Microplastics in marine environments: protocol for isolating natural biofilms from seawater-incubated particles v1

Microplastics, upon which microbial communities arise rapidly, have emerged as a significant source of pollution in the ocean1. Studying these communities is crucial to understand their potential impacts on marine ecosystems, as well as their role as vector for potential pathogens2, 3 , and reservoir for antimicrobial resistance genes4, 5. The proposed workflow delineates an approach for incubating microplastics particles in seawater, ensuring reproducibility while controlling the origin, incubation duration, and weathering conditions of microplastics. The workflow details the preparation of the incubation device, sample collection, and subsequent laboratory processes to obtain the natural marine biofilms associated with microplastics. Our processing method helps minimize contamination by naturally occurring microparticles in marine environments and allows for an accurate estimate of microplastics concentration by direct counting. The aim of this publication is to provide a standardized protocol to facilitate comparisons between natural communities associated with microplastic pollution. References 1. Bowley, J., Baker-Austin, C., Porter, A., Hartnell, R. & Lewis, C. Oceanic Hitchhikers – Assessing Pathogen Risks from Marine Microplastic. Trends Microbiol. 29, 107–116 (2021). 2. Kirstein IV., Kirmizi S., Wichels A., Garin-Fernandez A., Erler R., Löder M., Gerdts G., – Dangerous hitchhikers? Evidence forpotentially pathogenic Vibrio spp. on microplastic particles. Mar Environ Res .120, 1–8 (2016). 3. Pedrotti ML., De Figueiredo Lacerda AL., Petit S., Ghiglione JF., Gorsky G., Vibrio spp and other potential pathogenic bacteria associated to microfibers in the North-Western Mediterranean Sea. PLOS ONE 17:e0275284. (2022). 4. Wu X., Pan J., Li M., Li Y., Bartlam M., Wang Y., Selective enrichment of bacterial pathogens by microplastic biofilm. Water Res 165, 114979. (2019). 5. Liang H., de Haan WP., Cerdà-Domènech M., Méndez J., Lucena F., García-Aljaro C., Sanchez-Vidal A., Ballesté E., Detection of faecal bacteria and antibiotic resistance genes in biofilms attached to plastics from human-impacted coastal areas. Environ Pollut. 319:120983. (2023).