Electron acceptor-dependent duality of nitrous oxide metabolism in Thiobacillus during sulfur autotrophic denitrification.

Sulfur-driven autotrophic denitrification (SADN) is a promising biotechnology for nitrogen removal from low-carbon wastewater; however, nitrous oxide (N2O) emissions remain a significant environmental concern. This study systematically investigated the effects of different nitrogen oxide electron acceptors on denitrification performance, microbial community succession, distribution of the N2O reductase gene nosZ clade, and the ecological functions of Thiobacillus using sequential enrichment cultivation. Among the tested conditions, the nitrate ( NO 3 - )-fed system achieved the highest denitrification and sulfur oxidation rates, with the lowest net N2O accumulation, whereas the nitrite ( NO 2 - )-fed condition led to severe N2O accumulation due to an imbalance between N2O production and reduction. High-throughput sequencing and quantitative PCR analyses revealed that Thiobacillus became the primary sulfur-oxidizing denitrifier in the presence of NO 3 - , NO 2 - , and nitric oxide, accompanied by substantial enrichment of nirS and clade I nosZ genes. In contrast, N2O-fed conditions promoted a more functionally diverse community enriched with clade II nosZ bacteria, including Azonexus and Dechloromonas. Metagenomic analyses recovered three distinct Thiobacillus metagenome-assembled genomes (MAGs 10, 11, and 25), each with distinct denitrification and sulfur oxidation capacities. MAG 10 contains genes for complete sulfur oxidation and denitrification, including clade I nosZ and nirS genes. Conversely, the nosZ gene was not detected in MAG 11, whereas the norB/norC genes were present, indicating their potential role as an N2O producer. MAG 25 exhibits N2O-responsive functional enrichment upon N2O feeding, reflecting a specialized ecological strategy centered on sulfur oxidation coupled with N2O reduction. Overall, these findings show that electron acceptors play a key role in shaping microbial succession, nosZ clade distribution, and the dual roles of Thiobacillus in N2O cycling depending on conditions. This study provides new insights into microbial ecology and offers potential strategies for mitigating N2O emissions in SADN processes.