Availability of Nitrite and Nitrate as Electron Acceptors Modulates Anaerobic Toluene-Degrading Communities in Aquifer Sediments

Microorganisms are essential in the degradation of environmental pollutants. Aromatic hydrocarbons, e.g., benzene, toluene, ethylbenzene, and xylene (BTEX), are common aquifer contaminants, whose degradation in situ is often limited by the availability of electron acceptors. It is clear that different electron acceptors such as nitrate, iron, or sulfate support the activity of distinct degraders. However, this has not been demonstrated for the availability of nitrate vs. nitrite, both of which can be respired in reductive nitrogen cycling. Here via DNA-stable isotope probing, we report that nitrate and nitrite provided as electron acceptors in different concentrations and ratios not only modulated the microbial communities responsible for toluene degradation but also influenced how nitrate reduction proceeded. Zoogloeaceae members, mainly Azoarcus spp., were the key toluene degraders with nitrate-only, or both nitrate and nitrite as electron acceptors. In addition, a shift within Azoarcus degrader populations was observed on the amplicon sequence variant (ASV) level depending on electron acceptor ratios. In contrast, members of the Sphingomonadales were likely the most active toluene degraders when only nitrite was provided. Nitrate reduction did not proceed beyond nitrite in the nitrate-only treatment, while it continued when nitrite was initially also present in the microcosms. Likely, this was attributed to the fact that different microbial communities were stimulated and active in different microcosms. Together, these findings demonstrate that the availability of nitrate and nitrite can define degrader community selection and N-reduction outcomes. It also implies that nitrate usage efficiency in bioremediation could possibly be enhanced by an initial co-supply of nitrite, via modulating the active degrader communities.