this paper.

The results from the fermentation experiments clearly indicate that cells

immobilized in the FBB acquired an ability to tolerate and produce a higher propionate

concentration that could not be achieved by the original culture grown in free suspension.

Also, there was a significant shift in the metabolic pathway, leading to a higher

propionate yield from glucose and decreased production of acetate and succinate. Further

experiments were thus conducted to study the underlying changes or causes that allowed

the adapted culture in the FBB to produce more propionate at a higher concentration than

previously reported. To determine if there were any phenotypic changes in the FBB

culture, cells in the FBB were removed and grown as suspension culture to test their

propionate tolerance. The activities of several key enzymes in the acid-forming pathways

and cell membrane ATPase as well as membrane fatty acid composition were also

examined and compared with those of the wild type culture used in seeding the

bioreactor.

3.3.2 Propionic Acid Inhibition

Propionic acid is a strong growth inhibitor (Balamurugan et al., 1999) and 1%

(w/v) propionic acid in the medium could reduce the specific growth rate of

propionibacteria by more than 50% (Jin and Yang, 1998). Figure 3.4 compares the

77

specific growth rates for the wild type and the adapted culture from the FBB at various

initial concentrations of propionic acid in the growth media. Although the wild type had

a slightly higher growth rate in the absence of propionic acid, the adapted culture had a

significantly higher growth rate for the propionate concentrations tested between 5 and 60

g/L. Similar results have been reported for a propionate-tolerant P. acidipropionici

strain, which also had a slightly lower specific growth rate than the wild type strain

(0.185 vs. 0.199 h-1) in the absence of propionic acid but a higher growth rate at 8%

propionic acid (0.047 vs. 0.033 h-1) (Woskow and Glatz, 1991). In this study, the specific

growth rate reduced by 70% for the wild type and 50% for the FBB adapted culture,

respectively, as the propionate concentration increased to 10 g/L. There was minimal

growth when the propionate concentration was 80 g/L and higher.

The effect of propionic acid on cell growth can be described by the following

non-competitive product inhibition model:

PK

orPK

K

ii

i

maxmax

max 111µµµ

µµ +=+

= (3.1)

where µ is the specific growth rate (h-1), µmax is the maximum specific growth rate (h-1),

Ki is the inhibition rate constant (g/L), and P is the propionic acid concentration (g/L).

The values of µmax and Ki were determined from the linear plot of 1/µ vs. P shown in the

inset of Figure 3.4, and are given in Table 3.2. Compared to the wild type, the adapted

culture had a slightly lower µmax (0.133 ± 0.008 vs. 0.154 ± 0.017 h-1), but a much higher

Ki (8.93 ± 0.77 vs. 4.33 ± 0.67 g/L). It is clear that the adapted culture is less sensitive to

propionic acid inhibition.

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3.3.3 Acid-Forming Enzyme Activities

The cellular activities of several key enzymes in the dicarboxylic acid pathway

(see Figure 3.5) that would have major effects on the production of propionic acid, acetic

acid, and succinic acid were examined and compared between the mutant strain and the

wild type. As shown in Figure 3.6, the activities of both oxaloacetate transcarboxylase

and propionyl CoA: succinyl CoA transferase (CoA transferase), two key enzymes in the

pathway leading to propionic acid production, were significantly higher in the mutant

than in the wild type. Oxaloacetate transcarboxylase, a key enzyme in the pathway for

propionate synthesis, catalyzes a coupled reaction of pyruvate to oxaloacetate and

methylmalonyl CoA to propionyl CoA. It is responsible for supplying the carbon flux

from the central carbon metabolism toward the end product, propionate. CoA transferase

also catalyzes a coupled reaction of succinate to succinyl CoA and propionyl CoA to

propionate. It was found that CoA transferase was less active but more sensitive to

propionic acid inhibition than was transcarboxylase for both the mutant and wild type.

Clearly, the reaction catalyzed by CoA transferase was the rate-limiting step in the

pathway leading to propionic acid production in P. acidipropionici.

On the other hand, the activities of phosphotransacetylase (PTA) and acetate

kinase (AK) in the mutant were either about the same as or slightly lower than those of

the wild type (Figure 3.7). PTA and AK are two key enzymes in the pathway from

pyruvate to acetate, which is the major supply route for ATP. PTA catalyzes the reaction

of acetyl CoA to acetyl phosphate and AK catalyzes the conversion of acetyl phosphate

79

to acetate. Both PTA and AK were highly sensitive to propionic acid inhibition, but PTA

had a much lower activity and appeared to be the rate-limiting enzyme in the acetate-

formation pathway. However, it was not clear why the activity of PTA increased with

increasing the propionic acid concentration from the minimum level at 60 g/L of

propionic acid. The increased PTA activity at high propionic acid concentrations could be

an induced cell response to direct more pyruvate towards the acetate-forming pathway

instead of the propionate-forming pathway. Also, the increased PTA activity would

increase the cellular level of acetyl phosphate, which can function as a global regulator in

a metabolic network (McCleary et al., 1993). Acetyl phosphate can be used in the

phosphorylation of a group of proteins regulating cell’s responses to environmental

stimulations. The global phosphorylation could lead to acetate formation from acetyl

phosphate (Wanner and Willmes-Reisenberg, 1992) without the activity of acetate kinase.

PEP carboxylase is the enzyme that catalyzes the reaction from PEP directly to

oxaloacetate, thus leading to the production of succinate from glucose. As shown in

Figure 3.8, the activity of PEP carboxylase was generally lower in the mutant than in the

wild type. Furthermore, unlike other acid-forming enzymes, PEP carboxylase was

relatively stable in the presence of propionic acid up to ~80 g/L. In fact, the enzyme

activity in the wild type seemed to increase significantly when the propionic acid

concentration increased from 10 to 60 g/L. These characteristics could explain why

succinate production increased significantly later in the fed-batch fermentations when the

propionic acid concentration was higher (see Fig. 3.3) and why the adapted culture or

mutant in the FBB had much lower succinic acid production as compared to the wild

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type. This finding is also consistent with the result obtained in an extractive fermentation

where propionic acid was continuously removed from the fermentor and the production

of succinic acid was reduced to almost zero as a result of the low propionic acid

concentration maintained in the fermentation broth (Jin and Yang, 1998).

In summary, the enzyme activity assay results were in good accordance with the

fermentation results that the mutant produced more propionic acid and less acetic and

succinic acids than did the wild type. It is clear that changes in various key enzyme

activities in the dicarboxylic acid pathway led to a metabolic shift to favor more

propionic acid production over acetic and succinic acids. These results also provide

insights on how one could metabolically engineer the propionibacterium to further

increase propionic acid production and reduce byproduct formations.

Propionic acid is an inhibitor to oxaloacetate transcarboxylase, CoA transferase,

PTA, and AK (Fig. 3.6 and 3.7), and the inhibition can be modeled by the following non-

competitive inhibition kinetics equation:

PKvvv

orPK

Kvv

ii

i

maxmax

max 111 +=+

= (3.2)

where v is the specific activity of enzyme (U/mg), vmax is the maximum specific activity

(U/mg), Ki is the inhibition rate constant (g/L), and P is the propionic acid concentration

(g/L). The best values of vmax and Ki for these enzymes were determined from nonlinear

regression of the data and are listed in Table 3.2. The higher vmax indicates a more active

enzyme, whereas the higher Ki indicates that the enzyme is less sensitive to propionic

acid inhibition. Compared to the wild type, oxaloacetate transcarboxylase and CoA

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transferase in the mutant not only were more active but also less sensitive to propionic

acid inhibition, especially when the propionic acid concentration was lower than 60 g/L.

In fact, the mutant’s transcarboxylase activity was almost unaffected until the propionic

acid concentration was higher than 60 g/L (Fig. 3.6). In contrast, both PTA and AK in

the mutant and wild type had almost the same vmax and Ki, indicating their similar activity

and sensitivity to propionic acid inhibition. Compared to the wild type, the higher

propionate tolerance of the mutant was mainly attributed to the improvements in its

oxaloacetate transcarboxylase and CoA transferase that could maintain relatively high

activities and thus allow the cells to survive even at elevated propionate concentrations.

The alteration in the sensitivity to propionic acid inhibition of these two enzymes also

contributed to the increased final propionic acid concentration and yield obtained in the

fed-batch fermentation by cells immobilized in the FBB.

3.3.4 Membrane-Bound ATPase

The uncoupling effect of propionic acid on oxidative phosphorylation may

interfere with the establishment and maintenance of a functional pH gradient across the

cell membrane for the transport of metabolites (Gutierrez and Maddox, 1992). In order to

maintain a proper pH gradient, the extra protons must be pumped out at the cost of ATP

via membrane ATPase (Deckers-Hebestreit and Altendorf, 1996; Kobayashi, 1987;

O’Sullivan and Condon, 1999). Thus, an active ATPase is essential to prevent the

acidification of cytoplasm by propionic acid. The effects of propionic acid concentration

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on the membrane-ATPase activities of the mutant and wild type strains were studied with

cells harvested at exponential and stationary phases. As shown in Figure 3.9, the activity

of ATPase increased with increasing propionic acid concentration in the range between 0

and 10 g/L, but then decreased rapidly to zero at 30 g/L. Surprisingly, the ATPase

regained its activity when the propionic acid concentration was 60 g/L and increased with

increasing propionic acid concentration. Also, cells grown in the presence of 10 g/L of

propionic acid and harvested in the exponential phase had ~5% more ATPase activity as

compared with cells grown in the absence of propionic acid (data not shown). These

results are consistent with the fact that the ATPase activity increases as the acid

concentration increases (O’Sullivan and Condon, 1999; Zhu and Yang, 2003) or as the

extracelluar pH value decreases (Belli and Marquis, 1991; Miyagi et al., 1994;

O’Sullivan and Condon, 1999). In all cases studied, the effect of propionic acid on

ATPase was essentially the same for the mutant and wild type strains, but the former

appeared to have a consistently lower ATPase activity level, an indication that the mutant

was more efficient in pumping out protons and could survive in higher propionate

environment. The lower ATPase activity for cells in the stationary phase was attributed to

the lower ATP requirement, as cells were not actively growing in the stationary phase.

On the other hand, for cells in the exponential phase, a lower membrane-bound ATPase

activity can be an indication of a more efficient proton pump, as it has been reported that

the ATPase activity was lower in faster-growing cells than in more slowly growing cells

(O’Sullivan and Condon, 1999).

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3.3.5 Membrane Fatty Acid Composition

It has been reported that microorganisms such as Saccharomyces cerevisiae

(Casey and Ingledew, 1986) and Escherichia coli (Ingram, 1976) could increase their

tolerance to organic solvents by regulating their membrane lipid composition in response

to environmental stresses. Compared to the wild type, the membrane fatty acid

composition was significantly changed in the mutant, which had more longer-chain

saturated fatty acids (C17:0) and less unsaturated fatty acids (C18:1) (Table 3.3), both of

which could decrease membrane fluidity and thus might have contributed to the increased

propionate tolerance.

3.3.6 Morphological Change in Mutant

Cell adaptation and mutation are often accompanied with a significant change in

cell morphology as a response to environmental perturbations (Jan et al., 2001; Ye et al.,

1999). P. acidipropionici has a rod shape; however, the mutant appeared to be much

longer and thinner than the wild type as observed under SEM (Figure 3.10). The mutant

had an average length of 3.2 µm (vs. 1.1 µm for the wild type) and an average diameter of

0.50 µm (vs. 0.66 µm for the wild type). Compared to the wild type, the thinner and

longer appearance of the mutant had a ~10% increase in its specific surface area, which

should contribute to proportional increases in substrate uptake and metabolite excretion

rates. This could explain why the mutant might have a more efficient proton pump and

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can tolerate a higher propionic acid concentration. In the FBB high cell density

environment with relatively low glucose and high propionic acid concentrations, the

observed morphological change would be a natural response for cells to adapt and

survive. Interestingly, this dramatic morphological change was permanent and the mutant

preserved this morphology even after numerous subculturing in normal growth media for

one year. However, this finding is not a surprise as similar observations have also been

reported. P. freudenreichii lost its original rod shape and became shorter under an

extreme acidic pH of 2.0 (Jan et al., 2001). Lactobacillus plantarum immobilized in

chitosan treated polypropylene matrix experienced a morphological change from a

normal rod shape to a coccoid shape and a metabolic shift from homofermentative to

heterofermentative (Krishnan et al., 2001). Lactobacillus rhamnosus immobilized on

solid support changed from isolated rods to thinner, chain-linked consortia during

continuous lactic acid fermentation at high dilution rate (Goncalves et al., 1992).

3.3.7 Effects of Cell Immobilization in FBB

Clearly, adaptation of P. acidipropionici in the FBB provided an efficient method

to obtain a metabolically robust mutant that can better produce propionic acid from

glucose with a higher concentration and yield. It should be noted that free cell

fermentations carried out under similar fed-batch conditions could not achieve the same

adaptation effect and was not as effective in obtaining propionate tolerant mutants. It was

the unique environment in the FBB that facilitated rapid adaptation of cells and allowed

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mutants with higher propionate tolerance to survive when subjected to adverse

conditions. The FBB has also been successfully used for adapting and screening for acid-

tolerant strains of Clostridium formicoaceticum (Huang et al., 1998) and Clostridium

tyrobutyricum (Zhu and Yang, 2003). The ability to obtain acid-tolerant mutants in the

FBB can be attributed to: 1) the high cell density (>45 g/L) and viability (>70%)

maintained in the FBB and 2) distinct physiology and survivability of immobilized cells

resulting from their direct contact with a solid surface and with each other. None of these

conditions existed in the free cell fermentation. It should be noted that the mutant was

also tested in free-cell fed-batch fermentation and gave similar higher propionate yield

and lower succinate yield from glucose, but could not reach the same high level of

propionate concentration as in the FBB (see Table 3.1). This indicated that besides the

observed mutations in cell morphology, membrane fatty acid composition, and enzyme

activity and sensitivity to propionic acid, there were additional phenotypic changes for

cells immobilized in the FBB that could not be easily duplicated in the suspension

culture.

Immobilization mimics what occurs in nature when cells adhere and grow on

surfaces or within natural structures. The high cell density in the immobilization system

also provides conditions conducive to cell-to-cell communication. It is natural for cells to

modify their pattern of growth and replication as a result of direct contact with a solid

surface or with other cells. Immobilization also results in the change of physicochemical

properties of the microenvironment, including the presence of ionic charges, reduced

water activity, altered osmotic pressure, modified surface tension, and cell confinement.

86

Growth under this situation can induce responses at both biochemistry and genetic levels

that cause fundamental changes in the cells. As a cellular response to the environmental

stress, immobilization caused changes in cell metabolic pathway and morphology

(Krishnan et al., 2001). Cells in an immobilization system are usually maintained in a

non- or low-growing but metabolically active state (Paik and Glatz, 1994), which may

have allowed them to better survive adverse conditions. When subjected to a high ethanol

concentration, immobilized cells were able to maintain a higher cytoplasmic pH or a

more efficient proton pump (Groboillot et al., 1994; Loureiro-Dias and Santos, 1990).

All these factors might have contributed to the better adaptation and survivability of cells

in a high-density immobilized system such as the FBB.

In the free-cell fermentation, both acetate and succinate production increased with

increasing cumulative glucose consumption (see Fig. 3.3) or propionate concentration.

Apparently, free cells grown in suspension would need more energy (ATP) when the

propionic acid concentration was higher in order to maintain their intracellular pH. Thus,

there was a clear metabolic pathway shift; however, which could not continue to work at

higher concentrations of propionic acid and the fermentation ceased to produce propionic

acid at ~52 g/L. On the other hand, for the FBB fermentation, cells responded differently.

Instead of shifting the metabolic pathway, immobilized cells underwent some mutations,

including changes in morphology and membrane fatty acid content to increase their

tolerance to propionic acid. These changes allowed the immobilized cells to tolerate and

produce more propioinic acid at a higher concentration.

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3.4 Conclusion

Immobilization and adaptation of P. acidipropionici in the fibrous-bed bioreactor

provided an effective means to obtain a metabolically advantageous mutant with the

ability to tolerate and produce more propionic acid at higher concentration and yield from

glucose. The mutant had significant changes in its morphology, membrane lipid

composition, and key enzymes in the pathways leading to propionic acid and succinic

acid. The higher propionate yield was attributed to the higher activity levels of

oxaloacetate transcarboxylase and CoA transferase in the mutant, while the lower

succinate yield in the mutant resulted from the lower activity of PEP carboxylase. The

increased propionate tolerance of the mutant is possibly because of changes in its

membrane fatty acid composition and its slimmer morphology making the proton pump

more efficient as indicated by the lower ATPase activity. The decreased sensitivity to

propionic acid inhibition for the mutant’s oxaloacetate transcarboxylase and CoA

transferase also allowed the cells to survive and continue to produce propionate even

when the propionate concentration was high. The higher final product concentration,

coupled with the significantly reduced by-product and cell concentrations in the

fermentation broth should facilitate simple separations for product purification and

reduce the overall production cost of propionic acid by the FBB fermentation process.

88

3.5 References Balamurugan K, Venkata Dasu V, Panda T. 1999. Propionic acid production by whole

cells of Propionibacterium freudenreichii. Bioprocess Eng 20:109-116. Belli WA, Marquis RE. 1991. Adaptation of Streptococcus mutans and Enterococcus

hirae to acid stress in continuous culture. Appl Environ Microbiol 57:1134-1138. Casey GP, Ingledew WE. 1986. Ethanol tolerance in yeasts. Crit Rev Microbiol 13:219-

280. Deckers-Hebestreit G, Altendorf K. 1996. The F0F1-type ATP synthases of bacteria:

structure and function of the F0 complex. Annu Rev Microbiol 50:791-824. Emde R, Schink B. 1990. Enhanced propionate formation by Propionibacterium

freudenreichii subsp. freudenreichii in a three-electrode amperometric culture system. Appl Environ Microbiol 56:2771-2776.

Goncalves LMD, Barreto MTO, Xavier AMBR, Carrondo MJT, Klein J. 1992. Inert

supports for lactic acid fermentation–a technical assessment. Appl Microbiol Biotechnol 38:305-311.

Goswami V, Srivastava AK. 2000. Fed-batch propionic acid production by

Propionibacterium acidipropionici. Biochem Eng J 4:121-128. Groboillot A, Boadi DK, Poncelet D, Neufeld RJ. 1994. Immobilization of cells for

application in the food industry. Crit Rev Biotechnol 14:75-107. Gu Z, Glatz BA, Glatz CE. 1998. Effects of propionic acid on propionibacteria

fermentation. Enzyme Microb Technol 22:13-18. Gutierrez NA, Maddox IS. 1992. Product inhibition in a nonmotile mutant of Clostridium

acetobutylicum. Enzyme Microb Technol 14:101-105. Hsu ST, Yang S-T. 1991. Propionic acid fermentation of lactose by Propionibacterium

acidipropionici: effects of pH. Biotechnol Bioeng 38:571-578. Huang YL, Mann K, Novak JM, Yang S-T. 1998. Acetic acid production from fructose

by Clostridium formicoaceticum immobilized in a fibrous-bed bioreactor. Biotechnol Prog 14:800-806.

Huang YL, Wu Z, Zhang L, Cheung CM, Yang S-T. 2002. Production of carboxylic

acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor. Bioresource Technol 82:51-59.

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Huang Y, Yang S-T. 1998. Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor. Biotechnol Bioeng 60:498-507.

Ingram LO. 1976. Adaptation of membrane lipids to alcohols. J Bacteriol 125:670-678. Jan G, Leverrier P, Pichereau V, Boyaval P. 2001. Changes in protein synthesis and

morphology during acid adaptation of Propionibacterium freudenreichii. Appl Environ Microbiol 67:2029-2036.

Jin Z, Yang S-T. 1998. Extractive fermentation for enhanced propionic acid production

from lactose by Propionibacterium acidipropionici. Biotechnol Prog 14:457-465. Kobayashi H. 1987. Regulation of cytoplasmic pH in streptococci. In: Reizer J,

Peterkofsky A, editors. Sugar transport and metabolism in Gram-positive bacteria. London, UK: Ellis Harwood. p 255-269.

Krischke W, Schroder M, Trosch W. 1991. Continuous production of L-lactic acid from

whey permeate by immobilized Lactobacillus casei subsp. casei. Appl Microbiol Biotechnol 34:573-578.

Krishnan Sudha, Gowda LR, Misra MC, Karanth NG. 2001. Physiological and

morphological changes in immobilized L. plantarum NCIM 2084 cells during repeated batch fermentation for production of lactic acid. Food Biotechnol 15:193-202.

Lewis VP, Yang S-T. 1992. Continuous propionic acid fermentation by using immobilized

Propionibacterium acidipropionici in a novel packed-bed bioreactor. Biotechnol Bioeng 40:465-474.

Loureiro-Dias MC, Santos H. 1990. Effects of ethanol on Saccharomyces cerevisiae as

monitored by in vivo 31P and 13C nuclear magnetic resonance. Arch Microbiol 153:384-391.

McCleary WR, Stock JB, Ninfa AJ. 1993. Is acetyl phosphate a global signal in

Escherichia coli? J Bacteriol 175:2793-2798. Miyagi A, Ohta H, Kodama T, Fukui K, Kato K, Shimono T. 1994. Metabolic and

energetic aspects of the growth response of Streptococcus rattus to environmental acidification in anaerobic continuous culture. Microbiology 140:1945-1952.

Norton S, Lacroix C, Vuillemard J-C. 1994. Kinetic study of continuous whey permeate

fermentation by immobilized Lactobacillus helveticus for lactic acid production. Enzyme Microb Technol 16:457-466.

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O’Sullivan E, Condon S. 1999. Relationship between acid tolerance, cytoplasmic pH and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis. Appl Environ Microbiol 65:2287-2293.

Paik H-D, Glatz BA. 1994. Propionic acid production by immobilized cells of a

propionate-tolerant strain of Propionibacterium acidipropionici. Appl Microbiol Biotechnol 42:22-27.

Playne MJ. 1985. Propionic and butyric acids. In: Moo-Young M, editor.

Comprehensive Biotechnology, vol. 3. New York: Pergamon. p 731-759. Rickert DA, Glatz CE, Glatz BA. 1998. Improved organic acid production by calcium

alginate-immobilized propionibacteria. Enzyme Microb Technol 22:409-414. Senthuran A, Senthuran V, Mattiasson B, Kaul R. 1997. Lactic acid fermentation in a

recycle batch reactor using immobilized Lactobacillus casei. Biotechnol Bioeng 55:841-853.

Silva EM, Yang S-T. 1995. Kinetics and stability of a fibrous-bed bioreactor for

continuous production of lactic acid from unsupplemented acid whey. J Biotechnol 41:59-70.

Solichien MS, O'Brien D, Hammond EG, Glatz CE. 1995. Membrane-based extractive

fermentation to produce propionic and acetic acids: Toxicity and mass transfer considerations. Enzyme Microb Technol 17:23-31.

Van Hoek P, Aristidou A, Hahn JJ, Patist A. 2003. Fermentation goes large-scale. CEP

99:37S-42S. Wanner BL, Willmes-Riesenberg MR. 1992. Involvement of phosphotransacetylase,

acetate kinase and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli. J Bacteriol 174:2124-2130.

Woskow SA, Glatz BA. 1991. Propionic acid production by a propionic acid- tolerant

strain of Propionibacterium acidipropionici in batch and semicontinuous fermentation. Appl Environ Microbiol 57:2821-2828.

Yang S-T, Huang Y, Hong G. 1995. A novel recycle batch immobilized cell bioreactor

for propionate production from whey lactose. Biotechnol Bioeng 45:379-386. Yang S-T, Zhu H, Li Y, Hong G. 1994. Continuous propionate production from whey

permeate using a novel fibrous bed bioreactor. Biotechnol Bioeng 43:1124-1130.

91

Ye K, Shijo M, Miyano K, Shimizu K. 1999. Metabolic pathway of Propionibacterium growing with oxygen: enzymes, 13C NMR analysis, and its application for vitamin B12 production with periodic fermentation. Biotechnol Prog 15:201-207.

Zhu Y, Wu Z, Yang S-T. 2002. Butyric acid production from acid hydrolysate of corn

fiber by Clostridium tyrobutyricum in a fibrous-bed bioreactor. Process Biochem 38:657-666.

Zhu Y, Yang S-T. 2003. Adaptation of Clostridium tyrobutyricum for enhanced tolerance

to butyric acid in a fibrous-bed bioreactor. Biotechnol Prog 19:365-372.

92

Note: Mean ± standard deviation. aCells from the FBB after adaptation to high propionic acid concentration in the fed-batch fermentation. bYield was ~0.65 for propionic acid concentrations up to ~45 g/L and then decreased with increasing propionic acid concentration to ~0.40 at ~71 g/L. cIncluding all viable cells in suspension and immobilized in the fibrous matrix; based on the total liquid volume of 2 L. dBased on the assumption that 46.2% of the biomass was carbon. The amount of CO2 produced was estimated based on the assumption that one mole of CO2 was co-produced with each mole of acetic acid in the fermentation. Table 3.1 Comparison of product yields and maximum propionic acid concentrations from fed-batch fermentations with free cells and immobilized cells in the FBB.

Free-cell fermentations

Wild type Mutanta

Immobilized-cell

fermentation

Max. propionic acid conc. (g/L) 52.2 ± 1.1 51.5 ± 0.9 71.8 ± 0.8

Product yield (g/g)

Propionic acid

Acetic acid

Succinic acid

Total viable cell conc. (g/L)

Total carbon recovery (%)d

0.41 ± 0.02

0.12 ± 0.02

0.18 ± 0.03

2.41

88.2 ± 8.5

0.47 ± 0.01

0.11 ± 0.01

0.09 ± 0.01

5.67

87.1 ± 3.7

0.40 – 0.65 ± 0.02b

0.10 ± 0.01

0.09 ± 0.02

11.48c

95.7± 6.0

93

Note: Mean ± standard deviation; n = 2. Both wild type and mutant cells were grown in suspension cultures for specific growth rate determination and enzyme activity assays. Table 3.2 Comparison of rate constants for specific growth rate and several key enzymes in P. acidipropionici wild type and mutant from the FBB.

Table 3.3 Comparison of membrane fatty acid compositions of P. acidipropionici wild type and mutant from the FBB.

Wild type Mutant from FBB

µmax (h-1) or vmax

(U/mg)

Ki (g/L) µmax (h-1) or vmax

(U/mg)

Ki (g/L)

Specific growth rate 0.154 ± 0.017 4.33 ± 0.67 0.133 ± 0.008 8.93 ± 0.77

OAA transcarboxylase

CoA transferase

0.111 ± 0.001

0.027 ± 0.001

84.8 ± 4.9

10.1 ± 0.2

0.138 ± 0.014

0.059 ± 0.004

451.9 ± 42.9

13.5 ± 0.5

Phosphotransacetylase

Acetate kinase

0.0012 ± 0.0001

0.019 ± 0.001

18.4 ± 0.5

10.2 ± 0.8

0.0011 ± 0.0001

0.021 ± 0.002

19.0 ± 0.8

10.6 ± 0.5

Fatty Acid Wild Type Mutant

C15:0 70.76% 68.50%

C16:0 2.30% 2.90%

C17:0 14.75% 20.06%

C18:1 1.37% 1.24%

94

0

20

40

60

80

100

0 100 200 300 400 500 600 700 800 900Time (h)

Con

cent

ratio

n ( g

/L)

0

1

2

3

4

5

6

7

OD

Acetate

OD

Succinate

Propionate

Glucose

Free cells

A

0

20

40

60

80

100

0 100 200 300 400 500 600 700 800 900

Time (h)

Con

cent

ratio

n ( g

/L)

0

1

2

3

4

OD

Acetate

OD

Succinate

Propionate

Glucose

Immobilized cells

B

Figure 3.1 Fed-batch fermentations of glucose by P. acidipropionici at pH 6.5, 32°C. A. Free-cell fermentation; B. Immobilized-cell fermentation in the FBB.

95

0

1

2

3

4

5

6

0 20 40 60 80Propionic acid (g/L)

Prod

uctiv

ity (g

/L/d

ay)

Free cells

Immobilized cells

Figure 3.2 Effect of propionic acid on the volumetric productivity of propionic acid in fed-batch fermentations with free cells and immobilized cells.

96

0

10

20

30

40

50

60

70

0 20 40 60 80 100 120 140 160 180

Glucose consumption (g/L)

Prop

ioni

c ac

id p

rodu

ctio

n (g

/L)

Free cells

Immobilized cells

A

0

5

10

15

20

0 20 40 60 80 100 120 140 160 180

Glucose consumption (g/L)

Ace

tic a

cid

prod

uctio

n (g

/L)

Immobilized cells

Free cells

B

Continued Figure 3.3 Comparison of product yields from glucose in fed-batch fermentations with free cells and immobilized cells of P. acidipropionici. A. Cumulative propionic acid production vs. glucose consumption; B. Cumulative acetic acid production vs. glucose consumption; C. Cumulative succinic acid production vs. glucose consumption. The product yields can be estimated from the slopes of these plots.

97

Figure 3.3 (continued)

0

5

10

15

20

25

30

0 20 40 60 80 100 120 140 160 180

Glucose consumption (g/L)

Succ

inic

aci

d pr

oduc

tion

(g/L

)

C

Immobilized cells

Free cells

98

Figure 3.4 Effects of propionic acid on specific growth rates of P. acidipropionici wild type and mutant from the FBB. Inset shows the determination of rate constants in the non-competitive inhibition of propionic acid. The curves from the model predictions simulate the data (symbols) well.

0.00

0.05

0.10

0.15

0.20

0 10 20 30 40 50 60 70 80 90 100

Propionic acid (g/L)

Spec

ific

Gro

wth

Rat

e (h

-1)

adapted culture

wild type

y = 0.8397x + 7.4957R2 = 0.9951

y = 1.50x + 6.494R2 = 0.9862

0

5

10

15

20

25

0 4 8 12

Propionic acid (g/L)1/

u (h

)

99

Figure 3.5 The dicarboxylic acid pathway for the conversion of glucose to propionic, acetic, and succinic acids. The five key enzymes assayed in this study are labeled in the pathway.

NADH CoA

NAD+

CO2

Acetate

ADP

ATP

Acetyl CoA

Acetyl phosphate

Pi

CoA

Propionyl CoA

Pyruvate

Phosphoenolpyruvate ADP

ATP

Succinate

Methylmalonyl CoA

Succinyl CoA

Propionate

GDP

GTP

CO2

Oxaloacetate

NADH

Malate

Fumarate

NADH + ADP

FPH2

FP

FPH2

FP

NAD+

NAD+ + ATP

Phosphotransacetylase

Acetate kinase

CoA transferase

OAA transcarboxylase

PEP carboxylase

HMP

NAD+

NADH + CO2

Glucose

EMP

NAD+

NADH

100

Oxaloacetate Transcarboxylase

0.00

0.05

0.10

0.15

0.20

0 20 40 60 80 100Propionic acid (g/L)

Spec

ific

activ

ity (U

/mg)

mutant

wild type

A

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 20 40 60 80 100

Propionic acid (g/L)

Spec

ific

activ

ity (U

/mg)

mutant

wild type

CoA Transferase

B

Figure 3.6 Effect of propionic acid on oxaloacetate transcarboxylase (A) and propionyl CoA: succinyl CoA transferase (B) activities of P. acidipropionici wild type and mutant from the FBB. The curves from the non-competitive inhibition model simulate the data (symbols) wells.

101

0.0000

0.0003

0.0006

0.0009

0.0012

0.0015

0.0018

0 20 40 60 80 100

Propionic acid (g/L)

Spec

ific

activ

ity (U

/mg) Wild Type

Mutant

Phosphotransacetylase

A

0.000

0.005

0.010

0.015

0.020

0.025

0 20 40 60 80 100

Propionic acid (g/L)

Spe

cific

act

ivity

(U/m

g) Wild Type

Mutant

Acetate Kinase

B

Figure 3.7 Effect of propionic acid on phosphotransacetylase (A) and acetate kinase (B) activities of P. acidipropionici wild type and mutant from the FBB. The curves show the non-competitive inhibition model predictions that simulate the data (symbols) well for propionic acid concentrations up to 60 g/L.

102

0.0000

0.0005

0.0010

0.0015

0.0020

0 20 40 60 80 100

Propionic acid (g/L)

Spec

ific

Activ

ity (U

/mg)

wild type

mutant

PEP Carboxylase

Figure 3.8 Effect of propionic acid on PEP carboxylase activity of P. acidipropionici wild type and mutant from the FBB.

103

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0 20 40 60 80 100Propionic acid (g/L)

ATP

ase

(U/m

g ce

ll)

Wild type

Adapted cells from FBB

Exponential phase

A

0.000

0.005

0.010

0.015

0.020

0 20 40 60 80 100

Propionic acid (g/L)

ATPa

se (U

/mg

cell)

Wild type

Adapted cells from FBB

Stationary phase

B

Figure 3.9 Effect of propionic acid on membrane-bound ATPase activity of P. acidipropionici wild type and mutant from the FBB. A. Cells in the exponential phase; B. Cells in the stationary phase.

104

10 µmA

10 µmB

Figure 3.10 Scanning electron micrographs of P. acidipropionici showing morphological difference between the wild type and the mutant from the FBB. A. Wild type cells with a short rod shape; B. Mutant cells with an elongated slimmer rod shape.

105

CHAPTER 4

EFFECT OF CARBON SOURCES ON PROPIONIC ACID FERMENTATION BY

PROPIONIBACTERIUM ACIDIPROPIONICI

Summary

Glucose, sorbitol, gluconate, and xylose as carbon sources affected kinetics of

propionic acid fermentation by Propionibacterium acidipropionici due to cells responded

to a particular carbon source by redistributing the pattern of fermentation end-product

compositions for a redox balance. Sorbitol provided the highest propionic acid

production. As compared to glucose, the fermentation by P. acidipropionici wild type

using sorbitol achieved ~63%, ~37%, ~33%, and ~121% enhanced productivity (0.24 vs.

0.39 g/L/h), propionic acid yield (0.164 vs. 0.224 mol/mol C or 0.982 vs. 1.345 mol/mol

sorbitol), final propionic acid concentration (15.3 vs. 20.4 g/L), and propionate: acetate

(P/A) molar ratio (2.9 vs. 6.4), respectively with ~39% and ~13% reduced yields of

acetate (0.057 vs. 0.035 mol/mol C) and succinate (0.023 vs. 0.020 mol/mol C),

respectively. A P/A molar ratio of 8.1 was obtained from the fermentation by the wild

type using gluconate as the carbon source since succinic acid was produced as a main

byproduct instead of acetic acid. As compared to other carbon sources, xylose and

106

glucose gave the similar pattern of end-product compositions and much higher (~60-

200%) acetate yields, which resulted in a much lower P/A molar ratio of ~3.0. When the

adapted mutant from the FBB was compared to the wild type, glucose showed a ~17%

increase in propionate yield (from 0.164 to 0.191 mol/mol C) and a significant decrease

in succinate yield (~39%) (from 0.023 to 0.014 mol/mol C). In the fermentation by the

adapted mutant using gluconate, acetic acid production was not observed; however,

pyruvate was accumulated in the fermentation broth. The different metabolic patterns of

the propionic acid fermentation under feeding of different carbon sources were elucidated

by using metabolic stoichiometric analysis, indicating different controlling mechanisms.

Various acid-forming enzymes with significant changes in their activities and overall

protein expression pattern involved the controlling mechanism in the fermentation as

well. Both wild type and adapted mutant using xylose as the substrate possessed a unique

protein with a size of ~56 kDa, which could play a role in the xylose utilization. The ~45

kDa protein, only observed in the mutant, differentiated the mutant from the wild type

and this protein might be related to acid tolerance response. The enhanced propionic acid

production with reduced acetate and succinate formations obtained from the fermentation

by P. acidipropionici using sorbitol as the substrate could facilitate simple and

inexpensive downstream processing.

107

4.1 Introduction

Propionic acid is widely used in production of herbicides, pharmaceuticals,

artificial fruit flavors, cellulose plastics, and plasticizers. Propionate salts are widely used

in food and feed preservations due to their antimicrobial activity. Propionic acid

produced via fermentation by propionibacteria has been of interest since it can be used as

a natural preservative with increasing market demand. The improvement of propionic

acid yield (>0.9 g/g) has been achieved via the use of sophisticated processes operated

either under two atmospheres of hydrogen (Thompson et al., 1984) or with a three-

electrode amperometric culture system (Emde and Schink, 1990); however, these

processes are too complex to be practical for commercial application. The use of

immobilization systems (Huang et al., 2002; Paik and Glatz, 1994; Rickert et al., 1998;

Suwannakham and Yang, 2005; Yang et al., 1995; 1994) and the use of several types of

carbon sources as glucose (Lewis and Yang, 1992a), sucrose (Quesada-Chanto et al.,

1994), hemicellulose hydrolysate (Ramsay et al., 1998), lactose (Goswami and

Srivastava, 2000; Hsu and Yang, 1991; Jin and Yang, 1998), and whey lactose

(Colomban et al., 1993; Lewis and Yang, 1992b) have attained enhanced propionic acid

production; however, the high production of acetic acid as a main byproduct has been an

obstacle for separation.

Propionic acid fermentation is usually accompanied by the formation of acetate

for maintaining hydrogen and redox balances in cellular metabolism and for

stoichiometric reasons (Lewis and Yang, 1992a; Martínez-Campos and de la Torre,

108

2002). The use of a sole carbon source or a mixture of carbon sources has achieved

improved propionate yield with decreased acetate yield, leading to an enhanced P/A

molar ratio. A P/A molar ratio of 7.63 was obtained from the fermentation by P.

acidipropionici using a mixture of lactate and glucose at a molar ratio of 4 (Martínes-

Campos and de la Torre, 2002). Fermentations using glycerol as the substrate obtained a

high propionate yield of 0.844 mol/mol with low acetate production (0.023 mol/mol)

(Barbirato et al., 1997) and a propionate yield of 0.79 mol/mol with an acetate yield of

0.17 mol/mol (Himmi et al., 2000); however, the formation of other unconventional

byproducts, as formic acid and n-propanol, was accompanied. The NADH availability

and redox balance were used to explain the decreased acetate yield in propionic acid

fermentations obtained in these studies.

In bacterial catabolism, regeneration of NAD+ is required since the oxidization of

a carbon source occurs with the conversion of NAD+ to NADH (Berríos-Rivera et al.,

2003; San et al., 2002). Since propionibacterium is an anaerobe, the NAD+ regeneration

is achieved via the formation of reduced end products such as propionic and succinic

acids. As a result, the NADH availability in the metabolic network could impact

fermentation product formation. The effects of carbon sources with different oxidation

states on fermentation kinetics of several products such as ethanol by E. coli (San et al.,

2002) and 1,2-propanediol production by E. coli (Berríos-Rivera et al., 2003) were

studied. The increase in NADH availability by using a more-reduced carbon source as the

substrate influenced cells to redistribute their pattern of fermentation to achieve a redox

balance and resulted in enhanced ethanol production with lower acetate formation

109

(Berríos-Rivera et al., 2003; San et al., 2002). It could be of interest if a carbon source

could shift the metabolic pathway of P. acidipropionici toward propionic acid formation.

This could lead to enhanced propionic acid production with reduced formation of

byproducts, acetate and succinate, without the presence of other unconventional

byproducts, which could eventually facilitate simple and inexpensive downstream

processing. Although the improvement of propionic acid production by the use of several

carbon sources has been extensively reported, little is known about the factors controlling

the intermediary metabolism of propionic acid fermentation.

The goal of this study was to understand the physiological behaviors of P.

acidipropionici under the utilization of carbon sources with different oxidation states at

the biochemistry level. Four carbon sources used in this study were glucose (oxidation

state = 0), xylose (oxidation state = 0), sorbitol (oxidation state = -1), and gluconate

(oxidation state = +1). In this work, batch free-cell fermentations by P. acidipropionici

wild type and adapted mutant from a fibrous-bed bioreactor (FBB) were carried out to

study the effect of different carbon sources on propionic acid fermentation kinetics.

Possible pathways in P. acidipropionici for the utilization of several carbon sources were

proposed and metabolic stoichiometric analysis was determined to evaluate the metabolic

fluxes and the possible controlling mechanisms in the propionic acid fermentation under

feeding of different carbon sources. Finally, the activities of five major enzymes

involving in propionate, acetate, and succinate formations and the overall protein

expression pattern of cultures grown in different types of carbon sources were also

110

determined in order to understand the underlying mechanisms contributing to cell’s

response to change in types of carbon sources.

4.2 Materials and Methods

4.2.1 Culture and Media

P. acidipropionici ATCC 4875 wild type and adapted mutant from the FBB

(Suwannakham and Yang, 2005) were grown in a synthetic medium (Appendix A.1)

supplemented with 150 mM of a particular carbon source. The medium was sterilized by

autoclaving at 121°C, 15 psig for 30 min. The stock culture was kept in serum bottles

under anaerobic conditions at 4°C.

4.2.2 Batch Fermentation

Batch free-cell fermentations of P. acidipropionici wild type and mutant were

performed in a 5-L stirred-tank fermentor (BioFloII, New Brunswick, Edison, NJ)

containing 3 L of the synthetic medium supplemented with 150 mM of a carbon source,

at 32°C, pH 6.5, and 100 rpm for agitation. Anaerobiosis was established by sparging the

medium with N2 for ~45 min, and thereafter the fermentor headspace was maintained

under 5 psig N2. The fermentor was inoculated with ~150 mL of a fresh culture grown in

serum bottles (OD600 ~ 2.0), and liquid samples were withdrawn at regular time intervals.

111

4.2.3 Analytical Methods

The growth of cultures was measured by using a spectrophotometer (Sequoia-

turner, Model 340). One unit of optical density at 600 nm was equal to 0.435 g/L of dry

cell weight (Appendix B.1). The concentrations of substrates and acid products

(propionic, acetic, succinic, and pyruvic acids) were analyzed by high-performance liquid

chromatography (HPLC) as described in Appendix B.3.

4.2.4 Metabolic Stoichiometric Analysis

The dicarboxylic acid pathway of P. acidipropionici (starting at a PEP node) is

shown in Figure 4.5. The main biochemical reactions to various intermediates and

fermentation end products in various branching points of the metabolic network of P.

acidipropionici fermentation using glucose, sorbitol, gluconate, and xylose as substrate

were represented by stoichiometric equations shown in Tables 4.2, 4.3, 4.4, and 4.5,

respectively. Fluxes in various reaction branches were estimated solely by applying

measured accumulation rates of extracellular metabolites and the assumption of pseudo-

steady state for intracellular intermediates. Fluxes were calculated directly from the net

metabolite concentration change divided by the total period of experimental time and the

net optical density obtained from that experiment. The unit of fluxes was mM/h/OD. The

set of equations was expressed in a matrix form: A·X(t) = Y where A is a matrix of

stoichiometric coefficients, X(t) is a matrix containing individual branch flux vector, and

112

Y is the matrix of accumulation rates of metabolites. Maple V Release 3.0 was used to

solve the value of unknown coefficients. The fraction of intermediate ‘A’ to end product

‘B’ was calculated by the flux of ‘A’ toward ‘B’ divided by the total flux of ‘A’ at the

‘A’ node and multiplied by 100.

4.2.5 Preparation of Cell Extract

Cells, grown in the synthetic medium (50 mL) supplemented with 150 mM of a

particular carbon source at 32°C to the exponential phase (OD600 ~ 1.8), were harvested,

washed three times, and resuspended in 15 mL of 25 mM Tris/HCl (pH 7.4). The cell

suspension was then sonicated for 12 min to break cell walls and then centrifuged at

10,000 rpm, 4°C for 1 h to remove cell debris. The cell extracts were kept cold on ice

before they were used in the enzyme activity assays. The protein content of the cell

extract was determined in triplicate by Bradford protein assay (Bio-Rad) with bovine

serum albumin as the standard protein (Appendix B.4).

4.2.6 Enzyme Assays

The activities of oxaloacetate transcarboxylase, propionyl CoA: succinyl CoA

transferase (CoA transferase), phosphotransacetylase (PTA), acetate kinase (AK), and

phosphoenolpyruvate carboxylase (PEP carboxylase) were assayed in duplicate as

described in Appendix B.5.

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4.2.7 Protein Expression: SDS-PAGE

Gel preparation and SDS-PAGE setup are described in Appendix B.6. All protein

samples with a specific amount (~24 µg each) were loaded into wells and run, with Mini-

PROTEAN® 3 Cell (Bio-Rad), at a constant voltage of 110 V until the tracking dye

reached the gel bottom. The gels were stained with coomassie brilliant blue for 1 h and

then destained with a destaining solution containing 20% methanol and 10% acetic acid.

4.3 Results

4.3.1 Effect of Carbon Sources on Fermentation Kinetics

Figures 4.1, 4.2, 4.3, and 4.4 show fermentation kinetics by the wild type using

glucose, sorbitol, gluconate, and xylose, respectively. The use of these four carbon

sources for the fermentation by the wild type did not affect types of end products since

propionic acid production was still accompanied by acetate and succinate. However, it

was found that acetate and succinate were formed simultaneously in fermentations using

glucose (Figure 4.1) and sorbitol (Figure 4.2), while the succinate formation occurred

before acetate was produced when gluconate (Figure 4.3) and xylose (Figure 4.4) were

used. In the fermentation using gluconate, pyruvate was accumulated since the beginning

of fermentation period; however, it was completely consumed by the end of fermentation.

As seen in Table 4.1, the fermentation using sorbitol provided the highest propionic acid

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production including productivity (0.39 g/L/h), yield (0.224 mol/mol C), and final

concentration (20.4 g/L). As compared to glucose (0.164 mol/mol C), the fermentation

using xylose (0.196 mol/mol C) produced a ~20% higher propionic acid yield whereas

the use of gluconate (0.176 mol/mol C) provided a similar one. The fermentation using

gluconate produced the highest succinate yield (0.034 mol/mol C) but the lowest acetate

yield (0.022 mol/mol C) while fermentations using xylose and glucose obtained propionic

acid production with a much higher acetate yield (0.067 mol/mol C by xylose, 0.057

mol/mol C by glucose) than the use of other carbon sources. The highest P/A molar ratio

of 8.1 was obtained from the fermentation using gluconate (Table 4.1). The fermentation

with sorbitol also provided the higher P/A molar ratio of 6.4 than xylose (3.0) and

glucose (2.9). Specific growth rate of cells was the lowest with xylose (0.079 h-1) as

compared to other carbon sources.

4.3.2 Comparison of Fermentation Kinetics between Wild Type and Adapted

Mutant

Figures 4.1, 4.2, 4.3, and 4.4 show kinetics of fermentations by the adapted

mutant using glucose, sorbitol, gluconate, and xylose, respectively. When glucose,

sorbitol, and xylose were used as substrates, types of end products obtained from

fermentations by the mutant and the wild type were similar. Both acetate and succinate

were formed simultaneously when glucose (Figure 4.1) and sorbitol (Figure 4.2) were

used. Unlike the wild type, the fermentation by the mutant using xylose (Figure 4.4)

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simultaneously produced acetate and succinate. As seen in Figure 4.3, the pattern of end-

product compositions of the fermentation by the mutant using gluconate was changed to

propionic acid production with accumulations of succinate and pyruvate and the absence

of acetate formation. As compared to the wild type, a significant increase in P/A molar

ratio was achieved in the fermentation by the mutant utilizing gluconate (from 8.1 to

infinity) (Table 4.1) due to the zero acetate formation. It was found that when sorbitol,

xylose, and gluconate were used as carbon sources, there was no significant difference in

propionate yields obtained from the wild type and the mutant. Unlike the use of other

carbon sources, the fermentation by the mutant using glucose, as compared to the wild

type, provided a higher propionate yield (0.191 vs. 0.164 mol/mol C) and a lower

succinate yield (0.014 vs. 0.023 mol/mol C).

4.3.3 Metabolic Pathway Analysis

The pseudo-steady state assumption on intracellular metabolic intermediates (i.e.,

no net change in their intracellular amount with time) in each unbranched pathway was

applied; as a result, the beginning substrate(s) and the end products in each pathway were

solely considered to determine metabolic fluxes.

Propionic acid fermentation by P. acidipropionici is mainly via the dicarboxylic

acid pathway (Hettinga and Reinbold, 1972; Playne, 1985; Wood, 1981). Figures 4.5 and

4.6 show the dicarboxylic acid pathway and proposed pathways of sugar utilization,

respectively. For the fermentation using a particular carbon source, the main pathway of

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end-product formations is similar to the one shown in Figure 4.5 whereas the pathway for

substrate utilization is dependent on the type of the particular carbon source. At the PEP

node, PEP is distributed into 2 intermediates, pyruvate and oxaloacetate. Most of PEP is

converted into pyruvate, the main branch-point intermediate, after that pyruvate is

channeled toward 3 pathways leading to propionate, acetate, and biomass formations. The

remaining PEP is converted into oxaloacetate leading to succinate formation.

Glucose (Oxidation State = 0)

The key reactions in the metabolic pathway for the fermentation by P.

acidipropionici using glucose as substrate toward various intermediates at branching

points and final products can be represented by the equations given in Table 4.2. Both

Embden-Meyerhorf-Parnaz (EMP) pathway (eq. 1) and Hexose Monophosphate (HMP)

pathway (eq. 2) could participate in glucose glycolysis (Papoutsakis and Meyer, 1985)

(Figure 4.6). Via the EMP pathway, glucose is converted into PEP via glucose-6-

phosphate (G-6-P), fructose-6-phosphate (F-6-P), and then glyceraldehyde-3-phosphate

(glyceraldehydes-3-P) while, via the HMP pathway, glucose conversion to PEP is via G-

6-P and then glyceraldehydes-3-P and F-6-P. At the PEP node, the majority of PEP is

then distributed into pyruvate (eq. 4) while the remaining is converted into oxaloacetate

leading to succinate formation (eq. 3). At the pyruvate node, propionate (eq. 6), acetate

(eq. 5), and biomass (eq. 7) are products from a conversion of pyruvate, the key branch-

point intermediate, in propionate, acetate, and biomass formation pathways, respectively.

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Sorbitol (Oxidation State = -1)

Sorbitol utilization pathway in P. acidipropionici (Fig. 4.6) was proposed to be

similar to the pathway in E.coli (Berríos-Rivera et al., 2003; San et al., 2002). Table 4.3

shows equations representing main reactions in the metabolic pathway for the

fermentation by P. acidipropionici using sorbitol as the substrate toward various

intermediates at branching points and final products. As seen in Figure 4.6, the pathway

involves the utilization of sorbitol to sorbitol-6-phosphate (sorbitol-6-P) with ATP

consumption and the conversion of sorbitol-6-P to F-6-P with NADH production. Then

F-6-P enters the glycolysis for PEP production. Equation 1 represents PEP production via

sorbitol utilization. PEP is then converted to pyruvate (eq. 2) and oxaloacetate, which

leads to succinate formation (eq. 3). Pyruvate is directed toward pathways leading to the

formations of propionate (eq. 5), acetate (eq. 4), and biomass (eq. 6).

Gluconate (Oxidation State = +1)

Gluconate utilization pathways in several microorganisms Saccharomyces

cerevisiae (http://pathway.yeastgenome.org), Mycobacterium tuberculosis H37Rv

(http://biocyc.org/MTBRV), and Treponema pallidum (http://biocyc.org/TPAL) have

been reported. Since the proposed pathways for these different microorganisms are

similar, a similar gluconate utilization pathway may be assumed for P. acidipropionici.

As seen in Fig. 4.6, gluconate is consumed with an expense of ATP for the production of

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6-phosphogluconate, which is then converted into ribulose-5-P and CO2 with NADH

production. Then, ribulose-5-P finally enters the non-oxidative stage of the pentose-

phosphate pathway for PEP formation. The main reactions in the metabolic pathway for

the fermentation by P. acidipropionici using gluconate as the carbon source toward

various intermediates at branching points and final products can be represented by the

equations given in Table 4.4. Equation 1 represents the conversion of gluconate to PEP

via the gluconate utilization pathway. The majority of PEP is driven toward pyruvate

formation (eq. 2) while the remaining is directed into the succinate formation pathway

(eq. 3). Propionate (eq. 5), acetate (eq. 4), and biomass (eq. 6) are produced from

pyruvate.

Xylose (Oxidation State = 0)

The utilization of pentose sugars is generally by the HMP pathway. Xylose is

mainly oxidized by the HMP pathway; however, some of xylose can be directly oxidized

to CO2, with the production of NADH, if necessary (Papoutsakis and Meyer, 1985) (Fig.

4.6). Table 4.5 shows equations representing key reactions in the metabolic pathway for

the fermentation by P. acidipropionici using xylose as the substrate. Xylose is first

converted into PEP (eq. 1) via D-xylulose-5-phosphate and then to glyceraldehydes-3-P

and F-6-P (Papoutsakis and Meyer, 1985). Direct oxidization of xylose to CO2 with

NADH production is represented by equation 2. PEP is converted into pyruvate (eq. 3).

Succinate is the product from PEP in the succinate formation pathway (eq. 4), while

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propionate (eq. 6), acetate (eq. 5), and biomass (eq. 7) are products from pyruvate in

propionate, acetate, and biomass formation pathways, respectively.

For all carbon sources, the biomass synthesis equation proposed by Papoutsakis

and Meyer, 1985 was applied in this study. The equation representing the biomass

formation is based on a value of 0.462 of the weight fraction of carbon in the biomass and

a value of 4.291 of the reductance degree of biomass (Papoutsakis, 1984). The reductance

degree is defined as the number of electrons available in the compound per atom of

carbon.

4.3.4 Metabolic Stoichiometric Analysis (MSA)

The effect of different carbon sources on the metabolic pathway shift observed in

this study is evaluated with a stoichiometric analysis. To obtain the overall equations for

propionic acid fermentation, each stoichiometric equation shown in the Table for each

carbon source was multiplied with a coefficient (a, b, c, d, e, f, and g, respectively) and

then these equations were grouped together. The overall fermentation equation for each

carbon source is shown below:

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Glucose

(a+3b) glucose 3g (C4H4xO4yN4z) + f propionate + e acetate + c succinate + (3b-

c+e) CO2 + (d-e-f-4g) pyruvate + (2a+5b-c-d) PEP + (2a+11b-2c+e-2f-5.75g) NADH +

(2c+d+e+f-33.7g) ATP (4.1)

Sorbitol

a sorbitol 3f (C4H4xO4yN4z) + e propionate + d acetate + c succinate + (-c+d) CO2

+ (b-d-e-4f) pyruvate + (2a-b-c) PEP + (3a-2c+d-2e-5.75f) NADH + (b+2c+d+e-33.7f)

ATP (4.2)

Gluconate

a gluconate 3f (C4H4xO4yN4z) + e propionate + d acetate + c succinate + (a-c+d)

CO2 + (b-d-e-4f) pyruvate + ((5/3)a-b-c) PEP + ((8/3)a-2c+d-2e-5.75f) NADH +

(b+2c+d+e-33.7f) ATP (4.3)

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Xylose

(a+b) xylose 3g (C4H4xO4yN4z) + f propionate + e acetate + d succinate + (5b-d+e)

CO2 + (c-e-f-4g) pyruvate + ((5/3)a-c-d) PEP + ((5/3)a+10b-2d+e-2f-5.75g) NADH + (-

b+c+2d+e+f-33.7g) ATP (4.4)

For simplicity, the carbon sources from other nutrients such as yeast extract and

trypticase are neglected in the analysis. A pseudo-steady state is assumed for pyruvate,

PEP, and NADH (a net accumulation of intermediates or cofactors is zero). However,

only two of them would be needed in estimating the values of the remaining coefficients.

PEP is an energy-rich intermediate from the glycolytic pathway and it has the highest

phosphoryl-group-transfer potential. PEP always transfers its phosphoryl group to ADP, a

less energy-rich compound, yielding ATP (Horton et al., 2002). Since the existence of

PEP in the cellular metabolism would be in a short period of time, there would not be a

PEP accumulation; thus, the assumption of zero net change of PEP should be valid. The

accumulation of pyruvate may exist if the conversion of pyruvate to other intermediates

or end products is slower than the pyruvate formation. Therefore, the pseudo-steady state

assumption was applied to NADH production.

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4.3.5 Effect of Carbon Sources on MSA

Table 4.6 summarizes the MSA results for the fermentations with various carbon

sources. At the PEP node, the fraction of PEP flux to pyruvate was the highest (94.14)

when sorbitol was used as the substrate while the lowest fraction (87.82) was obtained in

the fermentation using gluconate. On the other hand, the highest and lowest fractions of

PEP to succinate were found in the fermentations using gluconate (12.18) and sorbitol

(5.86), respectively. At the pyruvate node, the high fractions of pyruvate to propionate

were found in both fermentations using gluconate (72.01) and sorbitol (71.43) while

others had lower fractions. Fractions of pyruvate toward acetate formation pathway were

found to be the highest and the lowest when xylose (22.82) and gluconate (9.04) were

used as carbon sources, respectively. The highest production of NADH (0.54 mol/mol

carbon) was found in the fermentation using sorbitol as substrate. The lowest fraction of

pyruvate toward biomass production was found in the fermentation using xylose (9.94).

Fermentations using gluconate and xylose produced the highest flux of CO2 (0.23

mM/h/OD), indicating ~16% and ~9% of carbon lost to the production of CO2,

respectively.

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4.3.6 Comparison of MSA between Wild Type and Adapted Mutant

With glucose as the carbon source, the fraction of pyruvate to propionate (61.93

by the mutant, 54.21 by the wild type) was ~14% higher while fractions of PEP to

succinate (4.26 by the mutant, 7.11 by the wild type) and pyruvate to biomass (10.68 by

the mutant, 13.26 by the wild type) were ~40% and ~20% lower, respectively for the

mutant as compared to the wild type. However, no significant difference in the carbon

flux distributions was observed in the fermentation using sorbitol and xylose. When

gluconate was used as the substrate, only fractions of pyruvate to acetate (9.04 by the

wild type, 0 by the mutant) and pyruvate to biomass (13.52 by the wild type, 10.00 by the

mutant) were significantly lower for the mutant (Table 4.6).

4.3.7 Effect of Carbon Sources on Activity of Acid-Forming Enzymes

Both wild type and adapted mutant, grown in serum bottles containing the

medium supplemented with a particular carbon source, were harvested and the

intracellular proteins were extracted and assayed for five major enzymes: two propionate-

forming enzymes, OAA transcarboxylase and CoA transferase, one succinate-forming

enzyme, PEP carboxylase, and two acetate-forming enzymes, PTA and AK.

As shown in Table 4.7, the activity of OAA transcarboxylase of cells treated with

sorbitol, as compared to cells using other carbon sources, was significantly higher while

the higher activity of CoA transferase was found in cells utilizing sorbitol and xylose as

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substrates as compared to cells treated with gluconate and glucose. Cells grown in

gluconate and xylose were found to have an increased activity of PEP carboxylase as

compared to cells grown in sorbitol and glucose. Moreover, PTA and AK activities of

cells treated with xylose were apparently higher than the activities of these two enzymes

in cells treated with other carbon sources. As compared to the wild type, the mutant

appeared to have higher activities of both propionate-forming enzymes and the reduced

activity of PEP carboxylase. However, there was no significant difference in activities of

PTA and AK in the wild type and the mutant for all carbon sources studied except for the

activity of AK in cells using sorbitol.

4.3.8 Effect of Carbon Sources on Protein Expression Pattern

The protein of ~56 kDa was observed in the wild type (Fig. 4.7, Lane 5) and the

adapted mutant (Fig. 4.8, Lane 5) grown in xylose. The unknown protein with a size of

~37 kDa was noticed, in both wild type (Figure 4.7) and mutant (Figure 4.8), with a

difference in intensity, from the highest to the lowest, when cells were grown in the

medium containing gluconate (Lane 2), xylose (Lane 5), glucose (Lane 1), and sorbitol

(Lane 4), respectively. The overall protein expression patterns observed in the wild type

(Figure 4.7) and the adapted culture (Figure 4.8) were similar except for the extra protein

with the size of ~45 kDa that was solely found in the adapted mutant.

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4.4 Discussion

4.4.1 Effect of Carbon Sources on Fermentation by P. acidipropionici

4.4.1.1 Production of Propionic Acid

The highest propionic acid production including productivity, final concentration,

and yield was achieved in the fermentation using sorbitol as a carbon source. Based on

the MSA (Table 4.6), the high fractions of PEP to pyruvate and pyruvate to propionate as

well as the highest production of NADH indicated the metabolic pathway shift toward

propionate formation, resulting in much increased propionic acid production obtained in

the fermentation using sorbitol as compared to the use of glucose or other carbon sources.

In addition, the higher activities of two propionate-forming enzymes in cells grown in

sorbitol, as compared to cells using other carbon sources, supported the metabolic

pathway shift toward propionate formation as well. Although the fractions of PEP to

pyruvate found in the fermentations by the wild type using xylose and glucose were as

high as the fraction obtained in the fermentation utilizing sorbitol, the fractions of

pyruvate to propionate were lower due to the ~70-100% increased fractions of pyruvate

to acetate, leading to lower propionate yields as compared to the fermentation using

sorbitol. The lower propionate yield was obtained from the fermentation using xylose as

substrate (Table 4.1) because of the lower fraction of pyruvate toward propionate

formation (Table 4.6) although the high production of NADH was obtained and the

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activities of CoA transferase in cells grown in xylose and sorbitol were similar (Table

4.7). The fraction of PEP to pyruvate was the lowest in the fermentation using gluconate

due to the very high fraction of PEP to succinate and the lost of carbon (~14%) to the

CO2 formation during the gluconate utilization as one mole of CO2 is produced per mole

of consumed gluconate. Although the fraction of pyruvate to propionate was as high as

the fraction obtained from the use of sorbitol due to less pyruvate was forced toward the

acetate formation pathway, the lower fraction of PEP to pyruvate, the much lower

activities of OAA transcarboxylase and CoA transferase, and a large amount of NADH

lost to succinate formation made the fermentation using gluconate unable to obtain high

propionic acid production as the fermentation using sorbitol did. The ~14% increased

fraction of pyruvate to propionate with ~40% and ~20% reduced fractions of PEP to

succinate and pyruvate to biomass, respectively could explain the ~17% increased

propionate yield and the ~39% decrease in succinate yield in the fermentation by the

mutant using glucose as compared to the wild type. It was noticed that ~18% more CO2

(35.0 vs. 29.7%) was produced from glucose utilization pathway (HMP pathway),

indicating the mutant utilized more glucose via the HMP pathway to enhance production

of NADH as observed by a slight increase in NADH production (from 0.43 to 0.46

mol/mol carbon). Moreover, the reduced biomass formation in the fermentation by the

mutant also required a lower amount of NADH utilized for the biomass formation. As a

result, a larger amount of NADH should be available for the formation of more propionic

acid as achieved as the ~17% enhanced yield of propionate. In addition to the MSA result

(Table 4.6), a significantly increased (~164%) activity of CoA transferase, a slight

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increase (~28%) in the OAA transcarboxylase activity, and a ~30% decreased activity of

PEP carboxylase, as seen in Table 4.7, could be responsible for the ~17% increased

propionate yield and the ~39% decrease in succinate yield. This result was consistent

with the finding reported by Suwannakham and Yang, 2005. As compared to the wild

type, the fed-batch free-cell fermentation by the adapted mutant from the FBB using

glucose as substrate provided the ~15% increased propionate yield and the ~50%

decreased succinate yield (Suwannakham and Yang, 2005).

4.4.1.2 Acetate Formation

The much higher (~70-150%) fraction of pyruvate to acetate and the much

increased activities of acetate-forming enzymes, both AK and PTA, resulted in the 60-

200% higher acetate yield and the much lower P/A molar ratio of ~3.0 obtained in the

fermentations by the wild type using xylose and glucose as compared to the

fermentations utilizing other carbon sources. On the other hand, fermentations by the

wild type using gluconate and sorbitol, as compared to the use of glucose, obtained ~61%

and ~39% decreased acetate yields, respectively. As a result, ~179% and ~121%

enhanced P/A ratios could be achieved in fermentations with the use of gluconate (8.1)

and sorbitol (6.4), respectively (Table 4.1). When sorbitol, glucose, and xylose were used

as substrates, the acetate production was similar or slightly different in the fermentations

by the wild type and the mutant. Surprisingly, no acetate accumulation was found in the

fermentation by the mutant using gluconate (Figure 4.3); however, some pyruvate was

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accumulated in the fermentation broth. It is possible that the ~26% lower fraction of

pyruvate to biomass in the mutant, as compared to the wild type (10.00 vs. 13.52),

resulted in the lower amount of NADH and ATP required for the biomass formation,

where a large amount of NADH and ATP would be consumed. As a result, the formation

of acetate, one of sources providing NADH and ATP, might not be required to produce

more NADH to the cell metabolism. Furthermore, as seen in Figure 4.3, succinate

formation began since the early period of the fermentation using gluconate before the

acetate was formed. In general, both succinate and propionate formations are

accompanied by a formation of ATP. It is possible that with sufficient NADH directly

produced from the gluconate utilization and ATP from formations of other end products,

acetate formation may be less or not required. As a result of the zero acetate formation,

the P/A ratio obtained in the fermentation by the mutant using gluconate was closed to

infinity.

4.4.1.3 Succinate Formation

In general, CO2 could be produced from either the acetate formation pathway or

the sugar utilization pathway or both, if a conversion of substrate to PEP is accompanied

by CO2 formation. Succinate is formed via a conversion of PEP and CO2 with an expense

of NADH; therefore, the formation of succinate would be dependent on the availability of

PEP, CO2, and NADH in the metabolic network. The highest succinate yield found in the

fermentation using gluconate could be explained by the high fraction of PEP to succinate,

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the high CO2 availability in the network, and the high activity of PEP carboxylase. As

previously mentioned, succinate was formed since the early fermentation period before

acetate was even produced (Fig. 4.3) when gluconate was used as a carbon source. This

indicated that succinate formation in the fermentation using gluconate utilized CO2

mainly produced from the gluconate utilization, which was >88% of total CO2 production

(Table 4.6).

Figure 4.4 shows fermentation kinetics of the wild type and the mutant using

xylose. In the wild type, succinate formation started before acetate formation whereas

both succinate and acetate were produced simultaneously in the mutant. As compared to

the mutant, the wild type gained ~14% more CO2 directly produced from xylose (Table

4.6). This indicated that succinate formation in the fermentation by the wild type using

xylose could start before the acetate formation because of the higher CO2 availability in

the system.

From the MSA result (Table 4.6), the sole source of CO2 in the fermentation with

a use of sorbitol was the acetate formation pathway. Due to one mole of CO2 and NADH

are produced per mole of formed acetate and succinate formation requires two moles of

NADH, the ratio of acetate: succinate would have to be two so that NADH would be

sufficient for succinate formation if only NADH produced from the acetate formation

would supposedly be utilized in the succinate formation. It was noticed that the molar

ratio of acetate: succinate was about 2.0 (~1.8 in the wild type, ~2.0 in the mutant).

Moreover, most of NADH produced from the sorbitol utilization may mainly be utilized

for the formation of propionate, a main product of the fermentation, due to the high

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fractions of PEP to pyruvate and pyruvate to propionate in the fermentation using

sorbitol. Thus, the acetate formation could be a sole source of CO2 and NADH for the

succinate formation in propionic acid fermentation using sorbitol.

In this study, sorbitol was found to be the best carbon source for improved

propionic acid production as compared to xylose, gluconate, and glucose. A more-

reduced carbon source as sorbitol would provide more reducing equivalents in the form

of NADH as compared to glucose, which is a more-oxidized carbon source (Berríos-

Rivera et al., 2003; San et al., 2002). As compared to the fermentation with a use of

glucose, the fermentation using sorbitol achieved ~63%, ~37%, ~33%, and ~121%

enhanced productivity (0.24 vs. 0.39 g/L/h), propionic acid yield ((0.164 vs. 0.224

mol/mol C) or (0.982 vs. 1.345 mol/mol sorbitol)), final propionic acid concentration

(15.3 vs. 20.4 g/L), and propionate: acetate (P/A) molar ratio (2.9 vs. 6.4), respectively.

Due to propionic acid is a main fermentation product used for the NAD+ regeneration

from NADH in P. acidipropionici and sorbitol could provide more NADH per mole of

substrate, more propionic acid could be produced from the fermentation using sorbitol as

compared to the use of glucose. In addition to the increased activities of two propionate-

forming enzymes, the reduced yields of byproducts, acetate (~39%) and succinate

(~13%), indicated that more pyruvate could be forced through the propionic acid

formation pathway, leading to the increase in propionic acid yield. Due to the higher

production of NADH was obtained from sorbitol and cells could obtain ATP from both

sorbitol utilization (two moles ATP/mole sorbitol) and propionate formation (one mole

ATP/mole propionate), a decrease in acetate production, one of sources where the

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regeneration of NADH from NAD+ (one mole NADH/mole acetate) and the production

of ATP (one mole ATP/mole acetate) occur, could be required for the redox balance. As

a result of the increased propionate yield and the reduced acetate yield, the significantly

enhanced P/A molar ratio (~121%) was achieved in the fermentation using sorbitol as

compared to the use of glucose as a carbon source. This result was consistent with the use

of sorbitol as substrate for the ethanol fermentation by E. coli (San et al., 2002) and the

ethanol formation in the 1,2-propanediol production by E. coli (Berríos-Rivera et al.,

2003). With a use of a more-reduced carbon source, as sorbitol, the significantly

increased ethanol yield and the considerably decreased yield of acetate, a key byproduct,

resulted in the highest ethanol: acetate (Et/Ac) ratio as compared to the use of glucose as

substrate. The increased Et/Ac ratio could serve as an indirect indicator of a higher

NADH: NAD+ ratio (Berríos-Rivera et al., 2003). Influenced by feeding a more-reduced

substrate to E. coli, the higher ratio of NADH: NAD+ could result in enhanced production

of reduced end-product metabolite as ethanol if the network of end-product formation is

limited or controlled by NADH (Berríos-Rivera et al., 2003). In this work, sorbitol was

proved to be a more-reduced carbon source and its use resulted in the shift in metabolic

pathway toward propionate formation as indicated by the much enhanced propionic acid

production and the decreased by-product formation. It could be possible that NADH may

play a key role in controlling or limiting propionic acid production.

The productivity of 0.18 g/L/h was obtained with the propionate yield of 0.844

mol/mol in the fermentation using glycerol (Barbirato et al., 1997). Himmi et al, 2000

reported that when glycerol was used as substrate in the fermentation by P.

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acidipropionici, 0.42 g/L/h productivity, 0.79 mol/mol propionate yield, and a P/A molar

ratio of 5.7 with 0.17 mol/mol acetate yield were attained. Since glycerol and propionic

acid had a similar reductance degree, production of another metabolite was not required

for the oxido-reduction balance (Himmi et al., 2000). In this work, the productivity of

0.39 g/L/h, the propionate yield of 1.345 mol/mol sorbitol (0.224 mol/mol C), and the

P/A molar ratio of 6.4 with the acetate yield of 0.212 mol/mol sorbitol (0.035 mol/mol C)

were achieved in the fermentation by P. acidipropionici utilizing sorbitol (Table 4.1). It is

possible that the ~39% reduced acetic acid yield, as compared to the fermentation using

glucose, was observed in the fermentation using sorbitol due to the redox states of

sorbitol and propionic acid are closed and lower production of more-oxidized end

product, as acetate, would require for the redox balance.

The fermentations using xylose and glucose showed a similar pattern of

fermentation end-product compositions as propionic acid production was accompanied

by formations of acetate and succinate as byproducts; however, the slightly higher

propionic acid yield was obtained when xylose was used as substrate. The low P/A molar

ratio of ~3.0 was obtained in the fermentations with a use of xylose and glucose due to

the high yield of acetic acid.

It was reported that the use of gluconate, a more-oxidized carbon source, in the

ethanol fermentation by E. coli provided the lower ethanol production, the higher acetate

yield, and the lowest ethanol: acetate ratio as compared to the use of sorbitol and glucose

(Berríos-Rivera et al., 2003; San et al., 2002). In this study, the opposite result was

observed. A similar propionic acid yield was obtained in the fermentations using

133

gluconate and glucose. Moreover, the fermentation using gluconate was found to provide

propionic acid production with very low production of acetic acid, resulting in the

significantly high P/A molar ratio of 8.1. It is possible that since the formation of ethanol

is not accompanied by the production of ATP, the higher acetate formation might be

required. On the other hand, in the propionic acid fermentation, ATP can be produced

from the propionate and succinate formations and the succinate formation significantly

increased in the fermentation using gluconate, resulting in the high ATP availability in

the metabolic network. As a result, a response of cells by lowering the production of

acetate, one of ATP sources, was observed.

4.4.2 Effect of Carbon Sources on Protein Expression Pattern

The extra protein of ~56 kDa found in the wild type and the mutant grown in

xylose could indicate that a newly expressed enzyme or protein was induced via a

utilization of xylose as a carbon source. The observation of different intensities of the

unknown protein with a size of ~37 kDa, from the highest to the lowest, was found in

both wild type and adapted mutant grown in the medium containing gluconate, xylose,

glucose, and sorbitol, respectively. This unknown protein might play a role in production

of succinate since the fermentation using gluconate produced higher succinate yield than

the fermentations using xylose, glucose, and sorbitol, respectively. The overall protein

expression patterns of the mutant and the wild type were similar except for the ~45-kDa

protein that was only found in the adapted mutant. As previously studied, the adapted

134

mutant from the FBB had higher propionate tolerance as compared to the wild type

(Suwannakham and Yang, 2005). This extra protein might be responsible for acid

tolerance response in the adapted mutant from the FBB.

4.5 Conclusion

The use of carbon sources with different oxidation states influenced the kinetics

of propionic acid fermentation. Metabolic stoichiometric analysis was used for

quantitatively evaluating the metabolic pathway shift influenced by the type of carbon

sources and for providing the better understanding on how cells responded to a particular

substrate and redistributed carbon flux as a change in the carbon source type was made.

The determination of activities of major enzymes in the dicarboxylic acid pathway and

the observation of overall protein expression patterns were also used to evaluate the

effect of different carbon sources on the propionic acid fermentation by P.

acidipropionici.

Sorbitol could be a carbon source of interest for enhanced propionic acid

production, including productivity, yield, final concentration, and P/A molar ratio with

low acetate and succinate yields. The cost of downstream processing would be reduced as

lower amount of byproducts present in the fermentation broth. Since sorbitol would be a

more-expensive carbon source as compared to glucose or others, sorbitol supplied from

upstream processes with the fermentation by other microorganisms or with enzymatic

reaction converting sorbitol from much cheaper carbon sources could be of interest in

135

order to make propionic acid fermentation using sorbitol as a carbon source to be more

feasible for commercial production of propionic acid.

136

4.6 References Barbirato F, Chedaille D, Bories A. 1997. Propionic acid fermentation from glycerol:

comparison with conventional substrates. Appl Microbiol Biotechnol 47:441-446. Berríos-Rivera SJ, San K-Y, Bennett GN. 2003. The effect of carbon sources and lactate

dehydrogenase deletion on 1,2-propanediol production in Escherichia coli. J Ind Microbiol Biotechnol 30:34-40.

Colomban A, Roger L, Boyaval P. 1993. Production of propionic acid from whey

permeate by sequential fermentation, ultrafiltration, and cell recycling. Biotechnol Bioeng 42:1091-1098.

Emde R, Schink B. 1990. Enhanced propionate formation by Propionibacterium

freudenreichii subsp. freudenreichii in a three-electrode amperometric culture system. Appl Environ Microbiol 56:2771-2776.

Gluconate utilization pathway in Mycobacterium tuberculosis H37Rv.

http://biocyc.org/MTBRV Gluconate utilization pathway in Saccharomyces cerevisiae.

http://pathway.yeastgenome.org Gluconate utilization pathway in Treponema Pallidum. http://biocyc.org/TPAL Goswami V, Srivastava AK. 2000. Fed-batch propionic acid production by

Propionibacterium acidipropionici. Biochem Eng J 4:121-128. Hettinga DH, Reinbold GW. 1972. Review: The propionic-acid bacteria II. Metabolism. J

Milk Food Technol 35:358-372. Himmi EH, Bories A, Boussaid A, Hassani L. 2000. Propionic acid fermentation of

glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. Appl Microbiol Biotechnol 53:435-440.

Horton HR, Moran LA, Ochs RS, Rawn JD, Scrimgeour KG. 2002. Principles of

biochemistry – 3rd ed. Upper Saddle River, NJ: Prentice-Hall, Inc. p 327-328. Hsu ST, Yang S-T. 1991. Propionic acid fermentation of lactose by Propionibacterium

acidipropionici: effects of pH. Biotechnol Bioeng 38:571-578. Huang YL, Wu Z, Zhang L, Cheung CM, Yang S-T. 2002. Production of carboxylic

acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor. Bioresource Technol 82:51-59.

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Jin Z, Yang S-T. 1998. Extractive fermentation for enhanced propionic acid production from lactose by Propionibacterium acidipropionici. Biotechnol Prog 14:457-465.

Lewis VP, Yang S-T. 1992a. Propionic acid fermentation by Propionibacterium

acidipropionici: effect of growth substrate. Appl Microbiol Biotechnol 37:437-442. Lewis VP, Yang S-T. 1992b. A novel extractive fermentation process for propionic acid

production from whey lactose. Biotechnol Bioeng 23:1517-1525. Martínez-Campos R, de la Torre M. 2002. Production of propionate by fed-batch

fermentation of Propionibacterium acidipropionici using mixed feed of lactate and glucose. Biotechnol Lett 24:427-431.

Paik H-D, Glatz BA. 1994. Propionic acid production by immobilized cells of a

propionate-tolerant strain of Propionibacterium acidipropionici. Appl Microbiol Biotechnol 42:22-27.

Papoutsakis ET. 1984. Equations and calculations for fermentations of butyric acid

bacteria. Biotechnol Bioeng 26:174-187. Papoutsakis ET, Meyer CL. 1985. Fermentation equations for propionic-acid bacteria and

production of assorted oxychemicals from various sugars. Biotechnol Bioeng 27:67-80.

Playne MJ. 1985. Propionic and butyric acids. In: Moo-Young M, editor.

Comprehensive Biotechnology, vol. 3. New York: Pergamon. p 731-759. Quesada-Chanto A, Afschar AS, Wagner F. 1994. Optimization of a Propionibacterium

acidipropionici continuous culture utilizing sucrose. Appl Microbiol Biotechnol 42:16-21.

Ramsay JA, Aly Hassan M-C, Ramsay BA. 1998. Biological conversion of hemicellulose

to propionic acid. Enzyme Microb Technol 22:292-295. Rickert DA, Glatz CE, Glatz BA. 1998. Improved organic acid production by calcium

alginate-immobilized propionibacteria. Enzyme Microb Technol 22:409-414. San K-Y, Bennett GN, Berríos-Rivera SJ, Vadali RV, Yang Y-T, Horton E, Rudolph FB,

Sariyar B, Blackwood K. 2002. Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metabolic Engineering 4:182-192.

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Suwannakham S, Yang S-T. 2005. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol Bioeng, in press.

Thompson TE, Conrad R, Zeikus JG. 1984. Regulation of carbon and electron flow in

Propionispira arboris: Physiological function of hydrogenase and its role in homopropionate formation. FEMS Microbiol Lett 22:265-271.

Wood HG. 1981. Metabolic cycles in the fermentation of propionic acid. In: Estabrook

and Srera RW, editors. Current Topics in Cellular Regulation, vol 18. New York: Academic Press. p 225-287.

Yang S-T, Huang Y, Hong G. 1995. A novel recycle batch immobilized cell bioreactor

for propionate production from whey lactose. Biotechnol Bioeng 45:379-386. Yang S-T, Zhu H, Li Y, Hong G. 1994. Continuous propionate production from whey

permeate using a novel fibrous bed bioreactor. Biotechnol Bioeng 43:1124-1130.

139

apyruvate yield = 0.017 ± 0.001 mol/mol carbon. bYield (mol/mol carbon) = Total moles of product/(Total moles of consumed substrate x The number of carbon atoms in the substrate). Table 4.1 Kinetics of fermentations by P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources.

Sorbitol Xylose Gluconate Glucose

WT Adapted Mutant WT Adapted

Mutant WT Adapted Mutanta WT Adapted

Mutant Max. propionate (g/L)

20.4 ± 0.5 21.9 ± 0.3 18.4 ± 0.7 19.5 ± 0.8 17.1 ± 0.5 19.0 ± 0.7 15.3 ± 0.2 17.2 ± 0.3

Yieldb (mol/mol C)

Propionate 0.224 ± 0.006 0.227 ± 0.003 0.196 ± 0.010 0.200 ± 0.012 0.176 ± 0.010 0.176 ± 0.008 0.164 ± 0.003 0.191 ± 0.005

Acetate 0.035 ± 0.003 0.032 ± 0.002 0.067 ± 0.005 0.073 ± 0.005 0.022 ± 0.002 0.000 ± 0.000 0.057 ± 0.001 0.065 ± 0.001

Succinate 0.020 ± 0.002 0.016 ± 0.001 0.025 ± 0.002 0.023 ± 0.001 0.034 ± 0.003 0.029 ± 0.002 0.023 ± 0.001 0.014 ± 0.001

Propionate: Acetate ratio (mol/mol)

6.4 ± 1.0 7.1 ± 0.7 3.0 ± 0.6 2.8 ± 0.5 8.1 ± 1.6 N/A 2.9 ± 0.1 3.0 ± 0.2

Productivity (g/L/h) 0.39 ± 0.03 0.30 ± 0.01 0.25 ± 0.01 0.33 ± 0.02 0.21 ± 0.02 0.28 ± 0.03 0.24 ± 0.01 0.26 ± 0.02

Sp. growth rate (h-1) 0.114 ± 0.013 0.125 ± 0.003 0.079 ± 0.009 0.109 ± 0.014 0.109 ± 0.007 0.158 ± 0.007 0.134 ± 0.015 0.113 ± 0.002

139

140

Table 4.2 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using glucose.

Embden-Meyerhof-Parnas Pathway (EMP)

1) Glucose + 2 NAD+ → 2 PEP + 2 NADH

Hexose Monophosphate Pathway (HMP)

2) 3 Glucose + 11 NAD+ → 5 PEP + 3 CO2 + 11 NADH

Acetic Acid Formation

5) Pyruvate + NAD+ + ADP → Acetate + NADH + ATP + CO2

Succinic Acid Formation

3) PEP + CO2 + 2 NADH + 2 ADP → Succinate + 2 NAD+ + 2 ATP

Pyruvic Acid Formation

4) PEP + ADP → Pyruvate + ATP

Propionic Acid Formation

6) Pyruvate + 2 NADH + ADP → Propionate + 2 NAD+ + ATP

Biomass Formation

7) 4 Pyruvate + 5.75 NADH + 33.7 ATP→ 3 C4H4xO4yN4z + 5.75 NAD+ + 33.7 ADP

141

Table 4.3 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using sorbitol.

Sorbitol Utilization

1) Sorbitol + 3 NAD+ → 2 PEP + 3 NADH

Acetic Acid Formation

4) Pyruvate + NAD+ + ADP → Acetate + NADH + ATP + CO2

Succinic Acid Formation

3) PEP + CO2 + 2 NADH + 2 ADP → Succinate + 2 NAD+ + 2 ATP

Pyruvic Acid Formation

2) PEP + ADP → Pyruvate + ATP

Propionic Acid Formation

5) Pyruvate + 2 NADH + ADP → Propionate + 2 NAD+ + ATP

Biomass Formation

6) 4 Pyruvate + 5.75 NADH + 33.7 ATP→ 3 C4H4xO4yN4z + 5.75 NAD+ + 33.7 ADP

142

Table 4.4 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using gluconate.

Gluconate Utilization

1) Gluconate + 8/3 NAD+ → 5/3 PEP + 8/3 NADH + CO2

Acetic Acid Formation

4) Pyruvate + NAD+ + ADP → Acetate + NADH + ATP + CO2

Succinic Acid Formation

3) PEP + CO2 + 2 NADH + 2 ADP → Succinate + 2 NAD+ + 2 ATP

Pyruvic Acid Formation

2) PEP + ADP → Pyruvate + ATP

Propionic Acid Formation

5) Pyruvate + 2 NADH + ADP → Propionate + 2 NAD+ + ATP

Biomass Formation

6) 4 Pyruvate + 5.75 NADH + 33.7 ATP→ 3 C4H4xO4yN4z + 5.75 NAD+ + 33.7 ADP

143

Table 4.5 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using xylose.

Hexose Monophosphate Pathway (HMP)

1) Xylose + 5/3 NAD+ → 5/3 PEP + 5/3 NADH

Direct Oxidization

2) Xylose + ATP + 10 NAD+ → 5 CO2 + ADP + 10 NADH

Acetic Acid Formation

5) Pyruvate + NAD+ + ADP → Acetate + NADH + ATP + CO2

Succinic Acid Formation

4) PEP + CO2 + 2 NADH + 2 ADP → Succinate + 2 NAD+ + 2 ATP

Pyruvic Acid Formation

3) PEP + ADP → Pyruvate + ATP

Propionic Acid Formation

6) Pyruvate + 2 NADH + ADP → Propionate + 2 NAD+ + ATP

Biomass Formation

7) 4 Pyruvate + 5.75 NADH + 33.7 ATP→ 3 C4H4xO4yN4z + 5.75 NAD+ + 33.7 ADP

144

a%A to B = (Total flux of A to B/Total flux of A) x 100. bNADH production = Total flux of produced NADH/(Total flux of consumed substrate x The number of carbon atoms in the substrate). c%CO2 = Total flux of produced CO2/(Total flux of consumed substrate x The number of carbon atoms in the substrate) x 100. d%CO2 produced from A = (Total flux of CO2 produced from A/Total flux of produced CO2) x 100. Table 4.6 Metabolic stoichiometric analysis of fermentations by P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources.

Sorbitol Xylose Gluconate Glucose WT Adapted

Mutant WT Adapted Mutant WT Adapted

Mutant WT Adapted Mutant

%PEP to Pyruvatea 94.14 95.32 92.25 92.81 87.82 89.76 92.89 95.74

%PEP to Succinate 5.86 4.68 7.75 7.19 12.18 10.24 7.11 4.26 %Pyruvate to Propionate 71.43 71.55 67.22 67.71 72.01 70.54 54.21 61.93

%Pyruvate to Acetate 11.28 10.12 22.82 24.54 9.04 0.00 19.04 21.01

%Pyruvate to Biomass 10.37 10.01 9.94 7.62 13.52 10.00 13.26 10.68 NADH productionb (mol/mol carbon) 0.54 0.53 0.48 0.48 0.47 0.44 0.43 0.46

CO2 (mM/h/OD) 0.06 0.04 0.23 0.37 0.23 0.32 0.12 0.20

%CO2c 1.6 1.7 9.2 9.3 15.5 13.8 5.9 8.6

%CO2 produced from substrated 0.0 0.0 42.9 37.6 88.3 100.0 29.7 35.0

%CO2 produced from acetate 100.0 100.0 57.1 62.4 11.7 0.0 70.3 65.0

%Carbon Recovery 93.5±3.2 92.1±1.7 105.3±4.8 104.5±5.0 96.0±4.6 90.4±3.2 87.8±1.5 94.1±2.1

144

145

Table 4.7 Specific activity of major enzymes involving in the dicarboxylic acid pathway of P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources.

Specific activity (U/mg)

OAA transcarboxylase

CoA transferase PEP carboxylase PTA AK

Sorbitol wild type 0.356 ± 0.004 0.072 ± 0.001 0.0008 ± 0.0005 0.0008 ± 0.0002 0.011 ± 0.001 adapted mutant 0.386 ± 0.008 0.116 ± 0.000 0.0004 ± 0.0000 0.0010 ± 0.0001 0.005 ± 0.001 Xylose wild type 0.158 ± 0.002 0.068 ± 0.003 0.0014 ± 0.0009 0.0017 ± 0.0000 0.027 ± 0.001 adapted mutant 0.187 ± 0.004 0.143 ± 0.005 0.0009 ± 0.0001 0.0020 ± 0.0001 0.028 ± 0.012 Gluconate wild type 0.097 ± 0.003 0.028 ± 0.001 0.0015 ± 0.0000 0.0008 ± 0.0000 0.015 ± 0.001 adapted mutant 0.163 ± 0.005 0.097 ± 0.001 0.0007 ± 0.0001 0.0010 ± 0.0001 0.011 ± 0.003 Glucose wild type 0.108 ± 0.003 0.025 ± 0.001 0.0010 ± 0.0001 0.0009 ± 0.0004 0.019 ± 0.001 adapted mutant 0.138 ± 0.004 0.066 ± 0.001 0.0007 ± 0.0001 0.0011 ± 0.0001 0.020 ± 0.002

145

146

Figure 4.1 Fermentations by P. acidipropionici using glucose. A. Wild type; B. Adapted mutant from the FBB.

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

6

7

OD

Glucose, adapted mutant

Succinate

Acetate

Propionate

OD

B

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 Time (h)

Con

cent

ratio

n (g

/L)

0 1 2 3 4 5 6 7 8 9

OD

Glucose, wild type

Succinate

Acetate

Propionate

OD

A

147

Figure 4.2 Fermentations by P. acidipropionici using sorbitol. A. Wild type; B. Adapted mutant from the FBB.

B 0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100 110 Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

6

OD

Sorbitol, adapted mutant

Succinate

Acetate

Propionate

OD

A 0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 Time (h)

0

1

2

3

4

5

6

OD

Sorbitol, wild type

Succinate

Acetate

Propionate

OD

Con

cent

ratio

n (g

/L)

148

Figure 4.3 Fermentations by P. acidipropionici using gluconate. A. Wild type; B. Adapted mutant from the FBB.

B 0

5

10

15

20

25

30

35

40

0 50 100 150 Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

OD

Gluconate, adapted mutant

Succinate

Propionate

OD

Pyruvate

A 0

5

10

15

20

25

30

35

40

0 50 100 150 Time (h)

0

1

2

3

4

5

6

OD

Gluconate, wild type

Succinate

Acetate

Propionate

OD

Pyruvate

Con

cent

ratio

n (g

/L)

149

Figure 4.4 Fermentations by P. acidipropionici using xylose. A. Wild type; B. Adapted mutant from the FBB.

B 0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100 110 Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

OD

Xylose, adapted mutant

Succinate

Acetate

Propionate

OD

A 0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100 110 Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

6

OD

Xylose, wild type

Succinate

Acetate

Propionate

OD

150

Figure 4.5 The dicarboxylic acid pathway for the formations of propionic, acetic, and succinic acids. The five key enzymes assayed in this study are labeled in the pathway.

NADH CoA

NAD+

CO2

Acetate

ADP

ATP

Acetyl CoA

Acetyl phosphate

Pi

CoA

Propionyl CoA

Pyruvate

Phosphoenolpyruvate ADP

ATP

Succinate

Methylmalonyl CoA

Succinyl CoA

Propionate

GDP

GTP

CO2

Oxaloacetate

NADH

Malate

Fumarate

NADH + ADP

FPH2

FP

FPH2

FP

NAD+

NAD+ + ATP

Phosphotransacetylase

Acetate kinase

CoA transferase

OAA transcarboxylase

PEP carboxylase

151

Figure 4.6 Proposed pathways of sugar utilization in P. acidipropionici.

HMP

glucose

EMP 2 PEP + 2 NADH

5/3 PEP + 11/3 NADH + CO2

glyceraldehyde-3-P

glyceraldehyde-3-P + fructose-6-P

sorbitol sorbitol-6-P fructose-6-P 2 PEP + 3 NADH

NAD+ NADH ADP ATP

gluconate 6-phosphogluconate ribulose-5-P 5/3 PEP + 8/3 NADH + CO2

NAD+ NADH + CO2 Non-oxidative stage Pentose phosphate

pathway

ADP ATP

xylose

D-xylulose-5-P 5/3 PEP + 5/3 NADH

5 CO2 + 10 NADH Direct Oxidization

HMP

152

Figure 4.7 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici wild type using different carbon sources. Lane 1: glucose; Lane 2: gluconate; Lane 3: molecular weight marker; Lane 4: sorbitol; Lane 5: xylose; Lane 6: molecular weight marker.

~37 kDa

97 66 45

30

20.1 14.4

~56 kDa

1 2 3 4 5 6

153

Figure 4.8 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici adapted mutant from the FBB using different carbon sources. Lane 1: glucose; Lane 2: gluconate; Lane 3: molecular weight marker; Lane 4: sorbitol; Lane 5: xylose; Lane 6: molecular weight marker.

~56 kDa

97 66

45

30 20.1 14.4

~37 kDa

1 2 3 4 5 6

~45 kDa

154

CHAPTER 5

CONSTRUCTION AND CHARACTERIZATION OF ACK GENE DELETED

MUTANTS OF PROPIONIBACTERIUM ACIDIPROPIONICI FOR PROPIONIC

ACID FERMENTATION

Summary

The heterofermentative Propionibacterium acidipropionici produces propionic

acid from glucose with byproducts including acetic acid, succinic acid, and CO2.

Inactivation of the ack gene, encoding acetate kinase (AK), by gene disruption and

integrational mutagenesis to reduce acetate formation in propionic acid fermentation was

studied. The partial ack gene of ~750 bp in P. acidipropionici was cloned using a PCR-

based method with degenerate primers and sequenced. The deduced amino acid sequence

had 88% similarity and 76% identity with the amino acid sequence of AK from Bacillus

subtilis and 77% similarity and 57% identity with that from Methanosarcina thermophila.

The linear fragment with an inserted tetracycline resistance cassette in the partial ack

gene and the non-replicative integrational plasmid containing the partial ack fragment

and the tetracycline resistance cassette were constructed and introduced into P.

acidipropionici by electroporation, resulting in two mutants, ACK-Tet and pTAT-ACK-

155

Tet, respectively. Southern hybridization confirmed that the ack gene was disrupted as

the tetracycline resistance gene was detected in the digested chromosomal DNA fragment

of the ack-deleted mutant. The activities of AK in the ACK-Tet and pTAT-ACK-Tet

mutants, as compared to the wild type, were 26% and 43% reduced, respectively. As

compared to that of the wild type (0.13 h-1), the specific growth rate of the ACK-Tet and

pTAT-ACK-Tet mutants decreased to 0.08 h-1 and 0.09 h-1, respectively, indicating AK

had a profound impact on cell growth. As compared to the wild type, both mutants

produced ~13% more propionate and ~14% less acetate from glucose. However,

inactivation of the ack gene alone did not completely eliminate acetate formation due to

the conversion of acetyl phosphate to acetate, suggesting that further genetic

manipulation of the acetate formation pathway in P. acidipropionici may be necessary to

further enhance propionic acid production.

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5.1 Introduction

Propionibacterium acidipropionici (ATCC 4875), a Gram-positive, non-spore

forming, anaerobic bacterium has been extensively studied for use in propionic acid

production from sugars (Blanc and Goma, 1987; Lewis and Yang, 1992; Quesada-Chanto

et al., 1994). Fermentation processes can reuse the byproducts from food manufacturing,

such as cheese whey and corn steep liquor, for propionic acid production. Not only does

this practice benefit the lower cost of food products, but it solves the waste disposal

problem for food manufacturers as well. Propionic acid production via fermentation is,

unfortunately, limited by low final propionic acid concentration, yield, and productivity

(Blanc and Goma, 1987; Boyaval et al., 1994; Lewis and Yang, 1992). A considerable

amount of work, as medium optimization, bioreactor design, and cell immobilization

system, has been performed to enhance the fermentation performance (Blanc and Goma,

1987; Boyaval et al., 1994; Huang et al., 2002; Paik and Glatz, 1994; Suwannakham and

Yang, 2005; Woskow and Glatz, 1991; Yang et al., 1994). However, the improved

propionic acid production has been accompanied by the formation of acetate as a main

byproduct. An enhanced propionic acid yield with a reduced acetate formation will

tremendously lower the final product cost by reducing the recovery cost and make the

fermentation process more feasible for commercial production (Van Hoek et al., 2003).

Although increasing selectivity for propionic acid production (>0.9 g/g) via sophisticated

fermentation processes such as operation under two atmospheres of hydrogen (Thompson

et al., 1984) or with a three-electrode amperometric culture system (Emde and Schink,

157

1990) has been demonstrated, it is of interest to improve propionic acid production by

eliminating acetate formation in propionic acid fermentation via genetic engineering.

To date, no work has been reported in respect to genetic manipulation of

eliminating the acetate formation in P. acidipropionici. Previous studies on mutants with

the deletion of the ack, encoding AK (acetate kinase, EC 2.7.2.1), and the pta, encoding

PTA (phosphotransacetylase, EC 2.3.1.8) suggested that the PTA - AK pathway is

responsible for the acetate secretion in various acid fermentations (Diaz-Ricci et al.,

1991; Kakuda et al., 1994b; Nyström, 1994). In most bacteria, acetyl CoA is usually

converted into acetyl phosphate by PTA, and then to acetate by AK. Therefore,

inactivation of the acetate-forming genes, either pta or ack gene, as a method to eliminate

or reduce the accumulation of acetate in propionic acid fermentation could direct more

carbon flow toward propionic acid production. It would be possible to approach the

theoretical maximum yield of 2 moles of propionic acid per mole of glucose (0.822 g/g)

via the EMP pathway if acetate formation and cell growth could be reduced to minimum.

Genetic engineering of propionibacteria has been studied (Jore et al., 2001;

Kiatpapan et al., 2000; Kaitpapan and Murooka, 2001; 2002; Piao et al., 2004a; b; van

Luijk et al., 2002). The expression vector harboring ALA synthase gene (hemA) from

Rhodobacter sphaeroides was used to transform Propionibacterium freudenreichii to

enhance production of 5-aminolevulinic acid (ALA), a common precursor of hemes,

tetrapyrroles, porphyrins, and vitamin B12 in all living organisms (Kiatpapan and

Murooka, 2001). The enhanced vitamin B12 production of ~1.7 mg/L was obtained from

the genetically engineered P. freudenreichii with the expression of multigenes, an

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exogenous hemA gene and endogenous hemB and cobA genes (Piao et al., 2004b).

Nevertheless, no work has done on the effect of pta or ack on propionic acid

fermentation.

The ack gene has been cloned, sequenced, and characterized from Escherichia

coli (Kakuda et al., 1994a; Matsuyama et al., 1989), Methanosarcina thermophila

(Latimer and Ferry, 1993), Bacillus subtilis (Grundy et al., 1993), and Clostridium

acetobutylicum (Boynton et al., 1996). In order to inactivate the undesired gene on the

host chromosome, two methods have been mainly used. Gene disruption is the first

method whose strategy is to insert an antibiotic resistance cassette, a selection marker,

into the middle of the cloned gene of interest and then this linear fragment will be used to

transform host cells and the original copy of the target gene on the host chromosome will

be replaced with the linear fragment via homologous recombination. The second method

is integrational mutagenesis using a non-replicative integrational plasmid containing the

fragment of the target gene and the antibiotic resistance cassette to transform host cells.

The partial gene fragment in the non-replicative plasmid can recombine with the internal

region of the original target gene on the parental chromosome, resulting in the insertional

inactivation of the target gene. The non-replicative plasmid (pJC4) with the partial pta

gene was constructed and integrated into the homologous region of the original pta gene

on the chromosome of C. acetobutylicum ATCC 824, resulting in a reduction of PTA and

AK activities and acetate production (Green and Bennett, 1998; Green et al., 1996).

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In this study, the partial ack gene from P. acidipropionici was cloned and

sequenced. The ack-deleted mutants, obtained by gene disruption and integrational

mutagenesis, were studied to investigate the impact of the gene inactivation on the

activity of the acetate-forming enzymes, the overall protein expression pattern, the cell

growth, and the kinetics of propionic acid fermentation with the mutants.

5.2 Materials and Methods

5.2.1 Bacterial Strains and Plasmids

All bacterial strains and plasmids used in this work are listed in Table 5.1.

5.2.2 Medium Preparation and Growth Conditions

P. acidipropionici (ATCC 4875), designated as the wild type, was grown

anaerobically at 32°C in a synthetic medium, described in Appendix A.1. The

transformants of P. acidipropionici were grown in the same medium supplemented with

10 µg/mL of tetracycline (Tet). E. coli was grown aerobically at 37°C in Luria-Bertani

(LB) medium, described in Appendix A.2, supplemented with 100 µg/mL ampicillin

(Amp) and 15 µg/mL of tetracycline.

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5.2.3 DNA Preparation

Genomic DNA of P. acidipropionici was isolated by using QIAGEN Genomic

DNA Kit (Qiagen, Valencia, CA) (Appendix D.1). Small-scale plasmid isolation from E.

coli was carried out by using QIAprep MiniPrep Plasmid Purification Kit (Appendix

D.2). QIAquick Gel Extraction Kit was used to purify DNA fragment from gel

(Appendix D.4). Taq DNA polymerase and dNTPs mixture were obtained from

Amersham Biosciences (Piscataway, NJ). Restriction enzymes were ordered from

Amersham and Stratagene (La Jolla, CA). Shrimp alkaline phosphatase (GIBCO/BRL),

T4 DNA ligase, and TOPO TA Cloning Kit were from Invitrogen (Carlsbad, CA).

5.2.4 PCR Amplification

Two degenerate primers (Integrated DNA Technologies, Coralville, IA) were

designed based on the homologous region of the known amino acid sequences of AK

among E. coli, M. thermophila, and B. subtilis. The sequences of the PCR forward and

reverse primers for the partial ack gene were 5’– AAG GAT CCA T(C)C(A)G IGT IGT

ICA T(C)GG IGG –3’ and 5’– AAG GAT CCT CIC CT(A/G) ATI CCI G(C)CI GTA(G)

AA –3’, respectively. The PCR amplification using the P. acidipropionici genomic DNA

as a template and the designed oligonucleotides as primers was conducted in a DNA

engine (MJ Research, Reno, NV) (Appendix D.6). The PCR product of the ack gene with

an expected size of 750 bp was cloned into a PCR vector and then sequenced.

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5.2.5 Gene Disruption: Construction of Disrupted ack

The disrupted ack gene was constructed by inserting a tetracycline resistance

(Tetr) cassette into the middle of the partial ack sequence obtained from P.

acidipropionici. The Tetr cassette was inserted at the XmaIII site whose sticky end was

complementary to that of the NotI, which was the site used to release the Tetr cassette

from the pBEST309 (Itaya, 1992). The pACK-Tet was obtained by a ligation of the Tetr

cassette and the XmaIII-cut plasmid containing the partial ack fragment. The insertion of

the Tetr cassette resulted in 350-bp and 400-bp ack fragments located at each end of the

inserted cassette. Since no commonly used restriction enzyme could release the intact

ACK-Tet sequence from the pACK-Tet and there were two EcoRI sites located at the

ends of the intact ACK-Tet sequence and one EcoRI site located inside the intact, partial

digestion with EcoRI was carried out to knock off the internal EcoRI site and the LAT4-

dE13 plasmid was obtained. The LAT4-dE13 was digested with EcoRI to release the

linear ACK-Tet with a size of ~2.7 kb, which was used for the transformation of P.

acidipropionici.

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5.2.6 Integrational Mutagenesis: Construction of Integrational Plasmid of ack

Figure 5.1 shows how the integrational plasmid was constructed. The partial ack

gene of ~500 bp containing BamHI ends was not directly cloned into pCR®2.1-TOPO®,

which required blunt ends with A’-overhang for ligation. To accomplish this, both ends

of the 500-bp partial ack fragment were filled with dNTPs by Klenow to create blunt

ends, then dephosphorylized by shrimp alkaline phosphatase to remove the phosphate

group at the 5’–end, and finally added with dATPs by Taq DNA polymerase to create A’-

overhang at the two blunt ends. The modified partial ack fragment was cloned into the

pCR®2.1-TOPO® (3.9 kb) (Invitrogen) to obtain the TOPOACK1 (4.4 kb) (Appendix

D.8). The 2.1-kb Tetr cassette was removed from the pDG1515 (Guérout-Fleury et al.,

1995) by a digestion with XbaI and ApaI and then ligated into XbaI-ApaI-digested

TOPOACK1. The resulting integrational plasmid was named as pTAT (6.5 kb) and used

for the transformation of P. acidipropionici to obtain the ack inactivation.

5.2.7 Transformation of P. acidipropionici

The transformation of P. acidipropionici with either the linear fragment of the

disrupted ack gene with the Tetr cassette or the integrational plasmid containing the

partial ack gene and the Tetr cassette was carried out using an Electrocell Manipulator

600 (BTX Inc., San Diego, CA). All manipulations were performed on the bench top

without any special anaerobic conditions, as described in Appendix D.10. Cells,

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cultivated to the stationary phase, were used to inoculate 50 mL of the synthetic medium.

Cells were grown at 32°C to the exponential phase (OD600 ~ 0.8-1.0), harvested, washed

three times with ice-cold sterile distilled water, and then resuspended with 0.5 mL of ice-

cold sterile distilled water. Either 5 µg of the linear fragment (ACK-Tet) or 8 µg of the

integrational plasmid (pTAT) was transferred into a pre-chilled 0.2-cm electroporation

cuvette (Bio-Rad, Hercules, CA) and then 200 µL of the competent cells were added into

the cuvette, mixed well with the DNA, and incubated on ice for 5 min. After a pulse (12.5

kV/cm, 50 µF, 129 Ω), the cells were diluted with 1 mL of the fresh synthetic medium,

transferred into a serum tube containing 5 mL of the synthetic medium, and incubated at

32°C for 9 h. After the incubation, transformants were harvested and then resuspended

with 0.5 mL of the fresh synthetic medium and a proper volume of the suspension was

plated on the synthetic medium agar containing 10 µg/mL Tet and incubated at 32°C for

10-14 days.

5.2.8 Southern Hybridization

The chromosomal DNA of the P. acidipropionici wild type and ack-deleted

mutant were completely digested with BamHI. The digested DNA was separated using

gel electrophoresis and then transferred to a Hybond-N+ nylon membrane (Amersham).

Pre-hybridization of the blotted nylon membrane was performed at 50°C for 1 h. Two

probes were used for the hybridization of the ack and Tetr genes. The ack probe was the

500 bp of the ack coding sequence from P. acidipropionici. The Tetr probe was the 1.9 kb

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of the tetracycline resistance cassette from the pBEST309. Both probes and the DNA-size

marker were labeled with alkaline phosphatase (Amersham). With gentle overnight

shaking at 62°C, the digested DNA was hybridized with the probes and HyperfilmTM

ECL (Amersham) was used for the detection.

5.2.9 Mutant Characterization

The activities of AK and PTA and the overall protein expression pattern of the

ack-deleted mutants (ACK-Tet and pTAT-ACK-Tet mutants) were compared to those of

the wild type. Moreover, fermentation kinetics of the mutants and the wild type were

determined.

5.2.9.1 Preparation of Cell Extract

Cells grown in the synthetic medium (50 mL) at 32°C to the exponential phase

(OD600 ~ 1.8) were harvested, washed, and resuspended in 15 mL of 25 mM Tris/HCl

(pH 7.4). The cell suspension was then sonicated to break cell walls and centrifuged at

10,000 rpm, 4°C for 1 h to remove cell debris. The cell extracts were kept cold on ice

before they were used in the enzyme activity assays. The protein content of the extracts

was determined in triplicate by Bradford protein assay (Bio-Rad) with bovine serum

albumin as the standard (Appendix B.4).

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5.2.9.2 Protein Expression: SDS-PAGE

Gel preparation and SDS-PAGE setup are described in Appendix B.6. All protein

samples with a specific amount (~23 µg each) were loaded into wells and run, with Mini-

PROTEAN® 3 Cell (Bio-Rad), at a constant voltage of 110 V until the tracking dye

reached the gel bottom. The gels were stained with coomassie brilliant blue for 1 h and

then destained with a destaining solution containing 20% methanol and 10% acetic acid.

5.2.9.3 Enzyme Assays

The activities of acetate kinase (AK) and phosphotransacetylase (PTA) were

assayed in duplicate as described in Appendix B.5.

5.2.9.4 Fermentation Kinetics

Batch fermentations of the P. acidipropionici wild type and mutants were

performed in duplicate in a 5-L stirred-tank fermentor (BioFloII, New Brunswick,

Edison, NJ) containing 3 L of the synthetic medium supplemented with 30 g/L glucose

and 10 µg/mL tetracycline as required, at 32°C, pH 6.5, and 100 rpm for agitation. The

anaerobic condition was established by sparging the medium in the fermentor with N2 for

~45 min and then the fermentor headspace was maintained thereafter under 5 psig N2.

The fermentor was inoculated with ~150 mL of a fresh culture grown in serum bottles

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(OD600 ~ 2.0), and liquid samples were withdrawn at regular time intervals. The

concentrations of glucose and acid products, including propionic, acetic, and succinic

acids, were analyzed by high-performance liquid chromatography (HPLC) with a Bio-

Rad HPX-87H (Appendix B.3).

5.3 Results

5.3.1 PCR Amplification and Sequence Analysis

A single DNA fragment with a size of ~750 bp was obtained from the P.

acidipropionici genome by using PCR amplification with two degenerate primers (Figure

5.2). The DNA fragment was cloned into a PCR vector and sequenced. Deposited in

GenBank database under accession number AY936474, the nucleotide sequence data of

the ack fragment showed 749 nucleotides (Figure 5.3). Figure 5.4 shows some important

restriction sites located on the cloned ack sequence. The amino acid sequence deduced

from this partial ack sequence was compared with the amino acid sequences of the AK

from E. coli, M. thermophila, and B. subtilis, respectively, and the alignment showed

conserved regions throughout the cloned sequence (Figure 5.5). The AK sequence

obtained from P. acidipropionici had 88% similarity and 76% identity with the AK

sequence from B. subtilis, 77% similarity and 57% identity with that from M.

thermophila, and 68% similarity and 48% identity with that from E. coli (Table 5.2). This

result demonstrated that the amino acid sequence of the AK from P. acidipropionici had

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higher similarity to those from the Gram-positive bacteria than to that from the Gram-

negative bacterium. The analysis of protein sequence alignment ensured that the obtained

PCR product was from the ack gene in P. acidipropionici.

5.3.2 Transformation of P. acidipropionici

The disrupted ack fragment with the tetracycline resistance cassette and the

integrational plasmid (pTAT) were constructed and used to transform P. acidipropionici

by electroporation to obtain transformants of ACK-Tet and pTAT-ACK-Tet,

respectively. The transformation had an efficiency of 5-10 colonies per µg DNA.

5.3.3 Disruption of ack

The genomic DNA of the ack-deleted-Tetr mutant (ACK-Tet) was isolated and

analyzed by southern blotting to confirm the ack disruption. When the ack-disrupted

fragment was introduced into P. acidipropionici, two types of mutants could occur: 1) the

ack original copy on the host chromosome is replaced with the ack-disrupted fragment

via homologous recombination and 2) the ack-disrupted fragment is randomly inserted

into the genome. For this study, the ack-disrupted mutant obtained from homologous

recombination was of interest, which was confirmed with southern hybridization

discussed later.

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5.3.4 Integrational Mutagenesis (pTAT)

If the ack fragment of the non-replicative plasmid was not integrated into the host

chromosome via homologous recombination, no transformants with tetracycline

resistance could be obtained since the Tetr gene would not be expressed in the

transformed cells. Therefore, pTAT must have been integrated into the host chromosome

by homologous recombination in the transformants and the original ack gene on the

chromosome should have been disrupted.

5.3.5 Southern Hybridization

As shown in Figure 5.6, the ack probe (~500 bp) hybridized to the BamHI-

digested chromosomal DNA fragment, with a size of ~850 bp, of the wild type and to the

BamHI-digested chromosomal DNA fragment, with a size of ~2.8 kb (the fragment of

850 bp + the 1.9 kb of the Tetr gene), of the ack-deleted mutant. With the Tetr probe, the

hybridization in the wild type was negative since no Tetr gene existed in the genome of

the wild type while the positive hybridization with the size of 2.8 kb was observed in the

mutant. The 2.8-kb band suggested that the chromosomal ack gene in the mutant was

replaced with the disrupted ack gene fragment carrying the Tetr gene and the partial ack

fragment.

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5.3.6 Overall Protein Expression Pattern

SDS-PAGE was used to evaluate the overall pattern of protein expression in the

wild type and the mutants with the inactivation of the ack gene. The overall protein

expression patterns of both mutants were changed. As can be seen in Figure 5.7, the

intensity of the protein band with the size of ~74 kDa was lower in both ACK-Tet (Lane

2) and pTAT-ACK-Tet (Lane 3) as compared to that of the wild type (Lane 1). The

protein band might represent the band of several proteins, possibly including AK, with

that particular molecular weight. With the absence of the AK, the band intensity could be

reduced.

5.3.7 Acid-Forming Enzyme Activities

The activities of AK and PTA were examined and compared between the mutants

and the wild type. It was expected that the activity of AK in both mutants would not be

detected; however, as shown in Figure 5.8, the activities of AK in the ACK-Tet and

pTAT-ACK-Tet mutants decreased by 26% and 43%, respectively. It is possible that the

detected activity was from other enzymes, which could consume the same substrate,

acetate, as AK does. It was also found that both mutants had a slight decrease in the PTA

activity. This could be related to a deficient conversion of acetyl phosphate to acetate

catalyzed by AK, since AK can no longer be expressed.

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5.3.8 Fermentation Kinetics

Figure 5.9 shows the kinetics of batch fermentations by the strains of wild type

(A), ACK-Tet (B), and pTAT-ACK-Tet (C). As shown in Table 5.3, the specific growth

rates of the mutants were lower (0.08 h-1 for ACK-Tet, 0.09 h-1 for pTAT-ACK-Tet) than

that of the wild type (0.13 h-1). This indicates that the ack deletion had a significant

impact on cell growth. The propionate yield increased from 0.404 g/g by the wild type to

0.452 g/g by ACK-Tet and 0.459 g/g by pTAT-ACK-Tet while the acetate yield

decreased from 0.115 g/g by the wild type to 0.099 g/g by ACK-Tet and 0.098 g/g by

pTAT-ACK-Tet (Table 5.3). As a result, the P/A ratio increased from 3.5 by the wild

type to 4.6 by ACK-Tet and 4.7 by pTAT-ACK-Tet. This indicates that the disruption of

the ack gene may play a limited role in the pattern of the fermentation end-product

formation.

5.4 Discussion

5.4.1 Genetic Manipulation of P. acidipropionici

Due to the lack of the known ack sequence of P. acidipropionici, PCR-based

amplification of the partial ack fragment using degenerate primers was used in order to

clone the partial ack gene in P. acidipropionici ATCC 4875. The obtained partial ack

gene was sequenced and the deduced amino acid sequence had high similarity and

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identity with the AK sequences from B. subtilis (88% and 76%) and M. thermophila

(77% and 57%) (Table 5.2). In order to inactivate the ack gene on the host chromosome

via homologous recombination, the methods of gene disruption using the linear fragment

containing the Tetr cassette in the partial ack gene and integrational mutagenesis using

the non-replicative integrational plasmid containing the partial ack gene and the Tetr

cassette were applied. Although many studies have focused on genetic manipulation on

propionibacteria (Jore et al., 2001; Kiatpapan et al., 2000; Kiatpapan and Murooka, 2001;

Piao et al., 2004b), none of them has worked on the improvement of propionic acid

production. In this study, genetically manipulated mutants of P. acidipropionici were

successfully created for propionic acid fermentation with improved propionic acid yield.

This work was the first one demonstrating the feasibility and patented benefit to disrupt

genes in the acetate forming pathway in P. acidipropionici.

PCR amplification is a key approach to obtain the partial ack gene from P.

acidipropionici. For successful PCR amplification, it is critical to obtain the optimal

condition of the ionic strength of the PCR reaction mixture and the primer annealing

temperature. In this work, the optimal MgCl2 concentration of 2 mM and the optimal

annealing temperature of 42°C were obtained. There was no amplification observed when

the annealing temperature was higher than 52°C and when very high or low MgCl2

concentrations were applied (data not shown).

Electroporation protocols have been developed in order to efficiently introduce

the desired DNA or plasmids into propionibacteria. The stage in which the competent

cells harvested and the electroporation gene pulser parameters are two important

172

variables that were critical to the transformation efficiency. As used in this study, cells

grown until the exponential phase were generally preferred for electroporation of

propionibacteria (Jore et al., 2001; Kiatpapan and Murooka, 2001). Several

electroporation gene pulser parameters (electric field strength, capacitance, and

resistance) including 6.0 kV/cm, 25 µF, and 129 Ω (Kiatpapan et al., 2000; Kiatpapan

and Murooka, 2001), 12.5 kV/cm, 25 µF, and 480 Ω (Piao et al., 2004b), and 20 kV/cm,

25 µF, and 200 Ω (Jore et al., 2001) were conducted to obtain the optimal conditions for

high transformation efficiency of propionibacteria. In this study, various sets of

electroporation parameters were tested. It was found that the field strength had a negative

impact on the survival of cells. As the field strength increased from 5.0 kV/cm to 12.5

kV/cm, less than 50% of cells could survive (data not shown). This finding was

consistent with the study by Kiatpapan et al., 2000. The use of high field strength or high

resistance or both decreased the transformation efficiency since the increased time

constant could result in less than 30% cell survival (Kiatpapan et al., 2000). As applied in

this work, the optimal condition was 12.5 kV/cm, 50 µF, and 129 Ω for the

transformation of P. acidipropionici.

5.4.2 Overall Protein Expression Pattern

The result from the SDS-PAGE showed a significant difference in the overall

protein expression patterns of the ack-deleted mutants and the wild type (Figure 5.7). It

was observed that the band of the ~74-kDa protein had lower intensity in both mutants as

173

compared to the wild type. It could be possible that there might be several proteins,

probably including AK, with this particular molecular weight. The absence of the AK, as

a result of the ack inactivation, might result in the decrease in the intensity of this specific

band as seen in Figure 5.7. However, it could not be identified in this work since no

proteomic data of P. acidipropionici are available.

5.4.3 Effect of ack Gene Inactivation

Gene inactivation has been used in studying mutagenesis in E. coli (Chan, 1993),

the analysis of the function of genes of interest in Thermococcus kodakaraensis KOD1

(Sato et al., 2003), and studying molecular genetics in C. acetobutylicum (Harris et al.,

2000). In addition, it was applied to disrupt metabolic pathways leading to by-product,

acetate and butyrate, formation in C. acetobutylicum ATCC 824 (Green et al., 1996).

However, it has not been used for the disruption of the acetate-forming genes in P.

acidipropionici. In this study, the ack gene in P. acidipropionici was inactivated via

homologous recombination. The activity of AK in both ack-deleted mutants (ACK-Tet

and pTAT-ACK-Tet) was reduced but not completely diminished (Figure 5.8). The

detected AK activity might probably result from the activity of other enzymes that could

consume the same substrate, acetate, as AK does. It is possible that the deletion of the ack

gene had caused the slightly lower PTA activity in the mutants. This might be to decrease

the conversion of acetyl CoA to acetyl phosphate, an intermediate compound that cannot

174

be accumulated to a high concentration inside the cells, since AK can no longer be

expressed and catalyze the reaction of acetyl phosphate to acetate.

Fermentation kinetics of both mutants showed that the propionate yield increased

~13% while the acetate yield decreased ~14% as compared to those of the wild type

(Table 5.3). It would be possible that the inactivation of the ack gene in both mutants

caused the deficient conversion of acetate from acetyl phosphate catalyzed by AK, which

would result in more pyruvate forced to flow toward the propionate formation pathway.

The slight decrease in the acetate yield indicated that AK might play a limited role in

controlling the acetate formation through the PTA - AK pathway since the acetate

accumulation could occur through the PTA - AK pathway with or without the AK

activity.

In the ack deletion mutant, acetyl phosphate might be formed as usual as long as

PTA, catalyzing the conversion of acetyl CoA to acetyl phosphate, functions normally.

Acetyl phosphate is an intermediate that usually is allowed to accumulate to a high

concentration level in the cells. Several ways could lead to acetate formation from the

accumulated acetyl phosphate. First of all, acetyl phosphate, a high-energy compound,

could be oxidized into acetate by transferring a phosphoryl group to ADP yielding ATP.

Secondly, acetyl phosphate could function as a phosphoryl-group donor to Enzyme I of

the bacterial phosphotransferase system, leading to acetate formation (Fox et al., 1986).

More recent evidences supported that acetyl phosphate could also function as a global

regulator in the metabolic network (McCleary et al., 1993). Acetyl phosphate can be used

in the phosphorylation of a group of proteins regulating cell’s responses to environmental

175

stimulations. The global phosphorylation could lead to the acetate formation from acetyl

phosphate (Wanner and Willmes-Riesenberg, 1992) without the activity of acetate kinase.

All of these possibilities indicate that acetate could be formed once acetyl phosphate was

accumulated in the cells. These results suggested that pta or pta and ack double deletion

mutant of P. acidipropionici could be of interest for studying the effect of gene

inactivation on acetate formation in propionic acid fermentation.

5.5 Conclusion

Genetic manipulation of P. acidipropionici for enhanced propionic acid

fermentation was first attained in this study. The developed mutant strains of P.

acidipropionici with the ack gene inactivation achieved improved propionic acid yield

with reduced acetate yield. This development could be an alternative means to lower the

downstream processing cost that is mainly affected by the presence of the byproduct,

acetate, in propionic acid fermentation.

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Chan E-C. 1993. Disruption of targeted gene in bacterial chromosome by using a

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180

a TOPOACK1 was constructed by the insertion of 0.5-kb ack fragment into pCR®2.1-TOPO®. b The construction was described in the Materials and Methods. Note: Abbreviations: ack -, acetate kinase gene deleted; Apr, ampicillin resistant; Kanr, kanamycin resistant; Tetr, tetracycline resistant; recA1, homologous recombination abolished. Table 5.1 Strains and plasmids.

Strain/Plasmid Relevant Characteristics Source/Reference

Strains

P. acidipropionici

ATCC 4875

wild type ATCC

ACK-Tet ack -, Tetr P. acidipropionici ack mutant

by Gene Disruption; this study

pTAT-ACK-Tet ack -, Tetr P. acidipropionici ack mutant

by Integrational Mutagenesis;

this study

E.coli, INVαF’ recA1, Apr Invitrogen

Plasmids

pBEST309 Tetr cassette Itaya, 1992 (Obtained from Bacillus Center,

The Ohio State University)

pDG1515 Tetr cassette Guérout-Fleury et al., 1995 (Obtained from Bacillus Center,

The Ohio State University)

pACK-Tet partial ack + inserted Tetr This study

LAT4-dE13 partial ack + inserted Tetr

without the inside EcoRI site

This study

pCR®2.1-TOPO® Cloning vector, Apr, Kanr Invitrogen

TOPOACK1a partial ack, Apr, Kanr This study

pTATb partial ack, Apr, Kanr, Tetr Non-replicative integrational

plasmid; this study

181

Bacteria Similarity (%) Identity (%)

Bacillus subtilis 88 76

Methanosarcina thermophila 77 57

Escherichia coli 68 48

Table 5.2 Comparison of amino acid sequences from ack gene of P. acidipropionici to the corresponding sequences from other microorganisms.

182

aBased on the assumption that 46.2% of the biomass was carbon. The amount of CO2 produced was estimated based on the assumption that one mole of CO2 was co-produced with each mole of acetic acid in the fermentation. Table 5.3 Comparison of fermentation kinetics from batch fermentations by P. acidipropionici wild type, ACK-Tet mutant, and pTAT-ACK-Tet mutant.

Wild Type ACK-Tet pTAT-ACK-Tet

Max. propionic acid conc. (g/L) 15.3 ± 0.2 16.5 ± 1.0 17.1 ± 0.4

Product yield (g/g)

Propionic acid 0.404 ± 0.007 0.452 ± 0.001 0.459 ± 0.001

Acetic acid 0.115 ± 0.002 0.099 ± 0.003 0.098 ± 0.003

Succinic acid 0.091 ± 0.002 0.063 ± 0.006 0.060 ± 0.006

Propionate: acetate ratio (g/g) 3.5 ± 0.1 4.6 ± 0.1 4.7 ± 0.2

Productivity (g/L/h) 0.24 ± 0.01 0.19 ± 0.04 0.19 ± 0.04

Specific growth rate (h-1) 0.13 ± 0.02 0.08 ± 0.01 0.09 ± 0.01

Total carbon recovery (%)a 87.5 ± 1.3 84.5 ± 1.3 84.8 ± 1.5

182

183

Figure 5.1 Construction of an integrational plasmid, pTAT, with a fragment of 0.5-kb ack gene cloned from P. acidipropionici. The arrows indicate the directions of a particular gene. Restriction sites of importance were shown. Note: Abbreviations: ack, a partial ack gene; f1 ori, f1 filamentous phage origin of replication with helper phage; Kanr, kanamycin resistance gene; Apr, ampicillin resistance gene; Tetr, tetracycline resistance gene; ColE1, compatibility group origin of replication in E. coli.

pCR®2.1-TOPO®

3.9 kb

Kanr

Partial ack 0.5 kb

Ampr

f1 ori

EcoRI EcoRI

ColE1

Tetr

f1 ori Kanr

Ampr

ColE1

ack

pTAT 6.5 kb

EcoRI EcoRI

XbaI

ApaI

XbaI and ApaI Digestion

ack

TOPOACK1 4.4 kb

ColE1

Ampr Kanr

f1 ori

EcoRI EcoRI

pDG1515

Tetr (2.1kb)

XbaI ApaI

184

Figure 5.2 PCR product of partial ack gene.

1 kb 700 bp 500 bp 200 bp

100 bp

PCR product ~750 bp

185

File : ack gene Range 1 – 749 Mode : Normal Codon Table : Universal 9 18 27 36 45 54 5' TGG ATC CAT AGG GTG GTG CAC GGG GAC GAA ATT TTC AAT GAT TCT GCT GTC GTC W I H R V V H G D E I F N D S A V V

63 72 81 90 99 108 AAT GAC CAG GTG CTC GCG CAA ATT GAA GAT TTG GCG GAA CTC GCG CCG CTT CAT N D Q V L A Q I E D L A E L A P L H

117 126 135 144 153 162 AAC CGG GCG AAC GCA ACG GGG ATC CGG GCG TTC CGT GCC GTG CTG CCG GAT GTG N R A N A T G I R A F R A V L P D V

171 180 189 198 207 216 GTT CAA GTG GCC GTG TTT GAT ACC GCT TTC CAC CAA ACG ATG CCG GAA AGT GCT V Q V A V F D T A F H Q T M P E S A

225 234 243 252 261 270 TTC TTA TAC AGC CTC CCC TAT GCA TAC TAC GAA AAA TAC CGG ATC CGC AAA TAC F L Y S L P Y A Y Y E K Y R I R K Y

279 288 297 306 315 324 GGG TTT CAT GGC ACT TCC CAT AAA TAT GTG GCG ATG CGT GCC GCT GAG CTG CTC G F H G T S H K Y V A M R A A E L L

333 342 351 360 369 378 GGC AGG CCA ATT GAA CAG CTG CGC CTG ATT TCA TGC CAT TTG GGC AAC GGG GCA G R P I E Q L R L I S C H L G N G A

387 396 405 414 423 432 AGC ATT GCG GCG ATC CAG GGC GGC CGG TCA ATC GAT ACG TCC ATG GGC TTT ACG S I A A I Q G G R S I D T S M G F T

441 450 459 468 477 486 CCA TTG GCC GGC GTG ACG ATG GGC ACG CGC TCC GGC AAT ATC GAC CCC GCA CTG P L A G V T M G T R S G N I D P A L

495 504 513 522 531 540 ATC CCG TTT ATT ATG GAG AAG ACA GGC AAA ACG GCA GAA GAA GTG CTC GAA GTG I P F I M E K T G K T A E E V L E V

549 558 567 576 585 594 TTA AAC AAA GAA TCC GGG CTT CTC GGC ATT TCG GGC GTT TCC AGC GAT TTG CGC L N K E S G L L G I S G V S S D L R

603 612 621 630 639 648 GAT ATC CAG GTG GCG GCG GAA CTC GAG CGG AAC AAG CGG GCT GAA CTG GCG CTT D I Q V A A E L E R N K R A E L A L

657 666 675 684 693 702 GAC ATT TTT GCA AGC CGC ATC CAT AAA TAC ATC GGT TCG TAT GCG GCA AAA ATG D I F A S R I H K Y I G S Y A A K M

711 720 729 738 747 GCC AGC GTC GAT GCG ATT ATT TTC ACC GCC GGC ATT GGC GAG GAT CC 3' A S V D A I I F T A G I G E D P

Figure 5.3 DNA and amino acid sequences of partial ack gene from P. acidipropionici.

186

Figure 5.4 Restriction enzyme map of partial ack gene from P. acidipropionici.

EagI = XmaIII

187

E. coli 1 MSSKLVLVLNCGSSSLKFAIIDAVNGEEYLSGLAECFHLPEARIKWKMDGNKQEAALGAG 60 M. thermophila 1 ---MKILVINCGSSSLKYQLIESKDGNVLAKGLAERIGINDSLLTHNANGEKIKIKKDM- 56 B. subtilis 1 --MSKIIAINAGSSSLKFQLFEMPSETVLTKGLVERIGIADSVFTISVNGEKNTEVTDI- 57 P. acidipropionici 1 ------------------------------------------------------------ 1 E. coli 61 AAHSEALNFIVNTILAQKP---ELSAQLTAIGHRIVHGGEKYTSSVVIDESVIQGIKDAA 117 M. thermophila 57 KDHKDAIKLVLDALVNSDYGVIKDMGEIDAVGHRVVHGGEYFTSSVLITDDVLKAITDCI 116 B. subtilis 58 PDHAVAVKMLLNKLT--EFGIIKDLNEIDGIGHRVVHGGEKFSDSVLLTDETIKEIEDIS 115 P. acidipropionici 1 --------------------------------HRVVHGDEIFNDSAVVNDQVLAQIEDLA 28 E. coli 118 SFAPLHNPAHLIGIEEALKSFPQLKDKNVAVFDTAFHQTMPEESYLYALPYNLYKEHGIR 177 M. thermophila 117 ELAPLHNPANIEGIKACYQIMPDVP--MVAVFDTAFHQTMPDYAYLYPIPYEYYTKYKYR 174 B. subtilis 116 ELAPLHNPANIVGIKAFKEVLPNVP--AVAVFDTAFHQTMPEQSYLYSLPYEYYEKFGIR 173 P. acidipropionici 29 ELAPLHNRANATGIRAFRAVLPDVV--QVAVFDTAFHQTMPESAFLYSLPYAYYEKYRIR 86 E. coli 178 RYGAHGTSHFYVTQEAAKMLNKPVEELNIITCHLGNGGSVSAIRNGKCVDTSMGLTPLEG 237 M. thermophila 175 KYGFHGTSHKYVSQRAAEILNKPIESLKIITCHLGNGSSIAAVKNGKSIDTSMGFTPLEG 234 B. subtilis 174 KYGFHGTSHKYVTERAAELLGRPLKDLRLISCHLGNGASIAAVEGGKSIDTSMGFTPLAG 233 P. acidipropionici 87 KYGFHGTSHKYVAMRAAELLGRPIEQLRLISCHLGNGASIAAIQGGRSIDTSMGFTPLAG 146 E. coli 238 LVMGTRSGDIDPAIIFHLHDTLGMSVDAINKLLTKESGLLGLTEVTSDCRYV--EDNYAT 295 M. thermophila 235 LAMGTRCGSIDPSIISYLMEKENISAEEVVNILNKKSGVYGISGISSDFRDLEDAAFKNG 294 B. subtilis 234 VAMGTRSGNIDPALIPYIMEKTGQTADEVLNTLNKKSGLLGISGFSSDLRDI-VEATKEG 292 P. acidipropionici 147 VTMGTRSGNIDPALIPFIMEKTGKTAEEVLEVLNKESGLLGISGVSSDLRDIQVAAELER 205 E. coli 296 KEDAKRAMDVYCHRLAKYIGAYTALMDGRLDAVVFTGGIGENAAMVRELSLGKLGVLGFE 355 M. thermophila 295 DKRAQLALNVFAYRVKT-IGSYAAAMGG-VDVIVFTAGIGENGPEIREFILDGLEFLGFK 352 B. subtilis 293 NERAETALEVFASRIHKYIGSYAARMSG-VDAIIFTAGIGENSVEVRERVLRGLEFMGVY 351 P. acidipropionici 206 NKRAELALDIFASRIHKYIGSYAAKMAS-VDAIIFTAGIGED------------------ 246 E. coli 356 VDHERNLAARFGKSGFIN--KEGTRPAVVIPTNEELVIAQDASRLTA----------- 400 M. thermophila 353 LDKEKNKVR--GEEAIISTAPDAKVRVFVIPTNEELAIARETKEIVETEVKLRSSIPV 408 B. subtilis 352 WDPALNNVR--GEEAFIS-YPHSPVKVMIIPTDEEVMIARDVVRLAK----------- 395 P. acidipropionici 246 ---------------------------------------------------------- 246

Figure 5.5 Alignment of amino acid sequences of AK from E. coli, M. thermophila, B. subtilis, and P. acidipropionici.

188

Figure 5.6 Southern blots of mutant ACK-Tet. A: 0.5-kb ack fragment as a probe; B: 1.9-kb Tetr as a probe. Lane 1: ack probe; Lane 2: wild type with a 0.85-kb DNA and without Tetr; Lane 3: ack-deleted mutant (ACK-Tet) with 2.8-kb fragment of 0.85-kb DNA and Tetr; Lane 4: Tetr probe.

B

A

Probe 1 (0.5 kb)

ack

2.8 kb 2.8 kb Probe 2(1.9 kb)

1 2 3 4 1 2 3 4

189

Figure 5.7 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici. Lane 1: wild type; Lane 2: ACK-Tet mutant; Lane 3: pTAT-ACK-Tet mutant; Lane 4: molecular weight marker.

1 2 3 4~ 74 kDa

97 66

45

30 20.1 14.4

190

Figure 5.8 Relative activities of AK and PTA in ACK-Tet and pTAT-ACK-Tet mutants as compared to the wild type. The activities of each enzyme in the mutants are represented as the percentage of the specific activities of the enzyme in the P. acidipropionici wild type.

0

20

40

60

80

100

120

140

160

AK PTA

Rel

ativ

e A

ctiv

ity (%

)

ACK-TetpTAT-ACK-Tet

191

Figure 5.9 Fermentations by P. acidipropionici. A: Wild type; B: ACK-Tet mutant; C: pTAT-ACK-Tet mutant.

0

5

10

15

20

25

30

35

40

0 20 40 60 80

Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

6

7

8

9

OD

Glucose

Succinate

Acetate

Propionate

OD

A

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100 120

Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

6

OD

Glucose

Succinate

Acetate

Propionate

OD

B

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100 120

Time (h)

Con

cent

ratio

n (g

/L)

0

1

2

3

4

5

6

7O

D

Glucose

Succinate

Acetate

Propionate

OD

C

192

CHAPTER 6

CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions

This research demonstrated the advantages of using the immobilized-cell

bioreactor FBB for propionic acid fermentation, the possibility of enhanced propionic

acid production with decreased by-product formations using a carbon source with a

reduced oxidation state, and the potential applications of gene inactivation in the acetate

formation pathway for further enhanced propionic acid production via fermentation by

Propionibacterium acidipropionici. The important results and conclusions obtained in

this study are summarized below.

6.1.1 Fermentation Kinetics

• The highest propionic acid concentration of 72 g/L and a high propionic acid

yield of up to 0.65 g/g with reduced formation of byproducts (acetate and

succinate) were achieved in the fermentation by P. acidipropionici immobilized

in a fibrous-bed bioreactor using glucose as the substrate.

193

• Free-cell fermentation using sorbitol as a carbon source improved propionic acid

production including productivity, yield, final concentration, and propionate:

acetate (P/A) molar ratio with reduced formation of byproducts (acetate and

succinate).

• Free-cell fermentation using gluconate produced propionic acid as a main product

and succinic acid, instead of acetic acid, as a key byproduct, resulting in a

significantly high P/A molar ratio. Moreover, acetate accumulation was not

observed in the fermentation by the adapted mutant from the FBB using

gluconate.

• Free-cell fermentations using xylose and glucose as carbon sources showed a

similar pattern of fermentation end-product compositions.

6.1.2 Fibrous-Bed Bioreactor

• The fibrous-bed bioreactor (FBB) could maintain a high density of active cells

(>45 g/L cell biomass and >70% cell viability), leading to the enhanced

productivity that was several-fold higher than that from a conventional free-cell

fermentation.

• The FBB provided an effective means to obtain a metabolically advantageous

mutant with improved fermentation capability.

194

• Distinct physiological characteristics have been developed for cells immobilized

in the FBB operated at the fed-batch mode. This could not be achieved in

conventional free-cell fermentations.

• The adapted mutant from the FBB had ~106% higher propionate tolerance as

compared to the wild type.

• In the adapted culture, the propionate-forming enzymes had higher activities and

were less sensitive to propionate inhibition while a significant decrease in activity

of the succinate-forming enzyme was observed.

• The adapted mutant had more longer-chain saturated fatty acids and less

unsaturated fatty acids in the cell membrane, which might have contributed to the

increased propionate tolerance.

• The adapted cell experienced a striking change in its morphology with a three-

fold increase in its length and a ~24% decrease in its diameter, resulting in a

~10% increase in its specific surface area. This might have contributed to more

efficient transports of substrate and metabolite across the cell membrane.

6.1.3 Metabolic Engineering

• Pathways of sorbitol and gluconate utilizations by P. acidipropionici and

stoichiometric equations of sugar utilization were proposed based on the batch

fermentation data.

195

• Batch free-cell fermentations with various carbon sources with different oxidation

states showed different patterns of fermentation product profiles.

• The metabolic shift toward propionate formation pathway was observed when

sorbitol was used as the substrate due to its high NADH availability for the redox

balance.

• A shift in the production of the main byproduct from acetate to succinate was

found in the fermentation with gluconate as the carbon source.

• The partial ack gene, encoding acetate kinase (AK) that is one of key acetate-

forming enzymes in P. acidipropionici, was cloned and sequenced. High degrees

of similarity and identity were found when the deduced amino acid sequence of

AK from P. acidipropionici was compared with the known amino acid sequences

from other microorganisms available in the database.

• Gene inactivation via gene disruption and integrational mutagenesis was applied

to develop the ack-deleted mutants. The ack inactivation in the mutants showed a

profound impact on cell growth rate. As compared to the wild type, a slight

increase in propionic acid yield and a slight decrease in acetic acid yield were

obtained from the ack-deleted mutants.

• Further molecular biology studies of P. acidipropionici and development of

mutants for enhanced propionic acid fermentations could be achieved by applying

genetic engineering techniques and gene inactivation methods developed in this

research.

196

6.2 Recommendations

This research used process engineering, metabolic engineering, and genetic

engineering methods to improve the bioprocess for propionic acid production. However,

many problems still remain unsolved and need to be studied in depth to provide the

fundamental understanding of the process nature. Two major areas are suggested for

future study.

6.2.1 Propionic Acid Fermentation by P. acidipropionici

• Several carbon sources, such as sorbitol and gluconate, can enhance propionic

acid production with reduced formation of byproducts; however, the raw material

cost would be much higher than the use of the commonly used substrate such as

glucose. Therefore, a fermentation process using microorganisms such as

Zymomonas mobilis for production of sorbitol and gluconate from fructose and

glucose, respectively or a process with the enzymatic reaction for production of

these two carbon sources by glucose-fructose oxidoreductase enzyme could make

propionic acid fermentation by P. acidipropionici with the use of sorbitol and

gluconate more feasible for commercial production of propionic acid.

197

• In this study, SDS-PAGE electrophoresis revealed significant difference in the

overall protein expression patterns between the adapted mutant and the wild type,

cells grown in carbon sources with different oxidation states, and the ack-deleted

mutants and the wild type. Two-dimensional electrophoresis could be an effective

tool for further analysis that would provide clearer observations, leading to a

better understanding of proteomics and facilitating further metabolic engineering

of P. acidipropionici.

• The immobilized-cell fermentation using the FBB could be applied for the ack-

deleted mutants in order to enhance propionate tolerance of these mutants.

6.2.2 Metabolic Engineering and Genetic Engineering of P. acidipropionici

• The stoichiometric analysis in this study was based on the simplified pathway.

Many intermediates, such as acetyl CoA, succinyl CoA, methylmalonyl CoA, and

propionyl CoA, were not included in the analysis. Data for intracellular

intermediates should be collected for an accurate description of the metabolic flux

in the fermentation pathway and a justification of assumptions used in the

analysis. Furthermore, activities of some key enzymes in the metabolic pathway

of P. acidipropionici such as pyruvate kinase and pyruvate dehydrogenase

complex should be measured in order to better understand the mechanisms of

carbon and electron flows in the complex metabolic network and identify the rate-

limiting step for propionic acid fermentation.

198

• Since the acetate formation could not be completely eliminated by the inactivation

of the ack gene, the inactivation of pta gene, encoding an upstream acetate-

forming enzyme, or both pta and ack genes is a better means to eliminate the

acetate formation. Further study on construction and characterization of pta- or

pta-ack-deleted mutants is thus recommended.

• Due to the reaction catalyzed by CoA transferase was the rate-limiting step in the

propionate formation pathway, the overexpression of CoA transferase is thus

recommended for improving propionic acid production.

• Since NADH availability is the key factor influencing propionic acid production

in P. acidipropionici, overexpression of genes involving the cofactor

regeneration, such as NAD+-dependent formate dehydrogenase, to increase the

availability of NADH in the metabolic network could improve propionic acid

production and is thus recommended for future study. The NAD+-dependent

formate dehydrogenase (FDH1), encoded by fdh1 gene from Candida boidinii,

catalyzes a conversion of formate to CO2 with a conversion of NAD+ to NADH.

In this preliminary work, the pSSF1, the expression vector containing fdh1 gene

was constructed and used to transform P. acidipropionici. Several types of

electroporation buffers, such as sterile distilled water, 1 mM HEPES, and 0.5 M

sucrose with 1 mM potassium acetate (pH 5.5), and several electroporation

parameters (12.5 kV/cm, 25 or 50 µF, 200 or 300 Ω) were used; however, no

transformants were obtained. Future study on optimizing conditions to obtain the

transformants is thus recommended.

199

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APPENDIX A

MEDIUM COMPOSITIONS

214

A.1 Medium Compositions for Propionibacterium acidipropionici

The synthetic medium contained per liter 10 g yeast extract, 5 g trypticase, 0.25 g

K2HPO4, and 0.05 g MnSO4. The pH of the medium was adjusted to 6.5.

A.2 Medium Compositions for Escherichia coli

The Luria-Bertani (LB) medium contained per liter 10 g tryptone, 10 g NaCl, and

5 g yeast extract. The pH of the medium was adjusted to 7.0.

215

APPENDIX B

ANALYTICAL METHODS

216

B.1 Cell Concentration

Optical Density (OD) was used to describe cell concentration. It was measured at

600 nm with a spectrophotometer (Sequoia-turner, Model 340). The value of OD was

proportional to the cell dry weight when the value was less than 0.6. One unit of OD600

was equivalent to 0.435 g/L of cell dry weight. To obtain the dry cell weight, cells in a

known volume of fermentation broth were harvested and washed three times with

distilled water and finally the cell suspension was dried at 105°C overnight.

B.2 Cell Viability

Cell viability assay followed the TTC method previously described by Glenner

(1977). 2,3,5-triphenyl-2H-tetrazolium chloride (final concentration: 1 g/L) was added to

the cell suspension and incubated at room temperature for 30 min for color development.

After centrifugation at 10,000 rpm for 10 min, the cell pellets were collected and then

suspended in methanol to extract the pink color. The supernatant (methanol extract) was

collected after centrifugation for 5 min. The absorbance of the methanol extract at 485

nm, which is proportional to the viable cell number, was measured with a

spectrophotometer (Shimadzu, UV-1601) using pure methanol as blank. The cell viability

(%) is reported with cells actively growing in serum bottles and harvested in the

exponential phase as the control with 100% viability.

217

B.3 High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) was used to analyze substrates,

mainly sugars, and acid products (mainly, propionic, acetic, succinic, and pyruvic acids)

present in the fermentation broth. Samples from the fermentation broth were centrifuged

and diluted with distilled water. For the sample dilution, the range of 6 to 20 times was

used depending on concentrations of the analyzed components. The HPLC system

(Shimadzu) consisted of a controller, a pump (LC-10Ai), an automatic injector (SIL-

10Ai), a column oven (CTO-10A), a refractive index detector (RID-10A), a Bio-Rad

Aminex HPX-87H organic acid analysis column (ion exclusion organic acid column;

300mm×7.8mm), and a computer with data analysis software (Shimadzu version 4.2).

The system was operated at 45°C using 0.01 N H2SO4 at 0.6 mL/min as the eluent. 15 µL

of each diluted particle-free sample were injected and the running time was 25 min. The

RI detector (Shimadzu-RID-10A) was set at the range of 200 to detect the organic

compounds. Peak height was used to calculate a concentration of each component based

on the height of 2 g/L of the component in the standard mixture. The standard and sample

HPLC chromatograms of propionic acid fermentation by P. acidipropionici using glucose

as the substrate are shown below.

218

Figure B.1 The HPLC chromatogram for standard containing glucose, succinic acid, lactic acid, acetic acid, and propionic acid.

219

Figure B.2 The sample HPLC chromatogram of propionic acid fermentation by P. acidipropionici using glucose as the substrate.

220

B.4 Preparation of Cell Extracts and Protein Assay

Cells grown in the synthetic medium (50 mL) at 32°C to the exponential phase

(OD600 ~ 1.8) were harvested, washed, and resuspended in 15 mL of 25 mM Tris/HCl

(pH 7.4). The cell suspension was then sonicated on ice (Cell Disruptor 350, Bransom

Sonic Power) to break cell walls for ~12 min, with 2 min interval between each 1.5 min

of ultrasonic shock. The suspension was observed under microscope to check the

complete cell disruption and then centrifuged at 10,000 rpm, 4°C for 1 h to remove cell

debris. The cell extracts were kept cold on ice before they were used in the enzyme

activity assays. The protein content of the extracts was determined in triplicate by

Bradford protein assay (Bio-Rad) with bovine serum albumin (BSA) as the standard.

Standard BSA with a concentration range from 0.05 to 0.5 mg/mL was used to

obtain the standard curve. 10 µL of standard BSA and sample were added into the 96-

well microplate followed by an addition of 200 µL of 5X-diluted dye reagent. The

absorbance was measured at 595 nm (SpectraMax 250) after 15-min incubation at 25°C.

The protein concentration of samples was determined based on the standard curve. Figure

B.3 shows a typical protein (BSA) standard curve.

221

Figure B.3 The standard curve of protein assay using bovine serum albumin. B.5 Enzyme Assays

Phosphotransacetylase was assayed by the method of Andersch et al. (1983). The

assay solution (final total volume: 200 µL) contained: 0.1 M potassium phosphate buffer

(pH 7.4), 0.2 mM acetyl CoA, 0.08 mM 5,5’-dithio-bis(2-nitrobenzoate) (DTNB), and the

cell extract (120 µL). The enzyme activity was determined at 32°C by measuring the

absorbance at 405 nm (SpectraMax 250) following the liberation of coenzyme A, which

has a molar extinction coefficient of 13.6 mM-1cm-1 (Ellman, 1959). One unit of activity

is defined as the amount of enzyme converting 1 µmole of acetyl CoA per minute.

Acetate kinase was assayed by the method of Allen et al. (1964). The assay

mixture (final total volume: 300 µL) contained: 81 mM Tris/HCl buffer (pH 7.4), 4 mM

ATP, 4 mM MgCl2, 1.6 mM phosphoenolpyruvate, 81 mM potassium acetate, 0.4 mM

y = 1.0678x + 0.0303R2 = 0.9853

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.1 0.2 0.3 0.4 0.5 0.6

BSA concentration (mg/mL)

Abso

rban

ce (5

95 n

m)

Abs

orba

nce

(595

nm

)

222

NADH, 0.4 U/mL pyruvate kinase, 0.4 U/mL lactate dehydrogenase, and the cell extract

(160 µL). The reaction rate at 25°C was followed by measuring the absorbance at 340

nm, which decreased linearly with time. A control without acetate was used as the blank

to correct for any ADP that might be present in the ATP preparation.

Oxaloacetate transcarboxylase was assayed by the method of Wood et al. (1969).

Briefly, the reaction mixture (final total volume: 300 µL) contained: 0.1 mM NADH, 0.2

mM methylmalonyl CoA, 10 mM sodium pyruvate, 350 mM potassium phosphate (pH

6.8), 2 U/mL malate dehydrogenase, and the cell extract (70 µL). The reaction rate at

25°C was followed by measuring the absorbance at 340 nm, which decreased linearly

with time, for at least 4 minutes. The reaction mixture without methylmalonyl CoA was

used as the blank.

Propionyl CoA: succinyl CoA transferase (CoA transferase) was assayed

following the method of Schulman and Wood (1975). The reaction mixture (250 µL)

consisted of: 100 µL of Mixture 1 (250 mM Tris/HCl buffer (pH 8.0), 1 mM sodium

malate, and 2.5 mM NAD), 10 µL of 1.5 M sodium acetate, 10 µL of Mixture 2 (220

units of malate dehydrogenase, 35 units of citrate synthase, and 0.1 M potassium

phosphate buffer (pH 6.8) to make up a 1-mL volume), 10 µL of 15 mM succinyl CoA,

and 60 µL of the cell extract. The reaction rate at 25°C was followed by measuring the

absorbance at 340 nm, which increased linearly with time, for 3-5 minutes.

223

Phosphoenolpyruvate carboxylase (PEP carboxylase) was assayed by the method

of Maeba and Sanwal (1969). The assay mixture, with a total volume of 300 µL,

contained: 66.67 mM Tris/HCl buffer (pH 9.0), 66.67 µM NADH, 10 mM MgCl2, 10

mM NaHCO3 (freshly prepared), 3.33 mM PEP, 6.25 U/mL malate dehydrogenase, and

the cell extract (180 µL). The reaction rate at 25°C was followed by measuring the

absorbance at 340 nm. A blank control had no PEP in the reaction solution.

Unless otherwise noted, the activities of above enzymes were determined on the

basis of the molar extinction coefficient of 6.2 mM-1cm-1 for NADH (van der Werf et al.,

1997) and one unit of activity is defined as the amount of the enzyme catalyzing the

reaction at a rate of producing or consuming 1 µmole of NADH per minute. The specific

enzyme activity is defined as the unit of enzyme activity per mg of total protein. All

enzyme activity assays were done in duplicate.

ATPase activity was determined based on the method of Gutierrez and Maddox

(1992). Cells were grown at 32°C in the synthetic medium (50 mL) containing 0.4%

(w/v) glycine to the exponential phase (OD600 = ~1.8) or stationary phase (OD600 = ~3.0),

harvested, and suspended in 2 mL of TE buffer (10 mM Tris/HCl, 1 mM EDTA (pH

8.0)). Then, mutanolysin (Sigma) at the final concentration of 100 µg/mL was added to

lyse the cells at 37°C for 40 min. The crude membrane fraction was obtained by

centrifugation at 4°C for 30 min and suspended in 2.5 mL of 100 mM PIPES buffer (pH

5.95). The ATPase activity was determined by measuring the release of inorganic

phosphate (Pi) from ATP in the reaction mixture (total volume: 500 µL) containing 5 mM

ATP, 10 mM MgCl2, and 50 µL of the crude membrane fraction. The mixture was

224

incubated at 37°C for 20 min and the reaction was stopped by adding 1 mL of 15% (w/v)

ice-cold trichloroacetic acid. The mixture was then centrifuged twice at 13,200 rpm for 5

min, and the inorganic phosphate (Pi) in the supernatant was determined

spectrophotometrically at 830 nm (Shimadzu UV-1601) following the molybdenum blue

method (Vogel, 1978) as described below.

The standard KH2PO4 solution containing 0.1 mg Pi per mL and 200-400 µL

reaction mixture from the ATPase assay were first neutralized with KOH and then added

into a centrifuge tube. Hydrazine sulphate solution (0.15% (w/v)) at a volume of 40 µL

and 100 µL of molybdate solution (0.25 g Na2MoO4·2H2O dissolved in 10 mL of 10 N

sulfuric acid) were added subsequently. Distilled water was finally added to make a total

volume of 1 mL. The reaction was carried out at 100 ºC in boiling water for 10 min. After

cooling down rapidly, the absorbance of the solution was measured

spectrophotometrically at 830 nm (Shimadzu UV-1601) using water as blank. The

standard KH2PO4 solution was added at different volumes to make a phosphate assay

curve in the range of Pi from 0.01 to 0.06 mg (Figure B.4) and the Pi amount of samples

could be found from the standard curve. One unit of activity is defined as the amount of

enzyme that releases 1 µmole of Pi per minute. Specific activity of ATPase is defined as

the unit of activity per mg biomass.

225

Figure B.4 An inorganic phosphate standard curve. B.6 SDS-PAGE

1. Carefully clean and dry glass plates and assemble casting stand following the

Mini-PROTEAN® 3 Cell (Bio-Rad) instruction manual.

2. Mix solutions for separating gels in order shown in Table B.1 (The addition of

10% (w/v) ammonium persulfate and TEMED was just prior to the gel casting.).

Pour the mixture into plates and leave ~2 cm at the top of the plate for comb

insertion.

3. Carefully overlay with distilled water and allow gel polymerization. Remove the

overlay water using filter papers.

y = 11.376xR2 = 0.9992

0

0.2

0.4

0.6

0.8

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

Inorganic Phosphate (mg)

Abs

orba

nce

(830

nm

) A

bsor

banc

e (8

30 n

m)

226

4. Mix solutions for stacking gels in order shown in Table B.1 (The addition of 10%

(w/v) ammonium persulfate and TEMED was just prior to the gel casting.). Insert

a comb and allow gel to set.

5. Prepare loading samples by mixing the protein extract with the loading sample

buffer containing bromophenol blue and boiling the mixture at 100°C for 5 min

while waiting for stacking gel to set.

6. Assemble the running unit following the instruction manual.

7. Load samples with a specific amount of protein (~23-24 µg each) into wells and

then overlay samples with 1X running buffer. Carefully flood the upper chamber.

8. Run gels at a constant voltage of 110 V until the tracking dye reached the gel

bottom (~1.5 h).

9. Stain gels in coomassie brilliant blue for 1 h and then destain in the destaining

solution containing 20% methanol and 10% acetic acid.

10. Visualize gels using Agfa FotoLook scanner.

227

Table B.1 Gel compositions for SDS-PAGE electrophoresis. B.7 Membrane Fatty Acid Composition

Fatty acid methyl ester (FAME) analysis was used to analyze cellular membrane

fatty acid compositions. The membrane fatty acids of cells, harvested at the exponential

phase, were extracted with solvents and then methylated before analysis with gas liquid

chromatography as done by Microcheck, Inc. (Northfield, VT). The analysis reports of P.

acidipropionici wild type and adapted mutant from the FBB are shown below.

Separating gel, 12% (2 gels)

Stacking gel, 4% (2 gels)

Distilled water (mL) 3.015 Distilled water (mL) 1.83 1.5 M Tris/HCl, pH 8.8 (mL)

2.25 0.5 M Tris/HCl, pH 6.8 (mL)

0.75

10% (w/v) SDS (mL) 0.09 10% (w/v) SDS (µL) 30 Acrylamide/Bis-acrylamide (30%(w/v)) (mL)

3.6 Acrylamide/Bis-acrylamide (30%(w/v)) (mL)

0.39

10% (w/v) ammonium persulfate(µL)

45 10% (w/v) ammonium persulfate (µL)

15

TEMED (µL) 4.5 TEMED (µL) 3 Total (mL) 9 Total (mL) 3

228

Continued

Figure B.5 The analysis report of membrane fatty acid content of P. acidipropionici wild type.

229

Figure B.5 (continued)

Continued

230

Figure B.5 (continued)

231

Continued

Figure B.6 The analysis report of membrane fatty acid content of P. acidipropionici adapted mutant from the FBB.

232

Figure B.6 (continued)

Continued

233

Figure B.6 (continued)

234

B.8 Scanning Electron Microscopy

Cells were laid by gentle filtration of cell suspension onto the filter paper

(Fisherbrand Grade P5, Fisher Scientific). The filter paper with overlaid cells was cut to

obtain pieces of samples with a size of 0.5 cm × 0.5 cm. The samples were fixed by

immersing in 2.5% glutaraldehyde solution for 15 h at 4°C and rinsed with distilled water

twice. The samples were processed through a progressive dehydration with 20 to 100%

ethanol at 10% increment for 20 min at each concentration. The samples were then

immersed in various concentrations of hexamethyl disilazane (HMDS) (ethanol: HMDS;

3:1, 1:1, and 1:3) for 15 min at each concentration and finally immersed in 100% HMDS

for 15 min for 3 times. Before the examination, the samples were coated with

gold/palladium using the spotter-coating machine in the presence of the medium

containing argon gas. The samples were scanned and photographed with a Philips XL 30

scanning electron microscope at 15 kV.

235

APPENDIX C

BIOREACTOR CONSTRUCTION AND OPERATION

236

C.1 Construction of Immobilized-Cell Bioreactor

Fibrous-bed bioreactor, with a working volume of ~690 mL, was constructed by

packing a wound cotton towel into a glass column bioreactor with a water jacket. A piece

of cotton towel laid with a stainless steel mesh was spirally wound and packed inside the

column for cell immobilization as seen in Figure C.1. The gap between the layers was

about 5 mm. A 1″ to 1.5″ layer of rasching rings was filled in the bottom of column to

support the spirally wound matrix and create a homologous flow distribution. The packed

glass column was sealed with rubber stoppers at both ends and connected to a 5-L

fermentor (Marubishi MD-300) through a recirculation loop (~1 m long, tubing ID: 3.1

mm; Microflex Norprene 06402-16, Cole Palmer, Chicago, IL) (Figure C.2). The fibrous-

bed bioreactor was operated under well-mixed conditions with pH by the recirculating

loop and temperature was controlled by circulating water with temperature control

through the water jacket in the glass column.

Stainless Steel Mesh

Fibrous Matrix

Liquid Outlet

Liquid Inlet Gas

Gas

Figure C.1 A fibrous-bed bioreactor construction.

237

Figure C.2 The system of propionic acid fermentation with a fibrous-bed bioreactor. C.2 Bioreactor Start-Up and Operation

To ensure complete sterilization, the bioreactor was autoclaved for 45 min, held

overnight, and then autoclaved again for 45 min before use. The sterile column was

aseptically connected to a 5-L fermentor (Marubishi MD-300) through a recirculation

loop as seen in Figure C.2. The total volume of 2 L of the medium was used for the entire

system. The system was maintained at 32°C, pH 6.5 by the addition of 6 M NaOH, and

100 rpm for agitation. Anaerobiosis was established by sparging the medium with N2 for

~30 min. After inoculation with ~100 mL of cell suspension (OD600 ~ 2.0) into the

fermentor, cells were grown for 3-4 days to reach an optical density (OD600) of ~3.5. The

N2

pH meter

6 M NaOH

Fibrous-bed bioreactor

pH probe

Circulation pump

Fermentor

CO2

238

fermentation broth was then circulated at a flow rate of ~30 mL/min through the FBB to

allow cells to attach and to be immobilized in the fibrous matrix. The process continued

for 60-72 h until most cells had been immobilized in the FBB. The medium circulation

rate was then increased to ~80 mL/min and the fermentation was run at a repeated-batch

mode to obtain a high cell density in the fibrous bed. After completion of start-up period,

the fermentation broth was replaced with the fresh sterilized medium containing glucose

as a carbon source to study the kinetics of propionic acid fermentation. Fed-batch

fermentation with pulse additions of concentrated glucose solution was then performed to

study the fermentation kinetics and to evaluate the achievable maximum propionic acid

concentration. Samples were taken at regular time intervals throughout the fermentation

for analyses.

239

APPENDIX D

GENETIC ENGINEERING PROTOCOLS

240

D.1 Preparation of Genomic DNA from P. acidipropionici with QIAGEN Genomic

DNA Kit

1. Inoculate 50 mL of the synthetic medium in a serum bottle with active P.

acidipropionici culture and grow at 32°C until the OD reached around 2.0.

2. Pellet down cells by centrifugation for 10 min at 13,200 rpm and save the pellet.

3. Suspend the pellet with 180 µL of lysis buffer containing 100 mg/mL lysozyme

and incubate at 37°C for at least 30 min.

4. Add 25 µL of Proteinase K solution and 200 µL of Buffer AL. Mix by vortexing.

Do not add Proteinase K into Buffer AL directly.

5. Incubate at 70°C for 30 min.

6. Add 200 µL of absolute ethanol to the sample. Mix thoroughly by vortexing to

obtain homogenous solution.

7. Apply the mixture into the DNeasy mini column with a 2-ml collection tube.

Centrifuge at 13,200 rpm for 1 min. Discard the flowthrough and collection tube.

Place the DNeasy mini column into a new 2-ml collection tube.

8. Add 500 µL of Buffer AW1. Centrifuge for 1 min. Discard the flowthrough and

collection tube. Place the DNeasy mini column into a new 2-ml collection tube.

9. Add 500 µL of Buffer AW2. Centrifuge for 3 min to dry the DNeasy membrane.

Discard the flowthrough and collection tube.

10. Place the DNeasy column in a clean, sterile 1.5-mL microcentrifuge tube. Pipette

200 µL of Buffer AE onto the center of DNeasy membrane. Incubate at room

temperature for 10 min and then centrifuge for another 1 min.

241

The gel electrophoresis of the genomic DNA from P. acidipropionici was shown in

Figure D.1.

Figure D.1 The genomic DNA isolated from P. acidipropionici. D.2 Preparation of Plasmid DNA by QIAprep Spin Miniprep Kit

1. Centrifuge 5 mL of overnight culture of E. coli at 13,200 rpm for 5 min.

2. Resuspend the pellet in 250 µL of Buffer P1.

3. Add 250 µL of Buffer P2 and gently invert the tube 4-6 times to mix. Do not

allow the lysis reaction to proceed for more than 5 min.

4. Add 350 µL of Buffer N3 and invert the tube immediately but gently 4-6 times.

Centrifuge at 13,200 rpm for 15 min.

5. Apply the supernatant to a QIAprep spin column by pipetting.

6. Centrifuge for 1 min and discard the flowthrough.

7. Add 500 µL of Buffer PB, centrifuge for 1 min, and discard the flowthrough.

8. Wash the column by adding 750 µL of Buffer PE. Centrifuge for 1 min.

1 kb

500 bp

Genomic DNA

242

9. Discard the flowthrough. Centrifuge for an additional 1 min.

10. Place the column in a clean, sterile 1.5-mL microcentrifuge tube. Add 50 µL of

Buffer EB or sterile distilled water to the center of each column to elute DNA. Let

stand for 10 min and centrifuge for 1 min.

The isolated plasmid was shown in Figure D.2.

D.3 Preparation of Plasmid DNA by HiSpeed Plasmid Maxi Kit

1. Harvest cells of E. coli grown in 250 mL of the LB medium at 37°C overnight by

centrifugation at 12,000 rpm, 4°C for 15 min.

2. Resuspend pellet in 10 mL of Buffer P1 by pipetting.

3. Add 10 mL of Buffer P2, gently invert 4-6 times, and incubate at room

temperature for exact 5 min.

4. Add 10 mL of pre-chilled Buffer P3. Mix immediately but gently by inverting 4-6

times.

5. Centrifuge at 12,000 rpm, 4°C for 20 min.

6. Transfer the supernatant into the QIAfilter Cartridge. Incubate at room

temperature for 10 min.

7. Equilibrate a HiSpeed Maxi Tip by applying 10 mL of Buffer QBT and allow the

column to empty by gravity flow.

8. Filter the cell lysate into the equilibrated Tip by gently inserting plunger into the

Cartridge.

9. Allow the cleared lysate to enter the resin by gravity flow.

243

10. Wash the Tip with 60 mL of Buffer QC. Elute DNA with 15 mL of Buffer QF.

11. Precipitate DNA by adding 10.5 mL of room-temperature isopropanol to the

eluted DNA. Mix and incubate at room temperature for 5 min.

12. Attach the QIAprecipitator Maxi Module onto the outlet nozzle. Place the

QIAprecipitator over the waste bottle, transfer the eluate/ isopropanol mixture

into the 30-mL syringe, and insert the plunger. Filter the mixture through the

QIAprecipitator using constant pressure.

13. Remove the QIAprecipitator from the 30-mL syringe and pull out the plunger.

Re-attach the QIAprecipitator and add 2 mL of 70% ethanol to the syringe. Wash

the DNA by inserting the plunger and pressing the ethanol through the

QIAprecipitator using constant pressure.

14. Remove the QIAprecipitator from the 30-mL syringe and pull out the plunger.

Attach the QIAprecipitator again, insert the plunger and dry the membrane by

pressing air through the QIAprecipitator quickly and forcefully. Repeat this step.

15. Dry the outlet nozzle of the QIAprecipitator with Kimwipes to prevent the ethanol

carryover.

16. Remove the plunger from a new 5-mL syringe and attach the QIAprecipitator

onto the outlet nozzle. Hold the outlet of the QIAprecipitator over a clean, sterile

1.5-mL microcentrifuge tube. Add 1 mL of Buffer TE or sterile distilled water to

the 5-mL syringe. Insert the plunger and elute the DNA into the tube using

constant pressure.

244

17. Remove the QIAprecipitator from the 5-mL syringe, pull out the plunger and

reattach the QIAprecipitator to the 5-mL syringe.

18. Transfer the eluate from the previous step to the 5-mL syringe and eluate for a

second time into the same tube.

D.4 DNA Purification by QIAquick Spin Gel Extraction Kit

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weigh the gel slice in 1.5-mL microcentrifuge tube (~300 mg). Add 3 volumes of

Buffer QG to 1 volume of gel (100 mg~100 µL).

3. Incubate at 50°C for 10 min.

4. Add 1 gel volume of isopropanol to the sample and mix.

5. Transfer the sample to a QIAquick column, centrifuge for 1 min at 13,200 rpm,

4°C, and discard flowthrough.

6. Add 500 µL of Buffer QG to the column, centrifuge for 1 min, and discard

flowthrough.

7. Add 750 µL of Buffer PE to the column, centrifuge for 1 min, and discard

flowthrough.

8. Centrifuge the column for an additional 1 min.

9. Place the column into a clean, sterile 1.5-mL microcentrifuge tube. Add 30-50 µL

of Buffer EB or sterile distilled water to the center of each column to elute DNA.

Let stand for 10 min and centrifuge for 1 min.

245

In some cases, the extraction of salt from DNA digestion reaction or DNA mixture was

required. The procedure was started with an addition of 3 volumes of Buffer QG and 1

volume of isopropanol to 1 volume of the DNA reaction mixture and then followed by

step 5 to 9.

D.5 DNA Electrophoresis

1. Use 0.8% and 1.0% agarose gels for genomic DNA and plasmid DNA

separations, respectively.

2. Prepare the agarose solution by heating a mixture of agarose powder and TAE

buffer.

3. Cool down the agarose solution to ~50-60°C, pour into a sealed gel casting

platform, and insert the gel comb.

4. Let the gel solidify. Remove the comb.

5. Place the platform in the electrophoresis tank and then add TAE buffer to cover

the gel (about 1mm in depth).

6. Use a pipette to load DNA samples into wells

7. Connect the Bio-Rad Mini Sub-Cell to the power supply (Bio-Rad PowerPac 300)

and run at constant voltage of 40 V for 1 h or until the bromophenol blue dye was

about 2/3 of the gel length.

8. Stain the gel with 0.5 µg/mL ethidium bromide solution for 5 min.

246

9. Destain the gel with distilled water for 5 min.

10. Visualize the DNA bands using gel documentation system (Bio-Rad Gel Doc

2000).

D.6 PCR Amplification of ack Gene from P. acidipropionici

1. In a clean, sterile 500-µL PCR tube, add

Reagent Volume (µL)

10X PCR buffer (Amersham Bioscience) 5 25 mM MgCl2 1 10 mM dNTPs (each) 1 Forward primer (10 µM) 1 Reverse primer (10 µM) 2 Genomic DNA 5 Taq DNA polymerase (5 U/µL) 0.5 Sterile water 34.5 Total volume 50

The primers used in this study are listed as follows:

Forward primer:

5’– AAG GAT CCA T(C)C(A)G IGT IGT ICA T(C)GG IGG –3’

Reverse primer:

5’– AAG GAT CCT CIC CT(A/G) ATI CCI G(C)CI GTA(G) AA –3’

2. Load the tubes of PCR reaction into a thermal cycler (MJ Research). Carefully

arrange the position of the tubes to ensure even heating.

247

3. Operate the thermal cycler by the following program.

Step 1 94°C ----- 3 min

Step 2 (x 15) 94°C ----- 50 s

42°C ----- 50 s

72°C ----- 1 min

Step 3 (x 30) 94°C ----- 50 s

50°C ----- 50 s

72°C ----- 1 min

Step 4 72°C ----- 4 min

4. Analyze 8 µL of the PCR product of each sample using DNA Electrophoresis.

5. Directly use PCR product for TOPO TA Cloning or purify the product from the

gel by QIAquick Gel Extraction Kit and then use the purified product for the

cloning.

D.7 TOPO TA Cloning Protocol (Invitrogen TOPO TA Cloning Kit)

1. Set up the 6 µL of ligation mixture as follows:

Reagent Volume (µL) Fresh PCR product X Salt Solution 1 Sterile water 4-X TOPO vector 1 Total volume 6

248

2. Incubate at room temperature for 5 min. Centrifuge the ligation reaction and place

it on ice.

3. Thaw on ice one 50-µL vial of frozen One Shot competent cells for each

transformation.

4. Pipette 2 µL of each ligation reaction into the competent cells and mix by stirring

gently with the pipette tip.

5. Incubate the vials on ice for 30 min. Store the remaining ligation mixtures at -20

°C.

6. Heat shock for exactly 30 s in the 42°C water bath. Immediately transfer the tubes

to ice.

7. Add 250 µL of SOC medium at room temperature to each tube.

8. Shake the vials at 37°C for 1 h at 225 rpm in a shaking incubator.

9. Evenly Spread 40 µL of 40 mg/mL X-gal stock solution onto each LB agar plate,

let dry 15 min.

10. Spread 10-100 µL from each transformation vial on separate LB agar plates

containing X-gal and 100 µg/mL ampicillin.

11. Invert the plates and place them in a 37°C incubator for 18 h. Store at 4°C for 2-3

h for color development.

12. Pick up ~10-20 white colonies to isolate the plasmids for analysis.

Figure D.2 shows the results of gel electrophoresis of undigested and EcoRI-digested

plasmids from a colony. The correct size of the insert demonstrated the positive colony

that contained the product of interest.

249

Figure D.2 EcoRI digestion of TOPOACK1. D.8 Construction of TOPOACK1

1. Prepare fresh ~500 bp of the ack fragment with BamHI ends.

2. Create blunt ends of this fragment using Fill-In Klenow. The reaction mixture

contained as follows:

Reagent Volume (µL) Fresh BamHI-ack fragment 30 10X buffer 4 0.5 mM dNTPs (each) 1 Sterile water 4 Klenow (5 U/µL) 1 Total volume 40

1 kb

1 2 3 4 5 6

1. 1 kb Ruler (Bio-Rad) 2. Undigested TOPOACK1 (positive) 3. Undigested negative plasmid 4. Precision Mass Standard Ruler (Bio-Rad) 5. EcoRI-digested TOPOACK1 6. EcoRI-digested negative plasmid 500 bp

250

3. Incubate at room temperature for 15 min. Stop the reaction by heating at 75°C for

10 min.

4. Dephosphorylize the created blunt-end fragment. The reaction mixture contained

as follows:

5. Incubate at 37°C for 1 h. Remove salt from the dephosphorylized fragment using

QIAquick Spin Gel Extraction Kit.

6. Add A’-overhang to the dephosphorylized fragment. The reaction mixture

contained as follows:

7. Incubate at 72°C for 25 min.

Reagent Volume (µL) Blunt-end fragment 40 10X buffer 5 Dilution buffer 4 Shrimp alkaline phosphatase (1 U/µL) 1 Total volume 50

Reagent Volume (µL) Dephosphorylized fragment 5 10X PCR buffer 1 1 mM dATPs 2 Sterile water 1 Taq DNA polymerase (1 U/µL) 1 Total volume 10

251

8. Ligate this blunt-end fragment with A’-overhang into a TOPO vector by

following TOPO TA Cloning protocol (Appendix D.7). The final product was

called TOPOACK1.

D.9 DNA Digestion and Ligation

1. Plasmid DNA digestion of 10 µL contained:

Reagent Volume(µL) 10X digestion buffer 1 Plasmid DNA 4 Restriction enzyme 1 Sterile water 4 Total volume 10

The incubation temperature (30°C or 37°C) and time (i.e., 1 h) were dependent on types

of restriction enzymes used in particular experiments. The digested products could be

visualized by gel electrophoresis as shown in Figure D.2.

2. DNA ligation of 20 µL contained:

Reagent Volume (µL) 5X ligase buffer (Invitrogen) 4 Two DNA fragments to be ligated x T4 DNA ligase 1 U/µL (Invitrogen) 0.2 Sterile water 15.8-x Total volume 20

252

A molar ratio of 3:1 (insert: vector) was used to calculate the amounts of the insert and

vector used in the reaction mixture. The reaction was incubated at 25°C for 1 h.

D.10 DNA Transformation in P. acidipropionici

1. All transformation experiments were performed on the bench top without any

special anaerobic conditions.

2. 2 mL of stationary-phase culture of P. acidipropionici was inoculated into 50 mL

of the fresh synthetic medium.

3. Cells were grown at 32°C until the exponential phase (OD ~ 0.8-1.0).

4. Harvest cells by centrifugation at 7,000 rpm, 4ºC for 5 min.

5. Wash pellet twice with 25 mL of ice-cold sterile distilled water. Gently resuspend

pellet with 500 µL of ice-cold sterile distilled water.

6. Transfer DNA (either 5 µg of the linear fragment or 8 µg of the non-replicative

integrational plasmid) into a pre-chilled 0.2-cm electroporation cuvette (Bio-Rad).

Add 200 µL of the competent cells and gently mix with a pipette. Incubate on ice

for 5 min.

7. Put the cuvette into the safety chamber (Electrocell Manipulator 600, BTX Inc.,

San Diego, CA) and apply a pulse (12.5 kV/cm, 50 µF, 129 Ω) with a time

constant of 4-5 ms.

8. Remove the cuvette from the chamber immediately. Add 1 mL of the fresh

medium.

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9. Transfer into a serum tube containing 5 mL of the fresh medium. Incubate at 32°C

for 9 h.

10. Harvest transformants and resuspend with 0.5 mL of the fresh medium prior to

plating on synthetic medium agar plates containing 10 µg/mL Tet. Incubate at

32°C for 10-14 days.

The transformation was performed with an efficiency of 5-10 colonies per µg DNA.

D.11 Construction of pSSF1

1. PCR amplification of fdh1 gene using pFDH1 (Sakai et al., 1997) as a template

The reaction mixture was prepared as shown below.

Reagent Volume (µL)

10X HiFi buffer (Invitrogen) 5 50 mM MgSO4 2 10 mM dNTPs (each) 2 Forward primer (FDH1(F)) (10 µM) 1 Reverse primer (FDH1 (R)) (10 µM) 1 pFDH1 (template) 1 Taq HiFi (5 U/µL) 0.4 Sterile water 37.6 Total volume 50

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The primers used in this study are listed as follows:

Forward primer:

5’– CGG GTC TCG CAT GAA GAT CGT TTT AGT CTT ATA TGA TGC –3’

Reverse primer:

5’– CCG ACC GCA TAT GTT ATT TCT TAT CGT GTT TAC CGT AAG –3’

The thermal cycler was operated by the following program.

Step 1 94°C ----- 2 min

Step 2 (x 35) 94°C ----- 1 min

55°C ----- 45 s

72°C ----- 2 min

Step 3 72°C ----- 10 min

2. Digest pKHEM01, an expression vector (Kiatpapan and Murooka, 2001) with a

size of 10.4 kb, with NcoI and NdeI restriction enzymes and the PCR product

(fdh1 gene, 1.1 kb) obtained from step 1 with BsaI and NdeI restriction enzymes

as following Appendix D.9.

3. Purify the digested expression vector and amplified fdh1 gene by following

Appendix D.4.

4. Ligate the purified cut vector (8.4 kb) and fdh1 gene by following Appendix D.9.

5. Transform DH5α E. coli competent cells (Library Efficiency DH5α, Invitrogen)

with the ligation mixture.

6. Positive colonies with Ampr (100 µg/mL) and HygBr (250 µg/mL) were selected.

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7. Isolate the plasmid from positive colonies and perform the restriction enzyme

digestion to confirm that the fdh1 gene was cloned into the expression vector.

pFDH1 and pKHEM01 were kindly provided by Dr. Yasuyoshi Sakai (Division of

Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan)

and Dr. Yoshikatsu Murooka (Department of Biotechnology, Graduate School of

Engineering, Osaka University, Osaka, Japan), respectively.

Figure D.3 shows the results of gel electrophoresis of the digested plasmid from a colony.

The correct size of the cut fragments (Lane 3: ~9.3 kb and ~0.2 kb) demonstrated the

positive colony that contained the product of interest. The constructed plasmid (9.5 kb)

consisting of the expression vector (8.4 kb) and the fdh1 gene (1.1 kb) was called pSSF1.

Figure D.3 Restriction enzyme digestion of pSSF1.

1 2 3 4 5

1. NdeI-digested pSSF1 2. BsaI-NdeI-digested pSSF1 3. NcoI-NdeI-digested pSSF1 4. 1 kb Ruler (Bio-Rad) 5. Precision Mass Standard Ruler (Bio-Rad)

1 kb 500 bp 100 bp

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APPENDIX E

REAGENTS AND BUFFERS

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ATP Solution, 0.1 M

Dissolve 0.055 g ATPNa2 in 800 µL of distilled water and adjust the pH to 7.0 with

NaOH. Mix well and add water to a final volume of 1 mL. Store at 4°C.

5, 5’-dithio-bis (2-nitrobenzoate) (DTNB) solution, 4 mM

Dissolve 0.016 g DTNB in 10 mL of 50 mM potassium phosphate buffer (pH 7.4).

EDTA (pH 8.0), 0.5 M

Dissolve 18.61 g EDTA in 80 mL of distilled water and adjust pH to 8.0 with NaOH

(pellet). Mix and add distilled water to 100 mL.

Ethidium Bromide, 1000x

Dissolve 0.05 g ethidium bromide in 100 mL of distilled water. Mix well and store in the

dark.

Magnesium Chloride, 1 M

Dissolve 2.03 g MgCl2·6H2O in 10 mL of distilled water.

NADH Solution, 15 mM

Dissolve 0.011 g NADH in 1 mL of distilled water. Store at 4°C.

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PEP, 0.2 M

0.0534 g PEP in 1 mL of distilled water. Store at -20°C.

PIPES Buffer, 100 mM

Dissolve 1.21 g PIPES (free-acid) in distilled water, adjust the pH to 5.95 with 1 M

NaOH, and add distilled water to 40 mL.

Potassium Phosphate Buffer (pH 7.4), 1 M

Dissolve 27.93 g K2HPO4 and 5.39 g KH2PO4 in 200 mL of distilled water.

TE Buffer

10 mM Tris/HCl with adjusted pH of 8.0 and 1 mM EDTA (pH 8.0).

TAE Electrophoresis Buffer, 50x

Dissolve 242.28 g Tris base in distilled water, add 100 mL of 0.5 M EDTA, and adjust

pH to 7.6 with 57.1 mL of glacial acetic acid. Mix and add distilled water to 1 L.

Tris/HCl, 1 M

Dissolve 12.114 g Tris base in 80 mL of distilled water and adjust to the desired pH with

concentrated HCl. Mix and add distilled water to 100 mL.