Characteristics of bacteria associated on biofilms in freshwater microcosms

Abstract

Freshwater pollution is an ever-growing concern, due to the increasing population growth combined with a rapidly decreasing natural resource capacity, such as water. The possibility of significant problems arising from water scarcity can lead to negative consequences for the environment and its inhabitants. One of the biggest water pollution concerns is microplastics. Microplastics can serve as vectors for potentially harmful bacterial groups and reservoirs for antibiotic-resistance genes. An increase in the water pollution rate, coupled with the overused and improper use of antibiotics, contributes towards the surge of antibiotic-resistant bacteria within the environment. In light of these concerns, this study aimed to determine the characteristics of bacteria associated with biofilms surrounding microplastics in a simulated freshwater environment. The first objective was to examine the bacterial colonisation on microplastics and wood substrates through scanning electron microscopy in microcosm experiments involving river water and wastewater treatment plant effluent obtained from the North West Province in South Africa. The second objective was to determine the characteristics of the bacterial isolates throughout the incubation period. The third objective was to perform Sanger sequencing of these purified isolates to aid in the identification of the bacteria found in biofilms surrounding the microplastics and wood substrate. The fourth objective was to perform next-generation sequencing targeting the 16S rRNA bacterial genes in order to characterise the bacterial community composition (BCC) within the plastisphere post-incubation. The microcosm study revealed that bacterial colonisation of various substrates occurred within 72 hours after incubation. Scanning electron microscopy images indicated that biofilm development happened within a 28-day period, revealing diverse microbial communities on the different substrates-the surface properties combined with the increased nutrient load assisted with the microbial attachment more readily. Scanning electron microscopy images revealed that the wood particles had a higher degree of colonisation than the microplastic particles due to the surface properties and organic composition. Among the 877 pure bacterial isolates tested for various extracellular enzymes, haemolysis was the most prevalent compared to DNase, resulting in 51 isolates used for further identification. Virulence testing revealed varying levels of antibiotic resistance across the 51 microbial isolates, with 16 presenting pathogenic potential. The 16 potentially pathogenic isolates underwent Sanger sequencing, and the phylogenetic relationship was determined, with a focus on eight bacterial species. Aeromonas sp. and Stenotrophomonas koreensis were among the potentially pathogenic bacteria detected. The microbial composition of the source water and plastisphere were ascertained and compared. The source water showed variation in species composition but a reduced species richness, while the plastisphere exhibited higher species richness and diversity. Next-generation sequencing revealed that Hyphomonadaceae was the dominant family within the plastisphere, belonging to the order Caulobacterales, within the Alphaproteobacteria class and Proteobacteria phylum. This study confirmed the presence of pathogenic bacteria and a significant bacterial composition on microplastics, highlighting their role as vectors for pathogens and the transfer of antibiotic-resistance genes within freshwater environments. Furthermore, the ongoing discharge of inadequately treated wastewater and the persistence of plastic particles can introduce waste-related pathogens into microplastic biofilms, thereby posing a significant concern for future generations. These findings highlight the urgent need for improved wastewater treatment practices and effective plastic waste management to protect freshwater ecosystems and public health.