Exploring the effects of carbon sources and exogenous electron donors on chain elongation in anaerobic mixed cultures

The carboxylate platform offers a sustainable bioconversion strategy for biorefineries, utilizing anaerobic mixed cultures to produce carboxylate mixtures, including medium-chain fatty acids (MCFAs), as valuable intermediates. The effects of carbon sources (glucose, glycerol, casein) and exogenous supplied electron donors (ethanol, methanol, propanol, pyruvate) on MCFA production via chain elongation were investigated to elucidate the role of external electron donors and assess the feasibility of self-sufficient MCFA production in their absence. For this purpose, all experimental sets included corresponding control conditions without external electron donor addition. Batch experiments were conducted without active pH control, allowing pH to evolve dynamically in response to substrate type and metabolic activity. Results showed that the carbon source significantly affected carboxylic acid production and composition. Glucose primarily yielded propionate, independent of the electron donor. Casein resulted in the lowest carboxylic acid and gas production but uniquely produced the highest MCFA. Acidic pH conditions (5.0–5.5), which developed primarily in glucose- and glycerol-fed systems, favoured short-chain fatty acid production, whereas near-neutral pH conditions (6.0–6.7), observed in casein-fed systems, enhanced MCFA formation. Electron donors significantly influenced the degradation rate of glycerol. Methane production was observed in glucose and glycerol sets but was absent in casein sets. Microbial community analysis revealed methanogen dominance across most sets, irrespective of substrate. These findings highlight the complex interactions between pH, electron donor/acceptor availability, and microbial community dynamics in anaerobic digestion. Future multi-omics and flux analyses are needed to elucidate the metabolic pathways governing chain elongation and anaerobic digestion. • The type of the carbon source affected acid yield and composition. • CaseIn produced the most medium-chain fatty acids but the least carboxylic acids. • Bio-isomerization primarily occurred during casein degradation. • Acidic pH promoted short-chain fatty acids, while neutral pH favoured longer chains. • Changes in the microbial community reflected effects on carboxylate formation.