Viral density and virus-to-bacterium ratio in deep-sea sediments of the Eastern Mediterranean

Viruses are now recognized as a key component in pelagic systems, but their role in marine sediment has yet to be assessed. In this study bacterial and viral densities were determined at nine deep-sea stations selected from three main sites (i.e., the Sporades Basin, the Cretan Sea, and the Ierapetra Trench at depths of 1,232, 1,840, and 4,235 m, respectively) of the Eastern Mediterranean. The three areas were characterized by different phytopigment and biopolymeric carbon concentrations and by changes in the protein and carbohydrate pools. A gradient of increasing trophic conditions was observed from the Sporades Basin (North Aegean) to the Ierapetra Trench (South Aegean). Viral densities (ranging from 1 x 10(9) to 2 x 10(9) viruses ml of sediment(-1)) were significantly correlated to bacterial densities (n = 9, r(2) = 0.647) and reached values up to 3 orders of magnitude higher than those generally reported for the water column. However, the virus-to-bacterium density ratio in deep-sea sediments was about 1 order of magnitude lower (range of 2 to 5, with a modal value of 2.6) than in pelagic environments. Virus density decreased vertically with depth in sediment cores at all stations and was below detection limits at the 10-cm depth of the abyssal sediments of the Ierapetra Trench. Virus density in the sediment apparently reflected a gradient of particle fluxes and trophic conditions, displaying the highest values in the Sporades Basin. The low virus-to-bacterium ratios and their inverse relationship with station depth suggest that the role played by viruses in controlling deep-sea benthic bacterial assemblages and biogeochemical cycles is less relevant than in pelagic systems.     

Fungi and Macroaggregation in Deep-Sea Sediments

Whereas fungi in terrestrial soils have been well studied, little is known of them in deep-sea sediments. Recent studies have demonstrated the presence of fungal hyphae in such sediments but in low abundance. We present evidence in this study that one of the apparent reasons for the poor detection of fungi in deep-sea sediments is their cryptic presence in macroaggregates. Fungal biomass carbon from different core sections of deep-sea sediments from approximately 5000 m depth in the Central Indian Ocean was estimated based on direct microscopic detection of fungal mycelia. Treatment of sediment samples with ethylenediamine tetra-acetic acid (EDTA) enabled more frequent detection and significantly higher biomass than in samples without such treatment. Treatment with EDTA resulted in detecting various stages of breakdown of aggregates in the sediments, gradually revealing the presence of fungal hyphae within them. Experimental studies of a deep-sea, as well as three terrestrial isolates of fungi, showed that all could grow at 200 bar and 5 degrees C in a nutrient medium and in deep-sea sediment extract. Hyphae of fungi grown in sediment extract under the above conditions showed various stages of accretion of particles around them, leading to the formation of aggregates. Such aggregates showed the presence of humic material, carbohydrate, and proteins. We suggest that fungi in deep-sea sediments may be involved in humic aggregate formation by processes very similar to those in terrestrial sediments. The importance of such a process in carbon sequestration and food web in the deep sea needs to be examined.     

Large-Scale Distribution and Activity of Prokaryotes in Deep-Sea Surface Sediments of the Mediterranean Sea and the Adjacent Atlantic Ocean

The deep-sea represents a substantial portion of the biosphere and has a major influence on carbon cycling and global biogeochemistry. Benthic deep-sea prokaryotes have crucial roles in this ecosystem, with their recycling of organic matter from the photic zone. Despite this, little is known about the large-scale distribution of prokaryotes in the surface deep-sea sediments. To assess the influence of environmental and trophic variables on the large-scale distribution of prokaryotes, we investigated the prokaryotic assemblage composition (Bacteria to Archaea and Euryarchaeota to Crenarchaeota ratio) and activity in the surface deep-sea sediments of the Mediterranean Sea and the adjacent North Atlantic Ocean. Prokaryotic abundance and biomass did not vary significantly across the Mediterranean Sea; however, there were depth-related trends in all areas. The abundance of prokaryotes was positively correlated with the sedimentary concentration of protein, an indicator of the quality and bioavailability of organic matter. Moving eastwards, the Bacteria contribution to the total prokaryotes decreased, which appears to be linked to the more oligotrophic conditions of the Eastern Mediterranean basins. Despite the increased importance of Archaea, the contributions of Crenarchaeota Marine Group I to the total pool was relatively constant across the investigated stations, with the exception of Matapan-Vavilov Deep, in which Euryarchaeota Marine Group II dominated. Overall, our data suggest that deeper areas of the Mediterranean Sea share more similar communities with each other than with shallower sites. Freshness and quality of sedimentary organic matter were identified through Generalized Additive Model analysis as the major factors for describing the variation in the prokaryotic community structure and activity in the surface deep-sea sediments. Longitude was also important in explaining the observed variability, which suggests that the overlying water masses might have a critical role in shaping the benthic communities.     

Pelagic-Benthic Coupling and Diagenesis of Nucleic Acids in a Deep-Sea Continental Margin and an Open-Slope System of the Eastern Mediterranean

Downward fluxes of nucleic acids adsorbed onto settling particles play a key role in the supply of organic phosphorus and genetic material to the ocean interior. However, information on pelagic-benthic coupling, diagenesis, and processes controlling nucleic acid preservation in deep-sea sediments is practically nonexistent. In this study, we compared nucleic acid fluxes, sedimentary DNA and RNA concentrations, and the enzymatically hydrolyzable fraction of DNA in a bathyal continental margin (North Aegean Sea) and an open-sea system (South Aegean Sea) of the Eastern Mediterranean. The two systems displayed contrasting patterns of nucleic acid fluxes, which increased significantly with depth in the North Aegean Sea and decreased with depth in the South Aegean Sea. These results suggest that in continental margin and open-ocean systems different processes control the nucleic acid supply to the sea floor. Differences in nucleic acid fluxes were reflected by nucleic acid concentrations in the sediments, which reached extremely high values in the North Aegean Sea. In this system, a large fraction of DNA may be buried, as suggested by the large fraction of DNA resistant to nuclease degradation and by estimates of burial efficiency (ca. eight times higher in the North than in the South Aegean Sea). Overall, the results reported here suggest that the preservation of DNA in deeper sediment layers may be favored in benthic systems characterized by high sedimentation rates.     

Properties of Bacteria Isolated from Deep-Sea Sediments

Thirty-eight isolates were subjected to taxonomic analysis by computer. Of the 38 isolates, 31 were from sediment samples collected at depths from 9,400 to 10,400 meters in the Philippine and Marianas Trenches of the Pacific Ocean, and 7 cultures were from seawater samples collected at various depths from surface to 4,000 meters and from several locations in the Pacific Ocean. A total of 116 characteristics were determined for each isolate, coded, and transferred to punch cards. Similarity values were obtained by computer analysis, with the use of two recently developed computer programs. Five distinct phenetic clusters were observed from the numerical analyses. Four of the clusters were identified as species of the genus Pseudomonas, and one, as an aerogenic species of Aeromonas. Group IV was identified as pigmented Pseudomonas fluorescens, and the major cluster, consisting of groups I and II, which merged at a species level of similarity, was treated as a new species of Pseudomonas. The 38 strain data were compared with data for 132 marine and nonmarine strains previously subjected to computer taxonomic analysis. The barotolerant deep-sea strains, with the exception of the deep-sea P. fluorescens isolates, clustered separately from all other marine strains.     

Microbial Community of a Hydrothermal Mud Vent Underneath the Deep-Sea Anoxic Brine Lake Urania (Eastern Mediterranean)

The composition of a metabolically active prokaryotic community thriving in hydrothermal mud fluids of the deep-sea hypersaline anoxic Western Urania Basin was characterized using rRNA-based phylogenetic analysis of a clone library. The physiologically active prokaryotic assemblage in this extreme environment showed a great genetic diversity. Most members of the microbial community appeared to be affiliated to yet uncultured organisms from similar ecosystems, i.e., deep-sea hypersaline basins and hydrothermal vents. The bacterial clone library was dominated by phylotypes affiliated with the epsilon-Proteobacteria subdivision recognized as an ecologically significant group of bacteria inhabiting deep-sea hydrothermal environments. Almost 18% of all bacterial clones were related to delta-Proteobacteria, suggesting that sulfate reduction is one of the dominant metabolic processes occurring in warm mud fluids. The remaining bacterial phylotypes were related to alpha- and beta-Proteobacteria, Actinobacteria, Bacteroides, Deinococcus-Thermus, KB1 and OP-11 candidate divisions. Moreover, a novel monophyletic clade, deeply branched with unaffiliated 16S rDNA clones was also retrieved from deep-sea sediments and halocline of Urania Basin. Archaeal diversity was much lower and detected phylotypes included organisms affiliated exclusively with the Euryarchaeota. More than 96% of the archaeal clones belonged to the MSBL-1 candidate order recently found in hypersaline anoxic environments, such as endoevaporitic microbial mats, Mediterranean deep-sea mud volcanoes and anoxic basins. Two phylotypes, represented by single clones were related to uncultured groups DHVE-1 and ANME-1. Thus, the hydrothermal mud of hypersaline Urania Basin seems to contain new microbial diversity. The prokaryotic community was significantly different from that occurring in the upper layers of the Urania Basin since 60% of all bacterial and 40% of all archaeal phylotypes were obtained only from mud fluids. The uniqueness of the composition of the active prokaryotic community could be explained by the complex environmental conditions at the site. The interaction of oxygenated warm mud fluids with the cold hypersaline brine of the Urania Basin seems to simultaneously select for various metabolic processes, such as aerobic and anaerobic heterotrophy, sulfide- and methane-dependent chemotrophy along with anaerobic oxidation of methane, sulfate- and metal-reduction.     

High Fungal Diversity and Abundance Recovered in the Deep-Sea Sediments of the Pacific Ocean

Knowledge about the presence and ecological significance of bacteria and archaea in the deep-sea environments has been well recognized, but the eukaryotic microorganisms, such as fungi, have rarely been reported. The present study investigated the composition and abundance of fungal community in the deep-sea sediments of the Pacific Ocean. In this study, a total of 1,947 internal transcribed spacer (ITS) regions of fungal rRNA gene clones were recovered from five sediment samples at the Pacific Ocean (water depths ranging from 5,017 to 6,986 m) using three different PCR primer sets. There were 16, 17, and 15 different operational taxonomic units (OTUs) identified from fungal-universal, Ascomycota-, and Basidiomycota-specific clone libraries, respectively. Majority of the recovered sequences belonged to diverse phylotypes of Ascomycota (25 phylotypes) and Basidiomycota (18 phylotypes). The multiple primer approach totally recovered 27 phylotypes which showed low similarities (≤97 %) with available fungal sequences in the GenBank, suggesting possible new fungal taxa occurring in the deep-sea environments or belonging to taxa not represented in the GenBank. Our results also recovered high fungal LSU rRNA gene copy numbers (3.52?×?10⁶ to 5.23?×?10⁷copies/g wet sediment) from the Pacific Ocean sediment samples, suggesting that the fungi might be involved in important ecological functions in the deep-sea environments.     

Links between Geographic Location, Environmental Factors, and Microbial Community Composition in Sediments of the Eastern Mediterranean Sea

The bacterial community composition of marine surface sediments originating from various regions of the Eastern Mediterranean Sea (12 sampling sites) was compared by parallel use of three fingerprinting methods: analysis of 16S rRNA gene fragment heterogeneity by denaturing gradient electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and analysis of phospholipid-linked fatty acid composition (PLFA). Sampling sites were located at variable depths (30–2860 m; water column depth above the sediments) and the sediments differed greatly also in their degree of petroleum contamination (0.4–18 μg g⁻¹), organic carbon (0.38–1.5%), and chlorophyll a content (0.01–7.7 μg g⁻¹). Despite a high degree of correlation between the three different community fingerprint methods, some major differences were observed. DGGE banding patterns showed a significant separation of sediment communities from the northern, more productive waters of the Thermaikos Gulf and the oligotrophic waters of the Cretan, S. Ionian, and Levantine Sea. T-RFLP analysis clearly separated the communities of deep sediments (>1494 m depth) from their shallow (<617 m) counterparts. PLFA analysis grouped a shallow station from the productive waters of the north with the deep oligotrophic sediments from the Ionian and Levantine Sea, with low concentrations of PLFAs, and hence low microbial biomass, as the common denominator. The degree of petroleum contamination was not significantly correlated to the apparent composition of the microbial communities for any of the three methods, whereas organic carbon content and sediment chlorophyll a were important in this regard.     

Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

Community Structure of Archaea from Deep-Sea Sediments of the South China Sea

Using the archaeal 16S rRNA gene, we determined the community structures of archaea of subseafloor sediments (～9-11 m below seafloor) from two geographically distant cores (MD05-2896, south, water depth 1,657 m; MD05-2902, north, water depth 3,697 m) in the South China Sea. Euryarchaeota accounted for 61.4% of total archaeal clone libraries at MD05-2896 and 56.2% at MD05-2902. At both locations, the Euryarchaeota-related sequences were dominated by Marine Benthic Group D, Terrestrial Miscellaneous Eryarchaeotal Group, and South African GoldMine Euryarchaeotal Group; the Crenarchaeota-related sequences were dominated by Marine Benthic Group B, Marine Group I, pSL12, and C3. The community structure showed no significant difference with depth at each location, suggesting the lack of stratification of archaeal populations in the deep-sea marine sediments in the South China Sea. On the other hand, the community structure is significantly different between the two sites, which may be related to geographical difference in the South China Sea.     

Reliability of CARD-FISH Procedure for Enumeration of Archaea in Deep-Sea Surficial Sediments

The enumeration of Archaea in deep-sea sediment samples is still limited, although different methodological procedures have been applied. Among these, catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH) technique is a promising tool for estimation of archaeal abundance in deep-sea sediment samples. Comparing different permeabilisation treatments, the best results obtained both on archaeal pure cultures and on natural assemblages were with hydrochloric acid (0.1 M) and proteinase K (0.004 U/ml) treatments. The application of CARD-FISH on deep-sea sediments revealed that Archaea reach up to 41% of total prokaryotic cells. Specific probes for planktonic Archaea showed that marine Crenarchaea dominated archaeal seafloor communities. No clear bathymetric trends were observed for archaeal abundances and the morphology of continental margin (slope vs. canyon) seems not to have a direct influence on archaeal relative abundances. The site-specific sediment habitat—both abiotic environmental setting and sedimentary organic matter quality—explain up to 65% of variance of archaeal, crenarchaeal and euryarchaeal relative abundance, suggesting a wide ecophysiological adaptation to deep-sea benthic ecosystems. The findings demonstrate that Archaea are an important component of benthic microbial assemblages so far neglected, and hence they lay the groundwork for more focused research on their ecological importance in the functioning of deep-sea benthic ecosystems.     

Fungal Community Analysis in the Deep-Sea Sediments of the Central Indian Basin by Culture-Independent Approach

Few studies have addressed the occurrence of fungi in deep-sea sediments, characterized by elevated hydrostatic pressure, low temperature, and fluctuating nutrient conditions. We evaluated the diversity of fungi at three locations of the Central Indian Basin (CIB) at a depth of ~5,000 m using culture-independent approach. Community DNA isolated from these sediments was amplified using universal and fungal-specific internal transcribed spacers and universal 18S rDNA primer pairs. A total of 39 fungal operational taxonomic units, with 32 distinct fungal taxa were recovered from 768 clones generated from 16 environmental clone libraries. The application of multiple primers enabled the recovery of eight sequences that appeared to be new. The majority of the recovered sequences belonged to diverse phylotypes of Ascomycota and Basidiomycota. Our results suggested the existence of cosmopolitan marine fungi in the sediments of CIB. This study further demonstrated that diversity of fungi varied spatially in the CIB. Individual primer set appeared to amplify different fungal taxa occasionally. This is the first report on culture-independent diversity of fungi from the Indian Ocean.     

Hydrocarbons in Deep-Sea Sediments following the 2010 Deepwater Horizon Blowout in the Northeast Gulf of Mexico

The Deepwater Horizon (DWH) spill released 4.9 million barrels of oil into the Gulf of Mexico (GoM) over 87 days. Sediment and water sampling efforts were concentrated SW of the DWH and in coastal areas. Here we present geochemistry data from sediment cores collected in the aftermath of the DWH event from 1000 – 1500 m water depth in the DeSoto Canyon, NE of the DWH wellhead. Cores were analyzed at high-resolution (at 2 mm and 5 mm intervals) in order to evaluate the concentration, composition and input of hydrocarbons to the seafloor. Specifically, we analyzed total organic carbon (TOC), aliphatic, polycyclic aromatic hydrocarbon (PAHs), and biomarker (hopanes, steranes, diasteranes) compounds to elucidate possible sources and transport pathways for deposition of hydrocarbons. Results showed higher hydrocarbon concentrations during 2010-2011 compared to years prior to 2010. Hydrocarbon inputs in 2010-2011 were composed of a mixture of sources including terrestrial, planktonic, and weathered oil. Our results suggest that after the DWH event, both soluble and highly insoluble hydrocarbons were deposited at enhanced rates in the deep-sea. We proposed two distinct transport pathways of hydrocarbon deposition: 1) sinking of oil-particle aggregates (hydrocarbon-contaminated marine snow and/or suspended particulate material), and 2) advective transport and direct contact of the deep plume with the continental slope surface sediments between 1000-1200 m. Our findings underline the complexity of the depositional event observed in the aftermath of the DWH event in terms of multiple sources, variable concentrations, and spatial (depth-related) variability in the DeSoto Canyon, NE of the DWH wellhead.     

Climate Change and Health Research in the Eastern Mediterranean Region

Anthropologically induced climate change, caused by an increased concentration of greenhouse gases in the atmosphere, is an emerging threat to human health. Consequences of climate change may affect the prevalence of various diseases and environmental and social maladies that affect population health. In this article, we reviewed the literature on climate change and health in the Eastern Mediterranean Region. This region already faces numerous humanitarian crises, from conflicts to natural hazards and a high burden of disease. Climate change is likely to aggravate these emergencies, necessitating a strengthening of health systems and capacities in the region. However, the existing literature on climate change from the region is sparse and informational gaps stand in the way of regional preparedness and adaptation. Further research is needed to assess climatic changes and related health impacts in the Eastern Mediterranean Region. Such knowledge will allow countries to identify preparedness vulnerabilities, evaluate capacity to adapt to climate change, and develop adaptation strategies to allay the health impacts of climate change.     

Bacterial Diversity in Deep-Sea Sediments from Afanasy Nikitin Seamount,Equatorial Indian Ocean

Deep-sea sediments can reveal much about the last 200 million years of Earth history, including the history of ocean life and climate. Microbial diversity in Afanasy Nikitin seamount located at Equatorial East Indian Ocean (EEIO) was investigated. Deep sea sediment collected at 150 cm depth (～67 ky BP) exhibited a dominance of clones clustering in the phylum Firmicutes (63%), followed by γ-Proteobacteria (37%). The bacterial diversity within sediment at 200 cm depth (～84 ky BP) was dominated by γ-Proteobacteria (66%) followed by β- Proteobacteria (34%), whereas no Firmicutes clones were retrieved. Notably, 29% clones from 150 cm and 18% of clones from 200 cm sediment showed high identity to sequences previously reported from mining sites, while 21% of clones from 150 cm depth and 10% from 200 cm sediments showed identity to sequences previously reported from volcanic areas. Further, many of the sequences were from uncultured clones. Overall, these data may help elucidate the nature of microorganisms inhabiting these sites and unravel the molecular diversity of the deep sea from an evolutionary perspective.     

Deep-Sea,Deep-Sequencing: Metabarcoding Extracellular DNA from Sediments of Marine Canyons

Marine sediments are home to one of the richest species pools on Earth, but logistics and a dearth of taxonomic work-force hinders the knowledge of their biodiversity. We characterized α- and β-diversity of deep-sea assemblages from submarine canyons in the western Mediterranean using an environmental DNA metabarcoding. We used a new primer set targeting a short eukaryotic 18S sequence (ca. 110 bp). We applied a protocol designed to obtain extractions enriched in extracellular DNA from replicated sediment corers. With this strategy we captured information from DNA (local or deposited from the water column) that persists adsorbed to inorganic particles and buffered short-term spatial and temporal heterogeneity. We analysed replicated samples from 20 localities including 2 deep-sea canyons, 1 shallower canal, and two open slopes (depth range 100–2,250 m). We identified 1,629 MOTUs, among which the dominant groups were Metazoa (with representatives of 19 phyla), Alveolata, Stramenopiles, and Rhizaria. There was a marked small-scale heterogeneity as shown by differences in replicates within corers and within localities. The spatial variability between canyons was significant, as was the depth component in one of the canyons where it was tested. Likewise, the composition of the first layer (1 cm) of sediment was significantly different from deeper layers. We found that qualitative (presence-absence) and quantitative (relative number of reads) data showed consistent trends of differentiation between samples and geographic areas. The subset of exclusively benthic MOTUs showed similar patterns of β-diversity and community structure as the whole dataset. Separate analyses of the main metazoan phyla (in number of MOTUs) showed some differences in distribution attributable to different lifestyles. Our results highlight the differentiation that can be found even between geographically close assemblages, and sets the ground for future monitoring and conservation efforts on these bottoms of ecological and economic importance.     

Bacterial Community Composition in Different Sediments from the Eastern Mediterranean Sea: a Comparison of Four 16S Ribosomal DNA Clone Libraries

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.