Enrichment and application of sulfur-dependent denitrifying microbes at low temperatures

Denitrification coupled to the oxidation of sulfur compounds has been proposed to remove residual nitrate in effluents of wastewater treatment plants due to the low demand of organic matter. However, low temperatures in high latitude and high-altitude areas will constrain its application. Diverse microbes simultaneously drive complete denitrification coupled to the oxidation of sulfur compounds, but how such consortia survive, and maintain denitrification in response to low temperatures remain unknown. Here, we enriched a set of efficient thiosulfate-dependent denitrifying cultures at 25 °C and 15 °C and thereafter inoculated these cultures to a continuous bioreactor. The reactor showed efficient denitrifying performance even at 10 °C with a very low accumulation of nitrite. Molecular analyses revealed that the five most abundant microorganisms constituted the denitrifying community, among which metabolic interactions existed that favored denitrification. A complete denitrifier, Hydrogenophilaceae, dominated at 25 °C and along with Thiobacillus_1 potentially fueled the community through carbon fixation. Our results showed that substrate exchange among the main community members improved the adaptation ability of the whole consortium at lower temperature. Nevertheless, Burkholderiaceae, the sole provider of pyridoxine and glutamine, could not grow fast enough at 5 °C, leading to the destabilization of metabolic interactions among main denitrifiers and ultimately causing nitrite accumulation. This suggests that low-abundance microorganisms might play important role in helping the microbial community to maintain denitrification activity at low temperatures. Finally, our study demonstrated that sulfur-dependent denitrification can function efficiently at temperatures as low as 10 °C, indicating its suitability for full-scale application.