Novel functional insights into the microbiome inhabiting marine plastic debris: critical considerations to counteract the challenges of thin biofilms using multi-omics and comparative metaproteomics.

Abstract Background Microbial functioning on marine plastic surfaces has been poorly documented, with only two studies using metaproteomics to unravel microbial genotype-phenotype linkages in the marine ‘plastisphere’. Here we provide a comprehensive methodological assessment for plastisphere metaproteomics, using multi-omics and data mining on thin plastic biofilms from an understudied cold environment, to provide unique insights into plastisphere metabolism. Our robust experimental design assessed DNA/protein co-extraction and cell lysis strategies, proteomics workflows, and diverse protein search databases, to encourage the more widespread application of these techniques to resolve plastisphere function. Results For the first time, we demonstrate the predominance and activity of hydrocarbonoclastic genera ( Psychrobacter , Flavobacterium , Pseudomonas ) within a primarily heterotrophic plastisphere. Correspondingly, oxidative phosphorylation, the citrate cycle, and carbohydrate metabolism were the dominant pathways expressed. We also identified quorum sensing and toxin-associated proteins in Streptomyces , stress response proteins expressed by Psychrobacter, Planococcus and Pseudoalteromonas , and xenobiotics degradation proteins in Psychrobacter and Pseudoalteromonas. Interestingly, a targeted search strategy identified plastic biodegradation enzymes, such as polyamidase, hydrolase, and depolymerase, expressed by rare taxa. In contrast to previous research, pathogenic genera were active, expressing virulence factors and mechanisms of antimicrobial resistance. Conclusion Our study demonstrates the power of multi-omics and comparative metaproteomics to resolve plastisphere functioning, to provide new bioengineering perspectives and improved assessment of the risks of plastic pollution.