Dual role of citric acid: facilitate the bioconversion of CO2 to CH4 and enhance bioavailability of zero-valent iron

Studies on the bioconversion of CO₂ to CH₄ using Fe⁰ have reported that a fraction of CO₂ is converted to FeCO₃, forming a passivation layer on the Fe⁰ surface, which reduces its reactivity and limits process efficiency. To mitigate FeCO₃ precipitation and enhance CO₂-to-CH₄ conversion using Fe⁰, this study evaluates the role of citric acid (CA) as a ligand to Fe²⁺, which helps maintain iron in a soluble form and prevents its rapid conversion to insoluble iron carbonate (FeCO₃). Batch experiments with 30 mM Fe⁰, anaerobic granular sludge (AGS), and NaHCO₃ with CA as a ligand showed that citrate biodegraded into acetic acid and CO₂ gas. To prevent citrate degradation and elucidate its effect on Fe⁰-mediated methanogenesis, 5 % v/v of antibiotic antimycotic solution (100x) (10,000 units penicillin, 10 mg streptomycin and 25 μg amphoctericin per ml) were introduced as a research tool, selectively inhibiting bacterial activity without affecting hydrogenotrophic methanogens. Methane production was notably enhanced only in the sample with NaHCO₃, CA, antibiotics, and Fe⁰, reaching 38 ml by day 50, whereas the corresponding sample without Fe⁰ produced only 22 ml, resulting in a net methane production of 16 ml. The results indicate that CA enhances methane production by maintaining iron solubility and inhibiting FeCO₃ precipitation, thereby facilitating continuous iron oxidation and sustained electron release, which supports the microbial reduction of CO₂ to CH₄. While antibiotics provided a controlled environment to uncover these mechanisms, their use is not a viable long-term solution. Based on the findings of this study, future work may explore short-term CA exposure (<12 h) or alternative ligands to minimize biodegradation without relying on antibiotics.