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
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
ii 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.
iii 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.
iv
Dedicated to my mother
v 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.
vi 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.
vii 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
viii TABLE OF CONTENTS
Page Abstract……………………………………………………………………...... ……ii Dedication………………………………………………………………………...... ……v Acknowledgments………………………………………………………………….....….vi Vita…………………………………………………………………………………..…viii List of Tables……………………………………………………………...... ….xiv List of Figures……………………………………………………………………….….xvi
Chapters:
1. Introduction……………………………………………………………………..……..1
2. Literature Review……………………………………………………………….….….9
2.1 Propionic Acid Fermentation…...... … …..9 2.1.1 Propionic Acid………………………………………………………..…. ….9 2.1.2 Microorganisms……………………………………………………….……12 2.1.3 Metabolic Pathway…………………………………………………………14 2.1.4 Fermentation Processes……………………………………………….……17 2.2 Cell Immobilization and Fibrous-Bed Bioreactor…………………………….….30 2.2.1 Cell Immobilization…………………………………………………...…..30 2.2.2 Immobilized-Cell Bioreactor……………………………………………. …31 2.2.3 Fibrous-Bed Bioreactor……………………………………………….……32 2.3 Immobilized-Cell Fermentation……………………………………………...……33 2.4 Metabolic Engineering……………………………………………………….……36 2.4.1 Metabolic Flux Analysis……………………………………………………36 2.4.2 Applications of Other Metabolic Engineering Techniques…………...……39 2.5 Genetic Engineering of Propionibacteria…………………………………….……40 2.5.1 Acetic Acid Formation in Propionic Acid Fermentation……………..……40 2.5.2 Acetic Acid Formation Pathway Genes and Enzymes………………..……42 2.5.3 Genetics and Molecular Biology of Propionibacteria………………...……43 2.6 References…………………………………………………………………………50
ix 3. Enhanced Propionic Acid Fermentation by Propionibacterium acidipropionici Mutant Obtained by Adaptation in a Fibrous-Bed Bioreactor……………...... ……64
Summary………………………………………………………………………....……64 3.1 Introduction…………………………………………………………………..……66 3.2 Materials and Methods……………………………………………………….……68 3.2.1 Culture and Media…………………………………………………….……68 3.2.2 Free-Cell Fermentation…………………………………………….....……68 3.2.3 Immobilized-Cell Fermentation……………………………………………69 3.2.4 Effect of Propionic Acid on Cell Growth………………………….....……70 3.2.5 Enzyme Assays…………………………………………………….....……70 3.2.6 Membrane-Bound ATPase Assay…………………………………….……71 3.2.7 Cell Membrane Fatty Acid Analysis………………………………….……72 3.2.8 Cell Viability Assay.………………………………………………….……72 3.2.9 Scanning Electron Microscopy……………………………………….…. …72 3.2.10 Analytical Methods…………………………………………………...……73 3.3 Results and Discussion……………………………………………………….……73 3.3.1 Fermentation Kinetics………………………………………………...……73 3.3.2 Propionic Acid Inhibition……………………………………………..……76 3.3.3 Acid-Forming Enzyme Activities…………………………………….…. …78 3.3.4 Membrane-Bound ATPase…………………………………………………81 3.3.5 Membrane Fatty Acid Composition…………………………………..……83 3.3.6 Morphological Change in Mutant…………………………………….……83 3.3.7 Effects of Cell Immobilization in FBB……………………………….……84 3.4 Conclusion…………………………………………………………………………87 3.5 References…………………………………………………………………………88
4. Effect of Carbon Sources on Propionic Acid Fermentation by Propionibacterium acidipropionici……………………………………………..….105
Summary……………………………………………………………………...…..…105 4.1 Introduction…………………………………………………………………..…107 4.2 Materials and Methods...... …110 4.2.1 Culture and Media…………………………………………………...... …110 4.2.2 Batch Fermentation………………………………………………...….…110 4.2.3 Analytical Methods………………………………………………...…..…111 4.2.4 Metabolic Stoichiometric Analysis………………………………………111 4.2.5 Preparation of Cell Extract……………………………………………….112 4.2.6 Enzyme Assays…………………………………………………….....…. .112
x 4.2.7 Protein Expression: SDS-PAGE……………….……………………..…..113 4.3 Results……………………………………………………………………...... …113 4.3.1 Effect of Carbon Sources on Fermentation Kinetics……...…………..….113 4.3.2 Comparison of Fermentation Kinetics between Wild Type and Adapted Mutant……………………………………….…114 4.3.3 Metabolic Pathway Analysis………………………………………….…115 4.3.4 Metabolic Stoichiometric Analysis (MSA)………………………………119 4.3.5 Effect of Carbon Sources on MSA……………………………….…...…122 4.3.6 Comparison of MSA between Wild Type and Adapted Mutant…...……123 4.3.7 Effect of Carbon Sources on Activity of Acid-Forming Enzymes...….…123 4.3.8 Effect of Carbon Sources on Protein Expression Pattern…………...... …124 4.4 Discussion………………………………………………………………….....…125 4.4.1 Effect of Carbon Sources on Fermentation by P. acidipropionici...….…125 4.4.2 Effect of Carbon Sources on Protein Expression Pattern……………..…133 4.5 Conclusion…………………………………………………………………….…134 4.6 References………………………………………………………………….....…136
5. Construction and Characterization of ack Gene Deleted Mutants of Propionibacterium acidipropionici for Propionic Acid Fermentation……….....…154
Summary………………………………………………………………………….…154 5.1 Introduction…………………………………………………………………..…156 5.2 Materials and Methods………………………………………………………..…159 5.2.1 Bacterial Strains and Plasmids………………………………………..…159 5.2.2 Medium Preparation and Growth Conditions……………………………159 5.2.3 DNA Preparation…………………………………………………………160 5.2.4 PCR Amplification………………………………………………………160 5.2.5 Gene Disruption: Construction of Disrupted ack……………………..…161 5.2.6 Integrational Mutagenesis: Construction of Integrational Plasmid of ack...... …162 5.2.7 Transformation of P. acidipropionici...... …162 5.2.8 Southern Hybridization…………………………………………….…..…163 5.2.9 Mutant Characterization……………………………………………….…164 5.3 Results………………………………………………………………………...…166 5.3.1 PCR Amplification and Sequence Analysis……………………….….…166 5.3.2 Transformation of P. acidipropionici...... …. .167 5.3.3 Disruption of ack…………………………………………………………167 5.3.4 Integrational Mutagenesis (pTAT)………………………………………168 5.3.5 Southern Hybridization………………………………………………..…168
xi 5.3.6 Overall Protein Expression Pattern…………...………………….…....…169 5.3.7 Acid-Forming Enzyme Activities………………………………….….…169 5.3.8 Fermentation Kinetics……………………………………………....……170 5.4 Discussion………………………………………………………………….……170 5.4.1 Genetic Manipulation of P. acidipropionici……………………….....…170 5.4.2 Overall Protein Expression Pattern…...…………………………….……172 5.4.3 Effect of ack Gene Inactivation……………………………………….…173 5.5 Conclusion………………………………………………………………………175 5.6 References………………………………………………………………….……176
6. Conclusions and Recommendations………………………………………….……192 6.1 Conclusions…………………………………………………………………..…192 6.1.1 Fermentation Kinetics……………………………………………………192 6.1.2 Fibrous-Bed Bioreactor……………………………………………….…193 6.1.3 Metabolic Engineering………………………………………………..…194 6.2 Recommendations………………………………………………………….……196 6.2.1 Propionic Acid Fermentation by P. acidipropionici……………………196 6.2.2 Metabolic Engineering and Genetic Engineering of P. acidipropionici…………………………………………………….…197
Bibliography………………………………………………………………………...…199
Appendices…………………………………………………………………………….213
Appendix A Medium Compositions……………………………………………...…213 A.1 Medium Compositions for Propionibacterium acidipropionici…….....…214 A.2 Medium Compositions for Escherichia coli…………………………...…214 Appendix B Analytical Methods……………………………………………….……..215 B.1 Cell Concentration……………………………………………...….…..…216 B.2 Cell Viability…………………………………………………………...…216 B.3 High-Performance Liquid Chromatography……...……...………….……217 B.4 Preparation of Cell Extracts and Protein Assay…………...…………...…220 B.5 Enzyme Assays………………………………………………...….…..….221 B.6 SDS-PAGE………………..…………………………...…………….……225 B.7 Membrane Fatty Acid Composition……………...………………….……227 B.8 Scanning Electron Microscopy…………………………………..…….…234 Appendix C Bioreactor Construction and Operation………………………….……235 C.1 Construction of Immobilized-Cell Bioreactor…………..……….…………236
xii C.2 Bioreactor Start-Up and Operation…………………………………….…237 Appendix D Genetic Engineering Protocols……………………………………..…239 D.1 Preparation of Genomic DNA from P. acidipropionici with QIAGEN Genomic DNA Kit…………..………………………...…240 D.2 Preparation of Plasmid DNA by QIAprep Spin Miniprep Kit…...………241 D.3 Preparation of Plasmid DNA by HiSpeed Plasmid Maxi Kit……...…..…242 D.4 DNA Purification by QIAquick Spin Gel Extraction Kit………...………244 D.5 DNA Electrophoresis…………………………...……………………...…245 D.6 PCR Amplification of ack Gene from P. acidipropionici…………….…246 D.7 TOPO TA Cloning Protocol (Invitrogen TOPO TA Cloning Kit)…….…247 D.8 Construction of TOPOACK1………………………..…………….…..…249 D.9 DNA Digestion and Ligation…………………………...………….…..…251 D.10 DNA Transformation in P. acidipropionici…………………...…………252 D.11 Construction of pSSF1………………...…………………………….……253 Appendix E Reagents and Buffers………………………………………………..…256
xiii LIST OF TABLES
Table Page
2.1 Development of fermentation processes for improved propionic acid production…………………………………………....….60
2.2 Genetic manipulation in propionibacteria……………………………………..……61
3.1 Comparison of product yields and maximum propionic acid concentrations from fed-batch fermentations with free cells and immobilized cells in the FBB……………………………………………..……92
3.2 Comparison of rate constants for specific growth rate and several key enzymes in P. acidipropionici wild type and mutant from the FBB……………………………………………………………93
3.3 Comparison of membrane fatty acid compositions of P. acidipropionici wild type and mutant from the FBB……………………….……93
4.1 Kinetics of fermentations by P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources……..………….…..139
4.2 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using glucose…………………………………………….…….140
4.3 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using sorbitol…………………………………………………..141
4.4 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using gluconate………………………………………………. .142
4.5 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using xylose………………………………………………..…..143
xiv 4.6 Metabolic stoichiometric analysis of fermentations by P. acidipropionici wild type and adapted mutant from the FBB using different carbon sources…………………………………………………….144
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…..…………….….145
5.1 Strains and plasmids……………………………………………………….…..….180
5.2 Comparison of amino acid sequences from ack gene of P. acidipropionici to the corresponding sequences from other microorganisms……………….…….181
5.3 Comparison of fermentation kinetics from batch fermentations by P. acidipropionici wild type, ACK-Tet mutant, and pTAT-ACK-Tet mutant…………………………….……. .182
B.1 Gel compositions for SDS-PAGE electrophoresis………...………………….…227
xv LIST OF FIGURES
Figure Page
1.1 Research objectives and the scope of this study…………………………………. ….8
2.1 The dicarboxylic acid pathway of P. acidipropionici………………………………62
2.2 Integrational mutagenesis……………………………………………………...……63
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….………94
3.2 Effect of propionic acid on the volumetric productivity of propionic acid in fed-batch fermentations with free cells and immobilized cells……………..……95
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……………… …96
3.4 Effects of propionic acid on specific growth rates of P. acidipropionici wild type and mutant from the FBB…………………………………………….….98
3.5 The dicarboxylic acid pathway for the conversion of glucose to propionic, acetic, and succinic acids………………………………………..…..99
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 ………………………….100
3.7 Effect of propionic acid on phosphotransacetylase (A) and acetate kinase (B) activities of P. acidipropionici wild type and mutant from the FBB……………………………………………………….….…101
xvi 3.8 Effect of propionic acid on PEP carboxylase activity of P. acidipropionici wild type and mutant from the FBB……………………….…102
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………………103
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…………………………….104
4.1 Fermentations by P. acidipropionici using glucose. A. Wild type; B. Adapted mutant from the FBB…………..……………………………….……146
4.2 Fermentations by P. acidipropionici using sorbitol. A. Wild type; B. Adapted mutant from the FBB……………..…………………………….……147
4.3 Fermentations by P. acidipropionici using gluconate. A. Wild type; B. Adapted mutant from the FBB………………………..………………….……148
4.4 Fermentations by P. acidipropionici using xylose. A. Wild type; B. Adapted mutant from the FBB……………………..………………….………149
4.5 The dicarboxylic acid pathway for the formations of propionic, acetic, and succinic acids……………………………………….….…150
4.6 Proposed pathways of sugar utilization in P. acidipropionici……………………151
4.7 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici wild type using different carbon sources…………………………………………152
4.8 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici adapted mutant from the FBB using different carbon sources…………..…….…153
5.1 Construction of an integrational plasmid, pTAT, with a fragment of 0.5-kb ack gene cloned from P. acidipropionici…………………………….……183
xvii 5.2 PCR product of partial ack gene…………………………………….……………184
5.3 DNA and amino acid sequences of partial ack gene from P. acidipropionici...…185
5.4 Restriction enzyme map of partial ack gene from P. acidipropionici……………186
5.5 Alignment of amino acid sequences of AK from E. coli, M. thermophila, B. subtilis, and P. acidipropionici………………...... …187
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…………………………………………………….....…188
5.7 SDS-PAGE gel electrophoresis of proteins from P. acidipropionici……...…..…189
5.8 Relative activities of AK and PTA in ACK-Tet and pTAT-ACK-Tet mutants as compared to the wild type……………………………………………….….…190
5.9 Fermentations by P. acidipropionici. A: Wild type; B: ACK-Tet mutant; C: pTAT-ACK-Tet mutant……………………………………………….………191
B.1 The HPLC chromatogram for standard containing glucose, succinic acid, lactic acid, acetic acid, and propionic acid..………………………218
B.2 The sample HPLC chromatogram of propionic acid fermentation by P. acidipropionici using glucose as the substrate…...…..……………………219
B.3 The standard curve of protein assay using bovine serum albumin…………..……221
B.4 An inorganic phosphate standard curve…………………...…………………..…225
B.5 The analysis report of membrane fatty acid content of P. acidipropionici wild type…………………………………..……………..……228
B.6 The analysis report of membrane fatty acid content of P. acidipropionici adapted mutant from the FBB………………...……….….…231
C.1 A fibrous-bed bioreactor construction……………...……………………….……236
xviii
C.2 The system of propionic acid fermentation with a fibrous-bed bioreactor…....…237
D.1 The genomic DNA isolated from P. acidipropionici……..………….….….……241
D.2 EcoRI digestion of TOPOACK1……………..………………….………….……249
D.3 Restriction enzyme digestion of pSSF1…………...………………………...... …255
xix 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
1 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
2 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.
3 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.
4 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.
5 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.
6 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.
7 Overall Objective
To develop a fermentation process for economical production of propionic acid
Process Engineering Genetic Engineering
* Fibrous-Bed Bioreactor * Gene Inactivation * Adaptation of • Gene Disruption P. acidipropionici • Integrational Mutagenesis
- Fermentation kinetics - Fermentation kinetics - Propionate tolerance - Acetate-forming enzyme (cell growth and enzymes) activity - Membrane ATPase - Protein expression pattern
- Membrane fatty acids (Chapter 5) - 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)
Figure 1.1 Research objectives and the scope of this study.
8 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
9 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.
10 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.
11 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;
12 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).
13 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
14 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
15 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).
16 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
17 (<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
18 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).
19 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
20 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
21 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
22 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).
23 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).
24 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
25 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
26 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).
27 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
28 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).
29 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).
30 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
31 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
32 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
33 (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
34 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.
35 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
36 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
37 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.,
38 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
39 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
40 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.
41 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
42 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.,
43 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).
44 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.
45 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
46 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).
47 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
48 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
activities and acetate production (Green and Bennett, 1998; Green et al., 1996).
Nevertheless, the insertional mutation by plasmid integration is not completely stable.
The two duplicated sequences flanking plasmid DNA are potential substrates for
homologous recombination, which could result in the loss of the integrated plasmid
(Wilkinson and Young, 1994).
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59
Propionic Acid Production Cell Density Final Fermentation Process/ Mode Substrate Yield Productivity References (g/L) Conc. (g/g) (g/L/h) (g/L)
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 corn meal batch - - 0.58 2.12 Huang et al., 2002 hydrolyzate de-lactose recycle-batch 34 65 0.5 0.22-0.47 Yang et al., 1995 whey permeate (0.68 with yeast extract)
60 Calcium- Alginate Beads semidefined 9.8x109 cells/g beads fed-batch 57 - 0.3 Paik and Glatz, 1994 medium 0.1 g beads/ mL medium (0.96: continuous mode) 2x1011 cells/g beads repeated-batch glucose 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
Table 2.1 Development of fermentation processes for improved propionic acid production.
57
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 - Kiatpapan et al., 2001 (Expression vector harboring Streptomyces choA )
61 5-Aminolevulinic acid (ALA) 8.3 mM ALA (Expression vector harboring hemA from Kiatpapan and Murooka, 2001 (comparable with that by recombinant E. coli) Rhodobacter spheroides)
Vitamin B12 (Expression vector harboring hemA from 1.7 mg/L Piao et al., 2004b R. spheroides and endogenous hemB and cobA)
Table 2.2 Genetic manipulation in propionibacteria.
58
Glucose + + NAD NAD EMP HMP
NADH + CO2 NADH CO2 Phosphoenolpyruvate ADP GDP (1) + NAD (5) ATP
Pyruvate GTP (6) NADH (2) CoA Oxaloacetate CO2 NADH Acetyl CoA Propionyl CoA (7) Pi + (3) NAD Methylmalonyl CoA CoA Malate
Acetyl phosphate (11) FP NADH + ADP ADP
(4) Succinyl CoA (8) ATP + FPH2 NAD + ATP Acetate (10) (9) Fumarate
Succinate FPH2 Propionate FP
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.
62
oriE CAM
P T
P CAM oriE T
Figure 2.2 Integrational mutagenesis.
63 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
64 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.
65 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,
66 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
67 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
68 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
69 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
70 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.
71 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.
72 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
73 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
74 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
75 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
76 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:
µ K 1 1 1 µ = max i or = + P (3.1) + µ µ µ K i P max max K i
-1 -1 where µ is the specific growth rate (h ), µmax is the maximum specific growth rate (h ),
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
-1 culture had a slightly lower µmax (0.133 ± 0.008 vs. 0.154 ± 0.017 h ), 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.
77 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
78 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
79 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:
v K 1 1 1 v = max i or = + P (3.2) + K i P v vmax vmax K i
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
80 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
81 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).
82 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
83 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
84 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.
85 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.
86 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.
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91 Free-cell fermentations Immobilized-cell
a fermentation Wild type Mutant
Max. propionic acid conc. (g/L) 52.2 ± 1.1 51.5 ± 0.9 71.8 ± 0.8
Product yield (g/g) Propionic acid 0.41 ± 0.02 0.47 ± 0.01 0.40 – 0.65 ± 0.02b Acetic acid 0.12 ± 0.02 0.11 ± 0.01 0.10 ± 0.01 Succinic acid 0.18 ± 0.03 0.09 ± 0.01 0.09 ± 0.02 c Total viable cell conc. (g/L) 2.41 5.67 11.48 Total carbon recovery (%)d 88.2 ± 8.5 87.1 ± 3.7 95.7± 6.0 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. d Based 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.
92
Wild type Mutant from FBB
-1 -1 µmax (h ) or vmax Ki (g/L) µmax (h ) or vmax Ki (g/L) (U/mg) (U/mg)
Specific growth rate 0.154 ± 0.017 4.33 ± 0.67 0.133 ± 0.008 8.93 ± 0.77
OAA transcarboxylase 0.111 ± 0.001 84.8 ± 4.9 0.138 ± 0.014 451.9 ± 42.9 CoA transferase 0.027 ± 0.001 10.1 ± 0.2 0.059 ± 0.004 13.5 ± 0.5
Phosphotransacetylase 0.0012 ± 0.0001 18.4 ± 0.5 0.0011 ± 0.0001 19.0 ± 0.8 Acetate kinase 0.019 ± 0.001 10.2 ± 0.8 0.021 ± 0.002 10.6 ± 0.5 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.
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%
Table 3.3 Comparison of membrane fatty acid compositions of P. acidipropionici wild type and mutant from the FBB.
93 100 7 Free cells OD 6 80
Glucose 5
60 Propionate 4 OD 3 40
2 Concentration ( g/L) Succinate 20 1 Acetate
0 0 0 100 200 300 400 500 600 700 800 900 A Time (h)
100 4 Immobilized cells
80 Propionate 3 Glucose 60
OD 2 OD 40
Concentration ( g/L) 1 20 Acetate
Succinate
0 0 0 100 200 300 400 500 600 700 800 900 B Time (h)
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.
94 6
5
4 Immobilized cells
3
2
Productivity (g/L/day) Free cells
1
0 0 20406080 Propionic acid (g/L)
Figure 3.2 Effect of propionic acid on the volumetric productivity of propionic acid in fed-batch fermentations with free cells and immobilized cells.
95 70
60 Immobilized cells 50
40 Free cells
30
20
10 Propionic acid production (g/L) production acid Propionic
0 0 20 40 60 80 100 120 140 160 180 A Glucose consumption (g/L)
20
Free cells 15
10 Immobilized cells
5 Acetic acid production (g/L) production acid Acetic
0 0 20 40 60 80 100 120 140 160 180 B Glucose consumption (g/L)
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.
96 Figure 3.3 (continued)
30
25 Free cells 20
15
10 Immobilized cells
5 Succinic acid production (g/L)
0 0 20 40 60 80 100 120 140 160 180 C Glucose consumption (g/L)
97 0.20 25 20 y = 1.50x + 6.494 R2 = 0.9862 0.15 15 )
-1
1/u (h) 10 y = 0.8397x + 7.4957 R2 = 0.9951
5
0.10 adapted 0 culture 04812 Propionic acid (g/L)
Specific Growth Rate (h 0.05
wild type
0.00 0 102030405060708090100 Propionic acid (g/L)
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.
98
Glucose + + NAD NAD EMP HMP NADH + CO2 NADH CO2 Phosphoenolpyruvate
ADP GDP
+ NAD PEP carboxylase ATP OAA Pyruvate transcarboxylase GTP NADH CoA Oxaloacetate CO2 NADH Acetyl CoA Propionyl CoA Pi + NAD Phosphotransacetylase Methylmalonyl CoA CoA Malate
Acetyl phosphate FP NADH + ADP ADP Acetate kinase Succinyl CoA ATP + NAD + ATP FPH2 Acetate CoA transferase Fumarate
Succinate FPH2 Propionate FP
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.
99 0.20 Oxaloacetate Transcarboxylase
0.15 mutant
0.10
wild type
Specific activity(U/mg) 0.05
0.00 0 20 40 60 80 100 A Propionic acid (g/L)
0.07
CoA Transferase 0.06
0.05
0.04
0.03
0.02 mutant Specific activity (U/mg) activity Specific
0.01 wild type 0.00 020406080100 B Propionic acid (g/L)
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.
100 0.0018
Phosphotransacetylase 0.0015 Wild Type 0.0012 Mutant
0.0009
0.0006 Specific activity (U/mg)
0.0003
0.0000 0 20406080100 A Propionic acid (g/L)
0.025
Acetate Kinase 0.020 Wild Type
Mutant 0.015
0.010 Specific activity (U/mg) activity Specific 0.005
0.000 0 20406080100 B Propionic acid (g/L)
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.
101 0.0020
PEP Carboxylase
0.0015 wild type
0.0010
mutant
Specific Activity (U/mg) Activity Specific 0.0005
0.0000 0 20406080100 Propionic acid (g/L)
Figure 3.8 Effect of propionic acid on PEP carboxylase activity of P. acidipropionici wild type and mutant from the FBB.
102 0.035
0.030 Exponential phase 0.025
0.020 Wild type
0.015 Adapted cells from FBB
0.010
ATPase (U/mg cell) (U/mg ATPase 0.005
0.000 0 20406080100 A Propionic acid (g/L)
0.020
Stationary phase 0.015
Wild type 0.010 Adapted cells from FBB
0.005 ATPase (U/mg cell) (U/mg ATPase
0.000 0 20406080100 B Propionic acid (g/L)
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.
103 A 10 µm
B 10 µm
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.
104 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
105 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.
106 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,
107 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
108 (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
109 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.
110 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
111 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.
112 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
113 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)
114 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
115 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.
116 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
117 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
118 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:
119 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)
120 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.
121 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.
122 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
123 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.
124 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
125 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
126 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
127 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,
128 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
129 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
130 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.
131 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
132 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
133 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
134 order to make propionic acid fermentation using sorbitol as a carbon source to be more feasible for commercial production of propionic acid.
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138 Sorbitol Xylose Gluconate Glucose Adapted Adapted Adapted Adapted WT WT WT WT Mutant Mutant Mutanta Mutant Max. propionate 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 (g/L)
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 ± 0.001 139
Propionate: Acetate ratio 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 (mol/mol)
Productivity 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 (g/L/h) Sp. growth 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 rate (h-1) 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. 139
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
Succinic Acid Formation → + 3) PEP + CO2 + 2 NADH + 2 ADP Succinate + 2 NAD + 2 ATP
Pyruvic Acid Formation
4) PEP + ADP → Pyruvate + ATP
Acetic Acid Formation + 5) Pyruvate + NAD + ADP → Acetate + NADH + ATP + CO 2
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
Table 4.2 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using glucose.
140
Sorbitol Utilization
1) Sorbitol + 3 NAD+ → 2 PEP + 3 NADH
Pyruvic Acid Formation
→ 2) PEP + ADP Pyruvate + ATP
Succinic Acid Formation → + 3) PEP + CO2 + 2 NADH + 2 ADP Succinate + 2 NAD + 2 ATP
Acetic Acid Formation
4) Pyruvate + NAD+ + ADP → Acetate + NADH + ATP + CO 2
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
Table 4.3 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using sorbitol.
141
Gluconate Utilization
+ 1) Gluconate + 8/3 NAD → 5/3 PEP + 8/3 NADH + CO2
Pyruvic Acid Formation
→ 2) PEP + ADP Pyruvate + ATP
Succinic Acid Formation → + 3) PEP + CO2 + 2 NADH + 2 ADP Succinate + 2 NAD + 2 ATP
Acetic Acid Formation
4) Pyruvate + NAD+ + ADP → Acetate + NADH + ATP + CO 2
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
Table 4.4 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using gluconate.
142
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
Pyruvic Acid Formation
3) PEP + ADP → Pyruvate + ATP
Succinic Acid Formation 4) PEP + CO + 2 NADH + 2 ADP → Succinate + 2 NAD+ + 2 ATP 2
Acetic Acid Formation
5) Pyruvate + NAD+ + ADP → Acetate + NADH + ATP + CO 2
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
Table 4.5 Stoichiometric equations of propionic acid fermentation by P. acidipropionici using xylose.
143
Sorbitol Xylose Gluconate Glucose Adapted Adapted Adapted Adapted WT WT WT WT Mutant Mutant Mutant 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 71.43 71.55 67.22 67.71 72.01 70.54 54.21 61.93 to Propionate
%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 0.54 0.53 0.48 0.48 0.47 0.44 0.43 0.46 (mol/mol carbon)
CO2 (mM/h/OD) 0.06 0.04 0.23 0.37 0.23 0.32 0.12 0.20
144 c %CO2 1.6 1.7 9.2 9.3 15.5 13.8 5.9 8.6 %CO produced from 2 0.0 0.0 42.9 37.6 88.3 100.0 29.7 35.0 substrated %CO produced from 2 100.0 100.0 57.1 62.4 11.7 0.0 70.3 65.0 acetate %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 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.
144
Specific activity OAA CoA PEP carboxylase PTA AK (U/mg) transcarboxylase transferase
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 ± 0.012 145
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
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.
145 40 9 Glucose, wild type 35 OD 8
30 7 6 25 5 OD 20 Propionate 4 15 3 10
Concentration (g/L) Acetate 2 5 1 Succinate A 0 0 0 10 20 30 40 50 60 70 80 Time (h)
40 7 35 Glucose, adapted mutant OD 6 30 5
25 4 20 Propionate OD 3 15 2 10 Concentration (g/L) Acetate 5 1 Succinate B 0 0 0 10 20 30 40 50 60 70 80 90 Time (h)
Figure 4.1 Fermentations by P. acidipropionici using glucose. A. Wild type; B. Adapted mutant from the FBB.
146 40 6
35 Sorbitol, wild type OD 5 30
4 25
Propionate OD 20 3 15 2 10 Concentration (g/L) Acetate 1 5 Succinate A 0 0 0 10 20 30 40 50 60 70 80 Time (h)
40 6 OD 35 Sorbitol, adapted mutant 5 30
4 25 Propionate OD 20 3 15 2 10 Concentration (g/L) Succinate 1 5 B 0 Acetate 0 0 10 20 30 40 50 60 70 80 90 100 110 Time (h)
Figure 4.2 Fermentations by P. acidipropionici using sorbitol. A. Wild type; B. Adapted mutant from the FBB.
147
40 6 OD Gluconate, wild type
35 5 30
4 25 OD 20 Propionate 3
15 2
Concentration (g/L) 10
Succinate 1 5 Acetate A Pyruvate 0 0 0 50 100 150 Time (h)
40 5 35 Gluconate, adapted mutant OD
4 30
25 3 OD 20 Propionate 15 2 10 Concentration (g/L) Succinate 1 5 Pyruvate B 0 0 0 50 Time (h) 100 150
Figure 4.3 Fermentations by P. acidipropionici using gluconate. A. Wild type; B. Adapted mutant from the FBB.
148 40 6 Xylose, wild type 35 OD
5 30
4 25 Propionate OD 20 3 15 2 10 Acetate Concentration (g/L) 1 5 Succinate A 0 0 0 10 20 30 40 50 60 70 80 90 100 110 Time (h)
40 5 Xylose, adapted mutant 35 OD 4 30
25 3
OD Propionate 20 15 2
10 Acetate Concentration (g/L) 1 5 Succinate B 0 0 0 10 20 30 40 50 60 70 80 90 100 110 Time (h)
Figure 4.4 Fermentations by P. acidipropionici using xylose. A. Wild type; B. Adapted mutant from the FBB.
149
CO2 Phosphoenolpyruvate
ADP GDP
+ NAD PEP carboxylase ATP OAA Pyruvate transcarboxylase GTP NADH CoA Oxaloacetate CO2 NADH Acetyl CoA Propionyl CoA Pi + NAD Phosphotransacetylase Methylmalonyl CoA CoA Malate
Acetyl phosphate FP NADH + ADP ADP Acetate kinase Succinyl CoA ATP + NAD + ATP FPH2 Acetate CoA transferase Fumarate
Succinate FPH2 Propionate FP
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.
150 EMP glyceraldehyde-3-P 2 PEP + 2 NADH glucose
glyceraldehyde-3-P + fructose-6-P 5/3 PEP + 11/3 NADH + CO2 HMP
ATP ADP NAD+ NADH sorbitol sorbitol-6-P fructose-6-P 2 PEP + 3 NADH
+ ATP ADP NAD NADH + CO2 Non-oxidative stage Pentose phosphate pathway gluconate 6-phosphogluconate ribulose-5-P 5/3 PEP + 8/3 NADH + CO2
HMP D-xylulose-5-P 5/3 PEP + 5/3 NADH
xylose 5 CO2 + 10 NADH Direct Oxidization
Figure 4.6 Proposed pathways of sugar utilization in P. acidipropionici.
151
~56 kDa
97
66
45
30
~37 kDa 20.1
14.4
1 2 3 4 5 6
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.
152
~56 kDa
97
66
~45 kDa 45
30
~37 kDa 20.1
14.4 1 2 3 4 5 6
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.
153 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-
154 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.
155 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,
156 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
157 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).
158 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.
159 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.
160 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.
161 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,
162 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
163 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).
164 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
165 (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
166 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.
167 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.
168 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.
169 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
170 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
171 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
172 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
173 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
174 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.
175 5.6 References
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179 Strain/Plasmid Relevant Characteristics Source/Reference Strains P. acidipropionici wild type ATCC ATCC 4875 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 This study without the inside EcoRI site 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 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.
180
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.
181
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 -1
182 Specific growth rate (h ) 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
a Based 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.
182
ack EcoRI EcoRI Partial ack 0.5 kb EcoRI EcoRI ColE1 f1 ori TOPOACK1 4.4 kb
Ampr Kanr ® ® f1 ori ColE1 pCR 2.1-TOPO 3.9 kb
XbaI and ApaI r Kan Digestion pDG1515 Ampr
ack EcoRI EcoRI XbaI ApaI XbaI ColE1
Tetr (2.1kb) pTAT Tetr 6.5 kb Ampr
ApaI Kanr f1 ori
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.
183 1 kb
700 bp PCR product 500 bp ~750 bp
200 bp
100 bp
Figure 5.2 PCR product of partial ack gene.
184 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.
185 EagI = XmaIII
Figure 5.4 Restriction enzyme map of partial ack gene from P. acidipropionici.
186 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.
187
1 2 3 4 1 2 3 4
2.8 kb 2.8 kb Probe 2 (1.9 kb)
ack
Probe 1 (0.5 kb) A B
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.
188
1 2 3 4 ~ 74 kDa
97
66
45
30
20.1
14.4
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.
189
160 ACK-Tet 140 pTAT-ACK-Tet 120
100
80
60
(%) Activity Relative 40 20
0 AK PTA
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.
190 40 9
35 Glucose 8 OD 7 30 6 25 5 20 Propionate 4 OD 15 3
Concentration (g/L) 10 2 Acetate 5 1 A Succinate 0 0 020406080
Time (h)
40 6
35 OD Glucose 5 30 4 25 20 Propionate 3 OD 15 2
Concentration (g/L) 10
Acetate 1 5 B Succinate 0 0 0 20 40 60 80 100 120 Time (h) 40 7
35 6 Glucose 30 OD 5 25 4 20 Propionate OD 3 15 2 Concentration (g/L) 10 5 Acetate 1
C 0 Succinate 0 0 20 40 60 80 100 120 Time (h)
Figure 5.9 Fermentations by P. acidipropionici. A: Wild type; B: ACK-Tet mutant; C: pTAT-ACK-Tet mutant.
191 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.
192 • 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.
193 • 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.
194 • 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.
195 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.
196 • 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.
197 • 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.
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212
APPENDIX A
MEDIUM COMPOSITIONS
213 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.
214
APPENDIX B
ANALYTICAL METHODS
215 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.
216 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.
217
Figure B.1 The HPLC chromatogram for standard containing glucose, succinic acid, lactic acid, acetic acid, and propionic acid.
218
Figure B.2 The sample HPLC chromatogram of propionic acid fermentation by P. acidipropionici using glucose as the substrate.
219 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.
220
0.6 ) 0.5
595 nm 0.4 ( 0.3 0.2 y = 1.0678x + 0.0303 R2 = 0.9853 0.1 Absorbance Absorbance nm) (595 0.0 0 0.1 0.2 0.3 0.4 0.5 0.6
BSA concentration (mg/mL)
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
221 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.
222 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
223 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.
224
0.8
) 0.6
830 nm ( 0.4 y = 11.376x R2 = 0.9992 0.2
Absorbance (830 nm) Absorbance 0 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 Inorganic Phosphate (mg)
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.
225 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.
226
Separating Stacking gel, 12% gel, 4% (2 gels) (2 gels) Distilled water (mL) 3.015 Distilled water (mL) 1.83 1.5 M Tris/HCl, pH 8.8 2.25 0.5 M Tris/HCl, pH 6.8 0.75 (mL) (mL) 10% (w/v) SDS (mL) 0.09 10% (w/v) SDS (µL) 30 Acrylamide/Bis-acrylamide 3.6 Acrylamide/Bis-acrylamide 0.39 (30%(w/v)) (mL) (30%(w/v)) (mL) 10% (w/v) ammonium 45 10% (w/v) ammonium 15 persulfate(µL) persulfate (µL) TEMED (µL) 4.5 TEMED (µL) 3 Total (mL) 9 Total (mL) 3
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.
227
Continued
Figure B.5 The analysis report of membrane fatty acid content of P. acidipropionici wild type.
228 Figure B.5 (continued)
Continued
229 Figure B.5 (continued)
230
Continued
Figure B.6 The analysis report of membrane fatty acid content of P. acidipropionici adapted mutant from the FBB.
231 Figure B.6 (continued)
Continued
232 Figure B.6 (continued)
233 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.
234
APPENDIX C
BIOREACTOR CONSTRUCTION AND OPERATION
235 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 Gas Liquid Outlet
Fibrous Matrix Gas Liquid Inlet
Figure C.1 A fibrous-bed bioreactor construction.
236
pH meter
pH CO2 probe Fibrous-bed bioreactor
6 M NaOH
Circulation pump
N2
Fermentor
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
237 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.
238
APPENDIX D
GENETIC ENGINEERING PROTOCOLS
239 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.
240 The gel electrophoresis of the genomic DNA from P. acidipropionici was shown in
Figure D.1.
Genomic DNA
1 kb
500 bp
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.
241 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.
242 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.
243 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.
244 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.
245 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
Volume Reagent (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.
246 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
247 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.
248 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 1kb 6. EcoRI-digested negative plasmid 500 bp
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
249 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:
Reagent Volume (µL) Blunt-end fragment 40 10X buffer 5 Dilution buffer 4 Shrimp alkaline phosphatase (1 U/µL) 1 Total volume 50
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:
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
7. Incubate at 72°C for 25 min.
250 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
251 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.
252 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.
Volume Reagent (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
253 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.
254 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.
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
Figure D.3 Restriction enzyme digestion of pSSF1.
255
APPENDIX E
REAGENTS AND BUFFERS
256 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.
257 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.
258