Microorganisms Enhance Denitrification of Low Carbon-to-Nitrogen Ratio Municipal Wastewater and Reduce N₂O Emission: Metabolites Promote Carbon Flux to Nitrate Bioreduction

Denitrifying carbon source is mainly used for microbial growth and proliferation, substrate transport, and nitrate bioreduction. This paper reported a new approach to dramatically promote denitrification and reduce N₂O emission of low carbon-to-nitrogen ratio (C/N) municipal wastewater by introducing two microorganisms to increase carbon flux to nitrate bioreduction without increasing the external carbon source. At a C/N of 3, the total nitrogen removal efficiency of the model denitrifier (<i>P. denitrificans</i>) was increased from 48.1% to 87.4%, with a 63.2% reduction in N₂O emission after adding <i>S. oneidensis</i> MR-1 and <i>B. subtilis</i>. Mechanism investigation revealed that the carbon flux for <i>P. denitrificans</i> growth and proliferation remained unchanged but decreased for substrate transport, resulting in more carbon flux to produce more NADH for nitrate bioreduction. Proteomics analysis showed that the decreased carbon flux for substrate transport was due to biosurfactants (surfactin, lichenysin, and fengycin) secreted by <i>B. subtilis</i>, increasing <i>P. denitrificans</i> cell membrane permeability and decreasing substrate uptake via active transport. Moreover, metabolites (riboflavin, heme, and cytochromes) from <i>S. oneidensis</i> MR-1 improved electron transfer, while lichenysin improved <i>P. denitrificans</i> surface hydrophilicity, which promoted the entry of metal ions associated with denitrification into the cell, increasing denitrifying enzyme activity, particularly N₂O reductase, ultimately enhancing denitrification and reducing N₂O emission.