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Correlating Physico-Chemical Process Parameters with Microbial Community Dynamics in Co-Digestion of Cattle Slurry and Grass Silage

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Correlating Physico-Chemical Process Parameters with Microbial Community Dynamics in Co-Digestion of Cattle Slurry and Grass Silage Correlating physico-chemical process parameters with microbial community dynamics in co-digestion of cattle slurry and grass silage Carl Warren Charles Samuel A thesis submitted for the degree of Doctor of Philosophy (PhD) School of Natural and Environmental Sciences (SNES) University of Newcastle March 2018 Abstract Improvements in biogas and methane production from on-farm anaerobic digesters (AD) are limited by the inherent complexity of the microbial community (MC) dynamics. The aim of this research was to improve CH4 production by investigating the impact of co-digesting of cattle slurry (CS) and grass silage (GS) on microbial community dynamics and the AD process. Batch and continuously stirred tank reactor (CSTR) laboratory experiments were conducted under mesophilic conditions (37◦C) with three feedstock mixtures of CS:GS ratios 100:0, 80:20 and 60:40. The microbial community was characterised using high throughput next generation sequencing of the V3-V4 region of the 16S rRNA amplicons on an Illumina MiSeq platform along with a novel high resolution denoising bioinformatics algorithm called DADA2 and the Sylva 123 reference database. The research findings indicate that: The co-digested treatment with the highest level of GS had the highest specific CH4 production. CH4 production from the 60:40 treatment was approximately 30% higher than the CS-only treatment. The CS-only and co-digested treatments both had similar hydrolysis constants therefore on-farm AD operators can co-digest CS with GS to increase CH4 production without having to alter the AD plant’s hydraulic retention time. Increased levels of CH4 production was associated with decreasing levels of total ammonia nitrogen (TAN), effluent volatile solids (VS), pH and total volatile fatty acids (TVFA). Over 80% of the phylogenetic differences observed were due to changes in these AD process parameters. Therefore TAN, %VS destroyed, pH and TVFA are important AD process parameters to monitor CH4 production when co-digesting CS with GS. The GS co-substrate enhanced CH4 production by influencing microbial community shifts. The relative abundance of the bacterial phylum Bacteroidetes was higher in the co-digested treatment (26%) than the CS-only treatment (19%) in the CSTR experiment. Novel bacteria from phylum Bacteroidetes namely Fermentimonas caenicola strain ING2-E5B and Petrimonas mucosa strain ING2-E5A, purported to have enhanced hydrolytic capabilities for complex carbohydrates, peaked in relative abundance in the co-digested treatment. i Process instability occurred when the loading rate of the CSTRs treating the CS-only and the co-digested treatments increased to 1g VS/m3 per day. This was likely due to increased levels of TAN during HRT 1 and 2 along with increases in the relative abundance of Dysgonomonas spp. which degrade carbohydrates without producing H2/CO2. Subsequent substrate limitation adversely affected the growth of hydrogenotrophic methanogens and resulted in propionate and butyrate accumulation. CH4 production increased by co-digesting cattle slurry (CS) with grass silage (GS). However, operators of on-farm AD plants should correlate microbial community changes with variations in the physico-chemical AD process parameters during reactor start-up and at regular intervals during normal operations to maximize digester performance. ii Acknowledgements I would like to express my appreciation to the Commonwealth Scholarship Fund for providing me with the opportunity to conduct my PhD. research. A very special thanks to my supervisors Dr. Paul Bilsborrow, Dr. Jan Dolfing and Dr. Matthew Wade, whose guidance and advice through a very challenging journey has been invaluable. I would also like to thank Dr. Paul Sallis for his advice and support in setting up and operating the laboratory-scale reactors. I would like to extend my heartfelt thanks and appreciation to Michael Botha at Cockle Park Farm for his assistance with sample collection. Special thanks to the laboratory technicians Peter Shotton, Wendy Bal, Chris Bulman, Roy Lamb and Fiona Maclachlan at the School of Agriculture and David Race, Amy Bell, Dave Earley and Sarah Jane Smith at the School of Civil Engineering and Geosciences for their hard work to ensure my samples were analysed and the chemicals and laboratory equipment were available for me to conduct my analyses. I would also like to thank Gloria Samuel, my mother whose support during the most challenging times made it possible for me to stay focused and my father, the late Joseph Samuel who always challenged me to achieve my goals. This PhD thesis is dedicated to them both. Lastly, I would like to thank my wife Kay Samuel. Without her support I would not have so easily overcome the challenges that I faced this past year. Upon the shoulders of the aforementioned and many more loved ones and with a thankful heart I can now exclaim with thanks to the Almighty God, “it is finished”. iii iv Table of Contents Chapter 1 Introduction ................................................................................... 1 1.1 Aims and Objectives ............................................................................................................. 6 Chapter 2 Literature Review ........................................................................... 8 2.1 Pretreatment Methods for Lignocellulosic Biomass ............................................................ 8 2.2 The Anaerobic Digestion Process ....................................................................................... 13 2.2.1 Hydrolysis ..................................................................................................................................... 13 2.2.2 Acidogenesis ................................................................................................................................. 15 2.2.3 Acetogenesis ................................................................................................................................ 16 2.2.4 Methanogenesis ........................................................................................................................... 18 2.3 Anaerobic Digestion Process Inhibition .............................................................................. 20 2.3.1 Ammonia ...................................................................................................................................... 20 2.3.2 Process Temperature ................................................................................................................... 22 2.3.3 Process pH .................................................................................................................................... 23 2.3.4 Trace elements ............................................................................................................................. 24 2.3.5 Hydrogen Sulphide ....................................................................................................................... 25 2.3.6 Organic and Hydraulic Overload .................................................................................................. 25 2.4 Types of Bioreactors used in the Co-digestion of Agricultural Waste ............................... 27 2.4.1 Batch versus Continuous Digesters .............................................................................................. 28 2.4.2 One versus Two-Stage Reactors ................................................................................................... 30 2.5 Process Indicators and the impact of Molecular Ecological Tools ..................................... 31 Chapter 3 Assessing the effect of the anaerobic fermentation of grass silage on AD process parameters in batch mode .................................................... 38 3.1 Introduction ........................................................................................................................ 38 3.2 Experimental Design ........................................................................................................... 39 3.2.1 Primary Feedstock Collection and Storage .................................................................................. 42 3.2.2 Apparatus Design and Assembly .................................................................................................. 43 3.2.3 Inoculum Preparation .................................................................................................................. 45 3.2.4 Treatment Preparation ................................................................................................................ 46 3.2.5 Data Handling ............................................................................................................................... 48 3.3 Methods .............................................................................................................................. 49 3.3.1 Total and Volatile Solids ............................................................................................................... 49 3.3.2 Biogas Production ........................................................................................................................ 49 v 3.3.3 Volatile Fatty Acids ....................................................................................................................... 52 3.3.4 Elemental Analysis ........................................................................................................................ 53 3.3.5 First
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