Upgrading dilute ethanol to odd-chain carboxylic acids by a synthetic co-culture of Anaerotignum neopropionicum and Clostridium kluyveri

Abstract

Background

Dilute 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, and Clostridium kluyveri, well-known for its chain-elongating metabolism. In this co-culture, A. neopropionicum grows on ethanol and CO₂ producing propionate and acetate, which are then utilised by C. kluyveri for chain elongation with ethanol as the electron donor.

Results

A co-culture of A. neopropionicum and C. kluyveri was 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⁻¹ d⁻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⁻¹ d⁻¹. In addition, up to 6.5 mM heptanoate was produced at a rate of 2.9 mmol L⁻¹ d⁻¹. Batch experiments were also conducted to study the individual growth of the two strains on ethanol. A. neopropionicum showed the highest growth rate when cultured with 50 mM ethanol (μ_max = 0.103 ± 0.003 h⁻¹) and tolerated ethanol concentrations of up to 300 mM. Cultivation experiments with C. kluyveri showed 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 by C. kluyveri during odd-chain elongation, where excessive ethanol was oxidised to acetate.

Conclusions

This 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 by C. kluyveri.