Contribution to deciphering the complexity of mixed acidogenic fermentation of agroindustrial waste for the production of carboxylates Doctoral Thesis Anastasios Perimenis Université catholique de Louvain, Earth & Life Institute, Team Bioengineering Supervisor Prof. Patrick Gerin (Université catholique de Louvain, Belgium) Accompanying committee Prof. Spiros Agathos (Université catholique de Louvain, Belgium & Yachay Tech, Ecuador) Prof. Jacques Mahillon (Université catholique de Louvain, Belgium) Jury members Prof. Claude Bragard (Université catholique de Louvain, Belgium) Prof. Philippe Delfosse (Luxembourg Institute of Science and Technology, Luxembourg) Prof. Frank Scholwin (University of Rostock and Institute of Biogas, Waste Management and Energy, Germany) Acknowledgements Many people have contributed intellectually, practically and mentally to the completion of this thesis. Prof. Gerin, in my eyes the ideal supervisor for a PhD candidate. His ability to provide answers to questions and his dedication to his students are unprecedented. Prof. Agathos, not only a supervisor but a mentor as well. Apart from his scientific advice, his mentality towards life and continued progress is a true guidance for me. Prof. Mahillon, always available for advice and one of the very few people I know that combine scientific excellence and coolness. Professors Delfosse and Scholwin, whose advice played a major role in improving the quality of the thesis. Prof. Bragard, who gave me the honour of presiding my jury. My colleagues, better said, my friends in GEBI, Matthieu, Thomas, Helene, Olivier (he is from another lab, but nobody is perfect) in the first place, whose presence and contribution was invaluable, both from a scientific and personal viewpoint. And all the colleagues in GEBI, past and present, who managed to create and maintain a friendly and collegial atmosphere. My students, Maya, Bruno, Mathilde, Sophie, Nicolas, Samuel, Eva, Sylvain. They worked with me (not for me) and we challenged each other as we were moving forward. A considerable amount of information in this thesis was possible also thanks to their work. I hope their stay with me had no adverse effect in their development. My current colleagues at ULB for respecting the particular circumstances and granting me the necessary flexibility to be able to complete this work. My parents Ioanna and Giorgos and my sister Penia, for their sacrifices and unconditional support throughout the years that allowed me to focus undivided on my constant improvement. What I have achieved, I owe it to them and I know that the best reward for them is that I do the same for my own children. My own family, Vanessa and our Vanestakia, Giorgos, Peter and Ilias. At the end of the day, their happiness is what truly matters and I am blessed to have them. Thank you all. 3 4 Foreword This thesis is situated within the context of sustainable exploitation of waste resources for the production of utilisable molecules for the benefit of the environment and the society. The motivation for a PhD in the domain was a growing personal interest on better understanding the fundamentals of complex biological processes. This desire was fuelled by my scientific background in chemical and environmental engineering, my fascination of how processes at the micro-scale can have a tangible effect at the macro-scale and my personal conviction that organic waste is not at all waste; on the contrary it can be source of added value. I have decided to pursue this interest through a PhD in bioengineering in the Bioengineering Team (GEBI) of the Earth and Life Institute of UCL. I am grateful to have received funding by the Walloon Region, through two collaborative research projects: • LIGNOFUEL (Convention Number: 716721). The purpose of the project was to develop integrated pathways (fermentation, catalysis, esterification) for the production of second generation biofuels from lignocellulosic residues. • WAL-AID (Convention Number: 6089). The purpose of the project was to create a platform of expertise to support the sustainability and competitiveness of the agri-food sector in the Walloon Region through the development of innovative and sustainable process for the valorisation of co- and by-products of the sector. As a consequence, certain scientific choices (e.g. types of waste tested, mechanical pre-treatment of waste) were partly imposed by the context of the projects and some analyses (e.g. waste composition) were conducted by the partners of the projects. The work conducted within this thesis resulted in three publications in peer- reviewed scientific journals (i.e. Waste Management, Biochemical Engineering Journal and Biomass Conversion and Biorefineries), one manuscript submitted for publication (Journal of the Institute of Brewing), two manuscripts under preparation, two poster presentations (National Symposium for Applied Biological Sciences 2013, 9th International Conference on Renewable Resources and Biorefineries 2013 ) and one oral presentation (ENVITAM and GEPROC Graduate school PhD day 2012; best presentation award). This work also fed a number of project proposals for research funding, the most elaborate of which being a H2020 collaborative proposal aiming to demonstrate sustainable downstream processing of waste-derived VFA streams (Acronym VOFAPURE; Reference SEP-210177779; Topic BIOTEC- 5 4-2014). GEBI would focus on the control of the pilot scale acidogenic fermentation and also assumed the role of the coordinator of the proposal, bringing together a group of 14 academic and industrial partners. I was personally responsible for the development of the project idea, the assembly of the consortium and the editing of the proposal combining inputs from all partners. The proposal passed the first stage of evaluation but was not retained for funding at the second stage. 6 Abbreviations COD: Chemical oxygen demand CODs: soluble COD CODt: total COD DGGE: Denaturing gradient gel electrophoresis DSP: Downstream processing FM: Fresh matter HRT: Hydraulic retention time ML: Mixed liquor OLR: Organic loading rate OTU: Operational taxonomic unit PCR: Polymerase chain reaction TS: Total solids VFA: Volatile fatty acids tVFA: total VFA VS: Volatile solids 7 8 Abstract This thesis focused on the production of short chain carboxylates by mixed acidogenic fermentation, as a possible option for the sustainable valorisation of agroindustrial waste. Acidogenic fermentation is an intermediate step of standard anaerobic digestion, a process that sequentially converts organic matter mainly into methane. The product of acidogenic fermentation are short chain carboxylic acids, like acetic, propionic, butyric, caproic and lactic acids, with a variety of uses in green chemistry, renewable energy and other environmental applications. “Mixed fermentation” indicates the existence of natural consortia of microbial species treating composite substrates like lignocellulose, contrary to single, pure cultures treating simple substrates like glucose. The advantages of this approach include, among others, higher conversion capacities and increased flexibility in terms of substrate type, but the challenge of mastering the complexity of the fermentation system and the downstream processing of the products increases. We examined a variety of agroindustrial waste, such as fruit pulps and brewery residues among others, to highlight the relevance of mixed acidogenic fermentation as a stand-alone bioconversion process. We employed a variety of techniques (including among others wet chemistry, chromatography, DNA extraction, amplification and sequencing) to analyse the metabolic and microbial profile of fermentations and decipher their complexity. We were able to achieve carboxylate (mainly acetic, butyric and caproic acids) concentrations of approximately 41 gCOD/kgmixed_liquor and substrate-to- carboxylate conversion yields of 0.64 gCOD_carboxylates/gCOD_substrate. We have shown that every substrate/inoculum combination leads to a unique and reproducible fermentation profile; a major factor for this uniqueness is the evolution and dynamics of the microbiome within each fermentation. We have highlighted, through the production of lactic acid from beer trub, the interest of mixed consortia not only as producers of target molecules but also as degraders of unwanted substances (inhibitory hop acids). We identified potential links between microbial species and metabolic products, but further research is needed to establish robust and direct correlations in a more systematic way. This would bring us a step closer to selective conversion of complex substrates into specific carboxylates and enable industrial use of mixed acidogenic fermentation. 9 10 Contents ACKNOWLEDGEMENTS .............................................................................................. 3 FOREWORD ................................................................................................................ 5 ABBREVIATIONS ......................................................................................................... 7 ABSTRACT .................................................................................................................. 9 CONTENTS ................................................................................................................ 11 INTRODUCTION ............................................................................................... 15 1.1 Context and theoretical background .......................................................