Start‑up and functional performance assessment of electron-shuttling-driven microalgal-anammox process

The anaerobic ammonium oxidation (anammox) process has received extensive attention because of its low energy consumption and high nitrogen removal efficiency. Nevertheless, its long start-up period limits practical application. In this study, a microalgae-bacteria symbiotic system (ABSS) based on Chlorella vulgaris (C. vulgaris) was established to investigate its effects on anammox start-up and nitrogen and phosphorus removal. The introduction of C. vulgaris shortened the start-up time from 68 to 46 days, corresponding to a 32 % reduction. After 210 days of operation, the nitrogen removal rate (NRR) and phosphorus removal rate (PRR) in the microalgae-bacteria symbiotic reactor (RAM) were 21 % and 25 % higher than those in the control reactor (RA), respectively. The concentration of extracellular polymeric substances (EPS) derived from C. vulgaris, as quantified via total organic carbon measurement, was 297.73 ± 8.55 mg/L. Spectroscopic analyses suggested the presence of humic-like substances and quinone-related structures in the EPS, indicating a potential role in electron transfer. At 100 mg/L, EPS increased hydrazine dehydrogenase (HDH) activity by 49 % relative to the control group. After CoQ extraction, EPS restored HDH activity more effectively than exogenous coenzyme Q (CoQ). Metagenomic analysis showed higher abundances of genes associated with photosynthesis, carbon fixation, and nitrogen metabolism. These findings imply that EPS-mediated electron transfer and strengthened HDH activity may have contributed to the establishment of anammox activity and the shortened start-up period. Overall, this study highlights microalgal integration as a potential strategy for accelerating anammox startup and improving simultaneous nitrogen and phosphorus removal.