BUTYRIC ACID PRODUCTION FROM RENEWABLE RESOURCES By Adam Marschall Jaros A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Food Science – Doctor of Philosophy 2014 ABSTRACT BUTYRIC ACID PRODUCTION FROM RENEWABLE RESOURCES By Adam Marschall Jaros Butyric acid is globally marketed with uses ranging from flavor additive to cattle feed enhancer to chemical feedstock for construction materials. Naturally found in milks, butter and numerous cheeses, butyric acid imparts a buttery flavor to food products. The manufacture and sale of butyric acid as a food flavoring for human consumption is subject to market demand for all-natural products. The “all-natural” labeling requirements preclude the use of synthetic or petro-chemical sourced compounds as a component of the product. In order for producers to cater to the demand for all-natural products alternative methods for generating butyric acid must be developed and evaluated. The fermentation production of butyric acid is an all-natural method utilizing the anaerobic bacteria Clostridium tyrobutyricum from hydrolyzed lignocellulosic biomass, a renewable resource. The overall hypothesis of this dissertation is that real-world lignocellulosic hydrolysates are feasible fermentation substrates for generating butyric acid and challenges from using lignocellulosics, such as acetate inhibition, can be overcome. Hydrolysis of hemicellulose, a component of lignocellulose, through hot water extraction provides a low-cost source of xylose for the fermentation production of butyric acid but also yields acetate, a microbial inhibitor. This dissertation provides a characterization of the effects of acetate on the fermentation production of butyrate (dissociated form of butyric acid at fermentation pH) and reveals that the lowered productivity is due to an extended lag-phase in bacterial growth. However, once the acetate induced lag-phase is overcome, acetate (26.3 g/L) challenged batch fermentations of C. tyrobutyricum demonstrate higher butyrate yields (12.6%) owing to an acetate re-uptake metabolic mechanism. Selective adaptation of C. tyrobutyricum cultures to the presence of 26.3 g/L acetate developed tolerant cultures with high butyrate productivity rates compared with non-adapted C. tyrobutyricum cultures under comparable batch conditions. Enzyme activity for the metabolic enzyme acetate kinase (responsible for acetate production) was evaluated and reduced activity was observed equally in both acetate tolerant and control C. tyrobutyricum in the presence of acetate. Lignocellulosic hydrolysate material having undergone pretreatments of either ammonia- fiber expansion (AFEX), alkaline hydrogen peroxide (AHP) or extracted ammonia-fiber expansion (E-AFEX) was obtained for fermentation trials on C. tyrobutyricum and all were adequate as fermentation substrates. A 1-liter batch fermentation of AFEX hydrolysate was conducted and similar sugar consumption and butyrate production was observed compared with a control batch. The C. tyrobutyricum culture fermenting AFEX hydrolysate experienced half the specific growth rate during log-phase growth than the control batch. Taken together, these results indicate that renewable lignocellulosic biomass is a feasible fermentation substrate for the fermentation production of butyric acid. ACKNOWLEDGEMENTS Foremost, I would like to thank my Ph.D advisor Professor Kris A Berglund for accepting me into his lab and providing me with the opportunity to undertake graduate research in both the Department of Food Science and Human Nutrition at Michigan State University and the Division of Sustainable Process Engineering at Luleå Technical University. Completing these programs has been a lot of hard work but Dr. Berglund has been an inspiration of ideas and research concepts the entire time. I thank him for his confidence in my work and motivation to follow through on my research. I would also like to thank my dissertation guidance committee members Dr. Strasburg, Dr. Linz and Dr. Hodge. All three have been there for me over the past several years to provide guidance and constructively challenge me. I appreciate the time I have spent with my lab-mates from the Berglund lab including Dr. Tim Petrik and John Jeffery. Conversations with the two of them were always inspiring and led to some interesting experimentation, especially in beverage distillation. Thanks also to fellow laboratory colleagues Jacob Rochte for always covering for me at the distillery and Stephen Peabody for tirelessly assisting with lab work. I thank my parents, Squire Jaros and Dr. Christel Marschall who have always supported me to follow my dreams and instilled the wisdom in me to pursue my education. I love you both and appreciate the opportunities you have granted me in life. iv I am grateful for my supportive friends, especially John Baer III, Nick and Lauren Noel, Christopher Singleton, Erica Clark, Dr. Lisa Harlow, Dr. Brenna Flannery and Annie Ledbury are always there for me when I need support and I am thankful for it. v TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………….…….ix LIST OF FIGURES……………………………………………………………………..………...x INTRODUCTION………………………………………………………………………………...1 Overall Hypothesis………………………………………………………….............................1 Chapter Summaries……………………………..…………..…………………………………3 REFERENCES ………………………………………….....………………………………..10 CHAPTER 1: LITERATURE REVIEW………………………………………………………...12 Motivation………………………………..…………………………………………………..12 Biorefinery………………………………………………………..………………………… 14 Lignocellulosic Chemistry and Pretreatments…………………………………………..…...17 Microbial Inhibition………………………………………………..………………………...20 Clostridial Fermentation…………………………………………………..…………………23 Production of Butyric Acid by Clostridia tyrobutyricum Fermentation………………..……24 REFERENCES………………………………………………………………….……….…..29 CHAPTER 2: THE EFFECT OF ACETATE ON THE FERMENTATION PRODUCTION OF BUTYRATE………………………………………………………………………………....36 Abstract………………………………………………………………………………………36 Introduction…………………………………………………………………………………..37 Experimental: Bacterial Strain, Media and Growth………………………………………….39 Fermentations………………………………………………………………………………...40 Analytical Methods…………………………………………………………………………..41 Cell Biomass Determination…………………………………………………………………41 Results and Discussion………………………………………………………………………41 Acetate Re-Utilization……………………………………………………………………….48 Conclusion…………………………………………………………………………………...51 Acknowledgements…………………………………………………………………………..52 APPENDIX………………………………………………………..…………………………53 REFERENCES………………………………………………………………………………60 CHAPTER 3: ACETATE ADAPTATION OF CLOSTRIDIA TYROBUTYRICUMFOR IMPROVED FERMENTATION PRODUCTION OFBUTYRATE….................................63 Abstract………………………………………………………………………………………63 Introduction……………………………………………………………..................................64 Methods………………………………………………………………………………………68 Microorganism and Adaptation……………………………………………………………...68 Fermentations………………………………………………………………………………...69 Analytical Methods…………………………………………………………….…………….70 Dry Cellular Weight Determination………………………………………..…………...……71 Specific Growth Rate (µnet)...……………………………………………………………….71 vi Acetate Kinase Assay………………………………………………………………………..71 Results………………………………………………………………………………………..72 Fermentation Kinetics………………………………………………………………………..72 Acetate Kinase Activity……………………………………………………………………...79 Discussion……………………………………………………………………………………81 Conclusion…………………………………………………………………………………...85 Acknowledgements…………………………………………………………………………..86 APPENDIX…………………………………………………………………………..………87 REFERENCES……………………………………………………………………….….…100 CHAPTER 4: BUTYRATE FERMENTATION PRODUCTION FROM PLANT BIOMASS HYDROLYSATE...........................................................................................................…...103 Introduction…………………………………………………………………………………103 Materials and Methods……………………………………………………………………...108 AFEX-CS…………………………………………………………………………………...108 E-AFEX-CS………………………………………………………………………………...109 AHP-CS……………………………………………………………………………….........109 Specific Growth Rate (µnet)………………………………………………………………...110 Fermentations……………………………………………………………………………….110 Analytical Methods…………………………………………………………………………111 Results and Discussion……………………………………………………………………..111 Conclusions…………………………………………………………………………………122 APPENDIX…………………………………………………………………………………124 REFERENCES……………………………………………………………………………..127 CHAPTER 5: ADAPTATION TO SUBSTRATE INHIBITION FROM HIGH INITIAL GLUCOSE CONCENTRATION……………………………………..……………….…130 Introduction…………………………………………………………………………………130 Methods……………………………………………………………………………………..134 Adaptation Selection………………………………………………………………………..134 Fermentations……………………………………………………………………………….135 Analytical Methods…………………………………………………………………………135 Dry Cellular Weight Determination………………………………………………………...136 Specific Growth Rate (µnet)………………………………………………………………...136 Results and Discussion……………………………………………………………………..136 Conclusions…………………………………………………………………………………139 APPENDIX…………………………………………………………………………………140 REFERENCES……………………………………………………………………………..144 CHAPTER 6: THE APPLICATION OF INDUCED WHOLE-CELL PICHIA PASTORIS IN A NATURAL BIOCONVERSION OF BUTANOL TO BUTYRALDEHYDE…………......146 Abstract……………………………………………………………………………………..147 Introduction…………………………………………………………………………………147 Background…………………………………………………………………………………148 The Whole-Cell Bioconversion System……………………………………………...……..149 Materials and Methods………………………………………………………….…..………151 Preparation of Induced Pichia pastoris Cells…………………..…………………….……151 vii Analytical Methods…………………………………………………………………………153 AOX Assay…………………………………………………………………………………153