Mixotrophics sulfur disproportionation enhables rapid and low-N2O denitrification in sulfur-packed systems through a novel symbiotic network

Elemental sulfur (S0) supports low-carbon denitrification in natural sediments and engineered reactors, yet nitrate reduction rates frequently far exceed those expected from S0’s slow abiotic dissolution. This kinetic discrepancy is often attributed to enzymatic sulfur disproportionation (SD). However, SD is considered as a predominantly autotrophic process and potentially suppressed by organic carbon in the prevalent mixotrophic conditions. This attribution appears insufficient to explain the pervasive occurrence of this kinetic discrepancy. Furthermore, the sulfide generated during SD process could inhibit nitrous oxide reductase (NosZ), promoting high emissions of N2O. Nevertheless, this effect is not always accompanied by a measurable N₂O accumulation, implying that additional regulatory mechanisms may be involved. Here, we combine long‑term reactor operation, targeted batch assays, DNA-stable isotope probing (DNA‑SIP) and genome-resolved metagenomics to identify the SD microbial mechanism under mixotrophy. A sulfur‑packed bed reactor operated for >300 days achieved >99% nitrate removal at a hydraulic retention time of 2 h, while accumulating both sulfate and sulfide and producing measurable ammonium, indicating concurrent cryptic sulfur cycling and DNRA signals. DNA‑SIP links carbon assimilation to distinct functional guilds and enriches sulfur oxidation, DNRA and N₂O‑reduction genes in isotopically heavy fractions. Metagenomics of active fractions reveal a novel thriving set of facultative mixotrophic sulfur-disproportionating denitrifiers (FMSDs) whose genomes consolidate SD, complete denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) within a single cellular framework. This unique integration facilitates an intrinsic dual-detoxification mechanism: the internal DNRA module acts as a sink for nitrite, mitigating the accumulation of inhibitory free nitrous acid (FNA), while robust sulfide oxidation modules concurrently detoxify the sulfide produced by SD’s reductive branch. By collectively safeguarding the terminal nitrous oxide reductase enzyme, this self-regulating network ensures profound N2O mitigation. This discovery redefines the microbial ecology of S-N coupling and provides a new blueprint for designing resilient, climate-friendly biotechnologies for water reclamation.