Adaptive Laboratory Evolution (ALE) enables carbon-negative mixotrophic fermentation and enhanced chain elongation in Clostridium sp. JS66

Improving carbon recovery during sugar fermentation remains a major challenge because a substantial fraction of substrate carbon is lost as CO ₂ 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 CO ₂ /H ₂ conditions to enhance H ₂ -assisted CO ₂ reassimilation during glucose fermentation. The evolved strain, ALECO2, exhibited CO ₂ consumption without a lag phase under autotrophic conditions and reached a 9.5-fold higher CO ₂ uptake rate than JS66. Under fed-batch conditions, glucose-only fermentation yielded a carbon molar yield (C _metabolite /C _sugar, C _M /C _S ) of 0.60, whereas H ₂ 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 CO ₂ supplementation, ALECO2 further assimilated externally supplied CO ₂ , increasing the C _M /C _S to 1.10 and demonstrating carbon-negative fermentation. Assimilation of externally supplied CO ₂ 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 H ₂ - and CO ₂ /H ₂ -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 CO ₂ reassimilation and external CO ₂ assimilation with chain elongation, enabling carbon-neutral and carbon-negative production of value-added C6 products from glucose.