Effect of Metal Bionanohybrids on Pre-formed Marine Biofilms

Marine biofouling causes significant ecological, environmental, and economic impacts worldwide and has driven extensive research into effective, cost-efficient, and environmentally sustainable solutions. In this study, we develop metal nanoparticle(NPs)-enzyme hybrids to mitigate a seven-week-old pre-formed <i>Cobetia marina</i> biofilm. These bionanohybrids consisted of copper or copper-silver NPs embedded in a protein network, and were synthesized under mild conditions using different protocols. The formation of nanostructures from 1.0 to 2.6 μm was observed in all bionanohybrids, as well as NPs with a diameter ranging from 3 to 60 nm. X-ray diffraction confirmed the presence of copper phosphate species in all hybrids, while silver phosphate or silver oxide species were identified depending on the synthesis method used. The antifouling activity of the metal bionanohybrids was evaluated against biofilms using a final bionanohybrid concentration of 250 ppm and a 6-h exposure period under dynamic conditions. Results showed a greater than 2 log decrease in culturable cell counts across all treatments. Confocal laser scanning microscopy analysis revealed significant structural disruption in the biofilms following bionanohybrid treatment, with an average reduction of 48% in biofilm thickness, 60% in total biovolume, and 71% in biovolume of viable cells. These findings confirm the significant antifouling and antibacterial activity of the bionanohybrids. Further investigation into their mechanism of action revealed that bionanohybrids induce changes in cell membranes and reduce bacterial metabolic activity.