Biological Caproate Production from Lignocellulosic Alkaline Hydrolysate via Chain Elongation: Deciphering the Role of Glycolate and Ethanol Supplementation

Glycolate, a key component of lignocellulosic alkaline hydrolysate, plays an unexplored role in biological caproate production via chain elongation (CE). This study first demonstrated that glycolate served as a more effective electron acceptor than xylose or xylan when coupled with ethanol as an electron donor. Varying ethanol-to-glycolate ratios significantly influenced the distribution of carboxylic products with caproate production peaking at the 2:1 ratio. Secondary ethanol supplementation further enhanced caproate yield, achieving a maximum concentration of 66.19 ± 0.00 mM at the 2:1 ratio, surpassing yields of 3:1 (40.82 ± 0.00 mM), 1:1 (22.30 ± 0.00 mM), and 1:2 (8.38 ± 1.98 mM). Distinct ethanol-to-glycolate ratios enriched specific CE genera, with Clostridium sensu stricto and Bacillus dominating at the 1:1 and 2:1 ratios. Metagenomic analysis revealed that increasing ratios boosted the abundance of specific genes involved in the ethanol oxidation, reverse β-oxidation (RBO), and fatty acid biosynthesis (FAB) pathways. The second addition of ethanol facilitated the abundance of the NAD(P)H-dependent gene involved in the RBO pathway, contributing to increased electron carrier availability and reinforced CE efficiency. These findings offered mechanistic insight into the role of glycolate in converting lignocellulosic biomass into caproate and underscored ethanol supplementation as an effective approach to optimize glycolate-driven CE efficiency, providing a novel strategy for the high-value utilization of lignocellulosic biomass.