oplankton communities, including the NPSG (see Fig. S6 in the supplemental material) (38, 46, 47).
The clades we observed associated with microplastics all appear well adapted to take advantage of niches created by surfaces. The abundant bacterial families we found on our microplastic samples, which were separated by as much as 1,700 km in the NPSG, were consistent with the clades observed on plastic debris in the Atlantic Ocean. These observations suggest that a predictable core group of clades occupies the niche created by small plastic debris in oligotrophic surface waters worldwide.
With respect to particular taxonomic groups, members of the Rhodobacteraceae clade are known to alternate between diverse lifestyles (e.g., planktonic and attached) and are also capable of rapid responses to various resources (48, 49). It is likely that these characteristics explain their observed frequency as early colonizers of artificial surfaces, including glass, polyvinyl chloride, and Plexiglas surfaces, and explain why we observed them in high abundances across our samples (27, 50, 51). Likewise, the marine Bacteroidetes groups Flavobacteriaceae and Saprospiraceae have a known preference for growth on particles, surfaces, and algae (52, 53) and members of the family Hyphomonadaceae are considered oligotrophs that readily form biofilms on surfaces (54). Together, these findings suggest that the microbial communities we observed seem more indicative of a general proclivity for surface attachment, as opposed to any specific selection of microbiota by the chemical composition of the plastic substratum itself.
We were unable to determine which bacteria might have been attached to the eukaryotic organisms (like the Bryozoa that can cover much of the substrate surface area) versus directly attached to the plastic substrate. Many of the bacterial groups we observed have been previously documented living in association with eukaryotic organisms, including marine invertebrates, corals, and sponges (55–57). The genus “Tunicatimonas,” which we observed was most abundant on the plastic particle with the highest number of hydrozoan reads, was first isolated from a sea anemone (58). The Flavobacteria clade Tenacibaculum (also observed by Zettler et al. [29] and Oberbeckmann et al. [30]) has been isolated from a variety of marine organisms, including bryozoans (59), and a few members of this genus are known fish pathogens (60, 61). Leptolyngbya and Phormidium sp. strains have been identified as members of the coral black band disease consortium (62).
The large number of KOs that were significantly more abundant in plastic-associated metagenomes than in free-living metagenomes provided further evidence that microplastics create a niche that is distinct from the niches utilized by the surrounding picoplankton. While it is possible that some differences are a result of differences in the sequencing technologies used to generate data from these two communities, previous studies suggest that this is unlikely. For example, a previous study comparing the 454 and Illumina platforms in an aquatic system showed that both platforms sample the same fraction of diversity and produce similar relative abundances of genes and genomes (63). Future gene or protein expression studies will provide additional information on the potential importance of the functions of these genes in plastic-associated habitats.
Not surprisingly, our results suggest that the microbial communities that develop on the plastics are enriched for traits necessary for a surface-attached lifestyle. Consistent with the stalked cells observed in the SEM image and the presence of Hyphomonadaceae across our samples, many of the Caulobacter-like cell cycle genes that are involved in transitions between a flagellated motile lifestyle and a sessile cell with a prosthecum were significantly more abundant in the plastic metagenomes (54) (Fig. 9; see Data Set S1 in the supplemental material). Similarly, methyl-accepting chemotaxis protein-encoding genes, the majority of the two-component system CheA family (chemotaxis-like) genes, the potABCD spermidine/putrescine transporter components, and KOs within the tight adherence export apparatus system were all more abundant in the plastic-associated metagenomes. These genes have been implicated in chemotaxis, signaling of swarming activity, surface motility, colonization, and biofilm formation (64–68) (Fig. 9; see Data Set S1 in the supplemental material).
KOs belonging to secretion system pathways, including numerous type IV secretion system (T4SS) genes and the majority of T6SS components were also more abundant in the plastic metagenomes. The most common function of T4SSs is to conjugate plasmid DNA, and hence, T4SS plays an important role in gene flow between cells (69). The T6SS transports effector proteins directly into neighboring eukaryotic or prokaryotic cells, thereby playing a key role in competition or pathogenesis (70). T4SSs have also been shown to mediate the transfer of toxins and other effector proteins in several pathogens.
Concerns have been raised that plastic debris could transport pathogens or other unfavorable organisms, including dinoflagellates that cause harmful algal blooms (23, 29). The presence of bacterial clades with some pathogenic members has been interpreted by some as evidence that plastic debris may act as a disease vector (29). We cannot, however, draw any definitive conclusions in this regard. Potentially pathogenic species, for example, frequently contain strains that are benign. Additionally, secretion systems are also used in many other processes, including nonpathogenic, nontoxic interbacterial interactions. Finally, little is known about the natural distribution and dispersal mechanisms of many pathogenic and nonpathogenic marine microbes and traits, so it is difficult to postulate how plastic debris impacts these natural processes (70, 71).
The significantly higher abundance of phycobilisome antenna protein-encoding genes in the plastic metagenomes compared to the increase of some Chl a/b-binding light-harvesting protein-encoding genes in the surrounding water column shows that the dominant cyanobacteria in the two habitats use different light-harvesting machinery (Fig. 9; see Data Set S1 in the supplemental material). The majority of cyanobacteria are believed to absorb photons for photosynthesis by using phycobilisome complexes, while Prochlorococcus bacteria, the dominant cyanobacteria in the surrounding water column, utilize Chl-binding complexes. It has been postulated that Prochlorococcus bacteria evolved the alternative light-harvesting mechanism to cope with the limited nutrients, including iron and nitrogen, in oligotrophic gyres (72). Assuming this, the prevalence of phycobilisome-utilizing cyanobacteria on plastics, in combination with the elevated rates of oxygen production and respiration on plastics relative to that in the background seawater, suggests that the nutrient limitation in the NPSG is less severe in plastic particle communities.
Consistent with this, the increased abundance of nitrogenase genes nifH, nifD, and nifK in the plastic-associated metagenomes suggests that nitrogen fixation could be reducing nitrogen limitation on the plastics. Additionally, key enzymes involved in phosphonate utilization were more abundant in plastic communities. Phosphonates are increasingly being recognized as an important source of phosphorus (73–76), and it has been suggested that some microbes use phosphonates when nitrogen fixation relieves nitrogen limitations (76). This is consistent with the possibility that oligotrophic conditions are reduced on plastic particles, at least in relation to nitrogen. Future studies focusing on biomass accumulation and nutrient fluxes on microplastics will clarify the extent to which microplastics are creating a eutrophic niche in oligotrophic waters.
A large number of membrane transporters were significantly more abundant on plastics. Notably, three genes encoding TonB-dependent iron complex outer membrane receptors that import chelated iron and two genes forming an inner membrane iron complex transport system involved in siderophore import were more abundant in plastic metagenomes (77, 78). Siderophore uptake is important to community dynamics on large marine particles, and siderophore biosynthesis is important for biofilm maturation in some taxa (79, 80).
Whether or not the microorganisms residing on plastic debris are degrading plastics and significantly contributing to the loss of plastic from marine surface waters is an ongoing question (81). It has been hypothesized that microbial communities associated with plastic debris could also be degrading organic pollutants adsorbed to plastic debris, as biofilms are often involved in remediation processes (81, 82). Similar to Zettler et al. (29), we observed SSU rRNA genes related to bacterial clades with hydrocarbon-degrading members or members that have sometimes been associated with oil-contaminated environments, including Phormidium, Muricauda, Hyphomonadaceae, and Rhodobacteraceae (54, 83–85). It has also been suggested that some Rhodobacteraceae strains isolated from coastal environments are capable of lignin degradation, an activity that may be associated with plastic degradation (86, 87).
Several putative xenobiotic biodegradation genes were more abundant on plastic particles, including homogentisate 1,2-dioxygenase, N-ethylmaleimide reductase, a cytochrome P450, and 2,4-dichlorophenol 6-monooxygenase (Fig. 9; see Data Set S1 in the supplemental material). In particular, homogentisate 1,2-dioxygenase is a ring-cleaving enzyme that has been implicated in the degradation of polycyclic aromatic hydrocarbons, as well as styrene (88). 2,4-Dichlorophenol 6-monooxygenase is a hydroxylase involved in the degradation of chlorinated aromatic pollutants (89, 90). The genes encoding the two subunits of protocatechuate 3,4-dioxygenase, an aromatic-ring-cleaving enzyme implicated in lignin degradation, were also observed in