Probiotic <i>Bacillus subtilis</i> enhances production efficiency in biofloc‐raised <i>Cherax quadricarinatus</i> via microbiome remodeling and host antioxidant capacity upregulation

Abstract Biofloc technology (BFT) presents a sustainable approach to aquaculture intensification by recycling nutrients and reducing water exchange. However, probiotic supplementation for optimization in emerging species such as redclaw crayfish ( Cherax quadricarinatus ) remains unexplored. This study investigated how Bacillus subtilis ( B. subtilis ) probiotic supplementation enhances BFT performance in a 70‐day redclaw crayfish culture trial. Two treatments—biofloc control (B) and biofloc + B. subtilis (BP)—were evaluated in quintuplicate ( n = 5), with systematic monitoring of water quality, biofloc dynamics, crayfish growth, digestive/antioxidant enzymes, and microbiome shifts using 16S rRNA sequencing and PICRUSt2 functional prediction. Probiotic amendment significantly boosted floc aggregation (TSS +40%, BFV +39%) and stabilized carbon‐to‐nitrogen ratios despite causing a transient elevation in nitrite. Microbial analysis revealed significant pathogen suppression ( Vibrio −50%, Aeromonas −61%) alongside enriched nitrifying taxa ( Nitrosomonas + 47%) and denitrifying taxa ( Thauera + 157%), with increased predicted functional diversity and inferred upregulation of key pathways. Crucially, crayfish in BP exhibited elevated digestive enzymes (α‐amylase, pepsin, lipase), antioxidant capacity (SOD, CAT, T‐AOC), and immune enzyme activity (AKP). These physiological enhancements, coupled with improved system stability, collectively were associated with a 15.8% increase in production yield, survival (13.9%), and a 12.7% lower feed conversion ratio (FCR). These results demonstrate that B. subtilis effectively optimizes BFT systems by concurrently modulating microbial ecology and host physiology, offering a practical strategy to advance sustainable redclaw crayfish aquaculture.