Impact of organic carbon on sulfide-driven autotrophic denitrification: Insights from isotope fractionation and functional genes

Additional organics are generally supplemented in the sulfide-driven autotrophic denitrification system to accelerate the denitrification rate and reduce sulfate production. In this study, different concentrations of sodium acetate (NaAc) were added to the sulfide-driven autotrophic denitrification reactor, and the S0 accumulation increased from 7.8% to 100% over a 120-day operation period. Batch experiments revealed a threefold increase in total nitrogen (TN) removal rate at an Ac−-C/N ratio of 2.8 compared to a ratio of 0.5. Addition of organic carbon accelerated denitrification rate and nitrite consumption, which shortened the emission time of N2O, but increased the N2O production rate. The lowest N2O emissions were achieved at the Ac−-C/N ratio of 1.3. Stable isotope fractionation is a powerful tool for evaluating different reaction pathways, with the 18ε/15ε values in nitrate reduction ranging from 0.5 to 1.0. This study further confirmed that isotope fractionation can reveal denitrifying nutrient types, with the 18ε (isotopic enrichment factor of oxygen)/15ε (isotopic enrichment factor of nitrogen) value approaching 1.0 for autotrophic denitrification and 0.5 for heterotrophic denitrification. Additionally, the 18ε/15ε values can indicate changes in nitrate reductase. There is a positive correlation between the 18ε/15ε values and the abundance of the functional gene napA, and a negative correlation with the abundance of the gene narG. Moreover, 18ε and 15ε were associated with changes in kinetic parameters during nitrate reduction. In summary, the combination of functional gene analysis and isotope fractionation effectively revealed the complexities of mixotrophic denitrification systems, providing insights for optimizing denitrification processes.