Introducing PHBV and controlling the pyrite sizes achieved the pyrite-based mixotrophic denitrification under natural aerobic conditions: Low sulfate production and functional microbe interaction

The pyrite-based autotrophic denitrification usually focused on the anaerobic/anoxic environment with high sulfate production and low denitrification efficiency. In this study, the oxygen limitation was broken and high-rate mixotrophic denitrification was realized via introducing poly-3-hydroxybutyrate-hydroxyvalerate (PHBV) and controlling the pyrite grain sizes compared with PHBV-heterotrophic and pyrite-autotrophic denitrification under natural aerobic conditions. Results showed that the optimal pyrite grain size was 30 meshes (0.45–0.55 mm) with the highest denitrification rate of 0.65 mg NO3−−N/(L•h) and sulfate production of <5 mg/L in pyrite-PHBV system. Its removal efficiency of nitrogen and phosphorus was 96% and 25%, respectively. According to functional genes and Raman analysis, the low SO42− production were attributed to the S reduction and polysulfide formation in the mixotrophic systems, which also enhanced denitrification efficiency by improving bioavailability and multi-stage utilization of sulfur matrix (i.e., S2−, poly-S and SO42−) in these mixotrophic systems. Thiobacillus and Acidovorax have been enriched on the pyrite and PHBV surface, respectively, which highlighted that the autotrophic-heterotrophic synergy was achieved via different functional microbes domesticated by substrates. The differential analysis among functional genes suggested that PHBV surface promoted the NO2− reduction and S oxidation, and pyrite surface enhanced the N2O reduction and S reduction. A closer connection between C degradation and N cycle indicated their copiotrophic nutritional patterns based on the network analysis. The key taxa co-occurrence revealed rare species involved in Fe cycle metabolism were the hidden drivers in the mixotrophic denitrification process. These findings provide an in-depth understanding on the multiple interactions among N, S, and Fe cycling, and improve the practical application of pyrite-PHBV mixotrophic systems.