Toward industrial C8 production: Oxygen intrusion drives renewablen-caprylate production from ethanol and acetateviaintermediate metabolite production

Abstract

Previous bioreactor studies achieved high volumetricn-caprylate (i.e., n-octanoate) production rates and selectivities from ethanol and acetate with chain-elongating microbiomes. However, the metabolic pathways from the substrates ton-caprylate synthesis were unclear. We operated twon-caprylate-producing upflow bioreactors with a synthetic medium to study the underlying metabolic pathways. The operating period exceeded 2.5 years, with a peak volumetricn-caprylate production rate of 190 ± 8.4 mmol C L-1d-1(0.14 g L-1h-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 with13C-labeled ethanol suggest acetyl-coenzyme A-based derived production ofn-butyrate (i.e., n-butanoate),n-caproate (i.e., n-hexanoate), andn-caprylate. Here, we postulate a trophic hierarchy within the bioreactor microbiomes based on metagenomics, metaproteomics, and metabolomics data, as well as experiments with aClostridium kluyveriisolate. First, the aerobic bacteriumPseudoclavibacter caeniand the facultative anaerobic fungusCyberlindnera jadiniiconverted part of the ethanol pool into the intermediate metabolites succinate, lactate, and pyroglutamate. Second, the strict anaerobicC. kluyverielongated acetate with the residual ethanol ton-butyrate. Third,Caproicibacter fermentansandOscillibacter valericigeneselongatedn-butyrate with the intermediate metabolites ton-caproate and then ton-caprylate. Among the carbon chain-elongating pathways of carboxylates, the tricarboxylic acid cycle and the reverse ß-oxidation pathways showed a positive correlation withn-caprylate production. The results of this study inspire the realization of a chain-elongating production platform with separately controlled aerobic and anaerobic stages to producen-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 isn-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, stimulatedn-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 ton-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 producen-caprylate renewably at an industrial scale.