Stable-Isotope Probing Reveals the Activity and Function of Autotrophic and Heterotrophic Denitrifiers in Nitrate Removal from Organic-Limited Wastewater

Combined heterotrophic and autotrophic denitrification (HAD) is a sustainable and practical method for removing nitrate from organic-limited wastewater. However, the active microorganisms responsible for denitrification in wastewater treatment have not been clearly identified. In this study, a combined microelectrolysis, heterotrophic, and autotrophic denitrification (CEHAD) process was established. DNA-based stable isotope probing was employed to identify the active denitrifiers in reactors fed with either ¹³C-labeled inorganic or organic carbon sources. The total nitrogen removal efficiencies reached 87.2-92.8% at a low organic carbon concentration (20 mg/L COD). Real-time polymerase chain reaction of the nirS gene as a function of the DNA buoyant density following the ultracentrifugation of the total DNA indicated marked ¹³C-labeling of active denitrifiers. High-throughput sequencing of the fractionated DNA in H¹³CO₃ ^-/¹²CH₃ ¹²COO^--fed and H¹²CO₃ ^-/¹³CH₃ ¹³COO^--fed reactors revealed that Thermomonas-like phylotypes were labeled by ¹³C-bicarbonate, while Thauera-like and Comamonas-like phylotypes were labeled by ¹³C-acetate. Meanwhile, Arenimonas-like and Rubellimicrobium-like phylotypes were recovered in the "heavy" DNA fractions from both reactors. These results suggest that nitrate removal in CEHAD is catalyzed by various active microorganisms, including autotrophs, heterotrophs, and mixotrophs. Our findings provide a better understanding of the mechanism of nitrogen removal from organic-limited water and wastewater and can be applied to further optimize such processes.