Ozone nanobubble treatment effectively reduced pathogenic Gram positive and negative bacteria in freshwater and safe for tilapia

Abstract

19High concentrations of pathogenic bacteria in water usually results in outbreaks of bacterial 20 diseases in farmed fish. Here, we explored the potential application of an emerging nanobubble 21 technology in freshwater aquaculture. Specifically, we aimed to determine if this technology was 22 effective at reducing the concentration of pathogenic bacteria in the water, and to assess whether 23 it was safe for fish. An ozone nanobubble (NB-O3) treatment protocol was established based on 24 examination of nanobubble size, concentration, disinfection property, and impact on fish health. 25A 10-min treatment with NB-O3 in 50 L water generated approximately 2-3 × 10 7 bubbles with 26 majority sizes less than 130 nm and ozone level of ~800 mV ORP. A single treatment with water 27 contaminated with either Streptococcus agalactiae or Aeromonas veronii effectively reduced 28 96.11-97.92 % of the bacterial load. This same protocol was repeated 3 times with 99.93-99.99 % 29 reduction in the bacterial concentration. In comparison, bacterial concentration the control tanks 30 remained the same level during the experiments. In fish-cultured water with the presence of 31 organic matter (e.g. mucus, feces, bacterial flora, feed, etc.), the disinfection property of NB-O3 32 was reduced i.e bacterial concentration was reduced by 42.94 %, 84.94 % and 99.27 % after the 33 first, second and third treatments, respectively. To evaluate the safety of NB-O3 to fish, juvenile 34Nile tilapia were exposed to NB-O3 treatment for 10 minutes. No mortality was observed during 35 the treatment or 48 h post treatment. Gill histology examination revealed that a single NB-O3 36 treatment caused no alteration morphology. However, damage in the gill filaments was noticed in 37 the fish receiving two or three consecutive exposures within the same day. Results of all the 38 experiments conducted in this study suggest that NB-O3 technology is promising for controlling 39 pathogenic bacteria in aquaculture systems, and may be useful at reducing the risk of bacterial 40 disease outbreaks in farmed fish. 41

Pre-treatment of Nile tilapia (Oreochromis niloticus) with ozone nanobubbles improve efficacy of heat-killed Streptococcus agalactiae immersion vaccine

“…Notably, the findings of our investigation revealed that better bacterial antigen uptake into the gill tissues after 3 and 6 h immunization is achieved with fish in the vaccinated group that received a pre-treatment with NB-O3 (NB-O3-VAC) compared with fish in the AT-VAC group. It is possible that ozone nanobubbles in the NB-O3-VAC groups activated innate immune system, which modulated immune cells in response to infections [25][26][27]. When these innate immune cells are stimulated, they perform several response mechanisms to antigens, including phagocytosis, degranulation, and production of cytokines, which may trigger and/or recruit other leukocytes (e.g., neutrophils, macrophages, and dendritic cells) to the fish gills, and it is highly tempting to speculate that pre-treatment with NB-O3 might cause a temporary disruption of epithelial cells, leading to reducing barrier, increasing antigen uptake, and producing a greater immune response.…”

“…In our previous investigations, ozone nanobubbles were found to be efficient in reducing bacterial concentrations in water and upregulating the innate immune system of fish, resulting in increased fish survival during pathogenic infections. When Nile tilapia were infected with a pathogenic multidrugresistant Aeromonas hydrophila, they displayed a higher survival rate when exposed to treatments with ozone nanobubbles (NB-O3) [25][26][27]. Given the stimulation of the innate immune system in the treated groups, we hypothesized that this technology might improve the efficacy of immersion vaccines.…”

Characteristics and Stability of Ozone Nanobubbles in Freshwater Conditions

“…While oxygen, nitrogen, and air are the most common types of gases studied as nanobubbles (NBs), research with ozone NBs is still emerging: (i) remediation of sludge, sediments, and groundwater, − (ii) removal of micropollutants, − (iii) reduction of bacterial pathogens, − and (iv) antimicrobial effectiveness for fresh vegetables . Ozone readily reacts with organic and inorganic compounds in water, and it has been widely used as an oxidant and/or disinfectant in water and wastewater treatment.…”