A new approach to extracting biofilm from environmental plastics using ultrasound-assisted syringe treatment for isotopic analyses

Plastics are one of the ubiquitous and artificial types of substrates for microbial colonization and biofilm development in the aquatic environment. Characterizing plastic-associated biofilms is key to the better understanding of organicmaterial and mineral cycling in the "Plastisphere"-the thin layer of microbial life on plastics. In this study, we propose a new method to extract biofilms from environmental plastics, in order to evaluate the properties of biofilm-derived organic matter through stable carbon (delta C-13) and nitrogen (delta N-15) isotope signatures and their interactions with radionuclides especially radiocesium (Cs-137). The extractionmethod is simple and cost-effective, requiring only an ultrasonic bath, disposable plastic syringes, and a freeze drier. After ultrasound-assisted separation from the plastics, biofilm samples were successfully collected via a sequence of syringe treatments, with less contamination from plastics and other mineral particles. Effective removal of small microplastics from the experimental suspension was satisfactorily achieved using the method with syringe treatments. Biofilm-derived organic matter samples (14.5-65.4 mg) from four river mouths in Japan showed Cs-137 activity concentrations of <75 to 820 Bq.kg(-1) biofilm (dw), providing evidence that environmental plastics, mediated by developed biofilms, serve as a carrier for Cs-137 in the coastal riverine environment. Significant differences in the delta C-13 and delta N-15 signatures were also obtained for the biofilms, indicating the different sources, pathways, and development processes of biofilms on plastics. We demonstrate here a straightforwardmethod for extracting biofilms from environmental plastics; the results obtained with this method could provide useful insights into the plasticassociated nutrient cycling in the environment.