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An Integrated Study on Microbial Community in Anaerobic Digestion Systems

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An Integrated Study on Microbial Community in Anaerobic Digestion Systems An Integrated Study on Microbial Community in Anaerobic Digestion Systems DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yueh-Fen Li Graduate Program in Environmental Science The Ohio State University 2013 Dissertation Committee: Dr. Zhongtang Yu, Advisor Dr. Brian Ahmer Dr. Richard Dick Dr. Olli Tuovinen Copyrighted by Yueh-Fen Li 2013 Abstract Anaerobic digestion (AD) is an attractive microbiological technology for both waste treatment and energy production. Microorganisms are the driving force for the whole transformation process in anaerobic digesters. However, the microbial community underpinning the AD process remains poorly understood, especially with respect to community composition and dynamics in response to variations in feedstocks and operations. The overall objective was to better understand the microbiology driving anaerobic digestion processes by systematically investigating the diversity, composition and succession of microbial communities, both bacterial and archaeal, in anaerobic digesters of different designs, fed different feedstocks, and operated under different conditions. The first two studies focused on propionate-degrading bacteria with an emphasis on syntrophic propionate-oxidizing bacteria. Propionate is one of the most important intermediates and has great influence on AD stability in AD systems because it is inhibitory to methanogens and it can only be metabolized through syntrophic propionate- oxidizing acetogenesis under methanogenic conditions. In the first study (chapter 3), primers specific to the propionate-CoA transferase gene (pct) were designed and used to construct clone libraries, which were sequenced and analyzed to investigate the diversity and distribution of propionate-utilizing bacteria present in the granular and the liquid portions of samples collected from four digesters of different designs, fed different ii feedstocks, and operated at different temperatures. The results suggest that propionate metabolism can be affected by feedstocks and partitions differently between the solid and the liquid phases in digesters. Additionally, cluster-specific real-time PCR assays were developed and used in quantifying the abundance of different types of propionate- metabolizing bacteria. The second study (chapter 4) focused on developing a propionate- specific whole-cell bacterial biosensor that can serve as an alternative tool to measure concentrations and bioavailability of propionate in digesters. The biosensor was based on the transcription fusion of luxCDABE operon under the control of the promoter PprpB. The biosensor was shown to be specific to propionate and to report signal in a dose- dependent manner. The utility of the biosensor was also tested on several digester samples following dilution. The second half of my research focused on the microbial ecology in different AD systems, particularly on community composition and succession in response to changes in feedstock, organic loading rate, and operation. One study (chapter 5) investigated the spatial and temporal patterns of microbial composition within a full-scale mixed plug- flow loop reactor (MPFLR) treating dairy manure using DGGE, 454 pyrosequencing, and genus-specific quantitative PCR. The results showed that small microbial and chemical gradients existed within the digester, and the digestion process occurred similarly throughout the MPFLR digester. In another study (chapter 6), the community comparison in two lab-scale solid-state anaerobic digesters (SS-AD) fed the same feedstock (i.e., corn stover) but operated at different temperature (mesophilic vs. thermophilic temperature) was examined and compared using Illumina sequencing of 16S rRNA gene amplicons. iii Temporal succession in the microbial communities and methanogen populations were also examined. Moreover, canonical correspondence analysis (CCA) was used to identify correlation between microbial populations and the environmental/performance factors of the digesters. This study demonstrated that a distinct microbiome was formed at each of the two operation temperatures, with a greater microbial diversity and evenness observed during the mesophilic SS-AD than during the thermophilic SS-AD and suggested that syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis may be an important pathway for biogas production from acetate in the SS-AD. Because the same seed sludge was used to start up both the mesophilic and the thermophilic SS-ADs, the difference in the microbial communities observed in these two digesters might be attributed to both selection and adaptive diversification. The last study of my research (chapter 7) investigated the community successions and population dynamics in the thermophilic and the mesophilic digesters of a lab-scale temperature-phased anaerobic digester (TPAD) system fed a mixture of dairy manure and whey for co-digestion using Illumina sequencing. The TPAD system was intentionally fed with increasing amounts of readily digestible substrates (whey) to mimic organic overloading and to create an opportunity to examine how organic overloading affects different bacteria and methanogens. CCA was used to elucidate the correlation between microbial groups and the digester conditions/performance. The results showed that the microbial composition was affected by the organic overloading and revealed dramatic successions from a stable community structure to another distinct one, especially in the thermophilic digester. The iv CCA revealed that temperature and pH were the most influential environmental factors that explained much of the variations of the microbial communities in this TPAD system. Collectively, this series of studies advanced our understanding of the microbial community that underpins the AD process and elucidated possible roles that some bacterial and methanogens play in anaerobic digesters. The pct-specific qPCR and the propionate biosensor may also be useful tools in future studies on AD processes and in improving operation of anaerobic digesters. v Dedication To my father and mother. vi Acknowledgements First, I sincerely thank my advisor Dr. Zhongtang Yu for his support in all ways. Without his acceptance of me being in his lab, his generous support and great guidance, my study and the completion of this dissertation would not be possible. I would also like to show my appreciation to my committee members: Drs. Brian Ahmer, Richard Dick, and Olli Tuovinen. Thank you for the constructive suggestions that improve my research. My special appreciation to Dr. Ahmer for his kindly sharing of bacterial strains, vectors, instruments, and for the assistance from his lab members. Secondly, I would like to thank all the current and past lab members, especially Neslon, Lingling, and Jill. Thank you for your technical support and assistance on my research. I would like to thank my friends in the department of Animal Sciences, TWSA, NTUAA, ESGP, and Buckeye table tennis club, especially Paonan, Judy, Danni, Cressman, Josie, Anita, Po-Hsu, Riu, and Maureen. Your accompany and friendship make my study in OSU and life in Columbus wonderful and enjoyable most of the time. Last, my greatest appreciation to my parents, brother, sister, family members, and to a special friend Andre. Thank you for being exemplary, thank you for being strong, thank you for being endless supportive and encouraging, and thank you for always being there for me. vii Vita 2000..........................................................National Kinmen Senior High School, Taiwan 2004..........................................................B.S. Bioenvironmental Systems Engineering, National Taiwan University, Taiwan 2006..........................................................M.S. Bioenvironmental Systems Engineering, National Taiwan University, Taiwan 2006 to 2008 ............................................Research Assistant/Lab Manager, Bioenvironmental Systems Engineering, National Taiwan University, Taiwan 2008 to present ........................................Graduate Research/Administrative Associate, Environmental Science Graduate Program, The Ohio State University Publications Li YF, Wei S, Yu Z. 2013. Feedstocks affect the diversity and distribution of propionate CoA-transferase genes (pct) in anaerobic digesters. Microb Ecol. 66(2): 351-362. Li YF, Li FY, Ho CL, Liao VH. 2008. Construction and comparison of fluorescence and bioluminescence bacterial biosensors for the detection of bioavailable toluene and related compounds. Environ Pollut. 152(1): 123-129. Fields of Study Major Field: Environmental Science Focus: Microbial Ecology viii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... vi Acknowledgements ........................................................................................................... vii Vita ................................................................................................................................... viii Table of Contents ............................................................................................................... ix List of Tables ....................................................................................................................
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