High-Resolution Screening for Marine Prokaryotes and Eukaryotes With Selective Preference for Polyethylene and Polyethylene Terephthalate Surfaces

Abstract

Marine plastic debris serve as substrates for the colonization of a variety of prokaryote and eukaryote organisms. Of particular interest are the microorganisms that have adapted to thrive on plastic as they may contain genes, enzymes or pathways involved in the adhesion or metabolism of plastics. We implemented DNA metabarcoding with nanopore MinION sequencing to compare the 1-month-old biomes of hydrolyzable (polyethylene terephthalate) and non-hydrolyzable (polyethylene) plastics surfaces vs. those of glass and the surrounding water in a Mediterranean Sea marina. We sequenced longer 16S rRNA, 18S rRNA, and ITS barcode loci for a more comprehensive taxonomic profiling of the bacterial, protist, and fungal communities, respectively. Long read sequencing enabled high-resolution mapping to genera and species. Using previously established methods we performed differential abundance screening and identified 30 bacteria and five eukaryotic species, that were differentially abundant on plastic compared to glass. This approach will allow future studies to characterize the plastisphere communities and to screen for microorganisms with a plastic-metabolism potential.

Keywords: DNA metabarcoding; MinION; differentially abundant species; marine fungi; nanopore; plastic microbiome; plastisphere.

FIGURE 1

Experiment location and set up. (A) Locations of the five replicates that were placed from south to north in Herzliya Marina, Israel (32° 09′ 38.8″ N 34° 47′ 35.0″ E). (B) Each replicate consists of two 17 × 26 mm flat pieces of each of the three materials (PE,PET, and glass) tied with a fishing line to a straw and secured with plastic beads. This “mobile” of materials was secured to the dock such that the surfaces were submerged ∼0.5 m below the surface and 6–8 m above the marina bottom. (C) The materials after they were submerged for 30 days in Herzliya Marina water.

FIGURE 2

Microscopy images of the microbial community on PE or PET surfaces from marine waters after 1 month. (A–H) SEM images; (A) Biological coverage on PE surface after 1 month, (B) bacterial colonies on PET surface, (C,D) diatoms associated with biofilm on PE, (C) Fragilariopsis sp. (D) Amphora sp. (E–H) Images from PET; (E) a single cell eukaryote, (F) Bryozoan, (G) fruiting body, (H) multicellular filaments. (I) Light microscopy image of a network of fungal hyphae specifically stained with Lactophenol Cotton Blue.

FIGURE 3

PCoA of the Bray-Curtis dissimilarities among the plastisphere communities of the samples (N = 5 per treatment). (A,B) 16S rRNA gene microbiomes with the water community (left) or without it (right). (C,D) 18S rRNA gene microbiomes with the water community (left) or without it (right). (E,F) ITS microbiomes with the water community (left) or without it (right). Results of PERMANOVA for significance between groups are shown on each plot.

FIGURE 4

Substrate specificity of bacteria based on relative abundance of 16S rRNA gene mapped reads. (A) Relative abundance of top 10 abundant genera within each treatment. Produced using MicrobiomeAnalyst (Chong et al., 2020). (B) Heatmap of the bacteria with higher relative mapped read abundance in the PE samples Limma – voom was used to identify OTUs that had FDR adjusted p-value < 0.05 and log- fold- change of >0 on PET or PE compared to glass. The relative abundance of their mapped reads was then plotted for all samples and replicates.

FIGURE 5

Substrate specificity of eukaryotes at the genus and species levels based on relative abundance of 18S rRNA gene mapped reads. (A) Relative abundance of top 10 abundant genera within each treatment. Produced using MicrobiomeAnalyst (Chong et al., 2020). (B) Eukaryotes with higher relative mapped read abundance in the plastic samples. Limma – voom was used to identify OTUs that had FDR adjusted p-value < 0.05 and log- fold- change of >0 on PET or PE compared to glass. The relative abundance of their mapped reads was then plotted for all samples.

FIGURE 6

ITS fungal metabarcoding analysis. (A) Top 10 abundant fungi genera based on ITS metabarcoding. (B) Shared and unique fungal OTUs among the surfaces and the water. (C) Fungi corresponding to the OTUs present only on PE, PET or PE, and PET.