Isotope analysis of nitrogen removal pathways and N2O production potential in the SDAD-anammox system under different N/S ratios.

This study explored the impact of varying nitrate to sulfide (N/S) ratios on nitrogen removal efficiency (NRE) in the sulfide-driven autotrophic denitrification and anammox (SDAD-anammox) system. Optimal nitrogen removal was observed at N/S ratios between 1.5 and 2.0. Isotope tracing results showed that the contribution of anammox to nitrogen removal was enhanced with increasing N/S ratios, reaching up to 37 % at the N/S ratio of 2.5. Additionally, complex nitrogen pathways were identified, including dissimilatory nitrate reduction to ammonium (DNRA). Furthermore, isotope tracing was innovatively applied to investigate N2O emissions, demonstrating that higher N/S ratios significantly reduced N2O emissions, with the lowest emissions at N/S ratio of 2.5. Gene expression analysis indicated that nitrogen and sulfide transformation genes decreased with increasing N/S ratios, while anammox-related genes first increased and then decreased, reflecting the system's microbial dynamics. These findings offer insights into nitrogen transformation pathways and N2O production mechanisms in the SDAD-anammox process.

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.

Relationship between functional bacteria in a denitrification desulfurization system under autotrophic, heterotrophic, and mixotrophic conditions.

The denitrification desulfurization system can be used to remediate wastewater containing carbon, nitrogen, and sulfur. However, the relationship between autotrophic and heterotrophic bacteria remains poorly understood. To better understand the roles and relations of core bacteria, an expanded granular sludge bed (EGSB) reactor was continuously operated under autotrophic (stage I), heterotrophic (stage II) and mixotrophic (stages III-VII) conditions with a 490-day period. Stage IV represented the excellent S0 recovery rate (69.5%). The different trophic conditions caused the obvious succession of dominant bacterial genera. Autotrophic environment (stage I) enriched mostly Thiobacillus, and heterotrophic environment (stage II) was dominated with Azoarcus and Pseudomonas. Thauera, Arcobacter and Azoarcus became the predominant genera under mixotrophic conditions (stage III-VII). Strains belonged to these core genera were further isolated, and all seven isolates were confirmed with denitrifying sulfur oxidation capacity. Heterotrophic strain HDD1 (genus of Thauera) possessed both the highest sulfide degradation and S0 recovery rates. Expression levels of cbbM and gltA genes were positively related with the autotrophic and heterotrophic conditions, respectively. NirK gene was highly expressed between log 3.7-log 4.3 during the entire run. Expression of both sqr and soxB genes were closely related with sulfur conversion. More than 57.5% of S0 recovery rate could be obtained as sqr gene expression was greater than log 3.2, and while, sulfate was the primary form as soxB gene expression higher than log 3.9. The correlation between core microbial genera was very low from network, indicating a complex and non-specific mutualistic network between bacterial functional groups under each nutrient condition, and a stable coexistence state was possibly formed through utilizing each the secondary or waste metabolites in the mixotrophic conditions. This relationship was beneficial to the stability of the microbial community structure in the denitrification desulfurization system.