Deep-sea Sediment Samples Research Articles

A New Cytotoxic Sesquiterpene Quinone Produced by Penicillium sp. F00120 Isolated from a Deep Sea Sediment Sample

A new fungal strain, displaying strong toxic activity against brine shrimp larvae, was isolated from a deep sea sediment sample collected at a depth of 1300 m. The strain, designated as F00120, was identified as a member of the genus Penicillium on the basis of morphology and ITS sequence analysis. One new sesquiterpene quinone, named penicilliumin A (1), along with two known compounds ergosterol (2) and ergosterol peroxide (3), were isolated and purified from the cultures of F00120 by silica gel column, Sephadex LH-20 column, and preparative thin layer chromatography. Their structures were elucidated by detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analysis as well as comparison with literature data. The new compound penicilliumin A inhibited in vitro proliferation of mouse melanoma (B16), human melanoma (A375), and human cervical carcinoma (Hela) cell lines moderately.

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 (0.1M) and proteinase K (0.004U/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.

Practical steps for improved estimates of calcium carbonate concentrations in deep sea sediments using coulometry

Coulometric titration is a commonly used method for determination of carbonate carbon, and hence calcium carbonate concentrations, in geological materials. Analyzes of 460 samples of pure CaCO3 powder demonstrate that the coulometer output data need to be recalculated using a multi‐point regression analysis. These analyses also demonstrate that to generate a precision of ±0.8%, at least 7 mg of 100% calcite is needed. A precision of ± 0.5% requires 12 mg pure calcite. We recommend that 35 mg samples are used for most deep sea sediment samples, which gives a precision of ±0.8% down to 20% calcite contents in the sample. Calcite contents <10% in samples require multiple runs to improve precision.

Cosmopolitanism and Biogeography of the Genus Manganonema (Nematoda: Monhysterida) in the Deep Sea

Simple SummaryThe deep sea comprises more than 60% of the Earth surface, and likely represents the largest reservoir of as yet undiscovered biodiversity. Nematodes are the most abundant taxon on Earth and are particularly abundant and diverse in the deep sea. Nevertheless, knowledge of their biogeography especially in the deep sea is still at its infancy. This article explores the distribution of the genus Manganonema in the deep Atlantic Ocean and Mediterranean Sea providing new insights about this apparently rare deep-sea genus.Spatial patterns of species diversity provide information about the mechanisms that regulate biodiversity and are important for setting conservation priorities. Present knowledge of the biogeography of meiofauna in the deep sea is scarce. This investigation focuses on the distribution of the deep-sea nematode genus Manganonema, which is typically extremely rare in deep-sea sediment samples. Forty-four specimens of eight different species of this genus were recorded from different Atlantic and Mediterranean regions. Four out of the eight species encountered are new to science. We report here that this genus is widespread both in the Atlantic and in the Mediterranean Sea. These new findings together with literature information indicate that Manganonema is a cosmopolitan genus, inhabiting a variety of deep-sea habitats and oceans. Manganonema shows the highest diversity at water depths >4,000 m. Our data, therefore, indicate that this is preferentially an abyssal genus that is able, at the same time, to colonize specific habitats at depths shallower than 1,000 m. The analysis of the distribution of the genus Manganonema indicates the presence of large differences in dispersal strategies among different species, ranging from locally endemic to cosmopolitan. Lacking meroplanktonic larvae and having limited dispersal ability due to their small size, it has been hypothesized that nematodes have limited dispersal potential. However, the investigated deep-sea nematodes were present across different oceans covering macro-scale distances. Among the possible explanations (hydrological conditions, geographical and geological pathways, long-term processes, specific historical events), their apparent preference of colonizing highly hydrodynamic systems, could suggest that these infaunal organisms are transported by means of deep-sea benthic storms and turbidity currents over long distances.

Ultra-deep sequencing of foraminiferal microbarcodes unveils hidden richness of early monothalamous lineages in deep-sea sediments

Deep-sea floors represent one of the largest and most complex ecosystems on Earth but remain essentially unexplored. The vastness and remoteness of this ecosystem make deep-sea sampling difficult, hampering traditional taxonomic observations and diversity assessment. This problem is particularly true in the case of the deep-sea meiofauna, which largely comprises small-sized, fragile, and difficult-to-identify metazoans and protists. Here, we introduce an ultra-deep sequencing-based metagenetic approach to examine the richness of benthic foraminifera, a principal component of deep-sea meiofauna. We used Illumina sequencing technology to assess foraminiferal richness in 31 unsieved deep-sea sediment samples from five distinct oceanic regions. We sequenced an extremely short fragment (36 bases) of the small subunit ribosomal DNA hypervariable region 37f, which has been shown to accurately distinguish foraminiferal species. In total, we obtained 495,978 unique sequences that were grouped into 1,643 operational taxonomic units, of which about half (841) could be reliably assigned to foraminifera. The vast majority of the operational taxonomic units (nearly 90%) were either assigned to early (ancient) lineages of soft-walled, single-chambered (monothalamous) foraminifera or remained undetermined and yet possibly belong to unknown early lineages. Contrasting with the classical view of multichambered taxa dominating foraminiferal assemblages, our work reflects an unexpected diversity of monothalamous lineages that are as yet unknown using conventional micropaleontological observations. Although we can only speculate about their morphology, the immense richness of deep-sea phylotypes revealed by this study suggests that ultra-deep sequencing can improve understanding of deep-sea benthic diversity considered until now as unknowable based on a traditional taxonomic approach.

Eukaryotic Richness in the Abyss: Insights from Pyrotag Sequencing

BackgroundThe deep sea floor is considered one of the most diverse ecosystems on Earth. Recent environmental DNA surveys based on clone libraries of rRNA genes confirm this observation and reveal a high diversity of eukaryotes present in deep-sea sediment samples. However, environmental clone-library surveys yield only a modest number of sequences with which to evaluate the diversity of abyssal eukaryotes.Methodology/Principal FindingsHere, we examined the richness of eukaryotic DNA in deep Arctic and Southern Ocean samples using massively parallel sequencing of the 18S ribosomal RNA (rRNA) V9 hypervariable region. In very small volumes of sediments, ranging from 0.35 to 0.7 g, we recovered up to 7,499 unique sequences per sample. By clustering sequences having up to 3 differences, we observed from 942 to 1756 Operational Taxonomic Units (OTUs) per sample. Taxonomic analyses of these OTUs showed that DNA of all major groups of eukaryotes is represented at the deep-sea floor. The dinoflagellates, cercozoans, ciliates, and euglenozoans predominate, contributing to 17%, 16%, 10%, and 8% of all assigned OTUs, respectively. Interestingly, many sequences represent photosynthetic taxa or are similar to those reported from the environmental surveys of surface waters. Moreover, each sample contained from 31 to 71 different metazoan OTUs despite the small sample volume collected. This indicates that a significant faction of the eukaryotic DNA sequences likely do not belong to living organisms, but represent either free, extracellular DNA or remains and resting stages of planktonic species.Conclusions/SignificanceIn view of our study, the deep-sea floor appears as a global DNA repository, which preserves genetic information about organisms living in the sediment, as well as in the water column above it. This information can be used for future monitoring of past and present environmental changes.

Evaluation of a deep-sea mesophilic bacteria exopolysaccharides in removal of low concentration Pb(II) from aqueous medium

Wangia profunda SM-A87, representative of a new genus of family Flavobacteriaceae, was isolated from deep-sea sediment samples near the southern Okinawa. The exopolysaccharides (EPS) produced by W. profunda SM-A87 was used as an absorbent of Pb(II) from low concentration solution in this paper. The effect of various parameters such as EPS concentration, pH, temperature, contact time, ionic strength and the competitive adsorption of Pb(II), Cu(II), Cd(II) were evaluated in batch technique. The adsorption kinetic and isotherm were also calculated. The preferences biosorption conditions were, EPS dosage at 0.1~0.2 mg/l, pH value at 5.5, temperature at 20~30°C and contact time for 60 min, respectively. Coexistent cations decreased Pb(II) uptake in the order of K+<Na+<Ca2+<Mg2+. The maximum adsorption capacity of Pb(II) was 357 mg/g at 30°C which was much higher than those of other biosorbents. The adsorption data fi tted better to the Langmuir equation than Freundlich equation. The pseudo second-order kinetic...

Levantilides A and B, 20-Membered Macrolides from a Micromonospora Strain Isolated from the Mediterranean Deep Sea Sediment

Two new 20-membered macrolides, levantilide A and B, were isolated from the Micromonospora strain M71-A77. Strain M71-A77 was recovered from an Eastern Mediterranean deep-sea sediment sample and revealed to produce the levantilides under in situ salinity of 38.6‰. The chemical structures of the levantilides were elucidated on the basis of different one- and two- dimensional NMR experiments. Levantilide A exhibits a moderate antiproliferative activity against several tumor cell lines.

Novel Lipolytic Enzymes Identified from Metagenomic Library of Deep‐Sea Sediment

Metagenomic library was constructed from a deep-sea sediment sample and screened for lipolytic activity. Open-reading frames of six positive clones showed only 33–58% amino acid identities to the known proteins. One of them was assigned to a new group while others were grouped into Families I and V or EstD Family. By employing a combination of approaches such as removing the signal sequence, coexpression of chaperone genes, and low temperature induction, we obtained five soluble recombinant proteins in Escherichia coli. The purified enzymes had optimum temperatures of 30–35°C and the cold-activity property. Among them, one enzyme showed lipase activity by preferentially hydrolyzing p-nitrophenyl palmitate and p-nitrophenyl stearate and high salt resistance with up to 4 M NaCl. Our research demonstrates the feasibility of developing novel lipolytic enzymes from marine environments by the combination of functional metagenomic approach and protein expression technology.

Response of bacteria in the deep-sea sediments and the Antarctic soils to carbohydrates: Effects on ectoenzyme activity and bacterial community

The response of bacteria to various carbohydrates in the deep-sea sediments and the Antarctic soils was investigated using cellulose, chitin, and olive oil. It was found that the carbohydrates significantly increased the corresponding specific ectoenzyme activity (β-glucosidase, β-N-acetylglucosaminidase, lipase) in the samples from deep-sea sediments. In the case of Antarctic soil samples, the cellulose or olive oil amendments had minor or no effect on β-glucosidase or lipase activity, except the chitin which stimulated β-N-acetylglucosaminidase production. The responses of the bacteria in the deep-sea sediment sample WP02-3 and the Antarctic soil sample CC-TY2 towards the chitin amendment were further analyzed. Chitin amendments were shown to stimulate the ectoenzyme activity in all the tested sediments and the soils. The bacterial response before and after the carbohydrates amendments were compared by denaturing gradient gel electrophoresis and quantitative competitive polymerase chain reaction. Significant changes were found in the structure and density of the bacterial community in the deep sea sediments as compared to the Antarctic soil sample, where the effects were relatively lower. There was no change in the bacterial population in both studied samples in response to carbohydrates amendments. These data indicate that the bacterial communities in the oligotrophic deep-sea sediments are more dynamic than that in the Antarctic soils as they respond to the nutrient sources efficiently by regulation of ectoenzyme activity and/or changing community structure.

Construction and Preliminary Analysis of a Deep-Sea Sediment Metagenomic Fosmid Library from Qiongdongnan Basin, South China Sea

Preliminary characterization of the microbial phylogeny and metabolic potential of a deep-sea sediment sample from the Qiongdongnan Basin, South China Sea, was carried out using a metagenomic library approach. An effective and rapid method of DNA isolation, purification, and library construction was used resulting in approximately 200,000 clones with an average insert size of about 36 kb. End sequencing of 600 individual clones from the fosmid library generated 1,051 sequences with an average sequence length of 619 bp. Phylogenetic ascription indicated that this library was dominated by Bacteria, predominantly Proteobacteria, though Planctomycetes were also relatively abundant. Sulfate-reducing and anaerobic ammonium-oxidizing bacteria, which play important roles in the cycling of sedimentary nutrients, were abundant in the library. Cluster of orthologous groups category analysis showed that most of the genes contained in the end sequences were related to metabolism, and with cellular processes and signaling. Functional groups assigned by SEED (subsystems-based annotations) highlighted the existence of 'one-carbon' metabolism within this community as well as identifying functional genes involved in methanogenesis. Furthermore, diverse genes involved in the biodegradation of xenobiotics were found using Kyoto Encyclopedia of Genes and Genomes metabolic pathway analysis.

Preferred use of bacteria over phytoplankton by deep-sea nematodes in polar regions

The present study explored the selective feeding properties of Antarctic and Arctic deep-sea nematodes within an experimental setup. Nematodes are assumed to play an important role in the carbon flux within the polar bathyal food webs, but knowledge about their natural diets is limited. For the first time, deep-sea multicore sediment samples from both polar regions were incubated aboard research vessels with either 13 C-labelled bacteria or diatoms to determine whether the nematode community prefers freshly settled phytodetritus to a bacterial food source. The cores were collected at 2112 to 2400 m water depth and incubated onboard for 1, 3 and 6 d in the Arctic (Hausgarten) and for 1, 7 and 14 d in Antarctica (Kapp Norvegia). Natural carbon isotope signals of nematodes and organic sedimentary carbon showed a clear average offset (+3.2‰). The contribution of bacteria to the diet of nematodes explained this 13 C offset and observed natural 13 C isotopic signatures. The nematodes showed a clear, relatively rapid (maximum at 6 to 7 d) and sig- nificant selective response to the pulse of 13 C enriched bacteria in surface sediments of both regions. This indicated that bacteria were preferred over fresh phytoplankton as a carbon source for both Arctic and Antarctic deep-sea nematode communities. The results also suggest that bacteria may provide a path through which unused detritus may enter the traditional metazoan food web by microbial reworking of organic matter. At the same time, uptake rates of nematode communities were minimal, which suggests the contribution of nematodes to benthic mineralisation of freshly deposited organic matter may be limited in deep polar seas.

Fungal diversity in deep-sea sediments – the presence of novel fungal groups

Abstract We investigated the diversity of fungal communities in 10 different deep-sea sediment samples by PCR-mediated internal transcribed spacer (ITS) regions of rRNA gene clone analysis. Although some of the amplified sequences were identified as common terrestrial fungal species, the majority were novel sequences that were not closely related to previously identified fungal ITS sequences in public databases. Phylogenetic analysis also suggested the novel phylogenetic affiliation of a group of predominant deep-sea phylotypes within the phylum Ascomycota. These phylotypes may be distributed widely in global deep-sea sediments. Another novel phylotype was placed in deep branches within the phylum Chytridiomycota with Rozella spp. as the closest related organisms. This study revealed the possible existence of previously unknown fungal components in various depths and sites of deep-sea sedimentary habitats. It is suggested that deep-sea environments are habitats hosting previously unexplored fungi, which may provide key insights into the early evolution of fungi and their ecological and physiological significance.

Ribosomal RNA gene fragments from fossilized cyanobacteria identified in primary gypsum from the late Miocene, Italy

Earth scientists have searched for signs of microscopic life in ancient samples of permafrost, ice, deep-sea sediments, amber, salt and chert. Until now, evidence of cyanobacteria has not been reported in any studies of ancient DNA older than a few thousand years. Here, we investigate morphologically, biochemically and genetically primary evaporites deposited in situ during the late Miocene (Messinian) Salinity Crisis from the north-eastern Apennines of Italy. The evaporites contain fossilized bacterial structures having identical morphological forms as modern microbes. We successfully extracted and amplified genetic material belonging to ancient cyanobacteria from gypsum crystals dating back to 5.910-5.816 Ma, when the Mediterranean became a giant hypersaline brine pool. This finding represents the oldest ancient cyanobacterial DNA to date. Our clone library and its phylogenetic comparison with present cyanobacterial populations point to a marine origin for the depositional basin. This investigation opens the possibility of including fossil cyanobacterial DNA into the palaeo-reconstruction of various environments and could also be used to quantify the ecological importance of cyanobacteria through geological time. These genetic markers serve as biosignatures providing important clues about ancient life and begin a new discussion concerning the debate on the origin of late Miocene evaporites in the Mediterranean.

Isolation and identification of Rhodosporidium diobovatum DS-0205 from deep-sea sediment of eastern Pacific Ocean

A facultative heterotrophic strain (DS-0205) was isolated from a deep-sea sediment sample collected at a depth of 5.2 km in the eastern Pacific Ocean, China. Strain DS-0205 is motile helmet-like single cell or pairs, forming hemisphere with the center sunken of variable size. It has a widespread carbon source and nitrogen source, including agarose, citric acid, salicin, D-glucitol nitrate, sodium nitrite and ethylamine. It can grow in the following environment: temperature 4–37 °C, pH 2.0–12.0, tolerance to NaCl⩽15%. Two phylogenetic trees, one based on the ITS and 5.8S rRNA sequences and the other based on the 18S rRNA sequences, unite strain DS-0205(=JCM 0205) to the type strain of Rhodosporidium diobovatum JCM 3787 through a considerable evolutionary distance. These results suggest that strain DS-0205 is a new strain of the Rhodosporidium diobovatum.

Verrucosispora sediminis sp. nov., a cyclodipeptide-producing actinomycete from deep-sea sediment

An actinomycete, designated MS426T, the culture broth of which showed potent antimicrobial activity, was isolated from a deep-sea sediment sample of the South China Sea. An almost-complete sequence of the 16S rRNA gene of strain MS426T was determined and aligned with those of representatives of the family Micromonosporaceae available in public databases. Phylogenetic trees were inferred by using three algorithms. Strain MS426T formed a branch adjacent to Verrucosispora lutea YIM 013T in a distinct cluster occupied only by strains of the genus Verrucosispora. Strain MS426T was distinguishable from the type strains of the two described Verrucosispora species by using a combination of chemical and morphological markers and by DNA-DNA relatedness. On the basis of these genotypic and phenotypic differences, the novel antimicrobial strain with pharmaceutical potential represents a novel species, for which the name Verrucosispora sediminis sp. nov. is proposed. The type strain is MS426T (=CGMCC 4.3550T =JCM 15670T).

Bhargavaea cecembensis gen. nov., sp. nov., isolated from the Chagos-Laccadive ridge system in the Indian Ocean

A novel Gram-positive, rod-shaped, non-motile, non-spore-forming bacterium, strain DSE10(T), was isolated from a deep-sea sediment sample collected at a depth of 5904 m from the Chagos-Laccadive ridge system in the Indian Ocean. Cells of strain DSE10(T) were positive for catalase, oxidase, urease and lipase activities and contained iso-C(14 : 0), iso-C(15 : 0), iso-C(16 : 0) and anteiso-C(15 : 0) as the major fatty acids. The major respiratory quinones were MK-6 and MK-8 and the major lipids were phosphatidylglycerol and diphosphatidylglycerol. The cell-wall peptidoglycan contained diaminopimelic acid as the diagnostic diamino acid. A blast sequence similarity search based on 16S rRNA gene sequences indicated that the genera Planococcus, Planomicrobium, Bacillus and Geobacillus were the nearest phylogenetic neighbours to the novel isolate with gene sequence similarities ranging from 94.9 to 95.2 %. Phylogenetic analyses using neighbour-joining, minimum-evolution and maximum-parsimony methods indicated that strain DSE10(T) formed a deeply rooted lineage distinct from the clades represented by the genera Planococcus, Planomicrobium, Bacillus and Geobacillus. Further, strain DSE10(T) could be distinguished from the above-mentioned genera based on the presence of signature nucleotides G, A, C, T, C, A, G, C and T at positions 182, 444, 480, 492, 563, 931, 1253, 1300 and 1391, respectively, in the 16S rRNA gene sequence. Based on the phenotypic and phylogenetic characteristics determined in this study, strain DSE10(T) was assigned as the type species of a new genus, Bhargavaea gen. nov., as Bhargavaea cecembensis sp. nov. The type strain of Bhargavaea cecembensis gen. nov., sp. nov. is DSE10(T) (=LMG 24411(T)=JCM 14375(T)). The genomic DNA G+C content of strain DSE10(T) is 59.5+/-2.5 mol%.

Croceicoccus marinus gen. nov., sp. nov., a yellow-pigmented bacterium from deep-sea sediment, and emended description of the family Erythrobacteraceae

A Gram-negative, aerobic, neutrophilic, coccoid bacterium, strain E4A9T, was isolated from a deep-sea sediment sample collected from the East Pacific polymetallic nodule region. 16S rRNA gene sequence analysis showed that the isolate was related to the type strain of Altererythrobacter epoxidivorans (96.0% sequence similarity). Lower 16S rRNA gene sequence similarities were observed with other members of the genera Altererythrobacter (94.7%), Erythrobacter (94.0-95.4%), Erythromicrobium (94.8%) and Porphyrobacter (94.6-95.1%) of the family Erythrobacteraceae. Phylogenetic analysis including all described species of the family Erythrobacteraceae and several members of the family Sphingomonadaceae revealed that the isolate formed a distinct phylogenetic lineage with the family Erythrobacteraceae. Chemotaxonomic analysis revealed ubiquinone-10 as the predominant respiratory quinone, anteiso-C15:0, iso-C14:0 and iso-C15:0 as major fatty acids, and phosphatidylglycerol as the major polar lipid. The DNA G+C content was 71.5 mol%. The isolate contained carotenoids, but no bacteriochlorophyll a. On the basis of phenotypic and genotypic data presented in this study, strain E4A9T represents a novel species in a new genus in the family Erythrobacteraceae for which the name Croceicoccus marinus gen. nov., sp. nov. is proposed; the type strain is E4A9T (=CGMCC 1.6776T=JCM 14846T).

Microorganisms Isolated from Deep Sea Low-temperature Influenced Oceanic Crust Basalts and Sediment Samples Collected along the Mid-Atlantic Ridge

Basalt and sediment samples were collected (at 1,460–2,996 m of water depth) along the Mid-Atlantic-Ridge. The microbial diversity was analysed by aerobic cultivation on three different media enriched for manganese- and sulphur-oxidising bacteria. Phylogenetic analyses of bacterial isolates revealed affiliations to the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria and Bacilli. Our data revealed significant differences in the occurrence and diversity of these communities between the respective deep seafloor biosphere sources. Among 138 genotypes, 12 putatively unknown isolates were detected. Characterisation of selected isolates revealed high tolerances against a broad range of temperatures, pH-values and salt concentrations.

Breviane Spiroditerpenoids from an Extreme-Tolerant Penicillium sp. Isolated from a Deep Sea Sediment Sample

Breviones F-H (1-3), three new bioactive breviane spiroditerpenoids, have been isolated from the crude extract of an extreme-tolerant Penicillium sp. obtained from a deep sea sediment sample that was collected at a depth of 5115 m. The structures of these compounds were elucidated primarily by NMR spectroscopy. The absolute configurations of 1 and 3 were assigned by application of the modified Mosher method, and 3 is further confirmed by X-ray crystallographic analysis of its S-MTPA ester. Compound 2 could be the precursor for 3, which features a previously undescribed skeleton of mixed biogenesis.