Identification of the bacterial community that degrades phenanthrene sorbed to polystyrene nanoplastics using DNA-based stable isotope probing

Abstract

In the Anthropocene, plastic pollution has become a new environmental biotope, the so-called plastisphere. In the oceans, nano- and micro-sized plastics are omnipresent and found in huge quantities throughout the water column and sediment, and their large surface area-to-volume ratio offers an excellent surface to which hydrophobic chemical pollutants (e.g. petrochemicals and POPs) can readily sorb to. Our understanding of the microbial communities that breakdown plastic-sorbed chemical pollutants, however, remains poor. Here, we investigated the formation of 500 nm and 1000 nm polystyrene (PS) agglomerations in natural seawater from a coastal environment, and we applied DNA-based stable isotope probing (DNA-SIP) with the 500 nm PS sorbed with isotopically-labelled phenanthrene to identify the bacterial members in the seawater community capable of degrading the hydrocarbon. Whilst we observed no significant impact of nanoplastic size on the microbial communities associated with agglomerates that formed in these experiments, these communities were, however, significantly different to those in the surrounding seawater. By DNA-SIP, we identified Arcobacteraceae, Brevundimonas, Comamonas, uncultured Comamonadaceae, Delftia, Sphingomonas and Staphylococcus, as well as the first member of the genera Acidiphilum and Pelomonas to degrade phenanthrene, and of the genera Aquabacterium, Paracoccus and Polymorphobacter to degrade a hydrocarbon. This work provides new information that feeds into our growing understanding on the fate of co-pollutants associated with nano- and microplastics in the ocean.

Keywords: DNA-SIP; Marine snow; Microplastics; Nanoplastics; Phenanthrene.

Figure 1

Polystyrene nanoplastic 1000 nm particles observed under the light (A, C) and epifluorescence (B, D) microscope before and after exposure of the particles to phenanthrene. Particles exposed to phenanthrene (A, B) have a tendency to agglomerate, and the adsorption of the phenanthrene shows up well under epifluorescence (B). Particles not exposed to phenanthrene (C, D; non-exposed controls) showed no visible presence of phenanthrene on the surface of the particles, especially when viewed under the epifluorescence microscopy (D). Scale bars, 5 µm.

Figure 2

Non-metric multi dimensional scaling (nMDS) plot showing the dissimilarity between bacterial communities for each of the Firth of Forth (FoF) samples analysed. Symbols in red represent the communities associated with agglomerations; symbols in blue are background water controls. Closed symbols represent communities from incubations with nanoplastics with sorbed phenanthrene; open symbols represent communities from incubations that did not contain phenanthrene. Seawater controls (squares), 500 nm nanoplastics (triangles) and 1000 nm nanoplastics (diamonds) are indicated. Light grey contour lines indicate H′ diversity.

Figure 3

Heatmap showing the relative abundance of the major taxa (> 2% relative abundance) in incubation experiments with seawater from the Firth of Forth (FoF). X-axis labels describe ‘matrix-plastic size-presence of phenanthrene’. The blue scale represents the abundance of taxa, from highly abundant (dark blue) to less abundant (light blue). The heatmap is separated into three columns to represent the three possible influencing factors—seawater only/agglomerate formation (water/agg), nanoplastic presence/absence and size [0 (not present)/500/1000), and presence/absence of phenanthrene (yes/no)]. Columns and rows are separated based on Pearson correlation co-efficient.

Figure 4

Distribution of the ‘heavy’ and ‘light’ DNA in separated SIP fractions. The top panel shows the DGGE profile of bacterial PCR products from separated [¹³C]-phenanthrene fractions (12 to 34) with decreasing densities from left to right. The distribution of qPCR-quantified total 16S rRNA gene sequences in fractions from the [¹³C]-phenanthrene incubation is shown below the DGGE image (black filled circle). Fractions 18–21 (left shaded area) were determined to represent ¹³C heavy DNA and were combined for further analysis. DGGE banding patterns for a given fraction are aligned with the corresponding gene abundance data below.

Figure 5

Relative distribution of taxa found in the heavy fractions from the SIP incubations with seawater from the Firth of Forth (FoF) exposed to polystyrene nanoparticles of size 500 nm primed with ¹³C-labelled phenanthrene. AH1 and AH2 represent agglomerate samples from the duplicate SIP incubations; WH1 and WH2 represent the corresponding samples of the water surrounding these agglomerates. The colour scale represents the relative abundance (%), from highly abundant (dark; 100%) to undetectable (white; 0%).