Lactate:propionate molar ratio and substrate concentrations shape valerate production in secondary lactate fermentations

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days at pH 5.5 to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (110-220 mM), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (330-440 mM; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis allowed to infer that under high-lactate conditions, up to 30% of lactate was used for propionate and acetate formation. Microbial community analysis revealed a stable dominance of Caproiciproducens spp. when valerate was the main product. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems.