bioRxiv2024
Previous bioreactor studies achieved high volumetric n-caprylate (i.e., n-octanoate) production rates and selectivities from ethanol and acetate with chain-elongating microbiomes. However, the metabolic pathways from the substrates to n-caprylate synthesis were unclear. We operated two n-caprylate-producing upflow bioreactors with a synthetic medium to study the underlying metabolic pathways. The operating period exceeded 2.5 years, with a peak volumetric n-caprylate production rate of 190 {+/-} 8.4 mmol C L-1 d-1 (0.14 g L-1 h-1). We identified oxygen availability as a critical performance parameter, facilitating intermediate metabolite production from ethanol. Bottle experiments in the presence and absence of oxygen with 13C-labeled ethanol suggest acetyl-coenzyme A-based derived production of n-butyrate (i.e., n-butanoate), n-caproate (i.e., n-hexanoate), and n-caprylate. Here, we postulate a trophic hierarchy within the bioreactor microbiomes based on metagenomics, metaproteomics, and metabolomics data, as well as experiments with a Clostridium kluyveri isolate. First, the aerobic bacterium Pseudoclavibacter caeni and the facultative anaerobic fungus Cyberlindnera jadinii converted part of the ethanol pool into the intermediate metabolites succinate, lactate, and pyroglutamate. Second, the strict anaerobic C. kluyveri elongated acetate with the residual ethanol to n-butyrate. Third, Caproicibacter fermentans and Oscillibacter valericigenes elongated n-butyrate with the intermediate metabolites to n-caproate and then to n-caprylate. Among the carbon chain-elongating pathways of carboxylates, the tricarboxylic acid cycle and the reverse {beta}-oxidation pathways showed a positive correlation with n-caprylate production. The results of this study inspire the realization of a chain-elongating production platform with separately controlled aerobic and anaerobic stages to produce n-caprylate renewably as an attractive chemical from ethanol and acetate as substrates. Broader contextNext to renewable electric energy, carbon-based chemicals have to be produced sustainably and independently from fossil sources. To meet this goal, we must expand the portfolio of bio-based conversion technologies on an industrial scale to cover as many target chemicals as possible. We explore the bioprocess of chain elongation to provide medium-chain carboxylates that can function as future platform chemicals in the circular economy. The most valuable medium-chain carboxylate produced with chain elongation is n-caprylate (i.e., n-octanoate). This molecule with eight carbon atoms in a row (C8) is challenging to produce renewably for the chemical industry. Previous reports elucidated that elevated ethanol-to-acetate ratios, which are found in syngas-fermentation effluent, stimulated n-caprylate production. Until now, studies have suggested that chain elongation from high concentrations of ethanol and acetate is a fully anaerobic process. We refine this view by showing a trophic hierarchy of aerobic and anaerobic microbes capable of facilitating this process. Appropriate oxygen supplementation enables the synthesis of succinate, lactate, and pyroglutamate that permit high-rate chain elongation to n-caprylate under anaerobic conditions. Given these results, future research should focus on the segregated study of aerobic and anaerobic microbes to further enhance the process performance to produce n-caprylate renewably at an industrial scale.