Response of exoelectrogens centered consortium to nitrate on collaborative metabolism, microbial community, and spatial structure

• Exoelectrogens centered biofilm performed DNRA transitorily with nitrate disturbance. • The mutualistic interaction between exoelectrogens and denitrifiers was established. • Co-existence of H 2 producer and consumer indicated H 2 mediated electron transfer. • Obvious aggregates was formed after nitrate addition facilitating interactions. • Proteins and humic acids secreted in EPS indicated interactions between cells. Exoelectrogens are widespread and participate in building up interspecies electron-sharing network between anaerobic microbes. However, the exoelectrogens-centered electron-sharing networks might be affected by over-released nitrate, which competes with solid minerals or electrodes as the electron acceptor. The knowledge on the mechanism on the response of exoelectrogens-centered community to nitrate disturbance is still limited. This study applied the microbial electrochemical system as a platform and provided insights into the response of electroactive biofilm to nitrate under a constant potential of + 0.2 V. Under a fixed carbon–nitrogen ratio of 6, the biofilm partially processed dissimilatory nitrate reduction to ammonium (DNRA) transitorily and gradually loses electron-output capability. After sufficient succession, the biofilm achieved complete denitrification without ammonia accumulation and started to acquire electrons from the electrode. With nitrate disturbance, the exoelectrogens-centered biofilm dominated by Geobacter sp. preferred to cooperate with highly enriched hydrogenotrophic denitrifiers for mutualistic growth with hydrogen as the electron mediator. Different scan rates of CVs also suggested the mediated electron transfer process. The extracellular polymer substrate (EPS) serving as a network contributed to aggregates formation. The enhanced humic acid and protein secretion in the EPS of nitrate-affected biofilm suggested frequent interactions between cells. Therefore, a mutualistic interaction was established between exoelectrogens and denitrifiers under nitrate stress.