Applications of Nanobubbles in Aquaculture: Pond Water Disinfection and its Impact on Microbial Community and Antimicrobial Resistance Genes

Abstract

Aquaculture is a rapidly growing industry worldwide. Intensive and semi-intensive production is replacing extensive farming, and this is resulting in an increase in frequency of disease outbreaks. The latter has led to more use of antibiotics, which in turn has been attributed to the proliferation of antibiotic resistant bacteria. The transmission of AMR pathogens between aquaculture and natural aquatic environments may pose a direct threat to human health. Hence, finding alternatives to reduce antibiotic usage is crucial. Nanobubbles were proposed as a potential alternative to antibiotics. These bubbles are characterized by their small size, long-term stability in the water column, and the ability to generate reactive free radicals when they collapse on themselves, which can kill bacteria. These bubbles also dissolve gases in water very efficiently due to their high surface area to volume ratio. Nanobubbles can be created using different types of gases.

Ozone is commonly employed by fish farmers to disinfect aquaculture systems due to its effective oxidizing potential. However, a major drawback of ozone disinfection is the substantial release of ozone gas, resulting in high gas consumption and toxicity to personnel. Notably, nanobubbles may significantly improve the dissolution and transfer of ozone in water given their physical characteristics, thereby enhancing disinfection efficiency.

Even though some emerging and potential applications of NBs with different gases in aquaculture have been explored to improve water parameters, reduce bacterial levels and improve aquatic animal health, there are several broad knowledge gaps that need to be addressed in order to more efficiently apply this technology in aquaculture. For example nanobubble technologies using innocuous gases like air and oxygen still required clarification regarding their potential bacteriostatic or bactericidal properties. Further, limited research has been done on the regrowth of bacterial communities after low-dose ozone disinfection in aquaculture systems, as well as the impact of ozone nanobubbles on overall bacterial diversity. Understanding the effects of ozone nanobubbles disinfection on antimicrobial resistance genes (ARGs) in aquaculture pond is also lacking.

To address these knowledge gaps, in the first chapter, we explored the applications of nanobubbles in aquaculture. In the second chapter, we conducted an investigation on the potential use of air or oxygen nanobubbles (with a mean diameter of 128 ± 44 nm) to decrease the concentration of the pathogenic bacteria Aeromonas hydrophila. This was carried out under controlled laboratory conditions using tanks. Even with the introduction of a substantial quantity of nanobubbles, twice a day for 15 minutes, into a relatively small volume of water (63L), a significant biological decrease in bacteria was not observed, suggesting that air and oxygen nanobubbles alone are insufficient to significantly reduce high levels of pathogenic bacteria in water. Therefore, in the third chapter, we utilized ozone as a resource to generate nanobubbles, and examined the impact of ozone macrobubbles (O3MB) and ozone nanobubbles (O3NB) on the microbial ecology of pond water and the health of fish. Remarkably, after treating our small pond water ecosystems with 0.15 mg/L ozone, we successfully eliminated between 90.9% and 99.4% of heterotrophic bacteria and 90.1% to 95.2% of bacterial DNA. Shotgun metagenomic sequencing revealed that O3MB and O3NB treatments reduced the relative abundance of all bacteria in our water sample, including the dominant bacterial species and Cyanobacteria, while the bacterial richness was maintained and the top ten bacterial species in the community underwent changes and exhibited a more even distribution within the water sample. We also observed a rebound in the bacterial community 24 hours after the ozone treatments. A major advantage of using ozone through nanobubbles rather than macrobubbles was the significantly reduced delivery time required (p < 0.0001) while also increasing the dissolved oxygen in the water. Furthermore, we evaluated the impact of ozone nanobubbles on jade perch and found no adverse effects on the fish at an exposure dose of 0.15 mg/L.

In the fourth chapter, we examined the impact of O3NB on the dynamics of AMR genes in pond water using the metagenomic data acquired in chapter three. The results revealed that ozone disinfection can lead to an increase in the relative abundance of bacteria with acquired antimicrobial resistance genes (ARGs) and intrinsic efflux-mediated ARGs within the resistance nodulation cell division (RND) family. Interestingly, we observed a co-occurrence of efflux and non-efflux ARGs within the same bacterial genera, with these genera dominating the bacterial population after ozone treatments, suggesting that ozone treatments may selectively promote the survival of bacterial genera that harbor efflux ARGs, some of which may also possess non-efflux ARGs.

These findings offer initial insights into the potential applications of nanobubble technology for "resetting" microbial communities during disease outbreaks. However, they also highlight the need to carefully consider the potential impacts of disinfection practices on the dissemination of ARGs, especially in aquaculture settings where disinfectants are commonly used at low levels. Future efforts should prioritize the evaluation of these strategies, as they may be associated with an elevated risk of AMR in aquaculture environments.

Date of Award	9 Sept 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Sophie Natasha ST-HILAIRE (Supervisor), Getchell RODMAN (External Co-Supervisor) & Wenlong CAI (Co-supervisor)