Influence of fragmentation techniques on the biotic aging of test microplasticsKlun, Barbara (Avtor) Ducoli, Serena (Avtor) Starin, Mark (Avtor) Novak, Janja (Avtor) Vaccari, Filippo (Avtor) Puglisi, Edoardo (Avtor) Rozman, Ula (Avtor) Čelan Korošin, Nataša (Avtor) Kalčíková, Gabriela (Avtor) Federici, Stefania (Avtor) agingbiofilmbiofoulingdegradationfragmentationplastic pollutionThis study investigates how preparing laboratory test materials with different mechanical fragmentation techniques — ultracentrifugal milling and mixer milling — affect the biotic aging of microplastics in natural freshwater, focusing on the development of biofilm, microbial colonization, and changes in physicochemical properties of microplastics. Using polypropylene (PP) and polystyrene (PS) as model polymers, we found that both polymer type and fragmentation method had a significant effect on biofilm accumulation, with mixer-milled PP supporting significantly more biofilm growth than its ultracentrifugally milled counterpart. Chlorophyll $a$ content and extracellular polymeric substances were consistently higher on ultracentrifugally milled microplastics, indicating increased phototrophic microbial activity. Metagenomic analysis showed a conserved community dominated by Brevibacillus, with fragmentation-dependent shifts. Brevibacillus and Clostridium enriched in samples fragmented with the ultracentrifugal mill, and Priestia fragmented with the mixer mill. Bradyrhizobium, a phototroph, maintained stable abundance, while chlorophyll levels varied, suggesting a fragmentation-dependent metabolic shift. Plastic-degrading gene profiles mirrored biofilm-associated trends, with oxidative enzymes (laccases and peroxidases) most represented. Fourier-transform infrared spectroscopy and scanning electron microscopy analysis confirmed functionalization and morphological differences, respectively, while density and crystallinity measurements revealed structural changes associated with biofilm colonization. These results emphasize the critical role of surface morphology and chemistry in the preparation and development of test materials for microplastic analysis, which depends on the type of polymer and fragmentation method and influences microbial attachment and aging processes. Our results underscore the need to carefully consider microplastic preparation techniques in environmental studies, as these methods significantly influence microplastic behaviour, ecological interactions, and environmental fate.20262026-04-08 14:00:10Neznano181469sl