CRANFIELD UNIVERSITY Robert Michael William Ferguson B.Sc
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CRANFIELD UNIVERSITY Robert Michael William Ferguson B.Sc.(Hons), M.Sc. FEEDSTOCKS INFLUENCE ON THE PROCESS PARAMETERS AND THE MICROBIAL COMMUNITY IN ANAEROBIC DIGESTION SCHOOL OF APPLIED SCIENCES DEPARTMENT OF ENVIRONMENTAL SCIENCE AND TECHNOLOGY Ph.D. THESIS CRANFIELD UNIVERSITY SCHOOL OF APPLIED SCIENCES DEPARTMENT OF ENVIRONMENTAL SCIENCE AND TECHNOLOGY Ph.D THESIS Academic Year 2012-2013 Robert M. W. Ferguson B.Sc.(Hons), M.Sc. FEEDSTOCKS INFLUENCE ON THE PROCESS PARAMETERS AND THE MICROBIAL COMMUNITY IN ANAEROBIC DIGESTION Supervisors: Dr Frédéric Coulon and Dr Raffaella Villa September 2013 This thesis is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy © Cranfield University 2004. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright owner. Abstract To improve our understanding into the key parameters controlling and regulating the microbial groups involved in the anaerobic digestion (AD) process, particularly over multiple changes in operational conditions, triplicate lab-scale digesters fed with sewage sludge were exposed to single and multiple changes in organic loading rate (OLR) using either glycerol waste (a by-product of biodiesel manufacture), or Fats oils and greace (FOG waste) collected from a restaurant grease trap. For the multiple changes in OLR, digesters were either exposed to repeated addition of glycerol waste or repeated addition of both glycerol waste and FOG waste. In all conditions tested, physicochemical variables including volatile fatty acids (VFA), alkalinity, pH, biogas production and composition were analysed. Molecular fingerprint techniques including lipid and ether lipid analysis and 454-pyrosequencing of 16S rRNA genes were used to characterise the microbial communities. These techniques were chosen as they complement each other providing information on the microbial biomass and in-depth phylogenetic analysis of the microbial community, respectively. The key question addressed here was how feedstock composition and variation in OLR would affect the microbial community structure and dynamics and relate this to the performance of the digesters in terms of methane production over a long-term period (> 120 days). Multiple changes in OLR with the same feedstock resulted in faster recovery of methane production (8-10 days faster) compared to digesters exposed to single changes in OLR. This finding was associated specifically with a higher proportion of Clostridia Incertae Sedis XV (closely related to Cloacibacillus genus (83% similarity), family Synergistaceae) in the pre- exposed digesters. It is speculated that members related to Clostridia Incertae Sedis XV play an important role in the syntrophic interactions with the methanogens. Analysis of the VFA profiles supported this by showing that the higher relative abundance of Cloacibacillus was related to higher acetic acid concentrations. The pyrosequencing analysis further showed that community evenness was correlated with the best biogas methane content and shifts in specific bacterial groups was clearly correlated with digester performance. Overall the findings of this PhD provide new insights into the relationships between microbial community structure and digester performance. It also provides new-evidence based knowledge on how molecular microbiological tools can be used in the future to optimise and manage AD plants. i ii Acknowledgments Firstly I would like to express my gratitude to my supervisors Dr Frédéric Coulon and Dr Raffaella Villa both of whom have been extremely generous with their time during my PhD and have given valuable advice on all aspects of this project. I would also like to thank the Questor Centre and the Engineering and Physical Sciences Research Council (EPSRC) for funding the project. I also wish to acknowledge the help of the laboratory support staff Jane Hubble, Rukhsana Ormesher, and Dr Paul Barton for their help in the lab. Many thanks also to Dr Sean Tyrrel my subject advisor for his advice on the project. On a more personal note I would like to thank my friends and family for their support. Thank you to my Parents Jacqueline and Christopher, sisters Lucy and Maud, and my Grandparents Bill and Molly. Many thanks to the building 40 crew, past and present, for enriching my PhD experience. In particular Francesco Ometto for his musical accompaniment in the lab, Philippa Douglas, my desk buddy (even though she did not acknowledge me in her thesis!) don't expect me to sing at your next wedding! and also Hayley Shaw for providing accommodation during the final week before submission. Thanks also to the members of the Bedford drinking club for their pastoral care. Most importantly I would also like to thank my wife Sophie Ferguson for her love and support. I hope that I can be as supportive during the final months of your PhD. I promise that I will finish digging up the hedge now that the excuse of "I got to write up my PhD" is no longer valid. Finally I would like to thank my cat Barabas for his casual indifference to my PhD. And last, but not least, thank you the social club sandwich team for feeding me for the last 3 years. iii iv Table of contents Abstract _________________________________________________________ i Acknowledgments ________________________________________________iii Table of contents _________________________________________________ v List of figures ___________________________________________________ ix List of Tables ___________________________________________________ xvi Abbreviations ________________________________________________ xviii Introduction ____________________________________________________ 1 Chapter 1: Bioengineering options and strategies for the optimisation of anaerobic digestion processes _____________________________________ 10 1.1. Introduction __________________________________________________________ 11 1.2. Overview of the microbial ecology in AD processes ___________________________ 13 1.3. Microbial diversity of anaerobic digestion __________________________________ 16 1.4. Influence of physicochemical parameters on microbial communities in AD ________ 19 1.5. Microbial optimisation of AD ____________________________________________ 28 1.6. Conclusions and research gaps ___________________________________________ 34 Chapter 2: The effect of feedstock and OLR change on AD performance during co-digestion ______________________________________________ 36 2.1. Introduction __________________________________________________________ 37 v 2.2. Methods _____________________________________________________________ 40 2.3. Results and discussion __________________________________________________ 45 3.4. Conclusions. __________________________________________________________ 62 Chapter 3: The Effect of feedstock and OLR changes on microbial community during co-digestion ____________________________________ 64 3.1. Introduction __________________________________________________________ 65 3.2. Methods _____________________________________________________________ 67 3.3. Results and discussion __________________________________________________ 73 3.4. Conclusions __________________________________________________________ 105 Chapter 4: The effect of feedstock and OLR change on volatile fatty acids (VFA) production and microbial community dynamics in anaerobic digestion. _____________________________________________________ 107 4.1. Introduction _________________________________________________________ 108 4.2. Methods ____________________________________________________________ 111 4.3. Results and discussion _________________________________________________ 115 4.4. Conclusions __________________________________________________________ 129 Chapter 5: Microbial community dynamics and anaerobic digester performance __________________________________________________ 131 5.1. Introduction _________________________________________________________ 132 5.2. Methods ____________________________________________________________ 134 vi 5.3. Results and discussion _________________________________________________ 137 5.4. Conclusions __________________________________________________________ 168 Chapter 6: Key findings and Implications __________________________ 170 6.1. Reminders of the PhD study aims ________________________________________ 170 6.2. Key results and implications ____________________________________________ 174 6.3. Recommendations for AD operators ______________________________________ 182 6.4. Wider implications of findings ___________________________________________ 185 6.5. Limitations __________________________________________________________ 186 6.6. Recommendations for further research ___________________________________ 188 References ____________________________________________________ 192 Appendix A - Initial experiments carried out with glycerol and FOGs co- digestion. _____________________________________________________ 212 Preliminary glycerol work __________________________________________________ 212 Preliminary work with FOGs ________________________________________________ 214 vii viii List of figures Figure 1.1. Theoretical response of a community to disturbance (adapted from Allison and Martiny, 2008) .................................................................................................................................................................... 15 Figure 1.2. Rarefaction curve of OTUs identified using Ribosomal Data Project (RDP)