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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]

A recent pyrosequencing study along the whole Baltic Sea salinity transect identified members of the V errucomicrobia class S partobacteria 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 S partobacteria comprise an important component, constituting up to 12% of all bacteria. The positive correlation and physical association of S partobacteria with phytoplankton suggest their involvement in the utilization of phytoplankton-derived organic matter in the Baltic Sea. [ABSTRACT FROM AUTHOR]

Summary: Microplastics in marine ecosystems are colonized by diverse prokaryotic and eukaryotic communities. How these communities and their functional profiles are shaped by the artificial surfaces remains broadly unknown. In order to close this knowledge gap, we set up an in situ experiment with pellets of the polyolefin polymer polyethylene (PE), the aromatic hydrocarbon polymer polystyrene (PS), and wooden beads along a coastal to estuarine gradient in the Baltic Sea, Germany. We used an integrated metagenomics/metaproteomics approach to evaluate the genomic potential as well as protein expression levels of aquatic plastic biofilms. Our results suggest that material properties had a minor influence on the plastic‐associated assemblages, as genomic and proteomic profiles of communities associated with the structurally different polymers PE and PS were highly similar, hence polymer‐unspecific. Instead, it seemed that these communities were shaped by biogeographic factors. Wood, on the other hand, induced the formation of substrate‐specific biofilms and served as nutrient source itself. Our study indicates that, while PE and PS microplastics may be relevant in the photic zone as opportunistic colonization grounds for phototrophic microorganisms, they appear not to be subject to biodegradation or serve as vectors for pathogenic microorganisms in marine habitats. [ABSTRACT FROM AUTHOR]

Abstract: We report on methane enrichments that were observed during summer in the upper water column of the Gotland Basin, central Baltic Sea. In the eastern part of the basin, methane concentrations just below the thermocline varied between 15 nM and 77 nM, in contrast to the western part where no methane enrichments could be detected. Stable carbon isotope ratios of methane (δ13C‐CH4 of −67.6‰) indicated its in situ biogenic origin from CO2 reduction, which was supported by clonal sequences that clustered with Methanomicrobiaceae, a family of methanogenic Archaea. Incubation experiments with a Temora longicornis dominated seston fraction obtained from the relevant depth showed a positive correlation between seston concentration and methane production rates. Our results, in combination with previous literature outcomes, suggest that the methane enrichment in the eastern basin might be sustained by a diet‐consumer relationship between the dinoflagellate Dinophysis norvegica and the copepod T. longicornis. However, our mass balance indicates that a local methane production of 110 pmol L−1 d−1 was needed to maintain the methane enrichment, and that the estimated production rate from our incubation experiments of 0.3 pmol CH4 d−1 per adult T. longicornis (about 1 pmol L−1 d−1) was too low to maintain the methane enrichment by zooplankton associated methane production only. These calculations also showed that methane was consumed below the thermocline and not transported into the upper‐ocean, suggesting that other sources in the mixed layer in the range of 95 pmol L−1 d−1 are needed to maintain the observed methane air–sea flux. [ABSTRACT FROM AUTHOR]

Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north-east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP-associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large. [ABSTRACT FROM AUTHOR]

Chemolithoautotrophic sulfur-oxidizing and denitrifying Gamma- (particularly the SUP05 cluster) and Epsilonproteobacteria (predominantly Sulfurimonas subgroup GD17) are assumed to compete for substrates (electron donors and acceptors) in marine pelagic redox gradients. To elucidate their ecological niche separation we performed 34S0, 15NO-3 and H13CO-3 stable-isotope incubations with water samples from Baltic Sea suboxic, chemocline and sulfidic zones followed by combined phylogenetic staining and high-resolution secondary ion mass spectrometry of single cells. SUP05 cells were small-sized (0.06-0.09 μm3) and most abundant in low-sulfidic to suboxic zones, whereas Sulfurimonas GD17 cells were significantly larger (0.26-0.61 μm3) and most abundant at the chemocline and below. Together, SUP05 and GD17 cells accumulated up to 48% of the labelled substrates but calculation of cell volume-specific rates revealed that GD17 cells incorporated labelled substrates significantly faster throughout the redox zone, thereby potentially outcompeting SUP05 especially at high substrate concentrations. Thus, in synopsis with earlier described features of SUP05/GD17 we conclude that their spatially overlapping association in stratified sulfidic zones is facilitated by their different lifestyles: whereas SUP05 cells are streamlined, non-motile K-strategists adapted to low substrate concentrations, GD17 cells are motile r-strategists well adapted to fluctuating substrate and redox conditions. [ABSTRACT FROM AUTHOR]

Knowledge on Actinobacteria rhodopsin gene ( actR) diversity and spatial distribution is scarce. The Baltic Sea is characterized by strong salinity gradients leading to the coexistence of marine and freshwater bacteria and hence is an ideal study area to elucidate the dispersion and phylogenetic affiliation of actR in dependence on salinity . ActR DGGE fingerprints in summer 2008 revealed between 3 and 19 distinct bands within a salinity range of 2.4-27 PSU. Environmental actR clone sequences were obtained from stations distributed along the whole salinity gradient. Overall, 20 different actR sequence groups (operational taxonomic units) were found, with up to 11 different ones per station. Phylogenetically, the actR sequences were predominantly (80%) affiliated with freshwater acI- Actinobacteria whose 16 S rRNA gene accounted for 2-33% of total 16 S rRNA genes in both the Bothnian Sea and central Baltic Sea. However, at salinities above 14 PSU, acI-16 S rRNA gene accounted for less than 1%. In contrast, the diversity of actR remained high. Changes in actR gene diversity were significantly correlated with salinity, oxygen, silica or abundance of Synechococcus sp. Our results demonstrate a wide distribution of freshwater actR along the Baltic Sea salinity gradient indicating that some freshwater Actinobacteria might have adapted to higher salinities. [ABSTRACT FROM AUTHOR]

Barrier zones between oxic and anoxic water masses (redoxclines) host highly active prokaryotic communities with important roles in biogeochemical cycling. In Baltic Sea pelagic redoxclines, Epsilonproteobacteria of the genus Sulfurimonas (subgroup GD17) have been shown to dominate chemoautotrophic denitrification. However, little is known on the loss processes affecting this prokaryotic group. In the present study, the protist grazing impact on the Sulfurimonas subgroup GD17 was determined for suboxic and oxygen/hydrogen sulphide interface depths of Baltic Sea redoxclines, using predator exclusion assays and bacterial amendment with the cultured representative ' Sulfurimonas gotlandica' strain GD1. Additionally, the principal bacterivores were identified by RNA-Stable Isotope Probing ( RNA-SIP). The natural Sulfurimonas subgroup GD17 population grew strongly under oxygen/hydrogen sulphide interface conditions (doubling time: 1-1.5 days), but protist grazing could consume the complete new cell production per day. In suboxic samples, little or no growth of Sulfurimonas subgroup GD17 was observed. RNA-SIP identified five active grazers, belonging to typical redoxcline ciliates ( Oligohymenophorea, Prostomatea) and globally widespread marine flagellate groups ( MAST-4, Chrysophyta, Cercozoa). Overall, we demonstrate for the first time that protist grazing can control the growth, and potentially the vertical distribution, of a chemolithoautotrophic key-player of oxic/anoxic interfaces. [ABSTRACT FROM AUTHOR]

Pelagic marine oxygen-depleted zones often exhibit a redox gradient, caused by oxygen depletion due to biological demand exceeding ventilation, and the accumulation of reduced chemical species, such as hydrogen sulfide. These redox gradients harbour a distinct assemblage of epsilonproteobacteria capable of fixing carbon dioxide autotrophically in the dark and potentially of utilizing hydrogen sulfide chemolithotrophically by oxidation with nitrate. Together, these two processes are referred to as chemolithoautotrophic denitrification. The focus of this study was the recently isolated and cultivated representative strain of pelagic epsilonproteobacteria, ' Sulfurimonas gotlandica' strain GD1, specifically dark carbon dioxide fixation and its substrate turnovers during chemolithotrophic denitrification. By connecting these processes stoichiometrically and comparing the results with those obtained for dark carbon dioxide fixation and nutrient concentrations measured in pelagic redox gradients of the Baltic Sea, we were able to estimate the role of chemolithoautotrophic denitrification in the environment. Evidence is provided for a defined zone where chemolithoautotrophic denitrification of these epsilonproteobacteria allows the complete removal of nitrate and hydrogen sulfide from the water column. This water layer is roughly equivalent in thickness to the average overlapping region of the two substrates, but slightly larger. Such a difference may be explained by a variety of reasons, including, e.g. utilization of substrates present at concentrations below the detection limit, alternative usage of other substrates as thiosulfate or nitrous oxide, or comparable activities of other microbes. However, the combined results of in vitro and in situ studies strongly suggest that epsilonproteobacteria are primarily responsible for hydrogen sulfide and nitrate removal from pelagic Baltic Sea redox gradients. [ABSTRACT FROM AUTHOR]

Marine pelagic redoxclines are zones of high dark CO2 fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO2 fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO2 incorporation in 13C-bicarbonate pulse experiments. The incorporation of 13C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in 13C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO2-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the 13C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO2-fixing Proteobacteria within this habitat. [ABSTRACT FROM AUTHOR]

Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchstädt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the ‘cattle manure’ itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore. [ABSTRACT FROM AUTHOR]

16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA. [ABSTRACT FROM AUTHOR]

Steep redoxclines form between oxic surface water and the stagnant, sulfidic hypolimnion in the eutrophied, brackish water Baltic Sea. Nitrification, denitrification, and anammox were measured at and below the redoxcline to quantify the role of water-column nitrogen processes in the overall magnitude of nitrogen removal in the Baltic Sea. Rates of nitrification were very high (up to 85 nmol N L-1 d-1) at the oxic-anoxic interface, but, surprisingly, nitrification was separated from the processes reducing nitrate to N2 (up to 810 nmol N L-1 d-1) by tens of meters in depth. N2 production was dominated by chemolithotrophic denitrification, with anammox playing only a negligible role, and limited to the water layers in which nitrite or nitrate coexisted with sulfide. The alternating oxygen concentrations in the basin induce irregular bursts of nitrogen removal. Nitrification takes place after mixing of ammonium-rich deep water with oxic water, and denitrification uses the formed nitrite and nitrate once anoxic conditions re-establish. Although removal rates can be high, conditions allowing such rates are likely short-lived. While the sedimentary denitrification rates at the shallower, oxic areas are lower, they are more constant in time, highlighting the need to avoid hypoxia, which would prevent sedimentary denitrification. [ABSTRACT FROM AUTHOR]

We determined the abundance and distribution of chemolithoautotrophic prokaryotes in the redoxcline in two basins (Gotland Deep, Landsort Deep) of the central Baltic Sea by combining dark CO2 fixation measurements with flow cytometric cell sorting. Maximum CO2 fixation rates were recorded in sulfidic waters about 20 m below the chemocline. Flow cytometric analyses of deoxyribonucleic acid (DNA)-stained bacterioplankton revealed the existence of at least five different prokaryotic clusters in water samples collected below the chemocline. Dark CO2 fixation in these clusters was determined by flow cytometric sorting after anoxic incubations with NaH14CO3 tracer. Two clusters, representing about 30% of total prokaryotes, were responsible for 65% to 100% of the total dark fixation. Calculated cell-specific CO2 fixation rates in the two basins ranged from 3.5 to 24.7 fg C cell-1 d-1 and suggested that these clusters are dominated by chemolithoautotrophic prokaryotes. Mean cell-specific fixation rates reached more than 10 fg C cell-1 d-1 in most cases, indicating relatively high growth rates (doubling times 1-2 d) of chemolithoautotrophic prokaryotes. Our results provide the first evidence of such high cell-specific CO2 uptake and abundance of chemolithoautotrophic prokaryotes in a pelagic marine environment. However, the identity of the organisms as well as the mechanisms fueling CO2 dark fixation in the anoxic zone remain unknown. [ABSTRACT FROM AUTHOR]