Hexanoate is a valuable chemical that can be produced by microorganisms that convert short-chain- to medium-chain carboxylic acids through a process called chain elongation. These microorganisms usually produce mixtures of butyrate and hexanoate from ethanol and acetate, but direct conversion of ethanol to hexanoate is theoretically possible. Steering microbial communities to ethanol-only elongation to hexanoate circumvents the need for acetate addition and simplifies product separation. The biological feasibility of ethanol elongation to hexanoate was validated in batch bioreactor experiments with a Clostridium kluyveri-dominated enrichment culture incubated with ethanol, acetate and butyrate in different ratios. Frequent liquid sampling combined with high-resolution off-gas measurements allowed to monitor metabolic behavior. In experiments with an initial ethanol-to-acetate ratio of 6:1, acetate depletion occurred after ± 35 h of fermentation, which triggered a metabolic shift to direct conversion of ethanol to hexanoate despite the availability of butyrate (± 40 mCmol L−1). When only ethanol and no external electron acceptor was supplied, stable ethanol to hexanoate conversion could be maintained until 60–90 mCmol L−1 of hexanoate was produced. After this, transient production of either acetate and butyrate or butyrate and hexanoate was observed, requiring a putative reversal of the Rnf complex. This was not observed before acetate depletion or in presence of low concentrations (40–60 mCmol L−1) of butyrate, suggesting a stabilizing or regulatory role of butyrate or butyrate-related catabolic intermediates. This study sheds light on previously unknown versatility of chain elongating microbes and provides new avenues for optimizing (waste) bioconversion for hexanoate production.@en

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-023-43682-x, published online 13 October 2023 The original version of this Article contained errors in the Discussion section, where reference 34 was incorrectly cited as reference 25. Consequently, “Spirito et al.25 also observed a decreased conversion rate when feeding a chain-elongating reactor microbiome with mainly ethanol. This could explain why previous studies with shorter incubation times reported no metabolic activity in the absence of acetate25,35. The mechanism that underlies this change in rate remains elusive. Spirito et al.25 proposed a thermodynamic constraint on the rate due to increased hydrogen partial pressures, but in our experiments pH2 was low due to continuous sparging and we still observed low rates.” now reads: “Spirito et al.34 also observed a decreased conversion rate when feeding a chain-elongating reactor microbiome with mainly ethanol. This could explain why previous studies with shorter incubation times reported no metabolic activity in the absence of acetate34,35. The mechanism that underlies this change in rate remains elusive. Spirito et al.34 proposed a thermodynamic constraint on the rate due to increased hydrogen partial pressures, but in our experiments pH2 was low due to continuous sparging and we still observed low rates.” The original Article has been corrected.@en