Rapid in-situ enrichment of anammox bacteria in a thiosulfate-driven partial denitrification and anammox biofilter packed with sponge iron

Anaerobic ammonium oxidation (anammox) is an efficient and low-consumption autotrophic nitrogen removal technology with significant engineering potential. However, the mainstream application of anammox has been constrained by slow start-up and the limited sources of anammox inoculum. This study developed a novel strategy for in-situ enrichment of anammox bacteria (AnAOB) by establishing a thiosulfate-driven partial denitrification and anammox biofilter (TDDA-BF). With the synergistic effect of sponge iron, the system achieved rapid start-up using conventional activated sludge and sulfur-autotrophic denitrifying sludge, without external anammox inoculation. Through optimized sulfur-to-nitrogen ratio (S/N) and hydraulic retention (HRT) time control, successful in-situ AnAOB enrichment was accomplished within 78 days. The system attained 85% total nitrogen removal, with the anammox contribution increasing remarkably from 4.52% to 81.24%. 15N isotope tracing confirmed the dominant role of anammox in nitrogen removal. Quantitative PCR demonstrated significant enrichment of AnAOB functional genes (hdh and hzsA), while microbial analysis revealed substantial increase in Candidatus_Brocadia abundance from 0.38% to 21.01%. PICRUSt2 prediction indicated that Fe (II) from sponge iron potentially enhanced key enzyme activity in AnAOB through promoted heme c synthesis. This work demonstrates the feasibility of rapid in-situ AnAOB enrichment in TDDA-BF, significantly shortening start-up period and advancing practical implementation of carbon-efficient nitrogen removal technologies.