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 (N 2 O) 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 N 2 O 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 N 2 O accumulation, whereas the nitrite ( NO 2 - )-fed condition led to severe N 2 O accumulation due to an imbalance between N 2 O 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, N 2 O-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 N 2 O producer. MAG 25 exhibits N 2 O-responsive functional enrichment upon N 2 O feeding, reflecting a specialized ecological strategy centered on sulfur oxidation coupled with N 2 O 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 N 2 O cycling depending on conditions. This study provides new insights into microbial ecology and offers potential strategies for mitigating N 2 O emissions in SADN processes.