Shallow Marine Hydrothermal Vent Research Articles

Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7–11) and temperature (40–80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Trace Element Signatures in Pyrite and Marcasite From Shallow Marine Island Arc-Related Hydrothermal Vents, Calypso Vents, New Zealand, and Paleochori Bay, Greece

Fluid conditions of shallow marine hydrothermal vent sites (<200 mbsl) in island arcs resemble those of subaerial epithermal systems. This leads to a distinct mineralization-style compared to deeper arc/back-arc (>200 mbsl) and mid-ocean ridge-related environments (>2000 mbsl). At Calypso Vents in the Bay of Plenty and Paleochori Bay at the coast of Milos Island, fluids with temperatures <200°C are emitted through volcaniclastic sediments in water depths <200 mbsl. The hydrothermal mineralization from these fluids is dominated by pyrite and marcasite showing diverse textures, including colloform alternations, semi-massive occurrences surrounding detrital grains, vein-type pyrite, and disseminated fine-grained assemblages. Pyrite and marcasite from Calypso SE show elevated concentrations of volatile elements (e.g., As, Sb, Tl, Hg) implying a vapor-rich fluid phase. By contrast, elements like Zn, Ag, and Pb are enriched in hydrothermal pyrite and marcasite from Calypso SW, indicating a high-Cl liquid-dominated fluid discharge. Hence, vapor-liquid element fractionation induced by fluid boiling is preserved in the seafloor mineralization at Calypso Vents. Hydrothermal mineralization at very shallow vent sites (<10 mbsl), like Paleochori Bay, are affected by wave action causing a seasonal migration of the seawater-fluid interface in the sediment cover. The δ34S composition of native S crusts and crystalline S (0.7–6.7‰) is indicative for host rock leaching and thermochemical reduction of seawater sulphate. By contrast, the highly negative δ34S signature of native S globules in sediments (−7.6 to −9.1‰) is related to microbial sulphate reduction or a subordinate magmatic fluid influx. Alunite-jarosite alteration (Paleochori Bay) and a mineral assemblage consisting of orpiment, realgar, and native S (Calypso Vents) may also suggest a contribution by an oxidised (sulphate-rich) low pH fluid of potential magmatic origin. However, fluid boiling is pervasive at Calypso Vents and Paleochori Bay, and the condensation of vapor-rich fluids in a steam-heated environment may produce a similar alteration and mineralization assemblage without a significant magmatic fluid influx, as known from some subaerial epithermal systems.

Sulfidization of 3.48 billion-year-old stromatolites of the Dresser Formation, Pilbara Craton: Constraints from in-situ sulfur isotope analysis of pyrite

This study reports in–situ sulfur isotope analyses (32S, 33S, 34S and 36S) of pyrite in strongly sulfidized stromatolites from the ~3.48 billion–year–old Dresser Formation, Pilbara Craton, Australia. These data shed light on sulfur reservoirs and sulfide precipitation processes and provide clues for the contribution of sulfur–cycling microbes to sulfidization.Sulfur isotope signatures derived from mass dependent fractionation (MDF; monitored by δ34S) and mass independent fractionation (MIF; here Δ33S and Δ36S) of pyrite in stromatolites, and of microscopic pyrite within associated barite, allow for the identification of distinctive sulfur sources: i) magmatic–hydrothermal sulfide (H2S) with δ34S and Δ33S ~ 0%; ii) magmatic–hydrothermal sulfate (SO42−) with a MDF signature (MDF–SO42−; δ34S ~ 10‰ and Δ33S ~ 0‰; iii) photochemically–derived sulfate with a MIF signature (MIF–SO4; δ34S ~ −6‰ and Δ33S ~ −3.0‰); iv) photochemically–derived elemental sulfur (S0) with δ34S ≪ 0 and Δ33S ≫ 0‰.The sulfur isotope data suggest that sulfidization was largely driven by reduction of intermixed MDF–SO42− and MIF–SO42− (bulk signature of δ34S ~ 5‰ and Δ33S ~ −1.4‰), and dilution of produced H2S (δ34S ~ −12‰ and Δ33S ~ −1.4‰) by native H2S in magmatic–hydrothermal fluids. The δ34S shifts (up to ~17‰) generated by sulfate reduction are consistent with both thermochemical reactions and influence of sulfate–cycling microbes, the latter which may have facilitated rapid pyrite precipitation and preservation of microbial remains that are entombed within the petrogenetically earliest pyrite generation of stromatolites.Collectively, our data are consistent with ancient stromatolite growth in proximity to shallow marine hydrothermal vents, where hydrothermal fluids contributed to sulfidization that may have been further influenced by sulfur–cycling microbes.

Extremely thermoactive archaeal endoglucanase from a shallow marine hydrothermal vent from Vulcano Island.

Already-characterized microbial cellulases have proven to be highly useful for industrial processes, since they can withstand harsh industrial conditions with characteristics such as high thermo- and acid stability. These properties provide promising features for the process of plant biomass degradation and biofuel generation. Nevertheless, the number of known extremely thermoactive archaeal cellulases is low. Hence, the discovery of archaeal cellulases with different characteristics is crucial for the development of efficient and sustainable biorefinery. In this work, the metagenome of a high-temperature enrichment culture from marine environment of Vulcano Island was screened for the presence of novel endoglucanase-encoding genes of archaeal origin. The ORF vul_cel5A was detected, and the deduced protein was characterized as the most thermoactive endoglucanase described to date. Vul_Cel5A was identified as a thermoactive glycoside hydrolase family 5 endoglucanase, with the highest sequence identity (72-75%) to putative endoglucanases from archaeal genera. Vul_Cel5A showed the highest activity at notable 115°C towards barley β-glucan (210.7U/mg), and lichenan (209.9U/mg), and further towards carboxymethyl cellulose (38.6U/mg) and locust bean gum (83.0U/mg). The endoglucanase exhibited a half-life time of 46min at 100°C and did not show any loss of activity after incubation for 48h at 75°C. Furthermore, Vul_Cel5A showed high affinity to barley β-glucan with a Km of 0.52mg/mL and showed tolerance against various chemical reagents. Due to the outstanding high thermoactivity and thermostability and tolerance to acidic conditions, Vul_Cel5A represents a promising novel archaeal endo-β-glucanase for application in biorefineries for an efficient biomass pre-treatment.

Corrigendum: Changes in Eukaryotic and Bacterial Communities along a 120 m Transect Associated with a Shallow Marine Hydrothermal Vent

Corrigendum on:Rubelmann H, Karlen DJ, and Garey JR (2017) Changes in eukaryotic and bacterial communities along a 120m transect associated with a shallow marine hydrothermal vent. Frontiers in Marine Science, 4:117, doi: 10.3389/fmars.2017.00177.Author Contribution StatementThere is an error in the Author contribution statement. The first sentence (JG was the PI of the grant that supported this project.) should be deleted. The second sentence should read: JG participated in the planning, field work, analysis, and writing of the manuscript. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. Acknowledgments StatementThere is an error in the Acknowledgement statement. The final sentence of the Acknowledgement should read:This work was supported by the U.S. National Science Foundation under BE/CBC grant CHE-0221834 to T. Pichler and by internal grants from the University of South Florida.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.

Changes in Eukaryotic and Bacterial Communities along a 120 m Transect Associated with a Shallow Marine Hydrothermal Vent

The biocomplexity of sediment communities along a 120 m transect near an arsenic-rich, shallow marine hydrothermal vent at Tutum Bay, Papua New Guinea was thoroughly examined. A count of macro- and meiofaunal organisms was combined with bacterial and eukaryotic SSU rRNA gene surveys to assess biodiversity. Each site along the transect had distinct microbial communities. Near-vent sites were more similar to each other than sites further from the vent. Some species, such as Ignavibacterium, Caldilinea, and Capitella were only found near-vent. Biodiversity generally increased with distance from the vent. The community composition responded to the presence of hydrothermal fluids with a clear correlation between temperature and thermophilic organisms. Primary production appeared to be a mix of chemo- and phototrophy. Association analyses suggest many potential interactions between organisms occur at certain sites, and that species distributions and interactions occur in the context of complex spatial relationships related to the geochemistry of the hydrothermal vent fluids. While Tutum Bay is heavily influenced by arsenic, no specific correlation between bacteria that metabolize arsenic and the concentration of different oxidation states of arsenic ions was observed, perhaps because very little of the arsenic present was bioavailable. The observed homogeneous distribution of arsenic reducers along the transect could be due to background arsenic metabolism. This study represents a holistic study of biocomplexity on a broad phylogenetic range across a 120m transect associated with a marine shallow-water hydrothermal vent.

Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island.

To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80°C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100°C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, ε-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature.

The Kallisti Limnes, carbon dioxide-accumulating subsea pools.

Natural CO2 releases from shallow marine hydrothermal vents are assumed to mix into the water column, and not accumulate into stratified seafloor pools. We present newly discovered shallow subsea pools located within the Santorini volcanic caldera of the Southern Aegean Sea, Greece, that accumulate CO2 emissions from geologic reservoirs. This type of hydrothermal seafloor pool, containing highly concentrated CO2, provides direct evidence of shallow benthic CO2 accumulations originating from sub-seafloor releases. Samples taken from within these acidic pools are devoid of calcifying organisms, and channel structures among the pools indicate gravity driven flow, suggesting that seafloor release of CO2 at this site may preferentially impact benthic ecosystems. These naturally occurring seafloor pools may provide a diagnostic indicator of incipient volcanic activity and can serve as an analog for studying CO2 leakage and benthic accumulations from subsea carbon capture and storage sites.

Organic Cu-complexation at the shallow marine hydrothermal vent fields off the coast of Milos (Greece), Dominica (Lesser Antilles) and the Bay of Plenty (New Zealand)

Hydrothermal vents at the seafloor release large volumes of metal-rich fluids into the ocean. Some of these metals are biologically essential (such as Fe), while others may be toxic (e.g. Cu). Organisms living at these habitats produce small organic molecules, ligands that are able to form complexes with different metals, to either enhance their bioavailability or to decrease their toxicity. While some deep-sea vents have been studied with respect to metal complexation, comparable studies at shallow marine hydrothermal vent systems are rather rare. In this study, total dissolved Cu concentrations ([Cu]T) and corresponding Cu binding ligand concentrations ([L]Cu) at the shallow water hydrothermal vent fields off the coast of Milos (Greece), Dominica (Lesser Antilles) and the Bay of Plenty (New Zealand) were examined by a voltammetric ligand titration, using competitive ligand equilibration–adsorptive stripping voltammetry (CLE–AdCSV).Milos, Dominica and the Bay of Plenty are three very different environments and our data show that they are relatively depleted in total dissolved Cu, compared to deep-sea hydrothermal vents where [Cu]T and [L]Cu concentrations up to 400nM and 4000nM have been found, respectively. In this data set, [Cu]T rises up to 21.53nM, however, most samples are in the range of 0.5nM–3nM. [L]Cu varies between 2.07nM and 23.9nM, with one exception, reaching 56.8nM. Conditional stability constants (Log K) range between 11.57 and 14.01.Assuming that the three investigated sites are representative for shallow marine hydrothermal vents, the Cu-flux into the ocean due to complexation appears to be relatively low compared to deep-sea systems. Our data indicate a stable complexation of Cu in most cases, which may lower the toxic effect of Cu on these mixed photic–chemosynthetic communities. However one pore water sample from Dominica showed an excess [Cu]T over [L]Cu, which would create a highly toxic environment for most marine organisms. We further show that ligand–Cu ratios and complex stability constants are in a similar range as the ones at deep-sea vents, which may indicate similar complexation parameters and processes at shallow and deep-sea hydrothermal vents.

A Valanginian crustacean microcoprolite ichnofauna from the shallow-marine hydrothermal vent site of Zengővárkony (Mecsek Mts., Hungary)

A rich and diverse crustacean microcoprolite ichnofauna is reported from the Valanginian of the Mecsek Mts., Hungary. Localities from the Bolondut and Dezső Rezső valleys near Zengővarkony (Eastern Mecsek Mts.) provided Favreina aff. dispentochetarius, Palaxius darjaensis, P. triochetarius,P. salataensis, P. tetraochetarius, and P. decaochetarius. They occur in limonitic and partly metasomatized limestones, limestone olistoliths, and ammonite body-chambers. The remarkable richness of this ichnofauna plus the previous records from the same environment (altogether 11 ichnospecies of four ichnogenera from a single stratigraphic level) make it the most diverse crustacean ichnofauna of the Mesozoic. The former ecosystem of the ichnofauna is considered a deeper shallow-marine (water depth <300 m) hydrothermal vent on which the producers of the microcoprolites lived. The related brachiopod fauna shows a remarkable size and is dominated by Lacunosella. This fauna is not similar to typical fossil deep-sea vent faunas; however, it offers a thorough understanding of other fossil shallow-marine hydrothermal vent faunas and the role of crustaceans in these ecosystems. This is the first record of crustacean coprolites from such a fossil site and documents that crustaceans were diverse and played an important role in shallow-marine, non photosynthesis-based ecosystems in the geological past.

Enhanced geochemical gradients in a marine shallow-water hydrothermal system: Unusual arsenic speciation in horizontal and vertical pore water profiles

The shallow marine hydrothermal vents near Ambitle Island in eastern Papua New Guinea discharge hot, slightly acidic, As-rich, chemically reduced fluid into cool, slightly alkaline, oxygenated seawater. Gradients in temperature, pH, and total As (AsT), among others, are established as the two aqueous phases mix. The hydrothermal fluid contained ∼900 μg/L AsT, almost exclusively present as the reduced AsIII, while local seawater measured between 1.2 and 2.4 μg/L As, with approximately equal levels of AsIII and AsV. Of particular interest in this study was As speciation and abundance in pore waters as a function of sediment depth and as a function of distance from the area of focused venting. With increasing distance, AsT concentration in the pore water decreased rapidly, but remained elevated up to 300 m from the area of focused venting when compared to a non-hydrothermal control site. As a function of depth (to ∼100 cm) AsT concentration in the pore water profiles was elevated and generally increased with depth. Surprisingly, aqueous AsV far exceeded aqueous AsIII at almost all distances and depths investigated, while at the control site the AsIII concentration exceeded that of AsV. In the Tutum Bay hydrothermal system, chemical disequilibria among As species provide potential metabolic energy for arsenite oxidizing microorganisms where hydrothermal fluid mixes with seawater near the vent orifice, and for arsenate reducing microorganisms with increasing distance and depth from the hydrothermal point source.

Metabolism of organic compounds in anaerobic, hydrothermal sulphate-reducing marine sediments.

Previous studies of hot (>80 degrees C) microbial ecosystems have primarily relied on the study of pure cultures or analysis of 16S rDNA sequences. In order to gain more information on anaerobic metabolism by natural communities in hot environments, sediments were collected from a shallow marine hydrothermal vent system in Baia di Levante, Vulcano, Italy and incubated under strict anaerobic conditions at 90 degrees C. Sulphate reduction was the predominant terminal electron-accepting process in the sediments. The addition of molybdate inhibited sulphate reduction in the sediments and resulted in a linear accumulation of acetate and hydrogen over time. [U-14C]- acetate was completely oxidized to 14CO2, and the addition of molybdate inhibited 14CO2 production by 60%. [U-14C]-glucose was oxidized to 14CO2, and this was inhibited when molybdate was added. When the pool sizes of short-chain fatty acids were artificially increased, radiolabel from [U-14C]-glucose accumulated in the acetate pool. L-[U-14C]-glutamate, [ring-14C]-benzoate and [U-14C]-palmitate were also anaerobically oxidized to 14CO2 in the sediments, but molybdate had little effect on the oxidation of these compounds. These results demonstrate that natural microbial communities living in a hot, microbial ecosystem can oxidize acetate and a range of other organic electron donors under sulphate-reducing conditions and suggest that acetate is an important extracellular intermediate in the anaerobic degradation of organic matter in hot microbial ecosystems.

Thermaerobacter nagasakiensis sp. nov., a novel aerobic and extremely thermophilic marine bacterium.

A novel, extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent at depth of 22 m in Tachibana Bay, Nagasaki Prefecture, Japan. Cells were gram-negative, non-spore-forming, motile rods. Growth was observed between 52 and 78 degrees C (optimum 70 degrees C), pH 5 and 8 (optimum pH 7) and 0-4.5% NaCl (optimum 1.0%). The isolate was a strictly aerobic heterotroph utilizing yeast extract and trypticase peptone. The G+C content of the genomic DNA is 69 mol%. Analysis of 16S rDNA sequences indicated that strain Ts1a is closely related to Thermaerobacter marianensis. The differences in physiology and DNA-DNA similarity between strain Ts1a and T. marianensis showed that strain Ts1a represents a new species of Thermaerobacter. The type strain of T. nagasakiensis is strain Ts1a (=JCM11223, DSM 14512).

Three novel halotolerant and thermophilic Geobacillus strains from shallow marine vents

During a polyphasic taxonomic analysis performed on isolates from shallow marine hydrothermal vents of Eolian Islands (Italy), three thermophilic, halotolerant bacilli, designated as strain 1bw, strain 5-2 and strain 10-1, could not be affiliated to any described species. Physiological and biochemical characteristics, membrane lipids composition, mol % G+C content, and phylogenetic relationships determined on the basis of the 16S rRNA gene sequence analysis, placed these strains within the genus Geobacillus. The three strains were only moderately related to species of Geobacillus and their relatives, members of Saccharococcus. Determination of the relatedness among each other at a higher taxonomic level by DNA-DNA reassociation experiments demonstrated the three isolates to represent three different novel Geobacillus genomospecies. The taxonomic novelty of these three marine strains was substantiated by their physiological properties and by fatty acid patterns that did not match closely those of any Geobacillus type strain. These three novel strains could be of interest to biotechnology because of their ability to produce exopolysaccharides and to adhere on polystirene, characteristics undescribed so far for other Geobacillus species. They are also able to utilise hydrocarbons such as gas oil, kerosene and mineral lubricating oil. Strain 5-2 is tolerant to zinc.

Bacillus vulcani sp. nov., a novel thermophilic species isolated from a shallow marine hydrothermal vent.

A thermophilic spore-forming bacterium was isolated from sediment of a shallow hydrothermal vent at Vulcano Island (Italy). After phenotypic and molecular analyses, it was identified as a novel Bacillus species, for which the name Bacillus vulcani is proposed. The type strain is strain 3s-1T (= DSM 13174T = CIP 106305T).

A molecular view of archaeal diversity in marine and terrestrial hot water environments

Molecular phylogenetic survey of naturally occurring archaeal diversities in hot water environments was carried out by using the PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. Mixed population DNA was directly extracted from the effluent hot water or sediment of a shallow marine hydrothermal vent at Tachibana Bay, or the acidic hot water of hot spring pools at Mt. Unzen, in Nagasaki Prefecture, Japan. Based on the partial rDNA sequences amplified with an Archaea-specific primer set, the archaeal populations of hot water environments consisted of phylogenetically and physiologically diverse group of microorganisms. The archaeal populations were varied in each sample and subject to its environmental conditions. In addition, the large number of archaeal rDNA sequences recovered from hot water environments revealed the distant relationship not only to the rDNA sequences of the cultivated thermophilic archaea, but also to the sequences of unidentified archaeal rDNA clones found in other hot water environments. The findings extend our view of archaeal diversity in hot water environments and phylogenetic organization of these organisms.

A molecular view of archaeal diversity in marine and terrestrial hot water environments

Molecular phylogenetic survey of naturally occurring archaeal diversities in hot water environments was carried out by using the PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. Mixed population DNA was directly extracted from the effluent hot water or sediment of a shallow marine hydrothermal vent at Tachibana Bay, or the acidic hot water of hot spring pools at Mt. Unzen, in Nagasaki Prefecture, Japan. Based on the partial rDNA sequences amplified with an Archaea-specific primer set, the archaeal populations of hot water environments consisted of phylogenetically and physiologically diverse group of microorganisms. The archaeal populations were varied in each sample and subject to its environmental conditions. In addition, the large number of archaeal rDNA sequences recovered from hot water environments revealed the distant relationship not only to the rDNA sequences of the cultivated thermophilic archaea, but also to the sequences of unidentified archaeal rDNA clones found in other hot water environments. The findings extend our view of archaeal diversity in hot water environments and phylogenetic organization of these organisms.

Extremely thermostable phosphoenolpyruvate carboxylase from an extreme thermophile, Rhodothermus obamensis.

Phosphoenolpyruvate carboxylase (PEPC) was purified from an extremely thermophilic bacterium, Rhodothermus obamensis, growing optimally at 80 degrees C, which had recently been isolated from a shallow marine hydrothermal vent in Japan. The native enzyme was a homotetramer of 400 kDa in molecular mass, as estimated by gel filtration chromatography, and the subunit exhibited an apparent molecular mass of 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum temperature for enzyme activity was 75 degrees C. The enzyme exhibited an absolute requirement for divalent cations and a pH optimum of 8.0. The enzyme was extremely thermostable and there was no loss of enzyme activity on incubation for 2 h at 85 degrees C. The enzyme exhibited a positive allosteric property with acetyl-CoA and fructose 1,6-bisphosphate, and a negative one with L-aspartate and L-malate. These effectors affected not only the thermophilicity but also the thermostability of the enzyme, and the substrate, co-factors, and salts increased the thermostability as well. The extrinsic thermostabilization might be a possible mechanism for adaptation of the enzyme to high temperature.