Effects of aerobic denitrifying Rhizobium pusense strain N7 on water quality, biofilm, and zebrafish gut microbiota in recirculating aquaculture systems

To investigate the effects of Rhizobium pusense N7 on water quality, microbial communities, and denitrification in recirculating aquaculture systems (RAS). Three concentrations (10 5 , 10 6 , and 10 7 CFU/mL) were tested alongside a control (CK). Results showed that R. pusense N7 significantly improved denitrification within 9 days, with 10 5 CFU/mL reducing ammonia/nitrite and 10 7 CFU/mL excelling in nitrate removal ( p < 0.05). At 10 6 CFU/mL, R. pusense N7 abundance in biofilms and zebrafish intestines surpassed the control ( p < 0.05). At 10 5 –10 6 CFU/mL, R. pusense N7 suppressed pathogens ( Flavobacterium , Polynucleobacter ) and enriched beneficial bacteria ( Arenimonas ) in water. In biofilms, it boosted nitrogen-fixing ( Ensifer , Brevundimonas ) and probiotic genera ( Haliangium , Lysobacter ). In zebrafish guts, it increased Cetobacterium and Hydrogenophaga abundance, while 10 7 CFU/mL made Rhizobium dominant. Optimal denitrification and survival rates occurred at 10 5 –10 6 CFU/mL. At 10 7 CFU/mL, R. pusense N7 reduced nitrate but raised conditionally pathogenic bacteria, impairing denitrification and increasing mortality. R. pusense N7 successfully colonized biofilms and zebrafish intestines, modulating microbial communities and improving water quality. The recommended concentration for efficacy and safety is 10 6 CFU/mL. • Rhizobium pusense N7 can colonize biofilms and zebrafish gut. • R. pusense N7 (10 6 CFU/mL) maximized denitrification and zebrafish survival. • Microbial diversity in biofilms and zebrafish gut become richer. • Higher inoculated strain N7 raised conditionally pathogenic bacteria.