Iron-enhanced rTCA cycle drives synergistic enhancement of inorganic carbon fixation and denitrification.

Iron-carbon autotrophic denitrification has shown potential for nitrogen removal from low-C/N wastewater. However, its application has been limited by slow Fe0 corrosion, increased alkalization, and insufficient inorganic carbon. CO2 induction represents a promising strategy for overcoming the above limitations. Here, a CO2 enhanced iron-carbon autotrophic denitrification process was established to elucidate how CO2 regulates iron corrosion, carbon metabolism, and denitrification. The results showed that the continuous CO2 supply maintained weakly acidic conditions (pH 6.0-6.5), accelerated Fe0 corrosion, increased Fe2+ release, and promoted poorly crystalline iron phases, thus enhancing electron transfer and nitrogen removal. The total nitrogen removal efficiencies increased and reached 97.95 ± 1.57%, with the concentrations decreasing to 0.83 ± 0.62 mg N L-1 in effluents. Metagenomic and enzymatic analyses revealed that CO2 activated two complementary carbon fixation pathways. Concurrent enrichment of the CBB-associated genes rbcL/rbcS and prk/prkB, together with increased apparent Rubisco activity, supported enhanced CBB-associated CO2 assimilation potential. Under Fe-rich reducing conditions, genes involved in ferredoxin-mediated carboxylation were enriched, potentially supporting the reductive tricarboxylic acid (rTCA) cycle. Fixed inorganic carbon was routed into central carbon metabolism, generating bioavailable intermediates that supported denitrification. Metagenome-assembled genome revealed a denitrifier-centered cooperative network linking carbon fixation, carbon turnover, and nitrate reduction. Collectively, CO2 promoted denitrification by coupling Fe0 corrosion, dual carbon fixation pathways, carbon turnover, and iron-dependent nitrogen transformation. These findings reveal an underappreciated role of iron-mediated carbon fixation in denitrification and provide a strategy for efficient nitrogen removal from low-C/N wastewater.