Probiotic Bacillus spp. modulate biofilm microbial assembly in Litopenaeus vannamei culture water

Shrimp aquaculture faces persistent challenges arising from microbial imbalances and pathogenic dominance, often linked to biofilm-forming bacterial communities. Probiotics, particularly Bacillus spp., offer a sustainable alternative to antibiotic and chemical management; however, their structural and ecological roles in biofilm modulation remain insufficiently understood. This study applied an integrative approach combining Scanning Electron Microscopy (SEM), 16S rRNA amplicon sequencing, and nutrient monitoring to evaluate the influence of Bacillus-based probiotic supplementation on biofilm communities in Litopenaeus vannamei culture water. Amplicon-based analysis revealed a distinct reconfiguration of bacterial assemblages: untreated biofilms were dominated by Vibrio spp., whereas probiotic supplementation reduced their relative abundance and transiently increased marine-associated genera such as Marivita, Pseudoalteromonas, and Marinobacter during early succession. Bacillus exhibited increased relative abundance under probiotic treatment, coinciding with a shift toward a more compositionally balanced community structure. SEM observations corroborated these findings, showing that probiotic application was associated with less compact and more heterogeneous biofilm architecture. Nutrient analyses demonstrated moderately lower concentrations of ammonia, nitrite, and nitrate in probiotic-treated systems, whereas phosphate concentrations remained consistently low across treatments. Although Bacillus abundance was negatively associated with inorganic nitrogen parameters, functional mechanisms were not directly measured. These findings therefore represent ecological associations rather than mechanistic confirmation of enhanced nutrient turnover. Overall, the results highlight the potential of Bacillus-based probiotics as environmentally compatible biofilm modulators in shrimp aquaculture systems.