Impact of ozone nanobubble on water quality, gut microbiota, and growth performance of white leg shrimp (Penaeus vannamei) in an intensive indoor farming system

• The first study on the impact of NB-O₃ on shrimp gut microbiota. • NB-O₃ supports sustainable shrimp farming by minimizing antibiotics use and promoting growth. • Vibrio spp. in water reduced 4-fold, resulting in lower V. parahaemolyticus in shrimp gut. • Water quality improved with 50% lower NH₃-N, reduced COD, and higher DO and ORP. • NB-O₃ did not negatively impact on shrimp health. This study evaluated the effects of ozone nanobubbles (NB-O₃) on water quality, growth performance, survival rate, and gut microbiota in intensive indoor farming of white-leg shrimp ( Penaeus vannamei ). The experiment lasted 12 weeks and included two groups: (1) NB-O₃ treatment at 0.3 mg/L ozone concentration and (2) a control group without NB-O₃ treatment. Water quality parameters including temperature, dissolved oxygen (DO), pH, and Oxidation Reduction Potential (ORP) were monitored daily. Weekly analyses were conducted for Chemical Oxygen Demand (COD), Biochemical Oxygen Demand after 5 days (BOD₅), Ammonia Nitrogen (NH₃-N), Nitrite Nitrogen (NO₂-N), and Vibrio counts. Shrimp weight was recorded weekly, while final biomass (FB), final body weight (FBW), survival rate (SR), feed conversion ratio (FCR), specific growth rate (SGR), and mean weekly weight gain (MWWG) were evaluated at the end of study. Gut microbiota samples were collected on days 1, 60, and 84. The results showed that NB-O₃ significantly reduced COD, total Vibrio , and NH₃-N levels compared to the control (p<0.05). Shrimp in the NB-O₃ group exhibited significantly higher weights from week 5 onwards (p<0.05), with improved FB, FBW, SGR, and MWWG compared to the control (p<0.05). However, SR and FCR were lower in the NB-O₃ group (p<0.05). Dominant gut microbiota phyla in NB-O₃-treated shrimp were Proteobacteria, Bacteroidota, and Acinobacteriota, with their proportions recorded as 72.27%, 13.82%, and 6.72% on day 1; 60.75%, 17.16%, and 18.64% on day 60; and 45.74%, 40.25%, and 8.44% on day 84, respectively. Significant differences in alpha and beta diversity were observed between groups on days 1 and 60 (p<0.05). Vibrio parahaemolyticus density was lower in NB-O₃-treated shrimp (p<0.05). These findings demonstrate that NB-O₃ improves water quality, enhances shrimp growth performance, and effectively regulates Vibrio spp. in intensive indoor farming systems. To the best of our knowledge, this is the first study to report the effects of NB-O3 on shrimp gut microbiota.