The increasing micro and nanoplastic pollution is of immense concern. Recent studies show that biofilm substances in contact with nanoplastics play an important role in aggregation and sedimentation of nanoplastics. Consequences of these processes are changes in biofilm formation and stability and changes in transport and fate of pollutants in the environment. Having a deeper understanding of the nanoplastics-biofilm interaction would help to evaluate the risks posed by uncontrolled nanoplastic pollution. These interactions are impacted by environmental changes due to climate change, such as e.g. acidification of surface waters. We apply fluorescence correlation spectroscopy (FCS) to investigate the pH-dependent aggregation tendency of non-functionalized polystyrene (PS) nanoparticles (NPs) due to intermolecular forces with model extracellular biofilm substances. Our biofilm model consists of bovine serum albumin (BSA), which serves as a representative for globular proteins, and the polysaccharide alginate, which is a main component in many biofilms, in solutions containing Na+ with ionic strength being realistic for fresh water conditions. Biomolecule concentrations ranging from 0.5 g/l up to at maximum 21 g/l are considered. We use non-functionalized polystyrene NPs as representative for mostly negatively charged nanoplastics. BSA promotes NP aggregation through adsorption onto the NPs and BSA mediated bridging. In BSA-alginate mixtures the alginate hampers this interaction, most likely due to alginate-BSA complex formation. Thus, the NP are more stable in the BSA-alginate mixtures compared to solutions containing solely BSA. In most BSA-alginate mixtures and alginate alone, other weaker attractive forces, mainly depletion forces, seem to cause NP aggregation. These forces are not electrostatic in nature and thus are less influenced by the pH-value. At high BSA contents the electrostatic BSA-BSA attraction is not sufficiently screened leading to a destabilization of the NP. This study clearly shows that it is crucial to consider correlative effects between multiple biofilm components to better understand the NP aggregation in the presence of complex biofilm substances. Single component biofilm model systems based on comparing the total organic carbon content of the extracellular biofilm substances, as usually considered, would have led to an misjudgment of the stability towards aggregation. Nevertheless, if the protein content is not too high, a simple model that only depends on polysaccharide concentration could be feasible to predict aggregation under environmental relevant conditions.
