Biodegradable plastics in Mediterranean coastal environments feature contrasting microbial succession.

Plastic pollution of the ocean is a top environmental concern. Biodegradable plastics present a potential "solution" in combating the accumulation of plastic pollution, and their production is currently increasing. While these polymers will contribute to the future plastic marine debris budget, very little is known still about the behavior of biodegradable plastics in different natural environments. In this study, we molecularly profiled entire microbial communities on laboratory confirmed biodegradable polybutylene sebacate-co-terephthalate (PBSeT) and polyhydroxybutyrate (PHB) films, and non-biodegradable conventional low-density polyethylene (LDPE) films that were incubated in situ in three different coastal environments in the Mediterranean Sea. Samples from a pelagic, benthic, and eulittoral habitat were taken at five timepoints during an incubation period of 22 months. We assessed the presence of potential biodegrading bacterial and fungal taxa and contrasted them against previously published in situ disintegration data of these polymers. Scanning electron microscopy imaging complemented our molecular data. Putative plastic degraders occurred in all environments, but there was no obvious "core" of shared plastic-specific microbes. While communities varied between polymers, the habitat predominantly selected for the underlying communities. Observed disintegration patterns did not necessarily match community patterns of putative plastic degraders.

Plastics select for distinct early colonizing microbial populations with reproducible traits across environmental gradients.

Little is known about early plastic biofilm assemblage dynamics and successional changes over time. By incubating virgin microplastics along oceanic transects and comparing adhered microbial communities with those of naturally occurring plastic litter at the same locations, we constructed gene catalogues to contrast the metabolic differences between early and mature biofilm communities. Early colonization incubations were reproducibly dominated by Alteromonadaceae and harboured significantly higher proportions of genes associated with adhesion, biofilm formation, chemotaxis, hydrocarbon degradation and motility. Comparative genomic analyses among the Alteromonadaceae metagenome assembled genomes (MAGs) highlighted the importance of the mannose-sensitive hemagglutinin (MSHA) operon, recognized as a key factor for intestinal colonization, for early colonization of hydrophobic plastic surfaces. Synteny alignments of MSHA also demonstrated positive selection for mshA alleles across all MAGs, suggesting that mshA provides a competitive advantage for surface colonization and nutrient acquisition. Large-scale genomic characteristics of early colonizers varied little, despite environmental variability. Mature plastic biofilms were composed of predominantly Rhodobacteraceae and displayed significantly higher proportions of carbohydrate hydrolysis enzymes and genes for photosynthesis and secondary metabolism. Our metagenomic analyses provide insight into early biofilm formation on plastics in the ocean and how early colonizers self-assemble, compared to mature, phylogenetically and metabolically diverse biofilms.

Sargasso Sea Vibrio bacteria: Underexplored potential pathovars in a perturbed habitat.

We fully sequenced the genomes of 16 Vibrio cultivars isolated from eel larvae, plastic marine debris (PMD), the pelagic brown macroalga Sargassum, and seawater samples collected from the Caribbean and Sargasso Seas of the North Atlantic Ocean. Annotation and mapping of these 16 bacterial genome sequences to a PMD-derived Vibrio metagenome-assembled genome created for this study showcased vertebrate pathogen genes closely-related to cholera and non-cholera pathovars. Phenotype testing of cultivars confirmed rapid biofilm formation, hemolytic, and lipophospholytic activities, consistent with pathogenic potential. Our study illustrates that open ocean vibrios represent a heretofore undescribed group of microbes, some representing potential new species, possessing an amalgam of pathogenic and low nutrient acquisition genes, reflecting their pelagic habitat and the substrates and hosts they colonize.

Diversity of the holopelagic Sargassum microbiome from the Great Atlantic Sargassum Belt to coastal stranding locations.

The holopelagic brown macroalgae Sargassum natans and Sargassum fluitans form essential habitats for attached and mobile fauna which contributes to a unique biodiversity in the Atlantic Ocean. However, holopelagic Sargassum natans (genotype I & VIII) and Sargassum fluitans (genotype III) have begun forming large accumulations with subsequent strandings on the western coast of Africa, the Caribbean and northern Brazil, threatening local biodiversity of coastal ecosystems and triggering economic losses. Moreover, stranded masses of holopelagic Sargassum may introduce or facilitate growth of bacteria that are not normally abundant in coastal regions where Sargassum is washing ashore. Hitherto, it is not clear how the holopelagic Sargassum microbiome varies across its growing biogeographic range and what factors drive the microbial composition. We determined the microbiome associated with holopelagic Sargassum from the Great Atlantic Sargassum Belt to coastal stranding sites in Mexico and Florida. We characterized the Sargassum microbiome via amplicon sequencing of the 16S V4 region hypervariable region of the rRNA gene. The microbial community of holopelagic Sargassum was mainly composed of photo(hetero)trophs, organic matter degraders and potentially pathogenic bacteria from the Pseudomonadaceae, Rhodobacteraceae and Vibrionaceae. Sargassum genotypes S. natans I, S. natans VIII and S. fluitans III contained similar microbial families, but relative abundances and diversity varied. LEfSE analyses further indicated biomarker genera that were indicative of Sargassum S. natans I/VIII and S. fluitans III. The holopelagic Sargassum microbiome showed biogeographic patterning with high relative abundances of Vibrio spp., but additional work is required to determine whether that represents health risks in coastal environments. Our study informs coastal management policy, where the adverse sanitary effects of stranded Sargassum might impact the health of coastal ecosystems.

Spatio-temporal patterns of Synechococcus oligotypes in Moroccan lagoonal environments.

Synechococcus are unicellular cyanobacteria susceptible to environmental fluctuations and can be used as bioindicators of eutrophication in marine ecosystems. We examined their distribution in two Moroccan lagoons, Marchica on the Mediterranean coast and Oualidia on the Atlantic, in the summers of 2014 and 2015 using 16S rRNA amplicon oligotyping. Synechococcus representatives recruited a higher number of reads from the 16S rRNA in Marchica in comparison to Oualidia. We identified 31 Synechococcus oligotypes that clustered into 10 clades with different distribution patterns. The Synechococcus community was mainly represented by oligotype 1 (clade III) in Marchica. Cooccurring clades IV and I had an important relative abundance in Marchica in the summer of 2014, which is unusual, as these clades are widespread in cold waters. Moreover, Clades VII and subcluster "5.3" formed a sizeable percentage of the Synechococcus community in Marchica. Notably, we found low Synechococcus sequence counts in the Atlantic Lagoon. These results showed that the relative abundance of Synechococcus reads is not constant over space and time and that rare members of the Synechococcus community did not follow a consistent pattern. Further studies are required to decipher Synechococcus dynamics and the impact of environmental parameters on their spatial and temporal distributions.

Daphnia magna's Favorite Snack: Biofouled Plastics.

The influence of biofouling on zooplankton ingestion rates of plastics in freshwater environments has received limited attention. We investigated how biofouling of microplastics in wastewater effluent and in fresh surface water influences Daphnia magna's microplastic consumption. The differences in ingestion of the biofouled as compared with the virgin microplastics were higher for the surface water by a factor of seven compared with a factor of two for the effluent. The intake of biofouled microplastics by D. magna was higher compared with virgin plastics, but the reason for this preference should be further investigated. Environ Toxicol Chem 2022;41:1977-1981. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Metals and metalloids concentrations in three genotypes of pelagic Sargassum from the Atlantic Ocean Basin-scale.

Since 2011, the Caribbean Islands have witnessed unprecedented massive stranding of a pelagic brown algal Sargassum spp. inducing damages for coastal ecosystems and economy. By accumulating heavy metals, Sargassum can play a role in contaminant transportation from offshore to the coast. In 2019, three genotypes of Sargassum (S. fluitans III, S. natans I, and VIII) were sampled in seven stations along a 3400 km transect in the Atlantic Ocean. Concentrations of 15 heavy metal(loid)s elements were analyzed by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). Mean metal concentrations were ranked following descending order: As >Fe > Al > Mn > Cd > Zn > Ni > V > Cu > Cr > Hg. The metalloid As was the most abundant contaminant with a maximum value of 115 ppm, previously observed in the Caribbean area (80-150 ppm). At Atlantic Ocean Basin-scale, metallic element concentrations do not present spatial longitudinal gradients. Genotypes S. fluitans III and S. natans (I and VIII), present differents metal(loid)s contamination distinct patterns.

Large quantities of small microplastics permeate the surface ocean to abyssal depths in the South Atlantic Gyre.

Hundreds of studies have surveyed plastic debris in surface ocean gyre and convergence zones, however, comprehensive microplastics (MPs, ≤5 mm) assessments beneath these surface accumulation areas are lacking. Using in situ high-volume filtration, Manta net and MultiNet sampling, combined with micro-Fourier-transform-infrared imaging, we discovered a high abundance (up to 244.3 pieces per cubic meter [n m-3 ]) of small microplastics (SMPs, characteristically <100 µm) from the surface to near-sea floor waters of the remote South Atlantic Subtropical Gyre. Large horizontal and vertical variations in the abundances of SMP were observed, displaying inverse vertical trends in some cases. SMP abundances in pump samples were more than two orders of magnitude higher than large microplastics (LMPs, >300 µm) concurrently collected in MultiNet samples. Higher-density polymers (e.g., alkyd resins and polyamide) comprised >65% of the total pump sample count, highlighting a discrepancy between polymer compositions from previous ocean surface-based surveys, typically dominated by buoyant polymers such as polyethylene and polypropylene. Contrary to previous reports stating LMP preferentially accumulated at density gradients, SMP with presumably slower sinking rates are much less influenced by density gradients, thus resulting in a more even vertical distribution in the water column, and potentially longer residence times. Overall, our findings suggest that SMP is a critical and largely underexplored constituent of the oceanic plastic inventory. Additionally, our data support that weak current systems contribute to the formation of SMP hotspots at depth, implying a higher encounter rate for subsurface particle feeders. Our study unveils the prevalence of plastics in the entire water column, highlighting the urgency for more quantification of the deep-ocean MP, particularly the smaller size fraction, to better understand ecosystem exposure and to predict MP fate and impacts.

Climate change facilitated the early colonization of the Azores Archipelago during medieval times.

Humans have made such dramatic and permanent changes to Earth's landscapes that much of it is now substantially and irreversibly altered from its preanthropogenic state. Remote islands, until recently isolated from humans, offer insights into how these landscapes evolved in response to human-induced perturbations. However, little is known about when and how remote systems were colonized because archaeological data and historical records are scarce and incomplete. Here, we use a multiproxy approach to reconstruct the initial colonization and subsequent environmental impacts on the Azores Archipelago. Our reconstructions provide unambiguous evidence for widespread human disturbance of this archipelago starting between 700-60+50 and 850-60+60 Common Era (CE), ca. 700 y earlier than historical records suggest the onset of Portuguese settlement of the islands. Settlement proceeded in three phases, during which human pressure on the terrestrial and aquatic ecosystems grew steadily (i.e., through livestock introductions, logging, and fire), resulting in irreversible changes. Our climate models suggest that the initial colonization at the end of the early Middle Ages (500 to 900 CE) occurred in conjunction with anomalous northeasterly winds and warmer Northern Hemisphere temperatures. These climate conditions likely inhibited exploration from southern Europe and facilitated human settlers from the northeast Atlantic. These results are consistent with recent archaeological and genetic data suggesting that the Norse were most likely the earliest settlers on the islands.

Diversity and predicted inter- and intra-domain interactions in the Mediterranean Plastisphere.

This study investigated the biogeography, the presence and diversity of potentially harmful taxa harbored, and potential interactions between and within bacterial and eukaryotic domains of life on plastic debris in the Mediterranean. Using a combination of high-throughput DNA sequencing (HTS), Causal Network Analysis, and Scanning Electron Microscopy (SEM), we show regional differences and gradients in the Mediterranean microbial communities associated with marine litter, positive causal effects between microbes including between and within domains of life, and how these might impact the marine ecosystems surrounding them. Adjacent seas within the Mediterranean region showed a gradient in the microbial communities on plastic with non-overlapping endpoints (Adriatic and Ligurian Seas). The largest predicted inter-domain effects included positive effects of a novel red-algal Plastisphere member on its potential microbiome community. Freshwater and marine samples housed a diversity of fungi including some related to disease-causing microbes. Algal species related to those responsible for Harmful Blooms (HABs) were also observed on plastic pieces including members of genera not previously reported on Plastic Marine Debris (PMD).

Biofouling impacts on polyethylene density and sinking in coastal waters: A macro/micro tipping point?

Biofouling causing an increase in plastic density and sinking is one of the hypotheses to account for the unexpectedly low amount of buoyant plastic debris encountered at the ocean surface. Field surveys show that polyethylene and polypropylene, the two most abundant buoyant plastics, both occur below the surface and in sediments, and experimental studies confirm that biofouling can cause both of these plastics to sink. However, studies quantifying the actual density of fouled plastics are rare, despite the fact that density will determine the transport and eventual fate of plastic in the ocean. Here we investigated the role of microbial biofilms in sinking of polyethylene microplastic and quantified the density changes natural biofouling communities cause in the coastal waters of the North Sea. Molecular data confirmed the variety of bacteria and eukaryotes (including animals and other multicellular organisms) colonizing the plastic over time. Fouling communities increased the density of plastic and caused sinking, and the plastic remained negatively buoyant even during the winter with lower growth rates. Relative surface area alone, however, did not predict whether a plastic piece sank. Due to patchy colonization, fragmentation of sinking pieces may result in smaller pieces regaining buoyancy and returning to the surface. Our results suggest that primarily multicellular organisms cause sinking of plastic pieces with surface area to volume ratios (SA:V) below 100 (generally pieces above a couple hundred micrometers in size), and that this is a "tipping point" at which microbial biofilms become the key players causing sinking of smaller pieces with higher SA:V ratios, including most fibers that are too small for larger (multicellular) organisms to colonize.

Correction: De Vogel, F.A., et al. Comparative Genomics of Marine Bacteria from a Historically Defined Plastic Biodegradation Consortium with the Capacity to Biodegrade Polyhydroxyalkanoates. Microorganisms 2021, 9, 186.

The authors wish to make the following corrections to this paper [...].

Comparative Genomics of Marine Bacteria from a Historically Defined Plastic Biodegradation Consortium with the Capacity to Biodegrade Polyhydroxyalkanoates.

Biodegradable and compostable plastics are getting more attention as the environmental impacts of fossil-fuel-based plastics are revealed. Microbes can consume these plastics and biodegrade them within weeks to months under the proper conditions. The biobased polyhydroxyalkanoate (PHA) polymer family is an attractive alternative due to its physicochemical properties and biodegradability in soil, aquatic, and composting environments. Standard test methods are available for biodegradation that employ either natural inocula or defined communities, the latter being preferred for standardization and comparability. The original marine biodegradation standard test method ASTM D6691 employed such a defined consortium for testing PHA biodegradation. However, the taxonomic composition and metabolic potential of this consortium have never been confirmed using DNA sequencing technologies. To this end, we revived available members of this consortium and determined their phylogenetic placement, genomic sequence content, and metabolic potential. The revived members belonged to the Bacillaceae, Rhodobacteraceae, and Vibrionaceae families. Using a comparative genomics approach, we found all the necessary enzymes for both PHA production and utilization in most of the members. In a clearing-zone assay, three isolates also showed extracellular depolymerase activity. However, we did not find classical PHA depolymerases, but identified two potentially new extracellular depolymerases that resemble triacylglycerol lipases.

Microbial carrying capacity and carbon biomass of plastic marine debris.

Trillions of plastic debris fragments are floating at sea, presenting a substantial surface area for microbial colonization. Numerous cultivation-independent surveys have characterized plastic-associated microbial biofilms, however, quantitative studies addressing microbial carbon biomass are lacking. Our confocal laser scanning microscopy data show that early biofilm development on polyethylene, polypropylene, polystyrene, and glass substrates displayed variable cell size, abundance, and carbon biomass, whereas these parameters stabilized in mature biofilms. Unexpectedly, plastic substrates presented lower volume proportions of photosynthetic cells after 8 weeks, compared to glass. Early biofilms displayed the highest proportions of diatoms, which could influence the vertical transport of plastic debris. In total, conservative estimates suggest 2.1 × 1021 to 3.4 × 1021 cells, corresponding to about 1% of the microbial cells in the ocean surface microlayer (1.5 × 103 to 1.1 × 104 tons of carbon biomass), inhabit plastic debris globally. As an unnatural addition to sea surface waters, the large quantity of cells and biomass carried by plastic debris has the potential to impact biodiversity, autochthonous ecological functions, and biogeochemical cycles within the ocean.

Ecology of the plastisphere.

The plastisphere, which comprises the microbial community on plastic debris, rivals that of the built environment in spanning multiple biomes on Earth. Although human-derived debris has been entering the ocean for thousands of years, microplastics now numerically dominate marine debris and are primarily colonized by microbial and other microscopic life. The realization that this novel substrate in the marine environment can facilitate microbial dispersal and affect all aquatic ecosystems has intensified interest in the microbial ecology and evolution of this biotope. Whether a 'core' plastisphere community exists that is specific to plastic is currently a topic of intense investigation. This Review provides an overview of the microbial ecology of the plastisphere in the context of its diversity and function, as well as suggesting areas for further research.

Spatial structure in the "Plastisphere": Molecular resources for imaging microscopic communities on plastic marine debris.

Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the "Plastisphere"-the thin layer of life on the surface of PMD-has been technology-limited. Research into microbe-microbe and microbe-substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI-FISH (combinatorial labelling and spectral imaging - fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha-, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell-count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro-metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales.

Application of Matrix Scoring Techniques to evaluate marine debris sources in the remote islands of the Azores Archipelago.

Three-quarters of all marine debris (MD) consists of plastic, a reflection of their worldwide use, production and waste mismanagement. Data on MD distributions can improve our ability to effectively reduce debris that escapes onto shorelines and the ocean. In this study, the Matrix Scoring Technique (Marine Strategy Framework Directive Technical Group on Marine Litter) was applied as an approach to calculate the likelihood of single debris items originating from a series of potential sources. Factors considered were: identity and function of debris, beach location, influential activities, "mix" of debris found, presence of indicator items, and quantity of MD. The standing-stock (abundance and composition) of MD was investigated in two sandy beaches (Conceição and Porto Pim) of the Azores Archipelago (NE Atlantic) for the period 2012-2018. The results of this study show promise towards the implementation of a new classification method to determine beach debris sources in remote open-ocean areas.

Phylogenetic diversity in freshwater-dwelling Isochrysidales haptophytes with implications for alkenone production.

Members of the order Isochrysidales are unique among haptophyte lineages in being the exclusive producers of alkenones, long-chain ketones that are commonly used for paleotemperature reconstructions. Alkenone-producing haptophytes are divided into three major groups based largely on molecular ecological data: Group I is found in freshwater lakes, Group II commonly occurs in brackish and coastal marine environments, and Group III consists of open ocean species. Each group has distinct alkenone distributions; however, only Groups II and III Isochrysidales currently have cultured representatives. The uncultured Group I Isochrysidales are distinguished geochemically by the presence of tri-unsaturated alkenone isomers (C37:3b Me, C38:3b Et, C38:3b Me, C39:3b Et) present in water column and sediment samples, yet their genetic diversity, morphology, and environmental controls are largely unknown. Using small-subunit (SSU) ribosomal RNA (rRNA) marker gene amplicon high-throughput sequencing of environmental water column and sediment samples, we show that Group I is monophyletic with high phylogenetic diversity and contains a well-supported clade separating the previously described "EV" clade from the "Greenland" clade. We infer the first partial large-subunit (LSU) rRNA gene Group I sequence phylogeny, which uncovered additional well-supported clades embedded within Group I. Relative to Group II, Group I revealed higher levels of genetic diversity despite conservation of alkenone signatures and a closer evolutionary relationship with Group III. In Group I, the presence of the tri-unsaturated alkenone isomers appears to be conserved, which is not the case for Group II. This suggests differing environmental influences on Group I and II and perhaps uncovers evolutionary constraints on alkenone biosynthesis.