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Abstract

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.