Abstract:In this study, micro-oxygen was integrated into a rusty scrap iron (RSI)-loaded anaerobic digester. Under an optimal RSI dosage of 20 g/L, increasing O 2 levels were added stepwise in seven stages in a semi-continuous experiment. Results showed the average methane yield was 306 mL/g COD (chemical oxygen demand), and the hydrogen sulphide (H 2 S) concentration was 1933 ppmv with RSI addition. O 2 addition induced the microbial oxidation of sulphide by stimulating sulfur-oxidizing bacteria and chemical corrosion of iron, which promoted the generation of FeS and Fe 2 S 3 . In the 6th phase of the semi-continuous test, deep desulfurization was achieved without negatively impacting system performance. Average methane yield was 301.1 mL/g COD, and H 2 S concentration was 75 ppmv. Sulfur mass balance was described, with 84.0%, 11.90% and 0.21% of sulfur present in solid, liquid and gaseous phases, respectively. The Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis revealed that RSI addition could enrich the diversity of hydrogenotrophic methanogens and iron-reducing bacteria to benefit methanogenesis and organic mineralization, and impoverish the methanotroph (Methylocella silvestris) to reduce the consumption of methane. Micro-oxygen supplementation could enhance the diversity of iron-oxidizing bacteria arising from the improvement of Fe(II) release rate and enrich the sulphur-oxidising bacteria to achieved desulfurization. These results demonstrated that RSI addition in combination with micro-oxygenation represents a promising method for simultaneously controlling biogas H 2 S concentration and improving digestion performance.