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An Integrated Investigation of the Microbial Communities Underpinning Biogas Production in Anaerobic Digestion Systems DISSERTAT

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An Integrated Investigation of the Microbial Communities Underpinning Biogas Production in Anaerobic Digestion Systems DISSERTAT An Integrated Investigation of the Microbial Communities Underpinning Biogas Production 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 Michael Christopher Nelson Graduate Program in Environmental Science The Ohio State University 2011 Dissertation Committee: Dr. Mark Morrison, Advisor Dr. Zhongtang Yu, Co-Advisor Dr. Olli Tuovinen Copyrighted by Michael Christopher Nelson 2011 ABSTRACT Anaerobic digestion (AD) has been used for decades as a waste processing technology, however its ability to serve as a potential renewable energy resource has spurred increased attention into how the microbial communities in AD systems carryout the digestion process and what factors influence their activity. Previous analyses of the microbial diversity in AD were generally based on community analysis techniques such as DGGE or small sequencing libraries. Additionally, conflicting results have been reported regarding the diversity and abundance of Bacteria and Archaea in AD systems. The overall objective of my research was to further describe the microbial consortia that participate in the AD process as well as to investigate the patterns of their diversity. In the first study an initial baseline of the microbial diversity participating in the AD process is established using a meta-analysis approach. This removed the bias inherent in individual studies, allowing the global diversity of microbes in AD systems to be determined. The major bacterial groups were identified as the phyla Chloroflexi, Proteobacteria, Firmicutes, and Bacteroidetes, while the largest archaeal groups were the genus Methanosaeta and an as yet uncultured clade known as WSA2/ArcI. The second study was focused on determining the effects of feedstock and biomass fraction on the microbial communities in a sequential digester operation. Using large 16S rRNA clone libraries, both the bacterial and archaeal populations were examined. The resulting ii communities were found to be very distinct, indicating feedstock composition had a large effect on selecting a microbial community best suited to that particular feedstock. Differences were also observed between the granular and liquid biomass fractions, indicating potential differences in the metabolic role of these two microbial communities within a single AD system. Using pyrosequencing, the third study was designed to examine changes in microbial community structure in relation to known reactor operational parameters in an AD system operated for a yearlong period. Analysis of six time points showed a highly variable microbial community, with large shifts in microbial abundance observed at the phylum level for the Bacteria. Within the Archaea, a significant shift from Methanosaeta to Methanosarcina was observed due to additions of acetate to the system, with an increase of unclassified Euryarchaeota due to an increase in temperature. Exploratory use of the statistical analysis method canonical correspondence analysis (CCA) failed to fully associate the patterns of bacterial diversity to reactor performance, however the use of CCA itself was fundamentally justified and future analyses employing the method will require a larger number of samples and more complete performance data for proper analysis. The fourth study was carried out in tandem with the third and sought to examine the shared microbial communities between AD systems with vastly different operational designs and parameters. The results indicated the majority of OTUs were unique to the system from which they were recovered, with sequences corresponding to shared OTUs not being recovered in equal abundance for each sample. This result confirms that the microbial diversity of AD systems is highly variable. Finally, as no iii members of the class Methanomicrobia were recovered in the sequencing datasets, multiplex qPCR assays for five select genera of methanogens found to be abundant in AD systems was developed. The results of the qPCR assays showed that the hydrogenotrophic methanogen genus Methanobacterium was abundant in nearly all samples but not recovered using the archaeal primers used for 16S sequencing. This suggests that 16S sequencing libraries can miss important groups of Bacteria or Archaea, hindering the ability to link the microbial diversity to operational performance. The overall results of my research provide greater insight into the microbial communities that participate in the AD process than was previously documented. Incorporation of these results into the design and further optimization of AD systems will further improve the stability and performance of these systems in the future. iv To my father and mother. v ACKNOWLEDGMENTS I first would like to thank all of my mentors who have served on my dissertation committee, Drs. Mark Morrison, Zhongtang Yu, and Olli Tuovinen. You have all played a crucial role in my development as an independent researcher and your insight and guidance has been invaluable. To Drs. Morrison and Yu I wish to acknowledge their gracious acceptance of me into their lab and for serving as my advisor and co-advisor, respectively, throughout my graduate studies. It because of them that I have been able to participate in an area of research that I greatly enjoy, and cannot imagine myself not being a part of. I doubt that I would have achieved everything that I have without their generous support. A special acknowledgement goes to Dr. Yu for his patience and understanding while supervising me on a day to day basis. To Dr. Tuovinen I thank you for your support during my transition from microbiology to the environmental science graduate program and your enthusiastic support of both myself and my research. I wish to thank Dr. Floyd Schanbacher for his service on my candidacy committee, his generosity in providing many of the digester samples used in this study, and for sharing his knowledge of anaerobic digestion systems. Within the Morrison/Yu lab group I specifically thank Jill Stiverson for her technical support in training me when I first joined the lab and for her kindness and friendship during the 5 years that we have worked together. Of the other former and present members of my lab, of whom there are vi too many to recount here, I thank you all for your integral role in my research but more importantly for your friendship. To my fellow graduate students working in the Department of Animal Sciences, I thank you all for your friendship and serving as a source of often needed entertainment and diversion from my studies. May you all succeed in your own research. vii VITA June 2001 .......................................................Sanford High School 2005 ...............................................................B.A., Microbiology, Ohio Wesleyan University 2005 to August 2006 .....................................Graduate Teaching Associate, Department of Microbiology, The Ohio State University September 2006 to present ............................Graduate Research Associate, Environmental Science Graduate Program, The Ohio State University Publications Nelson, M.C., Morrison, M., Yu, Z., 2011. A meta-analysis of the microbial diversity observed in anaerobic digesters. Bioresource Technology, 102, 3730-3739 Cressman, M.D., Yu, Z., Nelson, M.C., Moeller, S.J., Lilburn, M.S., Zerby, H.N., 2010. Interrelations between the microbiotas in the litter and in the intestines of commercial broiler chickens. Appl Environ Microbiol, 76, 6572-6582. Fields of Study Major Field: Environmental Science Focus: Microbial Ecology viii TABLE OF CONTENTS Abstract ............................................................................................................................... ii Dedication. .......................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii List Of Tables ................................................................................................................... xv List Of Figures ................................................................................................................. xvi Chapter 1: Introduction ...................................................................................................... 1 Chapter 2: Review Of Literature ........................................................................................ 5 2.1 Anaerobic Digestion for Waste Treatment ................................................................ 5 2.1.1 System Designs................................................................................................... 6 2.1.2 The Anaerobic Digestion Process ....................................................................... 9 2.1.3 The Microbial Diversity of AD ........................................................................ 14 2.1.4 The Black Box .................................................................................................. 16 2.2 Molecular Analysis Methods as Applied to AD Systems: ...................................... 16 2.2.1 Community Profiling Methods: ........................................................................ 18 2.2.2 Species Specific and Quantitative
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