Characterization of Enrichment Cultures of Anammox, Nitrifying and Denitrifying Bacteria Obtained from a Cold, Heavily Nitrogen-Polluted Aquifer

SIMPLE SUMMARY: Heavy nitrogen pollution is a major ecological problem for cold aquifers near the zone of a radioactive waste surface repository. Unique microbial communities are formed in these groundwater ecosystems that could be used for sustainable bioremediation in situ. Prior to field experiments, in this work we obtained and analyzed enrichment cultures of N cycle bacteria to shed light on their metabolic capabilities. Despite high N conversion activity, surprisingly, no “conventional” nitrifying bacteria were found in enrichment cultures. Moreover, stable nitrite production could be achieved through heterotrophic denitrification, which implies great potential to produce nitrite for sustainable anammox-based bioremediation. Anammox bacteria were difficult to enrich in fed-batch culture. However, stable removal of ammonium and nitrite under anaerobic conditions could be achieved in a continuous flow reactor packed with nonwoven fabric. Intriguing was the high nonstoichiometric buildup of nitrate, which could be an adaptive mechanism of anammox bacteria to low temperature. Xanthomonadaceae was a common taxon for all enrichment cultures, indicating its exclusive role in this ecosystem. Moreover, its representatives, genera Thermomonas and Stenotrophomonas, have nitrite oxidoreductases (nxr), which may explain the observed high accumulation of nitrates. These findings may be important in anammox-based low-temperature wastewater treatment. ABSTRACT: Anammox bacteria related to Candidatus Scalindua were recently discovered in a cold (7.5 °C) aquifer near sludge repositories containing solid wastes of uranium and processed polymetallic concentrate. Groundwater has a very high level of nitrate and ammonia pollution (up to 10 and 0.5 g/L, respectively) and a very low content of organic carbon (2.5 mg/L). To assess the potential for bioremediation of polluted groundwater in situ, enrichment cultures of anammox, nitrifying, and denitrifying bacteria were obtained and analyzed. Fed-batch enrichment of anammox bacteria was not successful. Stable removal of ammonium and nitrite (up to 100%) was achieved in a continuous-flow reactor packed with a nonwoven fabric at 15 °C, and enrichment in anammox bacteria was confirmed by FISH and qPCR assays. The relatively low total N removal efficiency (up to 55%) was due to nonstoichiometric nitrate buildup. This phenomenon can be explained by a shift in the metabolism of anammox bacteria towards the production of more nitrates and less N(2) at low temperatures compared to the canonical stoichiometry. In addition, the too high an estimate of specific anammox activity suggests that N cycle microbial groups other than anammox bacteria may have contributed significantly to N removal. Stable nitrite production was observed in the denitrifying enrichment culture, while no “conventional” nitrifiers were found in the corresponding enrichment cultures. Xanthomonadaceae was a common taxon for all microbial communities, indicating its exclusive role in this ecosystem. This study opens up new knowledge about the metabolic capabilities of N cycle bacteria and potential approaches for sustainable bioremediation of heavily N-polluted cold ecosystems.