Adaptive laboratory evolution enables carbon-negative mixotrophic fermentation and enhanced chain elongation in...

Improving carbon recovery during sugar fermentation remains a major challenge because a substantial fraction of substrate carbon is lost as CO₂ during central metabolism. To overcome this limitation, Clostridium sp. JS66 (JS66), an acetogen producing hexanoic acid from glucose, was subjected to adaptive laboratory evolution under autotrophic CO₂/H₂ conditions to enhance H₂-assisted CO₂ reassimilation during glucose fermentation. The evolved strain, ALECO2, exhibited CO₂ consumption without a lag phase under autotrophic conditions and reached a 9.5-fold higher CO₂ uptake rate than JS66. Under fed-batch mixotrophic conditions, glucose-only fermentation yielded a carbon molar yield (C_metabolite/C_sugar, C_M/C_S) of 0.60, whereas H₂ supplementation increased C_M/C_S to 0.91 and redirected carbon flux toward C6 products (hexanoic acid and hexanol), which accounted for 49% of total C_output. With additional CO₂ supplementation, ALECO2 further assimilated externally supplied CO₂, increasing the C_M/C_S to 1.10 and demonstrating carbon-negative fermentation. Assimilation of externally supplied CO₂ further redirected carbon flux toward chain elongation, producing 7.14 g/L hexanoic acid and increasing the C6 carbon fraction to 57% of total C_output. Constraint-based flux analysis supported increased acetyl-CoA formation through the Wood-Ljungdahl pathway and enhanced flux toward reverse β-oxidation under H₂- and CO₂/H₂-supplemented conditions. Genome analysis identified mutations including genes encoding a putative HytB homolog and a LysR-type transcriptional regulator. These results establish ALECO2 as a promising evolved anaerobic non-photosynthetic (ANP) mixotrophy platform that links CO₂ reassimilation and external CO₂ assimilation with chain elongation, enabling carbon-neutral and carbon-negative production of value-added C6 products from glucose.