Exploration of microbial systems as biocatalysts for conversion of syngas to bio-based chemicals Martijn Diender Propositions 1. Synthetic communities offer better perspectives to optimize gas fermentation processes than pure- or complex undefined-mixed culture approaches. (this thesis) 2. The ability to convert a broad spectrum of wastes by syngas fermentation will eventually be more important than the cost, rate or yield of the overall process. (this thesis) 3. With the increasing use of ~omics and computational techniques, our microbial physiological knowledge is becoming increasingly based on assumptions. 4. Mathematics is always correct. 5. Work performed by robots should be taxed in order to keep human labour competitive. 6. With the integration of the internet in our society, education should focus less on knowledge transfer and more on development of analytical skills. 7. One learns most by explaining to another. Propositions belonging to the thesis entitled: “Exploration of microbial systems as biocatalysts for conversion of synthesis gas to bio-based chemicals” Martijn Diender Wageningen, 8 February 2019 Exploration of microbial systems as biocatalysts for conversion of synthesis gas to bio-based chemicals Martijn Diender Thesis committee Promotor Prof. Dr A.J.M. Stams Personal chair at the Laboratory of Microbiology Wageningen University & Research Co-promotor Dr D.Z. Machado de Sousa Associate professor, Laboratory of Microbiology Wageningen University & Research Other members Prof. Dr A.J.H. Janssen, Wageningen University & Research Prof. Dr R.K. Thauer, Max Planck Institute for Terrestrial Microbiology, Germany Prof. Dr W. Verstraete, Ghent University, Belgium Dr A.M. Lopez Contreras, Wageningen University & Research This research was conducted under the auspices of the Graduate School for Socio- Economic and Natural Sciences of the Environment (SENSE). Exploration of microbial systems as biocatalysts for conversion of synthesis gas to bio-based chemicals Martijn Diender Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 8 February 2019 at 4 p.m. in the Aula. Martijn Diender Exploration of microbial systems as biocatalysts for conversion of synthesis gas to bio-based chemicals, 204 pages. PhD thesis, Wageningen University, Wageningen, NL (2019) With references, with summaries in English and Dutch DOI: https://doi.org/10.18174/466065 ISBN: 978-94-6343-391-4 First step to tackle any problem: ‘Stay calm’ -A.H. Diender Table of contents Chapter 1. Introduction and thesis outline 11 Chapter 2. Pathways and bioenergetics of carbon monoxide fermentation 23 Chapter 3. Proteomics analysis of the hydrogen and carbon monoxide 51 metabolism of Methanothermobacter marburgensis Chapter 4. High rate biomethanation of carbon monoxide-rich gases via a 71 thermophilic synthetic co-culture Chapter 5. Production of medium-chain fatty acids and higher alcohols by a 89 synthetic co-culture grown on carbon monoxide or syngas Chapter 6. Clostridium kluyveri drives carboxydotrophic growth of 109 Clostridium autoethanogenum to ethanol production Chapter 7. A flexible alcohol metabolism inClostridium autoethanogenum 131 allows additional energy conservation. Chapter 8. General discussion 153 Appendices 171 References 172 English Summary 189 Dutch Summary 192 Co-author affiliations 195 List of publications 196 Acknowledgements 197 About the Author 201 SENSE Diploma 202 Chapter 1 General introduction and thesis outline Chapter 1 Syngas fermentation for the sustainable production of chemicals and fuels Our planet is currently dealing with a rapid increase in world population with a consequent increase in the demand for fuels and chemicals. To answer this increasing demand, and ensure a viable environment for future generations, production processes need to become more sustainable (IPCC, 2014). Currently, many industrial processes are driven by fossil sources, causing environmental damage and contributing to global warming due to the exhaust of greenhouse gases. One of the ways to create a more sustainable economy is to make it circular, enabling the reuse of waste as a resource for the production of new commodities. Bio-based technologies, that use microorganisms to produce commodity chemicals and fuels, offer great potential, but are often limited to utilizing materials with easy accessible sugar content (e.g. starchy biomass) (Naik et al., 2010). However, production of such biomass often competes with food and feed supply, making the sustainability of this process questionable (Naik et al., 2010). For this reason, processes utilizing ligno-cellulosic, non-food biomass are preferred. However, this type of biomass is more recalcitrant and requires intensive pre-treatment (e.g. steam explosion, enzymatic treatment) in order to release the sugar content for fermentation (Naik et al., 2010). Weight percentage of the highly recalcitrant lignin content in woody biomass was estimated on average at about 25%, while in agricultural biomass this might even be 30% (Vassilev et al., 2012). Therefore, in case of optimal conversion of all non-lignin content this will still result in significant losses of the initial substrate, decreasing the yield of the overall process and resulting in generation of additional waste streams. This limits the scope of starting materials that bio-based technologies can utilize and leaves many other types of abundant waste (e.g. municipal wastes) unutilized. Currently, wastes that cannot be processed are burned or buried, contributing to environmental pollution and exhaust of greenhouse gases (Wiedinmyer et al., 2014). An alternative to the fermentation of sugar is gas fermentation (figure 1). In gas fermentation, CO2 is used by microorganisms as carbon source while utilizing an external electron source (e.g. H2, CO) to generate added-value products. Gas fermentation processes can be fed with different gases originating from distinct sources, such as off-gases from industry (Molitor et al., 2016) and from the gasification of carbonaceous materials (Daniell et al., 2016; Drzyzga et al., 2015). In addition, electrochemical systems can be used for the reduction of H2O/ CO2 streams to form H2 and CO rich gases (Dubois and Dubois, 2009; Furler et al., 2012; Redissi and Bouallou, 2013). Gasification followed by syngas fermentation allows for the recovery of carbon from recalcitrant wastes, such as municipal waste, 12 General Introduction and thesis outline lignocellulose and plastics. Gasification is performed at high temperature (>700 ○C) and at low oxygen:biomass ratio. This results in the partial oxidation of the carbonaceous materials and the formation of a gaseous mix consisting mainly of CO, H2 and CO2 (synthesis gas or syngas) (Heidenreich and Foscolo, 2015). Syngas can be used as a substrate for microbial fermentation processes where H2 and CO will act as electron donor while CO and CO2 acts as carbon source. One of the main challenges with syngas as substrate is the toxicity of CO towards the microbial catalyst. Thus, suitable carboxydotrophic strains (i.e. strains able to thrive on CO) need to be employed in the fermentation process to make it run efficiently. Presence of CO does however also allow for the production of more reduced compounds (such as 2,3-butanediol or alcohols) compared to H2/CO2 feed gas (Diender et al., 2015, Chapter 2; Oelgeschläger and Rother, 2008). Fermentation of gaseous substrates, with main focus on synthesis gas fermentation, is a way to generate chemicals and fuels from a wide spectrum of wastes and is the main topic of this thesis. Sugar based fermentation Gas based fermentation Biomass HO/CO O-gasses Municipal waste Biomass electro-chemical thermo-chemical Gasication reduction Ash Non-digested biomass Pre-treatment Free sugars Gas cleaning Gas tuning CO Fermentation CO, H, CO Fermentation CO Down-stream Biomass Down-stream processing processing Biomass Commodities, fuels Commodities, fuels Figure 1. Simplified scheme of sugar based fermentation (left) vs. gas based fermentation (right). Colours indicate either pre-fermentation processing (green), microbial fermentation (orange) or down- stream processing (grey). 13 Chapter 1 Microbial syngas fermentation and its (current) limitations During fermentation of syngas, the CO and H2 are oxidized and the released electrons are used to reduce CO2. Microbes suitable for fermentation of syngas are mainly acetogens, hydrogenogens and methanogens. In all known cases the organisms involved in utilization of CO/H2 for fermentation processes are strict anaerobes. However, besides these fermentation types of metabolism, also respiratory metabolism can be involved in conversion of syngas (e.g. coupled to sulfate or oxygen reduction) (Diender et al., 2015, Chapter 2; Oelgeschläger and Rother, 2008). From an industrial perspective, acetogens are interesting due to their native end products: acetate and ethanol. Additionally, natural products from syngas conversion by acetogens are in some cases longer acids and alcohols (e.g. butyrate, butanol), lactic acid or 2,3-butanediol (Bengelsdorf et al., 2013; Köpke et al., 2011b, 2014; Phillips et al., 2015). The two most studied acetogens for the conversion of syngas are Clostridum autoethanogenum (Abrini et al., 1994) and Clostridium ljungdahlii (Tanner et al., 1993). However, many other strains