Mechanistic insights into electrode autotrophic denitrification: Isolation and electron transfer of Paracoccus versutus HD1 in denitrifying bioelectrochemical systems

Electrode autotrophic denitrifying bacteria (EADB) are critical in denitrifying bioelectrochemical systems (dBESs) for nitrate removal. However, effective methods for isolating EADB and understanding how they utilize electrode electrons for nitrate reduction remain unclear. This study aims to establish a reliable method for isolating EADB and to investigate the electron transfer mechanisms involved in their denitrification process. Paracoccus versutus HD1 (P. versutus HD1) was successfully isolated using an in-situ dBESs separation method combined with streak plate technique, and its denitrification capability was confirmed under heterotrophic, sulfur autotrophic, and electrode autotrophic conditions, respectively. P. versutus HD1 exhibits a reduction current of 37.1 mA/m², comparable to the model EADB Shewanella loihica PV-4. The nitrate nitrogen reduction rate could reach 183.8 mg/(m²·d) by utilizing electrode electrons, and be enhanced by 75.4 % and 60.6 % with exogenous electron shuttles of neutral red and resazurin added, respectively. However, riboflavin had no discernible effect, which implied the lack of outer membrane cytochrome c proteins and the Mtr respiratory pathway in HD1. Transcriptomic and proteomic analyses revealed that P. versutus HD1 fixes CO₂ in dBES via the Calvin cycle and accepts electrode electrons to reduce nitrate through denitrification pathway, with periplasmic cytochrome c protein associated with other outer membrane proteins playing an important role in the electron transfer processes. This study provides valuable insights into the molecular mechanisms of EADB, advancing both the theoretical understanding and practical implementation of electrode autotrophic denitrification for nitrate removal.