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

Abstract

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.

Keywords: Azoarcus; bioremediation; oxygenic denitrification; stable isotope probing; toluene degradation.

Figure 1

Activity of nitrate, nitrite reduction and toluene oxidation in all except the control treatment microcosms. The first 7 days data are the average of three replicate incubations, while the other data points are the average of the two rest incubations. The 8/0 panel represents the results of the microcosm contained 8/0 mM (nitrate/nitrite) as electron acceptors; the 5/7 panel represents the results of the microcosm contained 5/7 mM (nitrate/nitrite) as electron acceptors; the 2/12 panel represents the results of the microcosm contained 2/12 mM (nitrate/nitrite) as electron acceptors; and the 0/18 panel represents the results of the microcosm contained 0/18 mM (nitrate/nitrite) as electron acceptors.

Figure 2

The abundance distribution of all bacteria (16S rRNA) and oxygenic denitrifiers (nod) along the density gradients of the 8/0, 5/7, 0/18, and the control microcosms. For each microcosm, DNA recovered from the fractions with highest 16S rRNA or nod counts at both light and heavy densities were used for amplicon sequencing analysis.

Figure 3

ASVs (amplicon sequencing variants) diversity in light and heavy fractions of the 8/0, 5/7, 0/18, and the control microcosms. ASVs with relative abundance <2% were not individually displayed.

Figure 4

Bootstrapped neighbor-joining phylogeny of abundant (≥2%) ASVs and reference 16S rRNA sequences. Bootstrap support (1,000 replicates) >50% is indicated at the nodes. The scale bar represents 5% nucleotide sequence divergence. The phylogeny was calculated in MEGA-X based on the V4 region alignment using default parameters.