Isolation, identification, and characterization of the heterotrophic nitrification–aerobic denitrification Bacillus subtilis strain HL-1

Recirculating aquaculture systems are usually operated under high dissolved oxygen, which promotes nitrification but may lead to nitrate accumulation and transient nitrite toxicity. In this study, a functional strain, HL-1, was isolated from biofilter media of a largemouth bass recirculating aquaculture system and identified as Bacillus subtilis by 16S rRNA sequencing and phylogenetic analysis. Its heterotrophic nitrification–aerobic denitrification capacity was evaluated using nitrogen removal kinetics, reductase activities, nitrogen mass balance and response surface optimization. Under aerobic simulated aquaculture conditions, HL-1 rapidly removed NH4⁺-N and NO2⁻-N, reducing them from 3.0 mg/L to 0.10 mg/L and 0 mg/L within 24 h, respectively, while NO3⁻-N showed transient accumulation followed by decline. Nitrate reductase and nitrite reductase activities reached (0.131±0.003) and (0.200±0.005) U/(mg protein), respectively. Aerobic nitrogen balance showed that gaseous nitrogen accounted for 76.75%–81.70%, whereas intracellular assimilation contributed only 1.27%-2.07%; under anaerobic conditions, gaseous nitrogen decreased to 0.29%-2.31%. The optimized TN removal conditions were 160 r/min, C/N=15, 28 ℃ and pH 7.0, giving a validated TN removal efficiency of 87.6%. Fermentation optimization further produced a bacterial powder with 2×10¹¹ CFU/g. These results indicate that HL-1 is a promising aerobic bioaugmentation candidate for inorganic nitrogen control in high-DO recirculating aquaculture systems.