BackgroundDilute ethanol streams generated during fermentation of biomass or syngas can be used as feedstocks for the production of higher value products. In this study, we describe a novel synthetic microbial co-culture that can effectively upgrade dilute ethanol streams to odd-chain carboxylic acids (OCCAs), specifically valerate and heptanoate. The co-culture consists of two strict anaerobic microorganisms:Anaerotignum neopropionicum, a propionigenic bacterium that ferments ethanol, andClostridium kluyveri, well-known for its chain-elongating metabolism. In this co-culture,A. neopropionicumgrows on ethanol and CO2producing propionate and acetate, which are then utilised byC. kluyverifor chain elongation with ethanol as the electron donor.ResultsA co-culture ofA. neopropionicumandC. kluyveriwas established in serum bottles with 50 mM ethanol, leading to the production of valerate (5.4 ± 0.1 mM) as main product of ethanol-driven chain elongation. In a continuous bioreactor supplied with 3.1 g ethanol L−1d−1, the co-culture exhibited high ethanol conversion (96.6%) and produced 25% (mol/mol) valerate, with a steady-state concentration of 8.5 mM and a rate of 5.7 mmol L−1d−1. In addition, up to 6.5 mM heptanoate was produced at a rate of 2.9 mmol L−1d−1. Batch experiments were also conducted to study the individual growth of the two strains on ethanol.A. neopropionicumshowed the highest growth rate when cultured with 50 mM ethanol (μmax= 0.103 ± 0.003 h−1) and tolerated ethanol concentrations of up to 300 mM. Cultivation experiments withC. kluyverishowed that propionate and acetate were used simultaneously for chain elongation. However, growth on propionate alone (50 mM and 100 mM) led to a 1.8-fold reduction in growth rate compared to growth on acetate. Our results also revealed sub-optimal substrate use byC. kluyveriduring odd-chain elongation, where excessive ethanol was oxidised to acetate.ConclusionsThis study highlights the potential of synthetic co-cultivation in chain elongation processes to target the production of OCCAs. Furthermore, our findings shed light on to the metabolism of odd-chain elongation byC. kluyveri.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02336-w.