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Pao Yue-kong Library, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong http://www.lib.polyu.edu.hk 1,3-PROPANEDIOL AND CAPROATE CO-PRODUCTION THROUGH GLYCEROL FERMENTATION AND CARBOXYLATE CHAIN ELONGATION IN MIXED CULTURE LENG LING Ph.D The Hong Kong Polytechnic University 2018 The Hong Kong Polytechnic University Department of Civil and Environmental Engineering 1,3-PROPANEDIOL AND CAPROATE CO-PRODUCTION THROUGH GLYCEROL FERMENTATION AND CARBOXYLATE CHAIN ELONGATION IN MIXED CULTURE LENG Ling A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2017 CERTIFICATE OF ORIGINALITY I hereby declare that this thesis is my own work and that, to the best of my knowledge and belief, it reproduces no material previously published or written, nor material that has been accepted for the award of any other degree or diploma, except where due acknowledgement has been made in the text. (Signed) LENG Ling (Name of Student) I DEDICATION To my family and friends who love and trust me To my supervisor who inspires me and has supported me every step of the way To the fellows who devote to this research field To the microorganisms that complete the pathways Also to the endless nights accompanying my growth II ABSTRACT Mixed culture chain elongation of short chain fatty acids (SCFAs) for a medium chain fatty acid (MCFA), caproate, formation is an attractive option for resource recovery in anaerobic wastewater treatment. Caproate, a value-added chemical, is slightly soluble in water and can be used by various industries. Biological production of caproate with ethanol as electron donor has been successfully achieved in anaerobic mixed culture. However, the underlying metabolic pathways of microorganisms except Clostridium kluyveri are not well understood. Another potential electron donor is glycerol which is presently being generated in surplus with the rapid growth of the biodiesel industry. In the current approach, an industrial chemical, 1,3-propanediol (1,3-PDO) is produced from crude glycerol along with a formation of other soluble byproducts including ethanol and SCFAs, which necessitates a significant amount of energy input for separation and purification. To circumvent the energy sink requirement and upcycle both the wastewater treatment process and the biodiesel industry, it is highly beneficial to co-produce caproate from the byproducts of glycerol dissimilation along with 1,3-PDO. At first, thermodynamic and physiological insights gained into the co-production of 1,3-PDO and caproate from glycerol are investigated. Thermodynamics analysis demonstrated that a higher pH range is more favorable when either glycerol or ethanol acting as an electron donor, whereas a high partial pressure (27% at 1 atm) and a low pH (≤ 5.5) are advantageous for caproate formation with hydrogen. With III the glycerol-to-acetate molar ratio of 4 and pH of 7, the physiological experiments achieved a co-production of 1,3-PDO and caproate. However, the caproate yield was low and found to be kinetic-limited. Caproate formation was significantly increased by the intermediate ethanol addition with the optimal mono-caproate formation obtained at the ethanol-to-acetate molar ratio of 3. A synergistic relationship was evinced through microbial characterization, resulting in C. kluyveri and some bacteria with function of converting glycerol to SCFAs. Whilst the metabolic pathway of C. kluyveri in carboxylates chain elongation has been discovered, the role of other co-existing microbiomes which promote the elongation remained unclear in mixed culture. Thus, we conducted a fermentation experiment at optimal conditions which is inoculated with fresh anaerobic digestion (AD) sludge and fed with ethanol and acetate. Both 16S rRNA gene-based amplicon and shotgun metagenomics sequencing were employed to elucidate the mixed culture chain elongation by uncovering the microbes and functional pathways. Results revealed a synergistic relationship between C. kluyveri and three co-dominant species Desulfovibrio vulgaris, Fusobacterium varium and Acetoanaerobium sticklandii. The co-existence of these three species were able to boost the carboxylates chain elongation by C. kluyveri. Draft genomes of C. kluyveri, D. vulgaris and A. sticklandii were successfully recovered, revealing that butyrate and caproate can be directly produced from ethanol and acetate by C. kluyveri and indirectly produced through a syntrophic partnership between D. vulgaris and A. sticklandii with IV hydrogen serving as a reducing equivalent messenger. This study presents evidences of a syntrophic partnership between bacterial species and unveils an intricate and synergistic microbial network in mixed culture carboxylates chain elongation. Moreover, this study enriched a microbial community capable of efficiently co-producing 1,3-PDO and caproate via glycerol fermentation and carboxylate chain elongation. A co-production of 6.38 mM C 1,3-PDO d-1 and 2.95 mM C caproate d-1 was achieved in a 2-L semi-continuous fermenter with a glycerol-ethanol-acetate stoichiometric ratio of 4:3:1. Microbimes, E. limosum, C. kluyveri and M. senegalense, utilize a unique combination of metabolic pathways to facilitate the above conversion. Based on metagenomics, E. limosum is capable of converting glycerol to 1,3-PDO, ethanol and H2, and also redirecting the electron potential of H2 into acetate via the Wood–Ljungdahl pathway for chain elongation. C. kluyveri worked synergistically with E. limosum by consuming ethanol and acetate for caproate production. M. senegalense encodes for ethanol oxidation to acetate and butyrate, facilitating the caproate production by C. kluyveri. During the transition between fermentation and elongation, an unexpected phenomenon of poly-β-hydroxybutyrate (PHB) formation and reutilization by M. senegalense was observed, which may be associated with butyrate formation for further caproate generation. Significant ethanol production as an intermediate of glycerol dissimilation and the non-inhibiting level of 1,3-PDO production, which allows the dominance of C. kluyveri, are key to increasing caproate production. V Finally, a batch test of glycerol fermentation for the co-production with ethanol self-sufficiency inoculated by the fermenter-enriched microbial community was conducted. This study answers whether the enriched versatile glycerol degrader, E. limosum, could convert glycerol-derived energy to ethanol and H2 in a balance with 1,3-PDO and acetate and whether the ethanol could be further utilized by carpoate producer within the cultivation matrix. In addition, this study also investigated the electron flux of glycerol fermentation and chain elongation. The co-production of 1,3-PDO and caproate was achieved with a favorable glycerol/acetate stoichiometric ratio. Significant ethanol production from glycerol oxidation is the main reason for the caproate production enhancement. A dynamic balance of three dominant microbiomes, E. limosum, M. senegalense, and C. kluyveri, could complete the multiple stages co-production process. E. limosum dominated in the glycerol fermentation phase, while M. senegalense and C. kluyveri worked together for caproate production with ethanol and acetate in the carboxylates chain elongation phase. Redirection of the electron potential of H2 back into acetate for chain elongation by E. limosum and PHB formation and reutilization by M. senegalense were proved by electron flux calculation. The physiological performance and dynamic microbial community disclosed a unique combination of metabolic pathways successfully facilitated the co-production. The knowledge gleaned paves new avenues for both the wastewater treatment process and the biodiesel industry by upcycling their resources recovery. VI PUBLICATIONS ARISING FROM THE THESIS Journal Papers: 1. LENG, L., Yang, P., Mao, Y., Wu, Z., Zhang, T., & Lee P.-H. (2017). Thermodynamic and physiological study of caproate and 1,3-propanediol co-production through glycerol fermentation and fatty acids chain elongation. Water Res. 114, 200-209. 2. LENG, L., Yang, P., Singh, S., Zhuang, H., Xu L., Chen W.-H., Dolfing, J., Li, D., Zhang, Y., Zeng, H., Chu W., & Lee P.-H. (2017). A review on the bioenergetics of anaerobic microbial metabolism close to the thermodynamic limits and its implications for digestion applications. Bioresour Technol. 247, 1095-1106. 3. LENG, L., Nobu, M. K., Narihiro, T., Yang, P., Tan, G.-Y., & Lee P.-H. (2017). Biological co-production of