Influence of Fragmentation Techniques on the Biotic Aging of Test Microplastics

This study investigates how mechanical fragmentation techniques─ultracentrifugal and mixer milling─affect the biotic aging of polystyrene (PS) and polypropylene (PP) microplastics in freshwater, focusing on biofilm formation, microbial colonization, and changes in their physicochemical properties. Both polymer type and fragmentation methods significantly affected biofilm development. Mixer-milled PP supported greater biofilm accumulation than ultracentrifugally milled PP, while the latter exhibited higher chlorophyll a and extracellular polymeric substances levels, indicating increased phototrophic activity. Metagenomic analysis showed a conserved community dominated by Brevibacillus, with fragmentation-dependent shifts. Brevibacillus and Clostridium enriched in ultracentrifugally milled samples, while Priestia was associated with mixer-milled particles. Bradyrhizobium remained stable despite variations in chlorophyll levels, suggesting a fragmentation-dependent metabolic shift. Plastic-degrading gene profiles mirrored biofilm trends, with oxidative enzymes (laccases and peroxidases) predominating. Spectroscopic and microscopic analysis confirmed functionalization and morphological differences, while density and crystallinity measurements revealed structural changes associated with colonization. These results emphasize the importance of surface properties in the preparation of test materials for microplastic analysis, which depends on the type of polymer and fragmentation method and influences microbial attachment and aging processes. Selection of fragmentation techniques therefore affect microplastic behavior, interactions, and environmental fate.