Application of humin-immobilized biocathode in a continuous-flow bioelectrochemical system for nitrate removal at low temperature

Solid-phase humic substances (humin) can work as an additional electron donor to support the low temperature denitrification but the reducing capacity of its non-reduced form is limited. In this study, a continuous-flow denitrifying BES with a humin-immobilized biocathode (H-BioC) was established. Humin was expected to function as a redox mediator and be persistently reduced on the cathode to provide reducing power to a denitrifying biofilm. Results showed that the H-BioC maintained a stable denitrification capacity with low nitrite accumulation for more than 100 days at 5 °C, and the specific microbial denitrification rate and electron transfer rate were 3.97-fold and 1.75-fold higher than those of the unaltered cathode. The results of repeated cycles of humin reduction and oxidation experiments further suggested that the redox activity of humin was stable. Acidovorax was the most dominant genus in both H-BioC biofilm and unaltered cathodic biofilm, while Rhodocyclaceae (unclassified_f_) was more enriched in H-BioC biofilm. Further Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analyses indicated that biofilm formation, electron transfer, and nitrate reduction functions were more abundant in H-BioC, suggesting a possible enhancement mechanism by humin. The results of this study raise the possibility that immobilization of solid-phase humin may be a useful strategy for electrostimulated heterotrophic denitrification in groundwater where the indigenous bacteria have poor electroactivity.