Headspace-driven metabolic rerouting balances chain elongation and acetogenic pathways for high-efficiency carbon recovery.

In situ assimilation of off-gases (H2/CO2) into liquid metabolites during anaerobic fermentation presents a promising approach to maximize waste biomass valorization. However, mechanisms governing the interaction between microbial carbon fixation and chain elongation (CE) through headspace dynamics remain unclear. This study explored gas-liquid metabolic flux and microbial response by varying headspace pressure/composition in batch CE bioreactors. Autogenic pressure reshaped the physicochemical microenvironment, which potentially induced specific biological inhibition and constrained ethanol utilization. Accordingly, caproic acid production decreased by 40.50-51.68%, whereas acetic acid accumulation increased by 70.67-104.44% under both N2 and H2/CO2 headspace. This alteration reflected metabolic flux redistribution between CE and acetic acid-producing pathways driven mainly by electron donor oxidation with homoacetogenesis as a potential secondary contributor. Meanwhile, an initial H2/CO2 headspace curbed off-gas emissions and induced a final 17.62 kPa (abs) vacuum, enhancing soluble metabolite carbon retention. Consequently, liquid-phase carbon recovery increased to 84.29% under autogenic pressure and peaked at 89.68% following further H2/CO2 headspace replacement. Microbial profiling revealed that self-pressurized conditions suppressed Oscillibacter from 15.14% to 1.93%, whereas H2/CO2 exposure selectively enriched Caproiciproducens to 30.52% as the dominant CE driver. Metatranscriptomics demonstrated that autogenic pressure markedly upregulated the Wood-Ljungdahl pathway genes by up to 28.62-fold, accompanied by induction of stress-responsive genes for physiological adaptation. Additionally, the initial H2/CO2 headspace promoted the expression of gene korA, suggesting a potential partial reductive tricarboxylic acid (rTCA) pathway for carbon fixation. These insights provide a mechanistic framework for optimizing carbon recovery and product selectivity in engineered CE systems, advancing sustainable waste-to-biochemical platforms.