Butanediol Production by Paenibacillus Polymyxa DSM 365
Total Page：16
File Type：pdf, Size：1020Kb
Process development and metabolic engineering to enhance 2,3- butanediol production by Paenibacillus polymyxa DSM 365 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Christopher Chukwudi Okonkwo Graduate Program in Animal Sciences The Ohio State University 2017 Dissertation Committee: Thaddeus C. Ezeji, Advisor Ramesh Selvaraj Katrina Cornish Ana Alonso Copyrighted by Christopher Chukwudi Okonkwo 2017 Abstract 2,3-Butanediol (2,3-BD) is a platform chemical with vast industrial applications; particularly for its use in the production of 1,3-butadiene (1,3-BD), the monomer from which synthetic rubber is manufactured. Currently, 2,3-BD production is by chemical synthesis using petroleum-derived feedstocks such as propylene, acetylene, butene and butane. Microbial 2,3-BD fermentation is aimed at producing 2,3-BD renewably, and potentially reduce dependency on finite petroleum-derived feedstocks. However, fermentative production of 2,3-BD is hampered by (1) cost of food-based substrates; (2) low 2,3-BD titer, yield and productivity during 2,3-BD fermentation stemming from formation of competing products such as exopolysaccharides (EPS), ethanol, lactic, formic and acetic acids; and (3) high cost of 2,3-BD purification, due partly to additional purification steps necessary to remove 2,3-BD co-products especially EPS prior to 2,3- BD recovery. The objectives of this study were conceived to examine use of process design, alternative substrates and metabolic engineering, to enhance 2,3-BD production. Chapter 3 (objective 1) focused on identification of key fermentation parameters that influence 2,3-BD fermentation by Paenibacillus polymyxa and optimization of them for maximum 2,3-BD production. The study examined the impact of yeast extract, tryptone, ammonium acetate, ammonium sulfate, and crude glycerol concentration, and inoculum size and fermentation temperature on 2,3-BD production by P. polymyxa. The ii results showed that only three parameters (tryptone, temperature and inoculum size) had significant effects on 2,3-BD production by P. polymyxa. The three factors were optimized and 2,3-BD production by P. polymyxa increased from ~27 g/L to 51.1 g/L in batch bioreactor cultures and from 47 g/L to 68.5 g/L in fed-batch cultures. The improvement in 2,3-BD production by P. polymyxa was accompanied by 11% and 19% reduction in ethanol and EPS formation, respectively, when compared to the un- optimized fermentation medium and conditions. Due to the inability of P. polymyxa to produce more than 6% 2,3-BD in fed-batch cultures, and the attendant increase in the accumulation of acetoin (the precursor from which 2,3-BD is biosynthesized) in the bioreactor, chapter 4 (objective 2) focused on understanding 2,3-BD-mediated feedback inhibition during 2,3-BD fermentation. This study evaluated the response of P. polymyxa to high 2,3-BD concentrations during growth and 2,3-BD fermentation. Cultures of P. polymyxa were challenged with levo-2,3-BD (20, 40 and 60 g/L) at 0 h fermentation in a glucose medium. The inhibition of P. polymyxa growth by levo-2,3-BD was concentration dependent, triggering total growth inhibition when the concentration of 2,3-BD attained 60 g/L. Furthermore, when P. polymyxa was challenged with incremental 2,3-BD concentrations (20, 40 and 60 g/L at 12, 24 and 36 h, respectively) to mimic 2,3-BD accumulation during fermentation, 2,3-BD was reconverted to acetoin when its concentration reached 60 g/L, possibly to alleviate 2,3-BD toxicity. Chapter 5 (objective 3) evaluated the feasibility of using readily available non- food lignocellulosic biomass (LB) as substrate for 2,3-BD fermentation. Pretreatment of LB to release fermentable sugars is accompanied by the generation of lignocellulose- iii derived microbial inhibitory compounds (LDMICs) such as furfural, hydroxymethyl- furfural (HMF), and phenolic compounds which inhibit growth and pose a significant roadblock to LB use as substrates. The study investigated the ability of P. polymyxa to use LB-based agricultural residue, wheat straw hydrolysate (WSH), for the production of 2,3-BD. Prior to testing the fermentability of WSH to 2,3-BD, the ability of P. polymyxa to co-metabolize the representative mixed sugars (glucose, xylose and arabinose) of WSH was evaluated. The results show that P. polymyxa simultaneously co-metabolized the mixed sugars (glucose, xylose and arabinose) component of LB to 2,3-BD without exhibiting signs of carbon catabolite repression characteristics. Batch fermentations conducted using 60%, 80%, and 100% WSH, and a glucose-based control, showed that the growth of P. polymyxa increased 17%, 27% and 32% in 60%, 80% and 100% WSH, respectively, relative to the glucose control medium. 2,3-BD production in 60%, 80% and 100% WSH was 32, 31 and 23 g/L, respectively, which was comparable to the 32 g/L obtained in the glucose-based control. The enhanced growth in WSH suggests that P. polymyxa might have sequestered additional carbon from LDMICs. Hence, the ability of P. polymyxa to use LDMICs as sole carbon sources was investigated. The growth of P. polymyxa in HMF increased 2.4-fold relative to the control with no carbon source which suggested that P. polymyxa might have utilized HMF in WSH for cell biomass accumulation. In addition, P. polymyxa showed robust tolerance to furfural and phenolic compounds (coumaric acid, vanillic acid and vanillin) during fermentation. Chapter 6 (objective 4) explored a metabolic engineering strategy to deactivate the EPS production pathway of P. polymyxa and drastically reduce or eliminate EPS iv production during 2,3-BD fermentation. The study identified a levansucrase gene which encodes levansucrase, the enzyme responsible for EPS biosynthesis in P. polymyxa. The results showed that the levansucrase gene was successfully disrupted, and the resulting P. polymyxa levansucrase null mutant showed 34% and 54% increases in growth in sucrose and glucose media, respectively. Additionally, the P. polymyxa levansucrase null mutant grown in sucrose and glucose media produced 6.4- and 2.4-fold lower EPS, respectively, than that produced by the P. polymyxa wildtype. The observed decrease in EPS formation by the levansucrase null mutant may be a direct cause of the 4-27% increase in 2,3-BD yield, and 4-128% increase in 2,3-BD productivity observed during 2,3-BD fermentation. Interestingly, the levansucrase null mutant remained genetically stable over fifty generations with no observable decrease in growth, 2,3-BD and EPS formation. Collectively, our results show that P. polymyxa levansucrase null mutant has potential for improving the economics of large-scale microbial 2,3-BD production. v Acknowledgments I am deeply indebted to my advisor Dr. Thaddeus Ezeji for his support, guidance and encouragement throughout the course of my graduate program. I would like to thank Dr. Ezeji specially for providing me the privilege and opportunity to conduct research under his mentorship. Dr. Ezeji offered me impeccable support through difficult times, research wise and offered me a research assistantship for over three years, which allowed me grow as a scientist. The training I received under his tutelage and mentorship has transformed me. I am eternally grateful to Dr. Victor Ujor for guiding me through good laboratory practices, scientific writing and for his invaluable advice and suggestions towards overcoming experimental challenges during the course of my graduate program. I also wish to thank him for meticulously reading the first draft of my PhD dissertation. I wish to thank a friend and colleague, Dr. Chidozie Agu for introducing me to the laboratory during the first few weeks of my graduate program and for being a wonderful team mate. I wish to thank members of my committee; Dr. Ramesh Selvaraj, Dr. Katrina Cornish and Dr. Ana Alonso for agreeing to serve on my committee and for their invaluable advice and constructive comments that led to the completion of this research. I am honored to have you all in my committee. I appreciate all past and present graduate students, faculty members and staff in the Department of Animals Sciences, The Ohio vi State University. I specially thank the Department of Animal Sciences for the invaluable contribution to the research funding that made my research work possible. I thank OARDC Graduate Research Enhancement Competitive Grants Program (OARDC SEEDS Grant) for research funding. I greatly appreciate Drs. Gabriel and Jane Okafor for sharing information leading to this research opportunity and for their encouragement and good will. I wish to express my special gratitude to my friends, Mary Mbah, Mrs. Dana Ujor, Drs. Segun and Foluke Awe, Gloria Okpala and Sarah Emereonye for their moral support and encouragement. I would like to express my gratitude to Prof. Obioma Njoku, Prof. Lawrence Ezeanyika, Prof. Edwin Alumanah and Prof. Ferdinand Chilaka for their moral support. My heartfelt gratitude goes to my parents, Mr. and Mrs. Christopher C. Okonkwo for their love and support. I also wish to thank my siblings for their prayers and moral support. Above all, my profound gratitude goes to almighty God for giving me the grace and endurance to complete this research. vii Vita 2008…………………………………………B.Sc. Biochemistry, University of Nigeria 2012………………………………………... M.Sc. Biochemistry, University of Nigeria 2013 to 2017 ..................................................Graduate Research Associate, Department of Animal Science, The Ohio State University Publications Okonkwo CC, Ujor V, Mishra, P, and Ezeji TC (2017) Process development for enhanced 2,3-butanediol production by Paenibacillus polymyxa DSM 365. Fermentation (Accepted). Okonkwo CC, Ujor CV, and Ezeji TC (2017) Investigation of relationships between 2,3- butanediol toxicity and production during growth of Paenibacillus polymyxa. New Biotechnology, 34:23-31 viii Okonkwo CC, Azam, MM, Ezeji TC, and Qureshi N (2016) Enhancing ethanol production from cellulosic sugars using Scheffersomyces (Pichia) stipitis.