Microplastics in the ocean function as an artificial microbial reef, with unique and diverse communities of eukaryotic and bacterial microbiota colonizing its surface. It is not well understood if the communities in this biofilm, also termed “plastisphere”, are specific for the type of microplastic on which they develop. Here, we carried out a controlled 6-week long in situ incubation experiment of six common plastic polymer in Bocas del Toro, Panama. The community composition of prokaryotes based on 16S rRNA gene sequencing data, when judged under a null model analysis, show that neither plastic polymer type nor time exposed to the environment play a significant role in shaping biofilm communities. However, the null model analyses of eukaryotic communities based on 18S rRNA gene sequences reveal that they can be significantly influenced by plastic polymer type and time incubated. This was confirmed by  scanning electron microscopy, which allowed us to distinguish plastic-specific diatom communities by the end of the incubation period.

Microplastic pieces of varying plastic polymers were incubated in situ in a coastal bay in the Caribbean off of the coast of Bocas del Toro, Panama and sampled over a time series to assess biofilm formation. At each sampling point, 15 microplastic pieces of each plastic polymer type were randomly selected from their respective sachets and stored in ATL buffer at -20°C after proteinase K digestion prior to being transported and extracted for DNA as per the Qiagen DNeasy Blood and Tissue kit manufacturer’s protocol. The taxonomic composition of bacterial and eukaryotic communities were determined by the Illumina MiSeq 2x300 amplicon sequencing platform of 16S and 18S rRNA genes. PCR amplification was performed using primers 515F and 926R (Quince et al., 2011; Parada et al., 2016) to amplify the V4-V5 region of bacterial 16S rRNA genes and primers eukv4F and eukv4R (Stoeck et al., 2010) to amplify the V4 region of eukaryotic 18S rRNA genes.

Microplastics were preserved in glutaraldehyde (Sigma-Aldrich, 5% (v/v)), cooled at 4°C for 2-8 hours, then transferred into 50% (v/v) ethanol in Phosphate Buffer Solution (PBS) and stored at -20℃ until further preparation and imaging at the ASU laboratory. Samples were then dehydrated through a graded ethanol series and critical-point dried. The dried samples were mounted on aluminum stubs and sputter-coated with 10-15 nm of gold-palladium (60/40). Images were generated using a TESCAN VEGA³ scanning electron microscope (SEM) operated at 15kV. The diatom community, specifically, was taxonomically distinguished based on their morphology. We classified diatoms into 14 morphologically distinct groups denoted D1 through D14, with D14 comprising all “other” diatoms that were counted in low abundance (<2). Five fields under SEM were counted, which amounted to 1-58 cells for a taxonomic group depending on its density on the plastic piece. Each field had a SEM magnification of 1760x with working distances between 13.36-14.02 mm, making each field approximately 200 µm² in size. Diatom density on the plastic surfaces was determined in duplicates for each plastic type and expressed in cells/mm². 

Eukaryotic amplicon sequence variants (ASVs) classified as Metazoans were removed from the analyses.

For the microscopy-derived diatom community data, D14, given its variability, was not used in any analyses.