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.