Aeration-free microalgal-bacterial granular sludge for simultaneous nitrification, denitrification and phosphorus removal: Rapid start-up and functional differentiation

The simultaneous nitrification, denitrification, and phosphorus removal (SNDPR) process based on aeration-free microalgal-bacterial granular sludge (MBGS) offers a promising aeration-free pathway for sustainable wastewater treatment, but suffers from prolonged and unstable start-up. In this study, we co-inoculated aerobic granular sludge (AGS) capable of SNDPR with pre-cultivated MBGS to achieve rapid start-up of the aeration-free MBGS-SNDPR process in a photo-sequencing batch reactor. This strategy maintained a stable biomass concentration, improved physicochemical properties of the granules, and achieved efficient removal of COD (88% ± 4%), NH₄⁺-N (99% ± 1%), total nitrogen (TN, 85% ± 6%), and PO₄^3--P (91% ± 7%) throughout 100 days of reactor operation. Two morphologically and functionally distinct MBGS types that evolved from the AGS inoculum were identified: MBGS-S originated from small AGS (<1 mm) entangled with cyanobacteria, whereas MBGS-L originated from large AGS (>1 mm) colonized by phototrophs. MBGS-S was dominated by nitrification and phosphorus removal, whereas MBGS-L was dominated by denitrification. Typical cycle tests and mass balance calculations demonstrated that the reactor achieved efficient SNDPR performance, produced less net CO₂ production than active sludge, and retained phosphorus in harvested biomass as a potential recovery pool. Microbial community analysis further indicated that SNDPR-related functional microorganisms persisted and abundances of key functional genes increased throughout the operation, thereby ensuring stable and efficient pollutant removal. Overall, this study provides insights into strategies for the rapid start-up of the aeration-free MBGS-SNDPR process and for the successful integration of functional microorganisms into this sustainable process.