Molecular mechanisms through which different carbon sources affect denitrification by Thauera linaloolentis: Electron generation, transfer, and competition.

Characterizing the molecular mechanism through which different carbon sources affect the denitrification process would provide a basis for the proper selection of carbon sources, thus avoiding excessive carbon source dosing and secondary pollution while also improving denitrification efficiency. Here, we selected Thauera linaloolentis as a model organism of denitrification, whose genomic information was elucidated by draft genome sequencing and KEGG annotations, to investigate the growth kinetics, denitrification performances and characteristics of metabolic pathways under diverse carbon source conditions. We reconstructed a metabolic network of Thauera linaloolentis based on genomic analysis to help develop a systematic method of researching electron pathways. Our findings indicated that carbon sources with simple metabolic pathways (e.g., ethanol and sodium acetate) promoted the reproduction of Thauera linaloolentis, and its maximum growth density reached OD600 = 0.36 and maximum specific growth rate reached 0.145 h-1. These carbon sources also accelerated the denitrification process without the accumulation of intermediates. Nitrate could be reduced completely under any carbon source condition; but in the "glucose group", the maximum accumulation of nitrite was 117.00 mg/L (1.51 times more than that in the "ethanol group", which was 77.41 mg/L), the maximum accumulation of nitric oxide was 363.02 µg/L (7.35 times more than that in the "ethanol group", which was 49.40 µg/L), and the maximum accumulation of nitrous oxide was 22.58 mg/L (26.56 times more than that in the "ethanol group", which was 0.85 mg/L). Molecular biological analyses demonstrated that diverse types of carbon sources directly induced different carbon metabolic activities, resulting in variations in electron generation efficiency. Furthermore, the activities of the electron transport system were positively correlated with different carbon metabolic activities. Finally, these differences were reflected in the phenomenon of electronic competition between denitrifying reductases. Thus we concluded that this was the main molecular mechanism through which the carbon source type affected the denitrification process. In brief, carbon sources with simple metabolic pathways induced higher efficiency of electron generation, transfer, and competition, which promoted rapid proliferation and complete denitrification; otherwise Thauera linaloolentis would grow slowly and intermediate products would accumulate seriously. Our study established a method to evaluate and optimize carbon source utilization efficiency based on confirmed molecular mechanisms.