Biomass Estimation of Marine Biofilms on Plastic Surfaces

Plastics have become a major source of marine pollution, which threatens food safety and quality, human health, and marine ecosystems. Due to the drastic negative effects of plastics in a marine environment, alternative biodegradable plastics are being generated that are more eco-friendly and have less environmental impact. Though some of these plastics are known to biodegrade, the process of degradation for bioplastics has not been heavily studied in a marine environment. This pilot project sought to both quantify the process of biodegradation and compare across different methods for effectiveness of biomass estimation, which serves as an indicator of biodegradation. Plastics were provided by Danimer Scientific®, with polyhydroxybutyrate (PHB) as the main ingredient in the plastic formulations. PHB is known to serve as a substrate for bacterial growth, so DNA quantification, plate counts, and crystal violet staining were employed as methods of biomass estimation for the resulting biofilm. The marine biofilm was involved in the degradation of PHB samples, as each method demonstrated that PHB samples contained greater biofilm growth and degradation when compared to the negative control. Though each method had benefits and drawbacks, crystal violet staining was the most useful and consistent estimate for biomass while plate counts and DNA quantification were variable and inconsistent. Crystal violet staining was also the most straightforward assay, while plating and DNA extraction were more time-consuming, involved, and expensive in comparison. For these reasons, crystal violet assay is recommended as a consistent and cost-effective method for measuring biofilm growth over time. Though crystal violet staining assesses biofilm bound to the plastics, plating and other DNA based studies can provide additional information about the biodegrading microbes. The methods developed and used in this investigation can be adapted for future research in biofilm removal, biomass quantification, and testing for evidence of bacteria-mediated degradation in a variety of bioplastic formulations and environments