The factors affecting bacterial colonisation on microplastics and the impact of tertiary treatment of wastewater on the attached bacteria and microplastics

Microplastics (MPs) in aquatic environments have become an environmental concern globally. In addition to the direct impact of these plastics on aquatic organisms, their surfaces could serve as a unique habitat for various microbial communities through the formation of biofilms. Various factors could play a role in microbial attachment and biofilm formation in wastewater. This study aimed to assess potential factors that lead to biofilm formation on different types of MPs in wastewater and determine the impact of UV and chlorine treatment on these biofilms. In a laboratory scale experiment, MPs (low density polyethylene (LDPE), high density polyethylene (HDPE), and polypropylene (PP) were exposed to untreated wastewater under various conditions of temperature (20°C, 25°C and 35°C), light and dark conditions, as well as aerobic and anaerobic conditions for a period of five weeks. The formation of biofilms on MPs was quantified using optical density (OD660) measurements. The highest biofilm formation was observed in week 3, with an OD of 1.77. Thereafter, a decline in OD was observed, reaching an OD of 1.1 by week 5. This change in biofilm concentration over the week corresponded to changes in nutrient (nitrite, nitrate and ammonia) concentration in the media. A positive correlation was observed between the changes in biofilm concentration and nitrite (r = 0.824) and ammonia (r = 0.1) levels in the media. Meanwhile, a negative correlation observed for nitrate concentration (r=-0.673). Factors such as dark conditions, 25 C, and aerobic conditions presented the highest median biofilm formation with an OD value of 1.6, 1.7 and 1.6, respectively. It was also observed that polyethylene had higher biofilm concentrations compared to the polypropylene. Furthermore, rough MPs had higher biofilm formation than smooth MPs, with median ODs of 1.7 and 1.6 respectively. The microbial communities in the biofilms and wastewater medium were characterised by 16S rRNA amplicon sequencing. The results revealed that the alpha diversity (richness, evenness, and diversity) was lower in wastewater compared to the biofilms. It was observed that PP supported the most diverse bacterial community ( H’= 2.51138 and Simpson index= 11.096), while HDPE supported the least diverse bacterial community (H’= 0.88779 and Simpson index= 1.5324). Beta diversity using the Jaccard distance index revealed that the most similar communities were observed among biofilms from the three types of MPs while the most dissimilar communities were observed between the biofilm and wastewater medium communities. The most dominant phyla in both the biofilms and wastewater medium during the five weeks were Proteobacteria, Bacteroidetes and Planctomycetes. The bacterial communities, however, varied for each type of plastic and the wastewater medium. It was observed that Methylotenera, Hydrogenophaga, and Rhodanobacter was the most abundant genera in biofilms whereas C39(45.25%) and Luteimonas(18.96%) were the abundant genera in the wastewater medium. Methylotenera mobilis was the most common species among the three types of MPs. In addition, pathogenic species such as Mycobacterium arupense and Methylobacterium adhaesivum were detected in abundance on LDPE and PP. To assess the impact of UV treatment and chlorination on the attached biofilms, the microplastics with attached biofilm were exposed to UV-C and Chlorine (5 mg/L) treatment for 60 minutes. The biofilms were inactivated (100%) after 30 mins of UV treatment, whereas 10 min was sufficient to achieve 100% inactivation of biofilm by chlorine treatment. In conclusion, the research presented in this study has made substantial contributions to our understanding of the role that environmental factors play in the formation of biofilm on MP surfaces.