Unraveling the mechanisms of anammox coupled process with thiosulfate-driven denitrification: community succession and substrate competition.

Abstract

Thiosulfate-driven denitrification coupled with anammox (TDDA) has garnered interest for its efficient and innovative nitrogen removal capabilities. However, the intricate dynamics of the internal microbial community and the specific characteristics of anaerobic ammonium oxidizing bacteria (AnAOB) remain incompletely understood. This study combines experimental methods with density functional theory (DFT) calculations to address these gaps. The TDDA reactor was successfully started-up with an optimal S₂O₃^2--S/NO₃^--N ratio of 0.6, achieving a nitrogen removal efficiency of 89.6%. Throughout this process, the relative abundance of Candidatus Kuenenia decreased by 10.2%, while the relative abundance of Candidatus Brocadia increased by 9.6%. The elevated concentration of NO₃^--N inhibited Candidatus Kuenenia, and simultaneously stimulated the secretion of extracellular polymers, affecting Fe uptake by Candidatus Kuenenia. To further elucidate substrate competition, molecular docking simulations and DFT calculations were employed. The binding energy, compared with the electrostatic potential energy of the protein pocket, clearly demonstrated that Nir in AnAOB has a higher affinity for the substrate (E_AnAOB = -163.2 kJ/mol vs. E_SOB =-77.7 kJ/mol). By integrating molecular dynamics insights, this study overcomes experimental limitations and deepens the understanding of the mechanisms within the TDDA system.