Probiotics have been widely applied in aquaculture as a sustainable alternative to antibiotics, with numerous studies reporting their potential to improve growth performance, enhance immune responses, and improve water quality. In particular, bacterial strains belonging to the genus Bacillus are commonly used due to their ability to form spores, enabling them to withstand harsh environmental conditions and maintain biological activity. Previous studies suggest that probiotics may function through multiple mechanisms, including competition with pathogenic bacteria for nutrients, production of digestive enzymes and antimicrobial compounds, and stimulation of host immune responses (El-Saadony et al., 2021; Ringø et al., 2020).
In pangasius (Pangasianodon hypophthalmus) aquaculture, probiotics have been associated with improved production performance. Dietary supplementation with Bacillus spp. has been linked to enhanced growth rate, improved feed conversion ratio (FCR), and increased digestive enzyme activity (Nguyen et al., 2024). In addition, probiotics may improve intestinal morphology, promote beneficial microbial populations, and reduce the presence of pathogenic bacteria such as Edwardsiella ictaluri and Aeromonas hydrophila, thereby potentially enhancing disease resistance (Tran et al., 2021). The use of multi strain probiotic formulations has also been reported to produce synergistic effects, further improving water quality and immune responses (Zhang et al., 2023).
Beyond their direct effects on the host, probiotics play an important role in regulating and stabilizing microbial communities in aquaculture environments. Their application has been associated with enhanced biogeochemical processes, particularly nitrogen cycling, contributing to improved water quality and reduced toxicity of compounds such as ammonia and nitrite (Mang et al., 2024). Additionally, probiotic strains, especially Bacillus spp., have been shown to degrade organic matter, compete with pathogenic microorganisms, and help maintain microbial balance in aquaculture systems (El-Saadony et al., 2021; Ringø et al., 2020).
However, the effectiveness of probiotics in practical aquaculture systems depends on multiple factors, including viable cell density, strain selection, and application conditions. Several studies have suggested the existence of an optimal dosage range, while excessive application may not provide additional benefits and could potentially disrupt microbial balance or increase operational costs (Putra et al., 2017). These findings highlight the importance of controlling and optimizing microbial density to ensure consistent probiotic performance.
Previous research on probiotic fermentation has emphasized the influence of key factors such as medium composition, pH, temperature, inoculation ratio, and incubation time on microbial growth and activity. Optimization approaches, including response surface methodology (RSM), have demonstrated their effectiveness in increasing viable cell density (CFU) and improving probiotic performance (Chen et al., 2025; Xia et al., 2022). In addition, multi-strain fermentation strategies have been shown to enhance the functional properties of probiotic products (Zhang et al., 2025).
Although many studies have focused on the effectiveness of probiotics and the optimization of culture conditions under laboratory settings, there is still a lack of information regarding the fermentation process of probiotics under practical pond conditions, particularly the variation in microbial density and community structure throughout the fermentation period. Monitoring microbial dynamics during this stage is necessary to better understand the effectiveness of probiotics and to provide a basis for optimizing their application process.
In practice, the application of probiotics in pangasius farming is still limited due to the lack of appropriate usage protocols, while the large pond size may increase production costs if probiotic use is not optimized. Therefore, this experiment was conducted to optimize the probiotic fermentation process used in pangasius farming, while also evaluating changes in microbial density and diversity during fermentation through KYTOS microbial analysis technology. The results are expected to provide a scientific basis for improving the effectiveness of probiotic application under practical farming conditions.
