Quantifying the Contribution of Cryptic Sulfide-Driven Autotrophic Denitrification to N 2 O Production in a Seasonally Hypoxic River-Reservoir System

Sulfur-autotrophic denitrification (S-ADN) has been frequently reported in inland waters, yet its quantitative contribution to nitrous oxide (N 2 O) productions remains poorly constrained. By combining field sampling, enrichment cultures, stable isotopes, and metagenomic analysis, we quantified S-ADN-derived N 2 O productions in an oligotrophic river-reservoir system and validated the universality of our approach across diverse aquatic ecosystems. A Thiobacillus -dominated S-ADN enrichment culture was successfully established over 218 days of continuous supplementation with reduced sulfur compounds, yielding critical isotopic signatures for source partitioning (δ 15 N Bulk, average N isotopic composition; δ 18 O; and δ 15 N SP, site preference). Then, the multi-isotope Bayesian model revealed that S-ADN (14.8%) and nitrifier denitrification (NDN, 21.1%) rapidly occupied the ecological niche of heterotrophic denitrification (HDN, 8.6%) to total microbial N 2 O sources under organic carbon-limited conditions. The cryptic sulfur cycle supplies a considerable pool of electron donors for S-ADN under low-sulfide conditions. Autotrophic denitrifiers (e.g., Thiobacillus, Sulfuritalea ) exhibited significant synergistic interactions with ammonia-oxidizing archaea (AOA, Nitrosarchaeum ), while ammonia-oxidizing bacteria (AOB, Nitrosomonas ) and nitrite-oxidizing bacteria (NOB, Nitrospira ) jointly completed nitrification─with Nitrosomonas further competing for nitrite to drive NDN. This study advances the quantitative assessment of S-ADN’s role in N 2 O production and provides novel insights into microbial community interactions in oligotrophic aquatic systems.