Biochar-Facilitated Extracellular Electron Synergism Between Anammox and Denitrifying Bacteria for Enhanced Nitrogen Removal

Low temperature is one of the critical limiting factors for anammox. To address this, our study investigated whether biochar could enhance the nitrogen metabolism of anammox consortia at 15 °C by serving as an exogenous electron shuttle. Compared with the control, biochar addition effectively improved the total nitrogen removal (520 vs. 450 g-N/(m3·d), p < 0.05), with notable enhancement in nitrate elimination. Specifically, biochar increased the abundance of denitrifying bacteria and anammox bacteria (predominantly Candidatus Kuenenia). Correspondingly, the expression of denitrification functional enzymes from denitrifiers, as well as hydroxylamine oxidoreductase HAO and hydrazine dehydrogenase HDH from Ca. Kuenenia, was upregulated. However, interestingly, the nitrite reductase NirS and hydrazine synthase HZS of Ca. Kuenenia were downregulated, suggesting the presence of an unconventional anammox pathway. Based on the differential expression of nitrogen metabolism and electron transport proteins, an extracellular electron transfer (EET)-dependent anammox process and its associated electron transfer pathway in Ca. Kuenenia were proposed. Furthermore, biochar was considered to bridge EET-dependent denitrification and anammox: biochar was first oxidized by electroactive denitrifiers and subsequently served as the electron acceptor for nitrite-independent ammonium oxidation by Ca. Kuenenia. Through the redox cycling, biochar mediated interspecies electron syntrophy of the N-cycling functional bacteria, thereby enhancing nitrogen removal efficiency. This study broadens the current understanding of the physiology and ecology of anammox and provides a promising improvement strategy under low temperatures. Nevertheless, since both microbial activity and biochar properties are subject to more complex conditions in actual wastewater, this strategy requires further evaluation in practical applications.