Taxonomic diversity of microbial communities in sub-seafloor hydrothermal sediments of the active Santorini-Kolumbo volcanic field.

Introduction: Active hydrothermal vents of volcanic origin provide a remarkable manifestation of life on Earth under extreme conditions, which may have consequences for our understanding of habitability on other terrestrial bodies as well. Methods: Here, we performed for the first time Illumina sequencing of bacterial and archaeal communities on sub-seafloor samples collected from the Santorini-Kolumbo volcanic field. A total of 19 (3-m long) gravity corers were collected and processed for microbial community analysis. Results: From a total of 6,46,671 produced V4 sequences for all samples, a total of 10,496 different Operational Taxonomic Units (OTUs) were identified that were assigned to 40 bacterial and 9 archaeal phyla and 14 candidate divisions. On average, the most abundant phyla in all samples were Chloroflexi (Chloroflexota) (24.62%), followed by Proteobacteria (Pseudomonadota) (11.29%), Firmicutes (Bacillota) (10.73%), Crenarchaeota (Thermoproteota) (8.55%), and Acidobacteria (Acidobacteriota) (8.07%). At the genus level, a total of 286 known genera and candidate genera were mostly dominated by members of Bacillus, Thermoflexus, Desulfatiglans, Pseudoalteromonas, and Pseudomonas. Discussion: In most of the stations, the Chao1 values at the deeper layers were comparable to the surface sediment samples denoting the high diversity in the subsurface of these ecosystems. Heatmap analysis based on the 100 most abundant OTUs, grouped the sampling stations according to their geographical location, placing together the two hottest stations (up to 99°C). This result indicates that this specific area within the active Kolumbo crater create a distinct niche, where microorganisms with adaptation strategies to withstand heat stresses can thrive, such as the endospore-forming Firmicutes.

Structure and metabolic potential of the prokaryotic communities from the hydrothermal system of Paleochori Bay, Milos, Greece.

Introduction: Shallow hydrothermal systems share many characteristics with their deep-sea counterparts, but their accessibility facilitates their study. One of the most studied shallow hydrothermal vent fields lies at Paleochori Bay off the coast of Milos in the Aegean Sea (Greece). It has been studied through extensive mapping and its physical and chemical processes have been characterized over the past decades. However, a thorough description of the microbial communities inhabiting the bay is still missing. Methods: We present the first in-depth characterization of the prokaryotic communities of Paleochori Bay by sampling eight different seafloor types that are distributed along the entire gradient of hydrothermal influence. We used deep sequencing of the 16S rRNA marker gene and complemented the analysis with qPCR quantification of the 16S rRNA gene and several functional genes to gain insights into the metabolic potential of the communities. Results: We found that the microbiome of the bay is strongly influenced by the hydrothermal venting, with a succession of various groups dominating the sediments from the coldest to the warmest zones. Prokaryotic diversity and abundance decrease with increasing temperature, and thermophilic archaea overtake the community. Discussion: Relevant geochemical cycles of the Bay are discussed. This study expands our limited understanding of subsurface microbial communities in acidic shallow-sea hydrothermal systems and the contribution of their microbial activity to biogeochemical cycling.

Genomic adaptation of Pseudomonas strains to acidity and antibiotics in hydrothermal vents at Kolumbo submarine volcano, Greece.

Although the rise of antibiotic and multidrug resistant bacteria is one of the biggest current threats to human health, our understanding of the mechanisms involved in antibiotic resistance selection remains scarce. We performed whole genome sequencing of 21 Pseudomonas strains, previously isolated from an active submarine volcano of Greece, the Kolumbo volcano. Our goal was to identify the genetic basis of the enhanced co-tolerance to antibiotics and acidity of these Pseudomonas strains. Pangenome analysis identified 10,908 Gene Clusters (GCs). It revealed that the numbers of phage-related GCs and sigma factors, which both provide the mechanisms of adaptation to environmental stressors, were much higher in the high tolerant Pseudomonas strains compared to the rest ones. All identified GCs of these strains were associated with antimicrobial and multidrug resistance. The present study provides strong evidence that the CO2-rich seawater of the volcano associated with low pH might be a reservoir of microorganisms carrying multidrug efflux-mediated systems and pumps. We, therefore, suggest further studies of other extreme environments (or ecosystems) and their associated physicochemical parameters (or factors) in the rise of antibiotic resistance.

Plant and sediment properties in seagrass meadows from two Mediterranean CO2 vents: Implications for carbon storage capacity of acidified oceans.

Assessing the status of important carbon sinks such as seagrass meadows is of primary importance when dealing with potential climate change mitigation strategies. This study examined plant and sediment properties in seagrass meadows (Cymodocea nodosa (Ucria) Asch.) from two high pCO2-low pH Mediterranean vent systems, located at Milos (Greece) and Vulcano (Italy) Islands, providing insights on carbon storage potential in future acidified oceans. Contrary to what has been suggested, carbon content (both inorganic and organic) and its surficial accumulation decreased at high pCO2-low pH in comparison with controls. The decrease in inorganic carbon may result from the higher solubility of carbonates due to the more acidic conditions. At Vulcano, the seagrass properties (e.g., leaf area, biomass) appeared negatively affected by environmental conditions at high pCO2-low pH conditions and this may have had a detrimental effect on the organic carbon content and accumulation. At Milos, organic carbon decreased at high pCO2-low pH conditions, despite the increase in seagrass aboveground biomass, leaf length and area, probably as a consequence of site-specific features, which need further investigation and may include both biotic and abiotic factors (e.g., oligotrophic conditions, decreased sedimentation rate and input of allochthonous material). Results suggest that, in contrast to previous predictions based exclusively on the expected positive response of seagrasses to ocean acidification, carbon storage capacity of the seagrass C. nodosa may not increase at high pCO2-low pH conditions. This study emphasizes the need to investigate further the potential alteration in the climate mitigation service delivered by seagrass meadows in acidified oceans.

Microbial strains isolated from CO2-venting Kolumbo submarine volcano show enhanced co-tolerance to acidity and antibiotics.

As ocean acidification intensifies, there is growing global concern about the impacts that future pH levels are likely to have on marine life and ecosystems. By analogy, a steep decrease of seawater pH with depth is encountered inside the Kolumbo submarine volcano (northeast Santorini) as a result of natural CO2 venting, making this system ideal for ocean acidification research. Here, we investigated whether the increase of acidity towards deeper layers of Kolumbo crater had any effect on relevant phenotypic traits of bacterial isolates. A total of 31 Pseudomonas strains were isolated from both surface- (SSL) and deep-seawater layers (DSL), with the latter presenting a significantly higher acid tolerance. In particular, the DSL strains were able to cope with H+ levels that were 18 times higher. Similarly, the DSL isolates exhibited a significantly higher tolerance than SSL strains against six commonly used antibiotics and As(III). More importantly, a significant positive correlation was revealed between antibiotics and acid tolerance across the entire set of SSL and DSL isolates. Our findings imply that Pseudomonas species with higher resilience to antibiotics could be favored by the prospect of acidifying oceans. Further studies are required to determine if this feature is universal across marine bacteria and to assess potential ecological impacts.

A simple cleanup method for the removal of humic substances from soil protein extracts using aluminum coagulation.

Soil proteomics, the large-scale characterization of the entire protein complement in soils, provides a promising approach for deciphering the role of microbial functioning in terrestrial ecosystems. However, the extraction of soil proteins in sufficient quantities and of adequate purity remains a challenging task mainly due to the co-extraction of interfering humic substances. Up to now, the treatment of soil extracts with liquid phenol has been the "gold standard" for reducing humics, while the NoviPure cleanup kit was recently launched as a non-toxic approach. The present study describes an alternative method for delivering high-purity proteins based on humic coagulation with trivalent aluminum ions (Al3+). Various experimental parameters were optimized individually in order to maximize protein yield and diminish co-extracted humics. The optimized method was applied on a set of soil samples with diverse physicochemical characteristics and a comparison with the other two techniques was conducted. The amount of residual humics resulting from Al3+-based method was 26 and 35% higher than that from phenol treatment and NoviPure Kit, respectively, but these differences were of marginal statistical significance. With regard to extracted proteins, the average yields of the three methods were comparable, without showing any statistically significant differences. Overall, humic coagulation with Al3+ offers comparable cleanup performance in terms of protein yield and purity, but it is less toxic and less complex than the phenol-partitioning method, whereas it is far less expensive than the NoviPure Kit. The new technique is expected to facilitate the implementation of proteomic studies in soils.

Correction to: Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano, Hellenic Volcanic Arc.

In the original publication there is a mistake in the supplementary material. The correct supplementary material is provided in this correction article.

Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano, Hellenic Volcanic Arc.

Over the last decades, there has been growing interest about the ecological role of hydrothermal sulfide chimneys, their microbial diversity and associated biotechnological potential. Here, we performed dual-index Illumina sequencing of bacterial and archaeal communities on active and inactive sulfide chimneys collected from the Kolumbo hydrothermal field, situated on a geodynamic convergent setting. A total of 15,701 OTUs (operational taxonomic units) were assigned to 56 bacterial and 3 archaeal phyla, 133 bacterial and 16 archaeal classes. Active chimney communities were dominated by OTUs related to thermophilic members of Epsilonproteobacteria, Aquificae and Deltaproteobacteria. Inactive chimney communities were dominated by an OTU closely related to the archaeon Nitrosopumilus sp., and by members of Gammaproteobacteria, Deltaproteobacteria, Planctomycetes and Bacteroidetes. These lineages are closely related to phylotypes typically involved in iron, sulfur, nitrogen, hydrogen and methane cycling. Overall, the inactive sulfide chimneys presented highly diverse and uniform microbial communities, in contrast to the active chimney communities, which were dominated by chemolithoautotrophic and thermophilic lineages. This study represents one of the most comprehensive investigations of microbial diversity in submarine chimneys and elucidates how the dissipation of hydrothermal activity affects the structure of microbial consortia in these extreme ecological niches.

The sponge microbiome project.

Marine sponges (phylum Porifera) are a diverse, phylogenetically deep-branching clade known for forming intimate partnerships with complex communities of microorganisms. To date, 16S rRNA gene sequencing studies have largely utilised different extraction and amplification methodologies to target the microbial communities of a limited number of sponge species, severely limiting comparative analyses of sponge microbial diversity and structure. Here, we provide an extensive and standardised dataset that will facilitate sponge microbiome comparisons across large spatial, temporal, and environmental scales. Samples from marine sponges (n = 3569 specimens), seawater (n = 370), marine sediments (n = 65) and other environments (n = 29) were collected from different locations across the globe. This dataset incorporates at least 268 different sponge species, including several yet unidentified taxa. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA using standardised procedures. Raw sequences (total of 1.1 billion sequences) were processed and clustered with (i) a standard protocol using QIIME closed-reference picking resulting in 39 543 operational taxonomic units (OTU) at 97% sequence identity, (ii) a de novo clustering using Mothur resulting in 518 246 OTUs, and (iii) a new high-resolution Deblur protocol resulting in 83 908 unique bacterial sequences. Abundance tables, representative sequences, taxonomic classifications, and metadata are provided. This dataset represents a comprehensive resource of sponge-associated microbial communities based on 16S rRNA gene sequences that can be used to address overarching hypotheses regarding host-associated prokaryotes, including host specificity, convergent evolution, environmental drivers of microbiome structure, and the sponge-associated rare biosphere.

Ultrasensitive and high-throughput analysis of chlorophyll a in marine phytoplankton extracts using a fluorescence microplate reader.

Chlorophyll a (Chl a) is the predominant pigment in every single photosynthesizing organism including phytoplankton and one of the most commonly measured water quality parameters. Various methods are available for Chl a analysis, but the majority of them are of limited throughput and require considerable effort and time from the operator. The present study describes a high-throughput, microplate-based fluorometric assay for rapid quantification of Chl a in phytoplankton extracts. Microplate sealing combined with ice cooling was proved an effective means for diminishing solvent evaporation during sample loading and minimized the analytical errors involved in Chl a measurements with a fluorescence microplate reader. A set of operating parameters (settling time, detector gain, sample volume) were also optimized to further improve the intensity and reproducibility of Chl a fluorescence signal. A quadratic regression model provided the best fit (r 2 = 0.9998) across the entire calibration range (0.05-240 pg µL-1). The method offered excellent intra- and interday precision (% RSD 2.2 to 11.2%) and accuracy (% relative error -3.8 to 13.8%), while it presented particularly low limits of detection (0.044 pg µL-1) and quantification (0.132 pg µL-1). The present assay was successfully applied on marine phytoplankton extracts, and the overall results were consistent (average % relative error -14.8%) with Chl a concentrations (including divinyl Chl a) measured by high-performance liquid chromatography (HPLC). More importantly, the microplate-based method allowed the analysis of 96 samples/standards within a few minutes, instead of hours or days, when using a traditional cuvette-based fluorometer or an HPLC system. Graphical abstract TChl a concentrations (i.e. sum of Chl a and divinyl Chl a in ng L-1) measured in seawater samples by HPLC and fluorescence microplate reader.

Kolumbo submarine volcano (Greece): An active window into the Aegean subduction system.

Submarine volcanism represents ~80% of the volcanic activity on Earth and is an important source of mantle-derived gases. These gases are of basic importance for the comprehension of mantle characteristics in areas where subaerial volcanism is missing or strongly modified by the presence of crustal/atmospheric components. Though, the study of submarine volcanism remains a challenge due to their hazardousness and sea-depth. Here, we report (3)He/(4)He measurements in CO2-dominated gases discharged at 500 m below sea level from the high-temperature (~220 °C) hydrothermal system of the Kolumbo submarine volcano (Greece), located 7 km northeast off Santorini Island in the central part of the Hellenic Volcanic Arc (HVA). We highlight that the mantle below Kolumbo and Santorini has a (3)He/(4)He signature of at least 7.0 Ra (being Ra the (3)He/(4)He ratio of atmospheric He equal to 1.39×10(-6)), 3 Ra units higher than actually known for gases-rocks from Santorini. This ratio is also the highest measured across the HVA and is indicative of the direct degassing of a Mid-Ocean-Ridge-Basalts (MORB)-like mantle through lithospheric faults. We finally highlight that the degassing of high-temperature fluids with a MORB-like (3)He/(4)He ratio corroborates a vigorous outgassing of mantle-derived volatiles with potential hazard at the Kolumbo submarine volcano.

Metagenomic investigation of the geologically unique Hellenic Volcanic Arc reveals a distinctive ecosystem with unexpected physiology.

Hydrothermal vents represent a deep, hot, aphotic biosphere where chemosynthetic primary producers, fuelled by chemicals from Earth's subsurface, form the basis of life. In this study, we examined microbial mats from two distinct volcanic sites within the Hellenic Volcanic Arc (HVA). The HVA is geologically and ecologically unique, with reported emissions of CO2 -saturated fluids at temperatures up to 220°C and a notable absence of macrofauna. Metagenomic data reveals highly complex prokaryotic communities composed of chemolithoautotrophs, some methanotrophs, and to our surprise, heterotrophs capable of anaerobic degradation of aromatic hydrocarbons. Our data suggest that aromatic hydrocarbons may indeed be a significant source of carbon in these sites, and instigate additional research into the nature and origin of these compounds in the HVA. Novel physiology was assigned to several uncultured prokaryotic lineages; most notably, a SAR406 representative is attributed with a role in anaerobic hydrocarbon degradation. This dataset, the largest to date from submarine volcanic ecosystems, constitutes a significant resource of novel genes and pathways with potential biotechnological applications.

Biodiversity, Anti-Trypanosomal Activity Screening, and Metabolomic Profiling of Actinomycetes Isolated from Mediterranean Sponges.

Marine sponge-associated actinomycetes are considered as promising sources for the discovery of novel biologically active compounds. In the present study, a total of 64 actinomycetes were isolated from 12 different marine sponge species that had been collected offshore the islands of Milos and Crete, Greece, eastern Mediterranean. The isolates were affiliated to 23 genera representing 8 different suborders based on nearly full length 16S rRNA gene sequencing. Four putatively novel species belonging to genera Geodermatophilus, Microlunatus, Rhodococcus and Actinomycetospora were identified based on a 16S rRNA gene sequence similarity of < 98.5% to currently described strains. Eight actinomycete isolates showed bioactivities against Trypanosma brucei brucei TC221 with half maximal inhibitory concentration (IC50) values <20 µg/mL. Thirty four isolates from the Milos collection and 12 isolates from the Crete collection were subjected to metabolomic analysis using high resolution LC-MS and NMR for dereplication purposes. Two isolates belonging to the genera Streptomyces (SBT348) and Micromonospora (SBT687) were prioritized based on their distinct chemistry profiles as well as their anti-trypanosomal activities. These findings demonstrated the feasibility and efficacy of utilizing metabolomics tools to prioritize chemically unique strains from microorganism collections and further highlight sponges as rich source for novel and bioactive actinomycetes.

The ocean sampling day consortium.

Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world's oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits.

Metagenomics: tools and insights for analyzing next-generation sequencing data derived from biodiversity studies.

Advances in next-generation sequencing (NGS) have allowed significant breakthroughs in microbial ecology studies. This has led to the rapid expansion of research in the field and the establishment of "metagenomics", often defined as the analysis of DNA from microbial communities in environmental samples without prior need for culturing. Many metagenomics statistical/computational tools and databases have been developed in order to allow the exploitation of the huge influx of data. In this review article, we provide an overview of the sequencing technologies and how they are uniquely suited to various types of metagenomic studies. We focus on the currently available bioinformatics techniques, tools, and methodologies for performing each individual step of a typical metagenomic dataset analysis. We also provide future trends in the field with respect to tools and technologies currently under development. Moreover, we discuss data management, distribution, and integration tools that are capable of performing comparative metagenomic analyses of multiple datasets using well-established databases, as well as commonly used annotation standards.

Pyrosequencing analysis of microbial communities reveals dominant cosmopolitan phylotypes in deep-sea sediments of the eastern Mediterranean Sea.

The deep eastern basin of the Mediterranean Sea is considered to be one of the world's most oligotrophic areas in the world. Here we performed pyrosequenicng analysis of bacterial and archaeal communities in oxic nutrient-poor sediments collected from the eastern Mediterranean at 1025-4393 m depth. Microbial communities were surveyed by targeting the hypervariable V5-V6 regions of the 16S ribosomal RNA gene using bar-coded pyrosequencing. With a total of 13,194 operational taxonomic units (OTUs) or phylotypes at 97% sequence similarities, the phylogenetic affiliation of microbes was assigned to 23 bacterial and 2 archaeal known phyla, 23 candidate divisions at the phylum level and distributed into 186 families. It was further revealed that the microbial consortia inhabiting all sampling sites were highly diverse, but dominated by phylotypes closely related to members of the genus Pseudomonas and Marine Group I archaea. Such pronounced and widespread enrichment probably manifests the cosmopolitan character of these species and raises questions about their metabolic adaptation to the physical stressors and low nutrient availability of the deep eastern Mediterranean Sea.

Distribution of aliphatic hydrocarbons, polycyclic aromatic hydrocarbons and organochlorinated pollutants in deep-sea sediments of the Southern Cretan margin, Eastern Mediterranean Sea: a baseline assessment.

Deep sediments from the southern Cretan margin were analyzed to establish baseline levels for various types of organic pollutants before the anticipated intensification of anthropogenic activities. The total concentration of aliphatic hydrocarbons (ΣAH:326-3758ngg(-1), dry weight) was similar to those reported for deep sediments of the western Mediterranean Sea, while considerably lower levels were measured for polycyclic aromatic hydrocarbons (ΣPAH:9-60ngg(-1)). Source-diagnostic ratios suggested that the aliphatic hydrocarbons in sediments were mainly of terrestrial biogenic origin, while polycyclic aromatic hydrocarbons stemmed from the deposition of long-range transported combustion aerosols. Among the organochlorinated compounds analyzed, ß-hexachlorocyclohexane (ß-HCH:222-7052pgg(-1)), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT:37-2236pgg(-1)) and polychlorinated biphenyls (ΣPCB:38-1182pgg(-1)) showed the highest abundance in sediments. The presence of HCHs and PCBs was attributed to historical inputs that have undergone extensive weathering, whereas an ongoing fresh input was suggested for p,p'-DDT. Multiple linear regression analysis revealed that the levels of the various pollutants in sediments were controlled by different factors, but with organic carbon content playing a prominent role in most cases.

New insights into hydrothermal vent processes in the unique shallow-submarine arc-volcano, Kolumbo (Santorini), Greece.

We report on integrated geomorphological, mineralogical, geochemical and biological investigations of the hydrothermal vent field located on the floor of the density-stratified acidic (pH ~ 5) crater of the Kolumbo shallow-submarine arc-volcano, near Santorini. Kolumbo features rare geodynamic setting at convergent boundaries, where arc-volcanism and seafloor hydrothermal activity are occurring in thinned continental crust. Special focus is given to unique enrichments of polymetallic spires in Sb and Tl (±Hg, As, Au, Ag, Zn) indicating a new hybrid seafloor analogue of epithermal-to-volcanic-hosted-massive-sulphide deposits. Iron microbial-mat analyses reveal dominating ferrihydrite-type phases, and high-proportion of microbial sequences akin to "Nitrosopumilus maritimus", a mesophilic Thaumarchaeota strain capable of chemoautotrophic growth on hydrothermal ammonia and CO2. Our findings highlight that acidic shallow-submarine hydrothermal vents nourish marine ecosystems in which nitrifying Archaea are important and suggest ferrihydrite-type Fe(3+)-(hydrated)-oxyhydroxides in associated low-temperature iron mats are formed by anaerobic Fe(2+)-oxidation, dependent on microbially produced nitrate.

Carbon and chlorine isotope fractionation during microbial degradation of tetra- and trichloroethene.

Two-dimensional compound-specific isotope analysis (2D-CSIA), combining stable carbon and chlorine isotopes, holds potential for monitoring of natural attenuation of chlorinated ethenes (CEs) in contaminated soil and groundwater. However, interpretation of 2D-CSIA data sets is challenged by a shortage of experimental Cl isotope enrichment factors. Here, isotope enrichments factors for C and Cl (i.e., εC and εCl) were determined for biodegradation of tetrachloroethene (PCE) and trichloroethene (TCE) using microbial enrichment cultures from a heavily CE-contaminated aquifer. The obtained values were εC = -5.6 ± 0.7‰ (95% CI) and εCl = -2.0 ± 0.5‰ for PCE degradation and εC = -8.8 ± 0.2‰ and εCl = -3.5 ± 0.5‰ for TCE degradation. Combining the values for both εC and εCl yielded mechanism-diagnostic εCl/εC ratios of 0.35 ± 0.11 and 0.37 ± 0.11 for the degradation of PCE and TCE, respectively. Application of the obtained εC and εCl values to a previously investigated field site gave similar estimates for the fraction of degraded contaminant as in the previous study, but with a reduced uncertainty in assessment of the natural attenuation. Furthermore, 16S rRNA gene clone library analyses were performed on three samples from the PCE degradation experiments. A species closely related to Desulfitobacterium aromaticivorans UKTL dominated the reductive dechlorination process. This study contributes to the development of 2D-CSIA as a tool for evaluating remediation strategies of CEs at contaminated sites.