Characterisation and Evaluation of Bacillus Based Probiotics for Environmental Stress Tolerance in Tripura, India

Abstract

This study evaluated the verification of bacterial claims in commercial probiotic products marketed for biofloc-based fish farming, assessing their performance under conditions characteristic of the North Eastern region of India, where water commonly exhibits high iron content and low pH. Five commercial probiotic products (PR, PB, BG, FB, and LP) labelled as containing multiple Bacillus sp. were analysed. Heterotrophic plate count agar was used for bacterial enumeration, followed by isolation, identification, and 16S rRNA sequencing. The findings revealed discrepancies between the label claims and the actual bacterial composition of all five products. The probiotic product PR exhibited lower CFU counts than claimed, while PB closely matched the label claim. Isolated bacteria exhibited low mutual inhibition and low-to-moderate inhibition of pathogenic bacteria. All Bacillus isolates produced extracellular enzymes and exhibited varying antibiotic resistance, with B. pacificus showing the highest resistance. Probiotic bacteria with antimicrobial resistance are unsafe for aquaculture use because they pose risks of horizontal gene transfer. Significant bacterial growth was observed over a pH range of 4-9 and substantial growth at 15 °C and 20 °C. Iron tolerance assessments indicated that Brevibacillus borstelensis, B. subtilis, and B. pacificus exhibited significant growth at an iron concentration of 5 mg L^-1. Additionally, B. sonorensis, B. borstelensis, and B. subtilis demonstrated superior ammonia removal efficiency. These findings provide insights into the performance of probiotic bacteria under region-specific water conditions, highlighting potential candidates for biofloc-based fish farming in Tripura.