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Microbial Communities in Biogas Reactors and Their Link to Stability and Performance

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Microbial Communities in Biogas Reactors and Their Link to Stability and Performance Microbial communities in biogas reactors and their link to stability and performance Mikrobielle samfunn i biogass reaktorer og deres kobling til stabilitet og ytelse Philosophiae Doctor (PhD) Thesis Live Heldal Hagen Department of Chemistry, Biotechnology and Food Science Faculty of Veterinary Medicine and Bioscience Norwegian University of Life Sciences Ås (2016) Thesis number 2016:60 ISSN 1894-6402 ISBN 978-82-575-1380-1 Table of Contents ACKNOWLEDGEMENTS .................................................................................................................. III SUMMARY ........................................................................................................................................... V SAMMENDRAG ................................................................................................................................. VII ABBREVIATIONS .............................................................................................................................. XI LIST OF PAPERS ................................................................................................................................ IX 1 INTRODUCTION ............................................................................................................................... 1 1.1 BIOGAS PRODUCTION FOR A SUSTAINABLE BIO-ECONOMY........................................................... 1 1.2 Biogas in Europe and Norway .................................................................................................. 2 1.2 THE BIOGAS REACTOR ................................................................................................................... 4 1.2.1 Feedstock ............................................................................................................................... 5 1.2.2 Process parameters influencing the process stability ............................................................ 6 1.3 FROM WASTE TO ENERGY – A MICROBIAL PROCESS ...................................................................... 8 1.3.1 The study of complex microbial communities ...................................................................... 14 2 OUTLINE AND AIM OF THESIS ................................................................................................... 17 3 MAIN RESULTS AND DISCUSSION ............................................................................................ 19 3.1 DYNAMICS IN A MICROBIAL COMMUNITY UNDERGOING DISTURBANCE (PAPER I) ..................... 19 3.2 EFFECT OF STORAGE CONDITION ON INOCULUM MICROBIAL COMMUNITY COMPOSITION AND PERFORMANCE (PAPER II) ................................................................................................................. 21 3.3 EFFECT OF TEMPERATURE AND RECIRCULATION ON MICROBIAL COMMUNITY COMPOSITION AND PERFORMANCE (PAPER III) ................................................................................................................ 23 3.4 CHARACTERIZATION OF THE MICROBIAL COMMUNITY IN A STABLE FULL-SCALE BIOGAS PLANT (PAPER IV) ......................................................................................................................................... 25 4 CONCLUDING REMARKS ............................................................................................................. 28 5 APPLICATION AND FUTURE PERSPECTIVES .......................................................................... 30 REFERENCES ..................................................................................................................................... 32 PAPERS……………………………………………………………………………………I-IV ii ACKNOWLEDGEMENTS The present work was carried out during the period 2013-2016 in the Protein Engineering and Proteomics (PEP) group, Department of Chemistry, Biotechnology and Food Science at The Norwegian University of Life Sciences (NMBU). The PhD study was part of two projects: “Robust processes for biogas production using manure and by-products from agriculture and agro-industry” (RobuBiogas) and “Biogas from organic residues and livestock manure as a vehicle fuel” (BiogasFuel), both funded by the Research Council of Norway. First of all, I would like to express gratitude to my main supervisor, Professor Svein Jarle Horn for supporting and encouraging me. You always keep your door open, and I truly appreciate that you have taken the time to patiently answer my questions. I would also like to thank co-supervisor Dr. Phillip Pope for opening the door to the PEP-corridor in the first place and for sharing valuable advises. A special thanks to co-supervisor Professor Vincent Eijsink, your endless knowledge and enthusiasm is a true inspiration. I would also like to express my gratefulness to Vivekanand Vivekanand and Mirzaman Zamanzadeh. Thank you for sharing of your biogas processing knowledge, and for collecting samples for microbial analysis. A special thanks to Jeremy Frank, for helpful support with the bioinformatics. Anne-Cath and Ellen, you are the core of the PEP group, and how you can keep track on everyone and everything in the lab is a mystery to me! I would also like to thank all members of the PEP-group, with a very special thanks to Trine for always keeping the door open for me and my frustrations, both research-related and private matter, and Kasia for always keeping the door closed (literally), and for creating an energetic atmosphere in our office. You make work fun! A special thanks to Professor Jon Fredrik Hanssen, who opened my eyes to the intriguing microbial world. You are my hero. Thanks to my family for letting me be myself - it seems like my stubbornness paid off. A special thanks to my “Afghan family” for the sweetest cakes, the warmest hugs and perspective on life. And then, last but not least, a very special thanks to my friends. Throughout the years, you have given me good laughs and valuable memories. During the last period of this work, you have also given me a support that I am eternally grateful for. You are simply the best. Live iii iv SUMMARY The Anaerobic digestion (AD) of organic material gathers a great interest worldwide due to the global needs for waste recycling and renewable energy production. Biogas, the end product of an AD process, is a mixture of methane and carbon dioxide. Biogas can be used for heating, electricity or upgraded to pure methane for vehicle fuels. It could also serve as a part of the cycle in biorefineries. Although widely applied for energy production, an improved knowledge regarding the underlying microbial community is desired to ensure stable and efficient energy production. Therefore, the studies described in this thesis aimed to increase the knowledge base of microbial community in biogas reactors and relate this to stability and performance of the digestion process. We analyzed 16S rRNA gene sequences from samples collected of both unstable and stable laboratory-scale biogas reactors, and studied the community profile and dynamics of bacteria and archaea. In particular, we evaluated the balance between acidogenesis and methanogenesis in reactors fed with an easy degradable substrate. We also evaluated how the microbial composition linked to performance was affected by different storage conditions on inoculum, as well as temperature and recirculation of effluent. The microbial community collected from a stable thermophilic industrial-scale reactor was further exposed using a combined meta-omics approach. This allowed us to map quantitative metaproteomics data to phylotype-specific genomic bins, in order to study the microbial network and metabolic pathways. The interaction between different participating bacterial and archaeal groups have a significant impact on the stability of a biogas process. Volatile fatty acid (VFA) accumulation and a reduced biogas production was related to an unbalance between the acidogenesis and methanogenesis in both mesophilic and thermophilic reactors, caused either by a too fast hydrolysis due to overloading, or a too slow methanogensis due to ammonia inhibition. Our results related the presence of several microbial groups to the accumulation and depletion of fatty acids. We also observed that recirculation of effluent had a negative effect on the thermophilic processes, with severe accumulation of VFAs and ammonia. While recirculation had only minor effects on the biogas production and performance under mesophilic operation, the microbial community composition changed. This indicates a substantial functional redundancy in the mesophilic microbiome, making v the community more resilient. The results also indicate that the inoculum may be stored up to one month without severe loss of microbial activity. Importantly, the deep characterization of microbial community revealed a synergic network, including a putative novel syntrophic acetate oxidizing bacteria. Overall, the findings reported in this thesis provides increased insight into microbial community and the ecological roles of different microbial groups in relation to stability and performance of the AD process. Improved understanding about this intricate microbial network can be used to monitor and control the AD process, to ensure stable and efficient biogas production. vi SAMMENDRAG Anaerob nedbrytning av organisk materiale har stor interesse verden over på grunn av det globale behovet for resirkulering av avfall og generering av fornybar energi. Biogass, som er sluttproduktet etter er anaerob nedbrytningsprosess, består av en blanding metan og karbondioksid.
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