Open culture chain elongation for branched carboxylate formation

Abstract

In order to achieve a sustainable society it is paramount to develop technologies that can aid in recycling waste streams and in reducing the environmental footprint of human activity on this planet. The introduction of this thesis underlines the necessity of transitioning towards a circular economy and presents a technology that can help with recycling and valorising organic residues. Chain elongation fermentation allows the production of medium chain carboxylates (MCC) from complex organic waste. The fermentation products can be used for a wide range of applications within agriculture and the chemical industry. In this thesis new methods for chain elongation are discovered and researched that broaden the product spectrum of the technology. Besides straight molecular chains, branched chained MCCs have been shown as dominant products in these new fermentation types. The products with a different molecular structure inherently have different physical properties that might make them better suited for certain applications within society. The research chapters elaborated on how specific selection pressures in open culture fermentations can be used to enrich microbiomes to harbour desired biocatalytic capabilities. Two different types of chain elongation fermentation are the subject of these investigations: methanol-based and ethanol-based chain elongation. These two alcohols are used by the microbiomes as electron donors within the fermentation. In order to harvest energy and grow, the organism use a metabolism where they process the energy-rich electrons from the alcohols. The electrons are subsequently used to reduce a carboxylate electron acceptor, which is simultaneously elongated in the process. Within methanol-based chain elongation microbiomes, the elongation of acetate leads to mainly butyrate formation. Depending on the pH, the microbiome could be enriched to the point that isomerization of n-butyrate to isobutyrate occurred (Chapter 2). At a pH around 6.75 no isomerization happened, but at a pH around 5.5 it did. The ratio of the n-butyrate and isobutyrate concentrations were found to be coupled to the thermodynamic equilibrium of isomerization. The responsible microorganism for isobutyrate formation was found to be closely related to an earlier described Clostridium luticellarii. When propionate was used as electron acceptor, elongation to n-valerate occurred (Chapter 3). The enriched microbiome also contained C. luticellari as dominant microorganism. The microbiome was capable to simultaneously elongate both acetate and propionate to n-butyrate, isobutyrate, n-valerate as dominant products. Also small amounts of n-caproate were formed whenever n-butyrate was present within methanol-based chain elongation microbiomes. Based on literature a metabolic pathway for methanol-based chain elongation was proposed that could describe the experimentally observed stoichiometry. Microbiomes were also enriched to perform ethanol-based chain elongation, in particular for the elongation of branched electron acceptors. When isobutyrate was fed to the microbiome together with ethanol, elongation towards isocaproate was stimulated (Chapter 4). However, due to the nature of ethanol-based chain elongation in situ acetate formation always occurs. Additionally ethanol can in some situations be directly converted towards acetate and hydrogen by other microbes that compete for substrate. This leads to a situation where the chain elongators can use an increasing amount of acetate as electron acceptor, which seemed to be preferred over isobutyrate. Limiting acetate supply led to isocaproate production up to 20% of the total products. In an attempt to control the excessive ethanol oxidation in the reactor, conditions were adjusted to limited CO2 supply (Chapter 5). Limitation of CO2 leads to a deficiency for hydrogenotrophic methanogens; they need CO2 as electron acceptor for their energy-providing, methane-producing metabolism. However, the conditions of the reactor were such that an alternative route for ethanol oxidation was stimulated. High ethanol to acetate ratios, and high (other) carboxylate to corresponding alcohol ratios created the potential for carboxylate reduction coupled to ethanol oxidation. In turn in situ acetate formation persisted, whereby straight chain elongation remained the most dominant metabolic functionality. In the general discussion hypotheses are presented that could further mechanistically explain the observed metabolic functionalities. For methanol-based chain elongation the metabolic pathway is revised, using supporting evidence from the genome of C. luticellarii. Improvements on reactor operation are suggested to increase the performances. Additionally recommendations are given on how integrated bioprocess designs could circumvent downstream processing difficulties. Finally an outlook on ethanol-based chain elongation fermentation for branched carboxylate production is presented.