RICE UNIVERSITY ISOLATION, CHARACTERIZATION AND SUBSTRATE-TRANSPORT STUDIES OF A NEW, UNIQUE METHYLOTROPH by Thomas Alan Keuer A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF THE REQUIREMENTS FOR THE DEGREE Master of Science APPROVED, THESIS COMMITTEE: E. Terry Papoutsakis, Assistant Professor of Chemical Engineering LarryOf. Mclntire, Professor and Chairman of Chemical Engineering <03^ ' Roger Storck, Professor of Biology Houston, Texas April, 1984 3 1272 00289 0232 ABSTRACT Keuer, Thomas A. M.S. Rice University, April 1984. Isolation, Characterization and Substrate-Transport Studies of a New, Unique Methylotroph. Major Professor: E. T. Papoutsakis. Methylotrophic bacteria which assimilate carbon via the Ribulose Monophosphate Pathway are bioenergetically superior to other methylotrophs. The dehydrogenases which catalyze the oxidation of formaldehyde to formate and formate to CO2 in RMP bacteria produce much of the ATP required for biosynthesis. A strain, designated T15, has been isolated on the basis of high In vitro activities of the above two key enzymes, and has been biochemically characterized. The new strain exhibits high yields (up to 0.63 g cells/g MeOH) and growth rates (up to 0.46 hr“^) in batch culture? however, the yields and growth rates in continuous culture are significantly lower. Study of the transport mechanisms has provided valuable insight into the relationship between substrate uptake and the growth characteristics of T15. Experi¬ ments with radiolabelled substrates have indicated that methanol enters the cells primarily by diffusion? consequently, the bacteria are not able to accumulate methanol internally in order to support efficient Ill continuous growth. Formaldehyde, on the other hand, is accumulated by an active transport system which depends on the A pH component of the membrane proton-motive force. The formate uptake mechanism is also dependent on the ApH, but ‘is more complex, possibly due to the uncoupling effect of the organic acid on the cell membrane. ACKNOWLEDGMENTS I would like to recognize those whose assistance made the completion of this thesis possible. Dr. E. T. Papoutsakis, for support, guidance and encouragement during the course of this research. Dr. L. V. Inlntire and Dr. R. Storck, for serving on my thesis committee. Chris Bussineau, for his assistance in the early part of this research. Anil Diwan, for building the apparatus and develop¬ ing the procedure for the substrate-transport experiments, and for his generous help during the course of my work. Monsanto Corporation, for providing me with the opportunity and financial support to make this endeavor possible. TABLE OF CONTENTS Page LIST OF TABLES vi LIST OF FIGURES viii NOMENCLATURES ix ABSTRACT iii INTRODUCTION 1 MATERIALS AND METHODS 14 I. Chemicals and Biochemicals 14 II. Chemical Assays 15 III. Selection and Growth of the Microorgan¬ ism 16 IV. Dry Weight Determination 20 V. Preparation of Cell-Free Extracts ... 21 VI. Enzyme Assays 21 VII. Yield Measurements 23 VIII. Other Characterization Techniques ... 24 IX. Preparation of Whole Cell Suspensions . 26 X. Transport Studies 26 RESULTS AND DISCUSSION 32 I. Isolation and Selection of the Micro¬ organism .... 32 II. Other Enzyme Activities for T15 .... 35 i) 3-Hexulose Phosphate Synthase (HPS ) 36 ii) Methanol Dehydrognase (MDH) ... 37 iii) Glucose 6-phosphate Dehydrogenase (GPD) and 6-Phosphogluconate Dehydrogenase (PGD) ...... 38 III. T15 Batch Growth Experiments 40 i) pH Profile 40 ii) Growth on Pure Substrates .... 42 iii) Growth on Mixed Substrates. ... 46 V Page iv) Discussion: The Mechanism of Substrate Inhibition 48 v) T15 Batch Yields 52 IV. Systematics: General Characterization of T15 58 i) General Characterization 59 ii) Morphological Characterization . 59 iii) Physiological Tests. 60 V. Continuous Culture Experiments with T15. 62 VI. Formate Uptake by T15: Proton Transloca¬ tion Measurements 68 VII. Radiolabelled Substrate-Transport Studies 76 i) Measurement of T15 Cell Volume . 76 ii) ^C-labelled Methanol Uptake ... 80 iii) 1’C-labelled Formaldehye Uptake. 80 iv) l^C-labelled Formate Uptake. ... 84 v) Discussion 86 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK . 93 LIST OF REFERENCES 97 APPENDIX 102 LIST OF TABLES Table Page 1. NAD+-linked Formaldehyde and Formate Dehydrogenase Activities of Soil Isolates 34 2. T15 Growth on Various Carbon Sources 60 Appendix Table A1. Formaldehyde DH Activities for Isolated Strains 102 A2. Formate DH Activities for Isolated Strains 103 A3. T15 3-Hexulose Phosphate Synthase Activities 104 A4. T15 MeOH Dehydrogenase Activities 106 A5. T15 Glucose-6-Phosphate Dehydrogenase Activities 107 A6. T15 6-Phosphogluconate Dehydrogenase Activities 108 A7. T15 Batch Yield Data 110 A8. Continuous Culture Yields for T15 Ill A9. Glycerol Uptake Experiment #1 117 A10. Glycerol Uptake Experiment #2 118 All. Sucrose Uptake Experiment #1 119 A12. Sucrose Uptake Experiment #2 120 A13. Methanol Uptake Experiment #1 121 A14. Methanol UPtake Experiment #2 122 A15. Methanol Uptake Experiment #3 123 Table Page A16. Formaldehyde Uptake Experiment #1 124 A17. Formaldehyde Uptake Experiment #2 125 A18. Formaldehyde + KSCN Uptake Experiment .... 126 A19. Formaldehyde + FCCP Experiment 127 A20. Formate Uptake Experiment 128 A21. Formate + KSCN Experiment 129 A22. Formate + FCCP Experiment #1 130 A23. Formate + FCCP Experimetn #2 131 LIST OF FIGURES Figure Page 1. Oxidation Pathways in the Ribulose Monophosphate Cycle 4 2. The T15 pH profile 41 3. Batch growth curves for T15 at different initial methanol concentrations 43 4. The effect of fast transfers on T15 growth. 45 5. The inhibitory effect of formaldehyde on T15 growth 47 6. Batch growth curves with methanol and formate 49 7. Biomass yield as a function of initial methanol concentration from batch experi¬ ments 53 8. Formate-induced proton uptake by T15 cells as a function of formate concentration. 72 9. The pH dependence of formate-induced proton uptake 74 10. Glycerol uptake profile 78 11. Sucrose uptake profile 85 12. Methanol uptake profile 81 13. Formaldehyde uptake profile 83 14. Formate uptake profile 85 Appendix Figure Page Al. HPS activity as a function of reaction time . 105 A2. T15 cell dry weight determination 109 NOMENCLATURE ADP,ATP Adenosine di-triphosphate BTB Bromothymol blue indicator CCCP Carbonyl cyanide m-chlorophenyl hydrazone D Dilution rate DCPIP 2,6-dichlorophenol indophenol ddH20 Deionized, distilled water FCCP Carbonyl cyanide p-(triflouromethoxy)- phenylhydrazone FDDH Formaldehyde dehydrogenase FDH Formate dehydrogenase G PD Glucose-6-phosphate dehydrogenase HPS 3-hexulose phosphate synthase KSCN Thiocyanate MDH Methanol dehydrogenase NAD,NADH2 Nicotinamide adenine dinucleotide and its reduced form O.D. Optical density PGA 3-phosphoglycerate PGD 6-phosphogluconate dehydrogenase PGI Phosphoglucoisomerase PHI Phospho-3-hexulose isomerase pmf Proton-motive force PMS Phenazine methosulfate P/O Moles of inorganic phosphate recovered per atom of oxygen taken up RMP Ribulose monophosphate S Substrate concentration SCP Single cell protein X Biomass concentration X P/0 ratio for methanol oxidation X P/0 ratio for formaldehyde oxidation to formate Cell mass yield, g cells/ g substrate P/0 ratio for formate oxidation Specific growth rate 1 INTRODUCTION In the last twenty years, a vast amount of litera¬ ture has been generated on the physiology and biochemistry of methylotrophic bacteria. Methylotrophs are defined as microorganisms which are able to grow at the expense of reduced carbon compounds containing one or more carbon atoms but no carbon-carbon bonds (Colby and Zatman, 1972). Of special interest are those methylotrophic bacteria which grow on reduced one-carbon (C^) compounds, as they are ubiquitous in nature and are readily isolated from almost any sample of soil or water. Also, these bacteria play a vital role in the global biological carbon and nitrogen cycles. Aside from these fundamental character¬ istics, it was the appreciation of the potential of these bacteria for the production of single cell protein (SCP) from methanol which stimulated the isolation and study of many new species (Anthony, 1982). Methanol is superior to other substrates for SCP prodution due in part to the attractive industrial characteristics that it possesses. MeOH is inexpensive, is produced commercially in large amounts from a variety of chemical feedstocks, is very pure, and is completely miscible in water (Papoutsakis, 1976). Therefore, the methanol-utilizing 2 methylotrophic species are the most attractive for com¬ mercial exploitation. More than half of the operating cost in any SCP process is that of the carbon substrate (Cooney, 1975); consequently, maximization of the biomass yield (Yx/S) is the primary objective of any biochemical engineer working with this process. Perhaps the single most important factor which influences the biomass yield of a methylotrophic species is the biochemical pathway through which the substrate carbon is incorporated into biomass. Carbon assimilation occurs by means of three primary reaction schemes in methanol-utilizing methylo- trophs? the ribulose biphosphate, serine, and ribulose monophosphate pathways. Anthony (1982) has provided a detailed summary and description of the biochemical reactions in each of these pathways. In the ribulose biphosphate pathway, or Calvin cycle, all of the carbon is assimilated at the oxidation level of CO2. The serine pathway involves the formation of cell material from both formaldehyde and CO2. In the ribulose monophos¬