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While it is now appreciated that the millions of tons of plastic pollution travelling through marine systems carry complex communities of microorganisms, it is still unknown to what extent these biofilm communities are specific to the plastic or selected by the surrounding ecosystem. To address this, we characterized and compared the microbial communities of microplastic particles, nonplastic (natural and wax) particles, and the surrounding waters from three marine ecosystems (the Baltic, Sargasso and Mediterranean seas) using high-throughput 16S rRNA gene sequencing. We found that biofilm communities on microplastic and nonplastic particles were highly similar to one another across this broad geographical range. The similar temperature and salinity profiles of the Sargasso and Mediterranean seas, compared to the Baltic Sea, were reflected in the biofilm communities. We identified plastic-specific operational taxonomic units (OTUs) that were not detected on nonplastic particles or in the surrounding waters. Twenty-six of the plastic-specific OTUs were geographically ubiquitous across all sampled locations. These geographically ubiquitous plastic-specific OTUs were mostly low-abundance members of their biofilm communities and often represented uncultured members of marine ecosystems. These results demonstrate the potential for plastics to be a reservoir of rare and understudied microbes, thus warranting further investigations into the dynamics and role of these microbes in marine ecosystems. IMPORTANCE This study represents one of the largest comparisons of biofilms from environmentally sampled plastic and nonplastic particles from aquatic environments. By including particles sampled through three separate campaigns in the Baltic, Sargasso, and Mediterranean seas, we were able to make cross-geographical comparisons and discovered common taxonomical signatures that define the plastic biofilm. For the first time, we identified plastic-specific bacteria that reoccur across marine regions. Our data reveal that plastics have selective properties that repeatedly enrich for similar bacteria regardless of location, potentially shifting aquatic microbial communities in areas with high levels of plastic pollution. Furthermore, we show that bacterial communities on plastic do not appear to be strongly influenced by polymer type, suggesting that other properties, such as the absorption and/or leaching of chemicals from the surface, are likely to be more important in the selection and enrichment of specific microorganisms.

We have known for more than 45 years that microplastics in the ocean are carriers of microbially dominated assemblages. However, only recently has the role of microbial interactions with microplastics in marine ecosystems been investigated in detail. Research in this field has focused on three main areas: ( a ) the establishment of plastic-specific biofilms (the so-called plastisphere); ( b ) enrichment of pathogenic bacteria, particularly members of the genus Vibrio , coupled to a vector function of microplastics; and ( c ) the microbial degradation of microplastics in the marine environment. Nevertheless, the relationships between marine microorganisms and microplastics remain unclear. In this review, we deduce from the current literature, new comparative analyses, and considerations of microbial adaptation concerning plastic degradation that interactions between microorganisms and microplastic particles should have rather limited effects on the ocean ecosystems. The majority of microorganisms growing on microplastics seem to belong to opportunistic colonists that do not distinguish between natural and artificial surfaces. Thus, microplastics do not pose a higher risk than natural particles to higher life forms by potentially harboring pathogenic bacteria. On the other hand, microplastics in the ocean represent recalcitrant substances for microorganisms that are insufficient to support prokaryotic metabolism and will probably not be microbially degraded in any period of time relevant to human society. Because we cannot remove microplastics from the ocean, proactive action regarding research on plastic alternatives and strategies to prevent plastic entering the environment should be taken promptly.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

While paint particles are an important part of the microplastic sphere, they have, as yet, received much less research coverage, particularly regarding microplastic-microbiological interactions. This study investigated the biofilm communities of a variety of paint particles from brackish sediment using 16S rRNA gene sequencing. Paint particle biofilm communities appear to be distinct from natural (water and sediment), non-synthetic particle (cellulose) and common microplastic biofilm communities. Notably, there appears to be 1 group of sulphate-reducing bacteria from the Desulfobacteraceae family, Desulfatitalea tepidiphilia, that dominate certain paint biofilms. Of the 8 investigated paint-associated communities, four paints displayed this high Desulfobacteraceae presence. However, it is currently unclear from the chemical analysis performed of the paint surface chemistry (ATR FT-IR spectroscopy, Raman spectroscopy, SEM-EDX) what the drivers behind this might be. As such, this study provides important insights as the first to analyse microplastic-paint biofilm communities and paves the way for future research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Microbial communities are the main drivers of biogeochemical cycling of multiple elements sustaining life in the ocean. The rapidity of their response to stressors and abrupt environmental changes implies that even fast and infrequent events can affect local transformations of organic matter and nutrients. Modern molecular techniques now allow for monitoring of microbial activities and functions in the environment through the analysis of genes and expressed genes contained in natural microbial assemblages. However, messenger RNA turnover in cells can be as short as 30 seconds and stability varies greatly between transcripts. Sampling of in situ communities involves an inevitable delay between the collection of seawater and the extraction of its RNA, leaving the bacterial communities plenty of time to alter their gene expression. The characteristics of microbial RNA turnover make time-series very difficult because samples need to be processed immediately to limit alterations to the metatranscriptomes. To address these challenges we designed an autonomous in situ fixation multi-sampler (AFISsys) for the reliable sampling of microbial metatranscriptomes at frequent intervals, for refined temporal resolution. To advance the development of this instrument, we examined the minimal seawater volume necessary for adequate coverage of community gene expression, and the suitability of phenol/ethanol fixation for immediate and long-term preservation of transcripts from a microbial community. We then evaluated the field eligibility of the instrument itself, with two case studies in a brackish system. AFISsys is able to collect, fix, and store water samples independently at a predefined temporal resolution. Phenol/ethanol fixation can conserve metatranscriptomes directly in the environment for up to a week, for later analysis in the laboratory. Thus, the AFISsys constitutes an invaluable tool for the integration of molecular functional analyses in environmental monitoring in brackish waters and in aquatic environments in general., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A recent pyrosequencing study along the whole Baltic Sea salinity transect identified members of the Verrucomicrobia class Spartobacteria as an important component of Baltic Sea bacterioplankton. In this study, catalysed reporter deposition-fluorescence in situ hybridization was used for cellular quantification. The published probes VER47 and SPA714 were optimized for samples from the Baltic Sea and a new, specific probe (SPA476) was used to quantify the dominant spartobacterial lineage ‘LD29’. The results confirmed that in the brackish surface waters of the Baltic Sea Spartobacteria comprise an important component, constituting up to 12% of all bacteria. The positive correlation and physical association of Spartobacteria with phytoplankton suggest their involvement in the utilization of phytoplankton-derived organic matter in the Baltic Sea.

The biodegradability of terrigenous dissolved organic matter (tDOM) exported to the sea has a major impact on the global carbon cycle, but our understanding of tDOM bioavailability is fragmentary. In this study, the effects of preparative tDOM isolation on microbial decomposition were investigated in incubation experiments consisting of mesocosms containing mesohaline water from the Baltic Sea. Dissolved organic carbon (DOC) consumption, molecular DOM composition, bacterial activities, and shifts in bacterial community structure were compared between mesocosms supplemented with riverine tDOM, either as filtered, particle-free river water or as a concentrate obtained by lyophilization/tangential ultrafiltration, and those containing only Baltic Sea water or river water. As shown using ultra-high-resolution mass spectrometry (15 Tesla Fourier-transform ion cyclotron resonance mass spectrometry, FT-ICR-MS) covering approximately 4600 different DOM compounds, the three DOM preparation protocols resulted in distinct patterns of molecular DOM composition. However, despite DOC losses of 4-16% and considerable bacterial production, there was no significant change in DOM composition during the 28-day experiment. Moreover, tDOM addition affected neither DOC degradation nor bacterial dynamics significantly, regardless of the tDOM preparation. This result suggested that the introduced tDOM was largely not bioavailable, at least on the temporal scale of our experiment, and that the observed bacterial activity and DOC decomposition mainly reflected the degradation of unknown, labile, colloidal and low-molecular weight DOM, both of which escape the analytical window of FT-ICR-MS. In contrast to the different tDOM preparations, the initial bacterial inoculum and batch culture conditions determined bacterial community succession and superseded the effects of tDOM addition. The uncoupling of tDOM and bacterial dynamics suggests that mesohaline bacterial communities cannot efficiently utilize tDOM and that in subarctic estuaries other factors are responsible for the removal of imported tDOM.

The Baltic Sea receives large nitrogen inputs by diazotrophic (N2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of >10 and <10 μm size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 × 10(7) nifH copies l(-1). Abundant nifH transcripts (up to 3.2 × 10(4) transcripts l(-1)) within nifH cluster III and co-occurring N2 fixation (0.44±0.26 nmol l(-1) day(-1)) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N2 fixation could also be of importance in other suboxic regions of the world's oceans.

Salinity is a major factor controlling the distribution of biota in aquatic systems, and most aquatic multicellular organisms are either adapted to life in saltwater or freshwater conditions. Consequently, the saltwater-freshwater mixing zones in coastal or estuarine areas are characterized by limited faunal and floral diversity. Although changes in diversity and decline in species richness in brackish waters is well documented in aquatic ecology, it is unknown to what extent this applies to bacterial communities. Here, we report a first detailed bacterial inventory from vertical profiles of 60 sampling stations distributed along the salinity gradient of the Baltic Sea, one of world's largest brackish water environments, generated using 454 pyrosequencing of partial (400 bp) 16S rRNA genes. Within the salinity gradient, bacterial community composition altered at broad and finer-scale phylogenetic levels. Analogous to faunal communities within brackish conditions, we identified a bacterial brackish water community comprising a diverse combination of freshwater and marine groups, along with populations unique to this environment. As water residence times in the Baltic Sea exceed 3 years, the observed bacterial community cannot be the result of mixing of fresh water and saltwater, but our study represents the first detailed description of an autochthonous brackish microbiome. In contrast to the decline in the diversity of multicellular organisms, reduced bacterial diversity at brackish conditions could not be established. It is possible that the rapid adaptation rate of bacteria has enabled a variety of lineages to fill what for higher organisms remains a challenging and relatively unoccupied ecological niche.

The bacterial community in the sea surface microlayer (SML) (bacterioneuston) is exposed to unique physicochemical properties and stronger meteorological influences than the bacterial community in the underlying water (ULW) (bacterioplankton). Despite extensive research, however, the structuring factors of the bacterioneuston remain enigmatic. The aim of this study was to examine the effect of meteorological conditions on bacterioneuston and bacterioplankton community structures and to identify distinct, abundant, active bacterioneuston members. Nineteen bacterial assemblages from the SML and ULW of the southern Baltic Sea, sampled from 2006 to 2008, were compared. Single-strand conformation polymorphism (SSCP) fingerprints were analyzed to distinguish total (based on the 16S rRNA gene) and active (based on 16S rRNA) as well as nonattached and particle-attached bacterial assemblages. The nonattached communities of the SML and ULW were very similar overall (similarity: 47 to 99%; mean: 88%). As an exception, during low wind speeds and high radiation levels, the active bacterioneuston community increasingly differed from the active bacterioplankton community. In contrast, the particle-attached assemblages in the two compartments were generally less similar (similarity: 8 to 98%; mean: 62%), with a strong variability in the active communities that was solely related to wind speed. Both nonattached and particle-attached active members of the bacterioneuston, which were found exclusively in the SML, were related to environmental clones belonging to the Cyanobacteria, Bacteroidetes, and Alpha-, Beta-, and Gammaproteobacteria originally found in diverse habitats, but especially in water columns. These results suggest that bacterioneuston communities are strongly influenced by the ULW but that specific meteorological conditions favor the development of distinctive populations in the air-water interface.

Proteorhodopsins (PRs) are light-driven proton pumps that have been found in a variety of marine environments. The goal of this study was to search for PR presence in different freshwater and brackish environments and to explore the diversity of non-marine PR protein. Here, we show that PRs exist in distinctly different aquatic environments, ranging from clear water lakes to peat lakes and in the Baltic Sea. Some of the PRs observed in this study formed unique clades that were not previously observed in marine environments, whereas others were similar to PRs found in non-marine samples of the Global Ocean Sampling (GOS) expedition. Furthermore, the similarity of several PRs isolated from lakes in different parts of the world suggests that these genes are dispersed globally and that they may encode unique functional capabilities enabling successful competition in a wide range of freshwater environments. Phylogenomic analysis of genes found on these GOS scaffolds suggests that some of the freshwater PRs are found in freshwater Flavobacteria and freshwater SAR11-like bacteria.

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.

While the global distribution of microplastics (MP) in the marine environment is currently being critically evaluated, the potential role of MP as a vector for distinct microbial assemblages or even pathogenic bacteria is hardly understood. To gain a deeper understanding, we investigated how different in situ conditions contribute to the composition and specificity of MP-associated bacterial communities in relation to communities on natural particles. Polystyrene (PS), polyethylene (PE), and wooden pellets were incubated for 2 weeks along an environmental gradient, ranging from marine (coastal Baltic Sea) to freshwater (waste water treatment plant, WWTP) conditions. The associated assemblages as well as the water communities were investigated applying high-throughput 16S rRNA gene sequencing. Our setup allowed for the first time to determine MP-dependent and -independent assemblage factors as subject to different environmental conditions in one system. Most importantly, plastic-specific assemblages were found to develop solely under certain conditions, such as lower nutrient concentration and higher salinity, while the bacterial genus Erythrobacter, known for the ability to utilize polycyclic aromatic hydrocarbons (PAH), was found specifically on MP across a broader section of the gradient. We discovered no enrichment of potential pathogens on PE or PS; however, the abundant colonization of MP in a WWTP by certain bacteria commonly associated with antibiotic resistance suggests MP as a possible hotspot for horizontal gene transfer. Taken together, our study clarifies that the surrounding environment prevailingly shapes the biofilm communities, but that MP-specific assemblage factors exist. These findings point to the ecological significance of specific MP-promoted bacterial populations in aquatic environments and particularly in plastic accumulation zones. [ABSTRACT FROM AUTHOR]

Throughout the Baltic Sea redoxcline, virus production and the frequency of lytically-infected prokaryotic cells were estimated from parallel incubations of undiluted seawater and seawater that contained prokaryotes with substantially reduced numbers of viruses (virus dilution approach), effectively preventing viral reinfection during the incubation period. Undiluted seawater incubations resulted in much higher estimates of virus production (6–35×104 mL-1 h-1) and the frequency of infected cells (5–84%) than the virus dilution approach (virus production: 1–3×104 mL-1 h-1; frequency of infected cells: 1–11%). Viral production and the frequency of infected cells from both approaches, however, cannot be directly compared, as data obtained from undiluted incubations were biased by viral reinfection and other uncontrollable processes during the incubation period. High in situ viral abundance (1–2×107 mL-1) together with low virus production rates based on the virus dilution approach resulted in some of the longest viral turnover times (24–84 d) ever reported for the epipelagial. Throughout a wide range of environmental conditions, viral turnover time and burst size were negatively correlated. Given that viral decay estimated in ultra-filtered water was below the detection limit and the burst size was low (1–17), we conclude that prokaryotic viruses in the Baltic Sea redoxcline are investing most of their resources into stress defense (strong capsids) rather than proliferation (high burst size). In summary, the Baltic Sea redoxcline constitutes an environment where low virus production is found in combination with low viral decay, resulting in high viral abundance. [ABSTRACT FROM AUTHOR]

Molecular surveys of marine picoeukaryotes have revealed a large number of sequences unrelated to cultured organisms, such as those forming the marine stramenopile (MAST)-4 clade. Recent FISH (fluorescent in situ hybridization) data have shown that MAST-4 cells are uncultured heterotrophic flagellates of 2–3 μm in size that have a global distribution in non-polar marine waters. However, FISH is time-consuming and hard to apply to the many samples generated during oceanographic cruises, so we developed a real-time quantitative polymerase chain reaction (Q-PCR) protocol to determine rapidly the abundance of this group using environmental DNA. We designed a primer set targeting the 18S rRNA genes (rDNA) of MAST-4 and optimized and calibrated the Q-PCR protocol using a plasmid with the target sequence as insert. The Q-PCR was then applied to quantify MAST-4 rDNA molecules along three marine transects, longitudinal in the Indian Ocean, latitudinal in the Drake Passage and coastal–offshore in the Mediterranean Sea, and to a temporal study in a Mediterranean Sea coastal station. MAST-4 was detected in all samples processed (averaged abundances between 500 and 1000 rDNA molecules ml−1) except in mesopelagic and Antarctic samples, where it was virtually absent. In general, it was more abundant in the coast than offshore and in the deep chlorophyll maximum than at surface. A comparison of Q-PCR and FISH signals in well-controlled microbial incubations indicated that MAST-4 cells have around 30 copies of the rDNA operon. This Q-PCR assay quickly yielded quantitative data of uncultured MAST-4 cells and confirmed their wide distribution and putative ecological importance. [ABSTRACT FROM AUTHOR]

Members of the class Epsilonproteobacteria are known to be of major importance in biogeochemical processes at oxic-anosic interfaces. In pelagic redoxclines of the central Battic Sea, an uncultured epsilonproteobacterium related to Sulfurimonas denitrificans was proposed to play a key rote in chemolithotrophic denitrification (I. Brettar, M. Labrenz, S. Flavier. J. Bötel, H. Kuosa, R. Christen, and M. G. Höfle, AppI. Environ. Microbiol. 72:1364-1372, 2006). In order to determine the abundance, activity, and vertical distribution of this bacterium in high-resolotion profiles, 16S rRNA cloning and catalyzed reporter deposition and fluorescence in situ hybridization CARD-FISH) and quantitative PCR measurements were carried out. The results showed that 21% of the derived clone sequences, which in the present study were grouped together under the name GD17, had >99% similarity to the uncultured epsilonproteobacterium. A specific gene probe against GD17 (S-*-Sul-0090-a-4-18) was developed and used for enumeration hy CARD-FISH. In different pelagic redoxclines sampled during August 2003, May 2005, and February 2006, GD17 cells were always detected from the lower oxic area to the sulfidic area. Maximal abundance was detected around the chemocline, where sulfide and nitrate concentrations were close to the detection limit. The highest GD17 numbers 12 × 105 cells ml-1), representing up to 15% of the total bacteria, were comparable In those reported for Epsilonproteobacteria in pelagic redoxclines of the Black Sea and the Cariaco Trench (X. Lin, S. G. Wakeham, I. F. Putnam, Y. M. Astor, M. I. Scranton, A. Y. Chistoserdov, and G. T. Taylor, AppI. Environ. Microbiol. 72:2679-2690, 2006). However, in the Baltic Sea redoxclines, Epsilonproteobacteria consisted nearly entirely of cells belonging to the distinct GD17 group. This suggested that GD17 was the best-adapted epsilonproteobacterium within this ecological niche. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that chemoautotrophic Epsilonproteobacteria might play an important role, especially as anaerobic or microaerophilic dark CO[sub2]-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO[sub2]-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of Epsilonproteobacteria to dark CO[sub2] fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [[sup14]C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 x 10[sup5] [sup14]CO[sub2]-fixing prokaryotic cells ml[sup-1] were enumerated in the redoxcline of the central Baltic Sea and up to 9 x 10[sup4] [sup14]CO[sub2]-fixing cells ml[sup-1] were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance. [sup14]CO[sub2]-incorporating cells belonged exclusively to the domain Bacteria. Among these, members of the Epsilonproteobacteria were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the Sulfurimonas subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO[sub2]-fixing group. In conclusion, Epsilonproteobacteria were found to be mainly responsible for chemoautotrophic activity in the dark CO[sub2] fixation maxima of the Black Sea and central Baltic Sea redoxclines. These Epsilonproteobacteria might be relevant in similar habitats of the world's oceans, where high dark CO[sub2] fixation rates have been measured. [ABSTRACT FROM AUTHOR]