Developing an optimized method for biofilm extraction from microplastic surfaces for high-efficiency analysis of...

Microplastics (MiPs, ×5 mm in size) harbor complex biofilms that facilitate pathogen dissemination, yet standardized extraction protocols are lacking. Here, we developed and optimized a method for biofilm extraction from environmentally weathered MiPs. To reflect real-world conditions, the protocol was applied directly to bulk, heterogeneous, field-collected MiP mixtures (size range: 80 µm-5 mm) without prior sorting by polymer type or morphology. By optimizing extraction buffers, mechanical disruption, and MiP quantities (100-150 particles), we established an optimal protocol combining phosphate-buffered saline with 0.1% Tween 80, ultrasonication (40 kHz, 10 min), vortexing with glass beads, and a two-cycle extraction-disaggregation workflow. This approach involves an initial extraction followed by a repeated, exhaustive extraction step designed to maximize the recovery of recalcitrant biofilm residues. This protocol markedly enhanced recovery of viable, culturable cells, delivering a 2,950-fold enhancement in the recovery of viable, culturable cells (evaluated via CFU counts; 28,020 ± 11,034 CFU MiP⁻¹) vs. conventional PBS extraction (9.5 ± 3 CFU MiP⁻¹) and 102-fold vs. passive extraction (274 ± 59 CFU MiP⁻¹). The 10-min sonication empirically maximized viable cell recovery within the tested duration range. The two-step protocol with Tween 80-mediated disaggregation proved critical, increasing recovery 208-fold by disaggregating biofilm fragments. While DNA yields (26.5 ± 3.93 ng µL⁻¹) were sufficient for targeted PCR-based pathogen detection (Aeromonas spp., Salmonella enterica), the co-extraction of complex environmental matrices (A₂₆₀/A₂₈₀ ratio: 0.17-0.19) strictly requires an additional purification step prior to next-generation sequencing. Validation across contrasting aquatic environments confirmed the method's robustness. Comparative analysis demonstrates that conventional single-step approaches fail to recover the majority of viable cells trapped within weathered MiP biofilms. This optimized and validated protocol provides a critical methodological foundation for investigating plastisphere microbial ecology and pathogen transport dynamics, supporting evidence-based risk assessment of MiP contamination, especially public health risks associated with microplastic pollution.IMPORTANCEMicroplastic-associated biofilms (the "plastisphere") serve as vectors for waterborne pathogens and antibiotic resistance genes; however, the persistent use of inadequate extraction methods has systematically underestimated microbial abundance, presenting a critical barrier to global environmental risk assessment. By overcoming the limitations of conventional extractions-which fail to penetrate recalcitrant extracellular polymeric matrices on environmentally weathered microplastics-our standardized methodology liberates previously undetectable bacterial populations. The ability to accurately quantify these hidden communities, including key pathogens like Aeromonas spp. and Salmonella enterica, fundamentally transforms our understanding of microplastics as hidden biological reservoirs. Ultimately, this methodological advancement bridges a critical gap in microbial ecology, delivering the reliable, quantitative data strictly required by policymakers, environmental agencies, and public health officials to establish evidence-based guidelines mitigating the impacts of microplastic pollution on global water systems.