Antibiofilm, Antimicrobial, and Limited Ammonia-Reduction Activities of Bacillus cereus-Group Isolates From Hot Spring Sediment and Rubber Latex Serum for Aquaculture Water Management.

Spore-forming Bacillus species are increasingly explored for their potential applications in antimicrobial activity and environmental bioremediation. However, comparative functional characterization of closely related strains with respect to biofilm inhibition, pathogen suppression, and ammonia removal remains limited. In this study, two bacterial isolates (PWR04 and SHPS6) were characterized using morphological, biochemical, and 16S rRNA gene sequencing analyses. Spore-forming ability, stress tolerance (bile salt and pH), antibiotic susceptibility, antibacterial activity, biofilm inhibition, auto-aggregation, and ammonia-removal efficiency were systematically evaluated. Statistical analyses included t-test, one-way ANOVA, and two-way ANOVA. Both isolates were identified as members of the Bacillus cereus group, showing ≥ 99.80% sequence similarity. High spore survival was observed under bile salt (82.21%-95.99%) and acidic conditions (81.60%-99.48%). No inhibition zones were detected for ampicillin or cloxacillin, whereas inhibition zones reached up to 25.33 mm for chloramphenicol. Limited antibacterial activity was detected in agar well diffusion assays, whereas biofilm inhibition reached 91.79% in PWR04 and 90.00% in SHPS6 under specific conditions. Auto-aggregation increased over time, reaching 76.24% in PWR04 and 71.84% in SHPS6 at 24 h. Ammonia-removal efficiency was significantly affected by concentration and time (p < 0.01), with maximum values of 20.00% (PWR04) and 18.00% (SHPS6). Although both isolates showed multifunctional antimicrobial and bioremediation traits, each displayed β-hemolysis on sheep blood agar-a virulence-associated phenotype that constitutes a clear safety concern. Genome-based screening for hemolysin and enterotoxin genes and antimicrobial resistance determinants, together with in vivo validation, is therefore required before application.