Dose–dependent effect of Bacillus subtilis on aquaculture pond ecosystems

Bacillus subtilis has shown potential as a probiotic for water quality regulation in aquaculture, but its dose-dependent effects remain insufficiently studied. This study established low-concentration (2.25×105 CFU·mL-1, L) and high-concentration (4.50×105 CFU·mL-1, H) treatment groups, integrating water quality analysis, spectrophotometry, and 16S rRNA high-throughput sequencing to dynamically monitor physicochemical parameters (transparency, dissolved oxygen, pH, ammonia nitrogen, sediment redox potential), chlorophyll content, and microbial community changes at the aquaculture water and sediment at 0, 6, 24, and 48 h. Results demonstrated that the high dose delivered faster and larger declines in ammonia nitrogen and chlorophyll a/b/c, accompanied by a transient nitrite rise (6 h) and a brief DO dip that resolved by 24 - 48 h. The low dose produced steadier improvements with smaller oxygen perturbations and greater short-term stability. Sediment redox potential increased at 6 - 24 h under both doses, indicating a shift toward more oxidizing conditions at the sediment–water interface. In the water column, Firmicutes (with transient Exiguobacterium) were enriched while Proteobacteria remained stable; in sediment, Proteobacteria, Firmicutes, and Chloroflexi dominated, with Thiobacillus and Lysobacter increasing under both doses (more pronounced in H). Predicted functional profiles showed stronger enrichment in H at 6 h for nitrogen transformations (ureolysis; nitrite respiration, nitrate respiration; nitrate reduction), while categories annotated as animal parasites/symbionts declined and chemoheterotrophy and chitinolysis remained high. Collectively, these findings support a practical dosing strategy: high dose for rapid remediation (acute nitrogen removal and algal control) and low dose for routine maintenance with minimal disturbance.