Temporal analysis of the microbial communities in a nitrate-contaminated aquifer and the co-occurrence of anammox, n-damo and nitrous-oxide reducing bacteria

Groundwater-N pollution derives from agricultural and urban activities, and compromises water quality in shallow aquifers, putting human and environmental health at risk. Nonetheless, subsurface microbiota can transform dissolved inorganic nitrogen into N₂. In this study, we surveyed the microbial community of a shallow aquifer by sampling one well, one piezometer and a spring within an agricultural area that receives N-inputs of more than 700 kg/ha per year through irrigation with wastewater. The survey was conducted during a year with a 16S rRNA next-gen approach. In parallel, we quantified the number of gene copies and transcripts related to anaerobic ammonium oxidation (anammox, hzo), nitrite-dependent anaerobic methane oxidation (n-damo, nod and pmoA) and nitrous oxide reduction (last step of denitrification, nosZ), during the dry and rainy seasons. Our results showed that the groundwater samples had 17.7 to 22.5 mg/L of NO₃⁻-N. The bacterial and archaeal community structure was distinctive at each site, and it remained relatively stable over time. We verified the co-occurrence of N-transforming bacteria, which was correlated with the concentration of NO₂⁻/NO₃⁻ and ORP/DO values (DO: ~3.0 mg/L). Our analyses suggest that these conditions may allow the presence of nitrifying microorganisms which can couple with anammox, n-damo and denitrifying bacteria in interrelated biogeochemical pathways. Gene density (as the number of gene copies per litre) was lower in the rainy season than in the dry season, possibly due to dilution by rainwater infiltration. Yet, the numbers of hzo gene copies here found were similar to those reported in oceanic oxygen minimum zones and in a carbonate-rock aquifer. The transcript sequences showed that Candidatus Brocadia spp. (anammox), Candidatus Methylomirabilis spp. (n-damo) and autotrophic denitrifying Betaproteobacteria coexist in the groundwater environment, with the potential to attenuate the concentration of dissolved inorganic nitrogen by reducing it to N₂ rather than N₂O; delivering thus, an important ecosystem service to remove contaminants.