Putative ammonia-oxidizing Crenarchaeota in suboxic waters of the Black Sea: a basin-wide ecological study using 16S ribosomal and functional genes and membrane lipids

Within the upper 400 m at western, central and eastern stations in the world's largest stratified basin, the Black Sea, we studied the qualitative and quantitative distribution of putative nitrifying Archaea based on their genetic markers (16S rDNA, amoA encoding for the alpha-subunit of archaeal ammonia monooxygenase), and crenarchaeol, the specific glycerol diphytanyl glycerol tetraether of pelagic Crenarchaeota within the Group I.1a. Marine Crenarchaeota were the most abundant Archaea (up to 98% of the total archaeal 16S rDNA copies) in the suboxic layers with oxygen levels as low as 1 microM including layers where previously anammox bacteria were described. Different marine crenarchaeotal phylotypes (both 16S rDNA and amoA) were found at the upper part of the suboxic zone as compared with the base of the suboxic zone and the upper 15-30 m of the anoxic waters with prevailing sulfide concentrations of up to 30 microM. Crenarchaeol concentrations were higher in the sulfidic chemocline as compared with the suboxic zone. These results indicate an abundance of putative nitrifying Archaea at very low oxygen levels within the Black Sea and might form an important source of nitrite for the anammox reaction.     

Analysis of nitrite reductase (nirK and nirS) genes and cultivation reveal depauperate community of denitrifying bacteria in the Black Sea suboxic zone

Chemical profiles of the Black Sea suboxic zone show a distribution of nitrogen species which is traditionally associated with denitrification, i.e. a secondary nitrite maximum associated with nitrate depletion and a N(2) gas peak. To better understand the distribution and diversity of the denitrifier community in the Black Sea suboxic zone, we combined a cultivation approach with cloning and sequencing of PCR-amplified nitrite reductase (nirS and nirK) genes. The Black Sea suboxic zone appears to harbour a homogeneous community of denitrifiers. For nirK, over 94% of the sequences fell into only three distinct phylogenetic clusters, and for nirS, a single closely related sequence type accounted for 91% of the sequences retrieved. Both nirS and nirK genes showed a dramatic shift in community composition at the bottom of the suboxic zone, but overall, nirK-based community composition showed much greater variation across depths compared with the highly uniform distribution of nirS sequences throughout the suboxic zone. The dominant nirK and nirS sequences differed at the amino acid level by at least 17% and 8%, respectively, from their nearest database matches. Denitrifying isolates recovered from the suboxic zone shared 97% 16S rRNA gene sequence similarity with Marinobacter maritimus. Analysis of the recently discovered nirS gene from the anammox bacterium Candidatus'Kuenenia stuttgartiensis' revealed that mismatches with commonly used primers may have prevented the previous detection of this divergent sequence.     

Inorganic carbon fixation by sulfate-reducing bacteria in the Black Sea water column

The Black Sea is the largest anoxic water basin on Earth and its stratified water column comprises an upper oxic, middle suboxic and a lower permanently anoxic, sulfidic zone. The abundance of sulfate-reducing bacteria (SRB) in water samples was determined by quantifying the copy number of the dsrA gene coding for the alpha subunit of the dissimilatory (bi)sulfite reductase using real-time polymerase chain reaction. The dsrA gene was detected throughout the whole suboxic and anoxic zones. The maximum dsrA copy numbers were 5 x 10(2) and 6.3 x 10(2) copies ml(-1) at 95 m in the suboxic and at 150 m in the upper anoxic zone, respectively. The proportion of SRB to total Bacteria was 0.1% in the oxic, 0.8-1.9% in the suboxic and 1.2-4.7% in the anoxic zone. A phylogenetic analysis of 16S rDNA clones showed that most clones from the anoxic zone formed a coherent cluster within the Desulfonema-Desulfosarcina group. A similar depth profile as for dsrA copy numbers was obtained for the concentration of non-isoprenoidal dialkyl glycerol diethers (DGDs), which are most likely SRB-specific lipid biomarkers. Three different DGDs were found to be major components of the total lipid fractions from the anoxic zone. The DGDs were depleted in (13)C relative to the delta(13)C values of dissolved CO(2) (delta(13)C(CO2)) by 14-19 per thousand. Their delta(13)C values [delta(13)C(DGD(II-III))] co-varied with depth showing the least (13)C-depleted values in the top of the sulfidic, anoxic zone and the most (13)C-depleted values in the deep anoxic waters at 1500 m. This co-variation provides evidence for CO(2) incorporation by the DGD(II-III)-producing SRB, while the 1:2 relationship between delta(13)C(CO2) and delta(13)C(DGD(II-III)) indicates the use of an additional organic carbon source.     

The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes

Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described.     

Metabolic strategies of free-living and aggregate-associated bacterial communities inferred from biologic and chemical profiles in the Black Sea suboxic zone

The Black Sea is a permanently anoxic basin with a well-defined redox gradient. We combine environmental 16S rRNA gene data from clone libraries, terminal restriction fragment length polymorphisms, and V6 hypervariable region pyrosequences to provide the most detailed bacterial survey to date. Furthermore, this data set is informed by comprehensive geochemical data; using this combination of information, we put forward testable hypotheses regarding possible metabolisms of uncultured bacteria from the Black Sea's suboxic zone (microaerophily, nitrate reduction, manganese cycling, and oxidation of methane, ammonium, and sulfide). Dominant bacteria in the upper suboxic zone included members of the SAR11, SAR324, and Microthrix groups and in the deep suboxic zone included members of BS-GSO-2, Marine Group A, and SUP05. A particulate fraction (30?μm filter) was used to distinguish between free-living and aggregate-attached communities in the suboxic zone. The particulate fraction contained greater diversity of V6 tag sequences than the bulk water samples. Lentisphaera, Epsilonproteobacteria, WS3, Planctomycetes, and Deltaproteobacteria were enriched in the particulate fraction, whereas SAR11 relatives dominated the free-living fraction. On the basis of the bacterial assemblages and simple modeling, we find that in suboxic waters, the interior of sinking aggregates potentially support manganese reduction, sulfate reduction, and sulfur oxidation.     

Free-living and aggregate-associated Planctomycetes in the Black Sea

We examined the distribution of uncultured Planctomycetes phylotypes along depth profiles spanning the redox gradient of the Black Sea suboxic zone to gain insight into their respective ecological niches. Planctomycetes phylogeny correlated with depth and chemical profiles, implying similar metabolisms within phylogenetic groups. A suboxic zone sample was split into >?30 and ?30?μm fraction except for members of the Scalindua genus, which were apparently free-living. Sequences from Candidatus Scalindua, known to carry out the anammox process, formed two distinct clusters with nonoverlapping depth ranges. One cluster, only 97.1% similar to the named species, was present at high nitrite/nitrate and low ammonium concentrations in the upper suboxic zone. We propose this sequence type be named 'Candidatus Scalindua richardsii'. A second cluster, containing sequences more similar to 'Candidatus Scalindua sorokinii', was present at high ammonium and low nitrite conditions in the lower suboxic zone. Sequences obtained from the sulfidic zone (1000?m depth) yielded Planctomycetes from two uncharacterized Planctomycetacia clusters and three potentially new genera as well as sequences from the uncultured OP3 phylum.     

Detection of microbial biomass by intact polar membrane lipid analysis in the water column and surface sediments of the Black Sea

The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact polar membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact polar membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact polar membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the apolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact polar membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.     

Protist community composition in the Pacific sector of the Southern Ocean during austral summer 2010

Knowledge about the protist diversity of the Pacific sector of the Southern Ocean is scarce. We tested the hypothesis that distinct protist community assemblages characterize large-scale water masses. Therefore, we determined the composition and biogeography of late summer protist assemblages along a transect from the coast of New Zealand to the eastern Ross Sea. We used state of the art molecular approaches, such as automated ribosomal intergenic spacer analysis and 454-pyrosequencing, combined with high-performance liquid chromatography pigment analysis to study the protist assemblage. We found distinct biogeographic patterns defined by the environmental conditions in the particular region. Different water masses harbored different microbial communities. In contrast to the Arctic Ocean, picoeukaryotes had minor importance throughout the investigated transect and showed very low contribution south of the Polar Front. Dinoflagellates, Syndiniales, and small stramenopiles were dominating the sequence assemblage in the Subantarctic Zone, whereas the relative abundance of diatoms increased southwards, in the Polar Frontal Zone and Antarctic Zone. South of the Polar Front, most sequences belonged to haptophytes. This study delivers a comprehensive and taxon detailed overview of the protist composition in the investigated area during the austral summer 2010.     

Novel eukaryotic lineages inferred from small-subunit rRNA analyses of oxygen-depleted marine environments

Microeukaryotes in oxygen-depleted environments are among the most diverse, as well as the least studied, organisms. We conducted a cultivation-independent, small-subunit (SSU) rRNA-based survey of microeukaryotes in suboxic waters and anoxic sediments in the great Sippewisset salt marsh, Cape Cod, Mass. We generated two clone libraries and analyzed approximately 300 clones, which contained a large diversity of microeukaryotic SSU rRNA signatures. Only a few of these signatures were closely related (sequence similarity of >97%) to the sequences reported earlier. The bulk of our sequences represented deep novel branches within green algae, fungi, cercozoa, stramenopiles, alveolates, euglenozoa and unclassified flagellates. In addition, a significant number of detected rRNA sequences exhibited no affiliation to known organisms and sequences and thus represent novel lineages of the highest taxonomical order, most of them branching off the base of the global phylogenetic tree. This suggests that oxygen-depleted environments harbor diverse communities of novel organisms, which may provide an interesting window into the early evolution of eukaryotes.     

Ciliates along Oxyclines of Permanently Stratified Marine Water Columns

Studies of microbial communities in areas of the world where permanent marine water column oxyclines exist suggest they are “hotspots” of microbial activity, and that these water features and the anoxic waters below them are inhabited by diverse protist taxa, including ciliates. These communities have minimal taxonomic overlap with those in overlying oxic water columns. Some ciliate taxa have been detected in multiple locations where these stable water column oxyclines exist; however, differences in such factors as hydrochemistry in the habitats that have been studied suggest local selection for distinct communities. We compare published data on ciliate communities from studies of deep marine water column oxyclines in Caricao Basin, Venezuela, and the Black Sea, with data from coastal, shallower oxycline waters in Framvaren and Mariager fjords, and from several deep‐sea hypersaline anoxic basins in the Eastern Mediterranean Sea. Putative symbioses between Bacteria, Archaea, and ciliates observed along these oxyclines suggests a strategy of cooperative metabolism for survival that includes chemosynthetic autotrophy and exchanges of metabolic intermediates or end products between hosts and their prokaryotic partners.     

Composition,diversity and distribution of microbenthos across the intertidal zones of Ryazhkov Island (the White Sea)

The composition and distribution of the main unicellular eukaryotic groups (diatom algae, ciliates, dinoflagellates (DF), other phototrophic (PF) and heterotrophic flagellates (HF)) were investigated in sandy sediments at five stations allocated across the tidal sheltered beach of the White Sea. Overall, 75 diatoms, 98 ciliates, 16 DF, 3 PF and 34 HF species were identified; some are new records for the White Sea. Common species for each group are illustrated. Diatoms and ciliates showed high alpha-diversity (species richness per sample), whereas flagellates were characterized by high beta-diversity (species turnover across the intertidal flat). Each group demonstrated its own spatial pattern that was best matched with its own subset of abiotic variables, reflecting group-specific responses to environmental gradients. Species richness increased from the upper intertidal zone seaward for ciliates but decreased for HF, whereas autotrophs showed a relatively uniform pattern with a slight peak at the mid-intertidal zone. Across the littoral zone, all groups showed distinct compositional changes; however, the position of the boundary between “upper” and “lower” intertidal communities varied among groups. Most of the species found at Ryazhkov Island are known from many other regions worldwide, indicating a wide geographic distribution of microbial eukaryotic species.     

Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.     

The microplankton organisms at the oxic-anoxic interface in the pelagial of the Black Sea

Abstract The semi-permanent anoxia in the deep-waters of the Black Sea supported the formation of a unique microplankton community at the oxicanoxic interface. One group of ciliates ( Pleuronema marinum, Askenasia sp., species of the families Tracheliidae, Holophryidae and Amphileptidae) inhabited the water layer just above the upper boundary of H₂S, and was spatially associated with and fed on large colorless sulphur bacteria ( Thiovulum spp.). Other ciliates (species of order Scuticociliatida) populated the upper layer of the H₂S zone. A significant part of them possessed ectosymbiotic bacteria. Since the metazoans were not found in the O₂ and H₂S boundary layer, the protist community is considered to be the main factor in utilization of chemotrophic bacterial production.     

Suboxic zone in the Black Sea: real fact or analytical artefact

A reevaluation of the presence of a suboxic zone above the sulphide onset and the fine structure of the oxic/anoxic interface in the Black Sea is discussed. The results qualify this phenomenon as an analytical artefact and that the methods used for dissolved oxygen (DO) profiling are not applicable in the situation. Former and recent studies have established the stability of the oxic/anoxic interface structure with two principal components: the low DO gradient zone situated at the base of oxycline, and of the C-layer located beneath the sulphide onset boundary, where DO and sulphide coexist. The existence of the C-layer does not permit a suboxic zone void of perceptible DO and sulphide.     

Not all are free‐living: high‐throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa

Protists, the most diverse eukaryotes, are largely considered to be free‐living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High‐throughput sequencing (HTS) approaches now commonly replace cultivation‐based approaches in studying soil protists, but insights into common biases associated with this method are limited to aquatic taxa and samples. We created a mock community of common free‐living soil protists (amoebae, flagellates, ciliates), extracted DNA and amplified it in the presence of metazoan DNA using 454 HTS. We aimed at evaluating whether HTS quantitatively reveals true relative abundances of soil protists and at investigating whether the expected protist community structure is altered by the co‐amplification of metazoan‐associated protist taxa. Indeed, HTS revealed fundamentally different protist communities from those expected. Ciliate sequences were highly over‐represented, while those of most amoebae and flagellates were under‐represented or totally absent. These results underpin the biases introduced by HTS that prevent reliable quantitative estimations of free‐living protist communities. Furthermore, we detected a wide range of nonadded protist taxa probably introduced along with metazoan DNA, which altered the protist community structure. Among those, 20 taxa most closely resembled parasitic, often pathogenic taxa. Therewith, we provide the first HTS data in support of classical observational studies that showed that potential protist parasites are hosted by soil metazoa. Taken together, profound differences in amplification success between protist taxa and an inevitable co‐extraction of protist taxa parasitizing soil metazoa obscure the true diversity of free‐living soil protist communities.     

Microbial Community Structure and Dynamics in the Largest Natural French Lake (Lake Bourget)

We investigated the dynamics and diversity of heterotrophic bacteria, autotrophic and heterotrophic flagellates, and ciliates from March to July 2002 in the surface waters (0–50 m) of Lake Bourget. The heterotrophic bacteria consisted mainly of “small” cocci, but filaments (>2 μm), commonly considered to be grazing-resistant forms under increased nanoflagellate grazing, were also detected. These elongated cells mainly belonged to the Cytophaga-Flavobacterium (CF) cluster, and were most abundant during spring and early summer, when mixotrophic or heterotrophic flagellates were the main bacterial predators. The CF group strongly dominated fluorescent in situ hybridization–detected cells from March to June, whereas clear changes were observed in early summer when Beta-proteobacteria and Alpha-proteobacteria increased concomitantly with maximal protist grazing pressures. The analysis of protist community structure revealed that the flagellates consisted mainly of cryptomonad forms. The dynamics of Cryptomonas sp. and Dinobryon sp. suggested the potential importance of mixotrophs as consumers of bacteria. This point was verified by an experimental approach based on fluorescent microbeads to assess the potential grazing impact of all protist taxa in the epilimnion. From the results, three distinct periods in the functioning of the epilimnetic microbial loop were identified. In early spring, mixotrophic and heterotrophic flagellates constituted the main bacterivores, and were regulated by the availability of their resources mainly during April (phase 1). Once the “clear water phase” was established, the predation pressure of metazooplankton represented a strong top-down force on all microbial compartments. During this period only mixotrophic flagellates occasionally exerted a significant bacterivory pressure (phase 2). Finally, the early summer was characterized by the highest protozoan grazing impact and by a rapid shift in the carbon pathway transfer, with a fast change-over of the main predators contribution, i.e., mixotrophic, heterotrophic flagellates and ciliates in bacterial mortality. The high abundance of ciliates during this period was consistent with the high densities of resources (heterotrophic nanoflagellates, algae, bacteria) in deep layers containing the most chlorophyll. Bacteria, as ciliates, responded clearly to increasing phytoplankton abundance, and although bacterial grazing impact could vary largely, bacterial abundance seemed to be primarily bottom-up regulated (phase 3).     

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.     

Characterization of protistan assemblages in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.