Microbial community structure and diversity in deep-sea hydrothermal vent sediments along the Eastern Lau Spreading Centre

The aim of this study is to investigate microbial structures and diversities in five active hydrothermal fields＇ sediments along the Eastern Lau Spreading Centre （ELSC） in the Lau Basin （southwest Pacific）. Microbial communities were surveyed by denatured gradient gel electrophoresis （DGGE） and clone library analysis of 16S rRNA genes. The differences in microbial community structures among sediment samples from the five deep-sea hydrothermal sites were revealed by DGGE profiles. Cluster analysis of DGGE profiles sepa- rated the five hydrothermal samples into two groups. Four different 16S rRNA gene clone libraries, repre- senting two selected hydrothermal samples （19-4TVG8 and 19-4TVG11）, were constructed. Twenty-three and 32 phylotypes were identified from 166 and 160 bacterial clones respectively, including Proteobacteria, Bacteroidetes, Firmicutes, Nitrospirae and Planctomycetes. The phylum Proteobacteria is dominant in both bacterial libraries with a predominance of Gamma-Proteobacteria. A total of 31 and 25 phylotypes were obtained from 160 and 130 archaeal clones respectively, including Miscellaneous Crenarchaeotic Group, Marine Group Ⅰ and Ⅲ, Marine Benthic Group E, Terrestrial Hot Spring Crenarchaeota and Deep-sea Hy- drothermal Vent Euryarchaeota. These results show a variety of clones related to those involved in sulfur cycling, suggesting that the cycling and utilization of sulfur compounds may extensively occur in the Lau Basin deep-sea hydrothermal ecosystem.

On Optimal Frequencies of Acoustic in-situ Detector for Seafloor Hydrothermal Vents

The approach to determine working frequencies of acoustic in-situ detector for seafloor hydrothermal fluid is presented. Based on the research of deep-sea noise and the sound generated by mid-ocean ridge black smoker hydrothermal vents, and on the hydrothermal-vent animal hearing ranges, coupled with influences of suspended particles of hydrothermal on acoustic attenuation under different frequencies, the optimal frequency range for detection of acoustical signal near black smokers is determined. The optimal frequencies providing the maximum ratio of receiver signal to background noise are obtained. We have developed a laboratory experimental setup for the optimal frequencies selection. In particular, we evaluated time-of-flight performance with respect to the source signal parameters of center frequency and bandwidth. The experimental results confirm the effectiveness of our approach. Current results indicate that individual transducers operated in the range of 18 - 25 kHz are immune to most interfering sounds and suitable for our system.

Molecular diversity of Thermococcales isolated from Guaymas Basin hydrothermal vents

Members of Thermococcales are organoheterotrophic hyperthermophilic anaerobes consisting of three genera, Thermococcus, Pyrococcus, and Palaeococcus, among which Thermococcus are the most frequently isolated. In this study, a variety of Thermococcales strains were isolated from the Guaymas Basin hydrother- mal vents under different temperatures and pressures. Based on their 16S rRNA gene sequences, all the s- trains isolated from 85℃ or 95℃ enrichment cultures at ambient pressure were classified as Thermococcus, while strains isolated from 108℃-30 MPa enrichment cultures belonged to Pyrococcus. The combination of high pressure and high temperature for enrichment was shown to be an efficient method for isolating Pyro- coccus strains. Through selecting and analyzing with multiple molecular phylogenetic markers, the isolated Thermococcales strains were found quite diversified including several putative novel species. This demon- strates a vast genetic reservoir of Thermoccoccales in the deep-sea hydrothermal vents, and a fast evolving of the Thermococcales species adapting to the fast changing environment.

Microbial community structure and nitrogenase gene diversity of sediment from a deep-sea hydrothermal vent field on the Southwest Indian Ridge

A sediment sample was collected from a deep-sea hydrothermal vent field located at a depth of 2 951 m on the Southwest Indian Ridge. Phylogenetic analyses were performed on the prokaryotic community using polymerase chain reaction（PCR） amplification of the 16 S rRNA and nifH genes. Within the Archaea, the dominant clones were from marine benthic group E（MBGE） and marine group I（MGI） belonging to the phyla Euryarchaeota and Thaumarchaeota, respectively. More than half of the bacterial clones belonged to the Proteobacteria, and most fell within the Gammaproteobacteria. No epsilonproteobacterial sequence was observed. Additional phyla were detected including the Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Nitrospirae, Chloroflexi, Chlorobi, Chlamydiae, Verrucomicrobia, and candidate divisions OD1, OP11, WS3 and TM6, confirming their existence in hydrothermal vent environments. The detection of nifH gene suggests that biological nitrogen fixation may occur in the hydrothermal vent field of the Southwest Indian Ridge. Phylogenetic analysis indicated that only Clusters I and III NifH were present. This is consistent with the phylogenetic analysis of the microbial 16 S rRNA genes, indicating that Bacteria play the main role in nitrogen fixation in this hydrothermal vent environment.

Study on the High Precision Acoustic Measurement Techniques for Determining Temperature Field Around Seafloor Hydrothermal Vent

This paper presents the basis of acoustic method used for temperature field measurement of seafloor hydrothermal vent and two techniques of the parabolic interpolation and the bending compensation of propagation paths of acoustic signal are introduced. Experimental research is performed to exactly rebuild the temperature field around hot springs on the floor of Qiezishan Lake, Yunnan, China. The accuracy of the travel time estimation has been improved based on the aforementioned technique and method. At the same time, by comparison of the results of temperature field with different means, the max absolute error, the maximum relative error and the root mean square error are given. It shows that the technique and the method presented in the paper can be applied to the temperature field measurement detector around the seafloor hydrothermal vent. It also has a good accuracy.

Behaviors of ^(210)Pb and ^(210)Po around hydrothermal vents in the Okinawa Trough

Vertical profiles for the uranium-series radioisotopes 210Pb and 210Po were obtained at thetwo hydrothermal vent sites, the Iheya Ridge and the Minami-Ensei Knoll, in the Mid-Okinawa Trough in 1993 and 1994, respectively. In 1995, both radioisotopes were measured at the Minami-Ensei Knoll a-gain. At the Iheya Ridge, where the hydrothermal activity is not active as reflected by the CH_4 and 222Rn data, both the total 210Pb and 210Po activities show deficiency relative to their parents, and the mean residence time of 210Pb and 210Po is approximately equal to 20 and 2-5 a, respectively. At the Minami-Ensei Knoll, which is characterized by black smokers, the total 210Pb(0.167 × 10^(-3)-2.5 × 10^(-3) Bq/kg) around the plumes is deficient relative to 226Ra but the total 210Po activities (1.83 × 10^(-3) - 2.83 × 10^(-3) Bq/kg) are in excess relative to 210Pb. The 210Po activities are higher than those in the East China Sea and the Okinawa Trough and excess 210Po has been found. The 210Pb/226Ra and 210Po/10Pb activity ratios are 0.1-0.4 and 1.1-7.8, respectively. 210Pb is preferentially scavenged in the hydrothermal plumes. The possible mechanisms governing the concentration of 210Pb in the Minami-Ensei Knoll involve a lateral transport of 210Pb via diffusion. The high 210Po/210Pb activity ratios in the hydrothermal plumes suggest a depletion of 210Pb and addition of 210Po in the hydrothermal vent area.

Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata

DNA polymerase δ(Polδ)plays a crucial and versatile role in DNA replication and DNA repair processes.Vent shrimp Rimicaris exoculata is the primary megafaunal community living in hydrothermal vents.In this study,the Polδfrom shrimp Rimicaris exoculata was cloned,expressed and characterized.The results showed that the Polδcatalytic subunit(POLD1),852 amino acids in length,shared high homology with crayfish Procambarus clarkii and shrimp Oratosquilla oratoria.The recombinant POLD1 expressed in Escherichia coli showed that the enzyme was active in a range of 20℃ to 40℃ with an optimum temperature at 25℃ and in a wide range of p H with an optimum at pH 6.0.The activities of POLD1 were significantly enhanced in the presence of Triton-X 100,Tween 20 and Mn^(2+).The K_(m)(dNTP)value of POLD1 was 4.7μmol/L.The present study would be helpful to reveal the characterization of Polδof deep-sea vent animals.

Microorganisms from deep-sea hydrothermal vents

With a rich variety of chemical energy sources and steep physical and chemical gradients,hydrothermal vent systems offer a range of habitats to support microbial life.Cultivation-dependent and independent studies have led to an emerging view that diverse microorganisms in deep-sea hydrothermal vents live their chemolithoautotrophic,heterotrophic,or mixotrophic life with versatile metabolic strategies.Biogeochemical processes are mediated by microorganisms,and notably,processes involving or coupling the carbon,sulfur,hydrogen,nitrogen,and metal cycles in these unique ecosystems.Here,we review the taxonomic and physiological diversity of microbial prokaryotic life from cosmopolitan to endemic taxa and emphasize their significant roles in the biogeochemical processes in deep-sea hydrothermal vents.According to the physiology of the targeted taxa and their needs inferred from meta-omics data,the media for selective cultivation can be designed with a wide range of physicochemical conditions such as temperature,pH,hydrostatic pressure,electron donors and acceptors,carbon sources,nitrogen sources,and growth factors.The application of novel cultivation techniques with real-time monitoring of microbial diversity and metabolic substrates and products are also recommended.

Arsenic and sulfur nanoparticle synthesis mimicking environmental conditions of submarine shallow-water hydrothermal vents

Arsenic and sulfur mineralization is a natural phenomenon occurring in hydrothermal systems where parameters like temperature and organic matter(OM)can influence the mobilization of the toxic metalloid in marine environments.In the present study we analyze the influence of temperature and OM(particularly sulfur-containing additives)on As and S precipitation based on the recent discovery of As-rich nanoparticles in the hydrothermal system near the coast of the Greek island Milos.To this end,we experimentally recreate the formation of amorphous colloidal particles rich in As and S via acidification(pH 3–4)of aqueous precursors at various temperatures.At higher temperatures,we observe the formation of monodisperse particles within the first 24 h of the experiment,generating colloidal particles with diameters close to 160 nm.The S:As ratio and particle size of the synthetized particles closely correlates with values for As_(x)S_(y)particles detected in the hydrothermal system off Milos.Furthermore,organic sulfur containing additives(cysteine and glutathione,GSH)are a key factor in the process of nucleation and growth of amorphous colloidal As_(x)S_(y)particles and,together with the temperature gradient present in shallow hydrothermal vents,dictate the stabilization of As-bearing nanomaterials in the environment.Based on these findings,we present a simple model that summarizes our new insights into the formation and mobility of colloidal As in aquatic ecosystems.In this context,amorphous As_(x)S_(y)particles can present harmful effects to micro-and macro-biota not foreseen in bulk As material.

Metagenomic analysis reveals wide distribution of phototrophic bacteria in hydrothermal vents on the ultraslow-spreading Southwest Indian Ridge

Deep-sea hydrothermal vents are known as chemosynthetic ecosystems.However,high temperature vents emit light that hypothetically can drive photosynthesis in this habitat.Metagenomic studies have sporadically reported the occurrence of phototrophic populations such as cyanobacteria in hydrothermal vents.To determine how geographically and taxonomically widespread phototrophs are in deep-sea hydrothermal vents,we collected samples from three niches in a hydrothermal vent on the Southwest Indian Ridge and carried out an integrated metagenomic analysis.We determined the typical community structures of microorganisms found in active venting fields and identified populations of known potential chlorophototrophs and retinalophototrophs.Complete chlorophyll biosynthetic pathways were identified in all samples.By contrast,proteorhodopsins were only found in active beehive smoker diffusers.Taxonomic groups possessing potential phototrophy dependent on semiconductors present in hydrothermal vents were also found in these samples.This systematic comparative metagenomic study reveals the widespread distribution of phototrophic bacteria in hydrothermal vent fields.Our results support the hypothesis that the ocean is a seed bank of diverse microorganisms.Geothermal vent light may provide energy and confer a competitive advantage on phototrophs to proliferate in hydrothermal vent ecosystems.

Investigation into extremely acidic hydrothermal fluids off Kueishan Tao, Taiwan, China

Kueishan Tao (24°51′N, 121°55′E) is located at a tectonic junction of the fault system extension of Taiwan and the southern rifting end of the Okinawa Trough. A cluster of over 30 vents, at a water depth of about 10-20 m off the eastern tip of the tao emits hy-drothermal fluids and volcanic gases such as H2S. A sulfur chimney or mound, formed by condensation of the sulfur contained in the hydrothermal fluid, can usually be seen around the vents. The tallest chimney reaches 6 m. Vents discharging a yellowish fluid have temperatures between 92 and 116 ℃ and flow rates as high as 158 t/h; vents discharging a whitish fluid have lower temperatures of between 48 and 62 ℃ and lower flow rates of about 7.0 t/h. These world-record, breaking low pH (as low as 1.52) fluids are totally different from those found in the black and white-chimneys of the mid-ocean ridges. Magnesium and SiO2 data indicate that these hydrothermal fluids probably originate from a depth of 915-1 350 m below the surface.While the ratios of major ions relative to the sodium of these hydrothermal fluids are quite similar to open ocean water, the ratios of SO4 and chloride to sodium seem to be higher for some of the vents. It is suggested that the volcanic gases contribute SO4 and chlorine to the fluids, hence increasing their ratios relative to sodium. Some hydrothermal fluids, however, are found to be depleted of the major elements which can have been caused by phase separation. The concentrations of iron and manganese in the fluids are much lower than those found in the mid-ocean ridges, while the aluminium content is higher. Four species of benthos (Xenograpsus testudi-natus, a snail, a sea anemone, and a Sipuncala), 1 species of algae (Corallinaceae), and 1 species of fish (Siganus fusescens) were recorded near the hydrothermal vents. A mitochondria DNA sequence comparison of Xenograpsus testudinatus with 6 other decapod species shows the greatest number of nitrogen base differences in the DNA fragments from 225 to 270. The gene diversity in this DNA region possibly results from the unusual habitat. A sediment core was collected at a pond near the eastern side of the tao. No volcanic ash could be detected in the core sample spanning 3 620 a.

Key genes expression of reductive tricarboxylic acid cycle from deep-sea hydrothermal chemolithoautotrophic Caminibacter profundus in response to salinity, pH and O_2

CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid （rTCA） cycle. However, genes fragments of cbbL and cbbMencoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase （ACL）, pyruvate： ferredoxin oxidoreductase （POR） and 2-oxoglutarate： ferredoxin oxidoreductase （OOR） were also present in C. profun- dus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% （optimum 3.0%）, pH 5.0-6.5（optimum pH 6.5）, anaerobic to microaerobic conditions （optimum 1.0% 02）. Gene expression pat- terns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profun- dus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.

A Lagrangian Particle Random Walk Model for Simulating A Deep-Sea Hydrothermal Plume with both Buoyant and Non-Buoyant Features

This paper presents a computational model of simulating a deep-sea hydrothermal plume based on a Lagrangian particle random walk algorithm. This model achieves the efficient process to calculate a numerical plume developed in a fluid-advected environment with the characteristics such as significant filament intermittency and significant plume meander due to flow variation with both time and location. Especially, this model addresses both non-buoyant and buoyant features of a deep-sea hydrothermal plume in three dimensions, which significantly challenge a strategy for tracing the deep-sea hydrothermal plume and localizing its source. This paper also systematically discusses stochastic initial and boundary conditions that are critical to generate a proper numerical plume. The developed model is a powerful tool to evaluate and optimize strategies for the tracking of a deep-sea hydrothermal plume via an autonomous underwater vehicle （AUV）.

DESIGN OF NOVEL HIGH PRESSURE-RESISTANT HYDROTHERMAL FLUID SAMPLE VALVE

Sampling study is an effective exploration method, but the most extreme environments of hydrothermal vents pose considerable engineering challenges for sampling hydrothermal fluids. Moreover, traditional sampler systems with sample valves have difficulty in maintaining samples in situ pressure. However, decompression changes have effect on microorganisms sensitive to such stresses. To address the technical difficulty of collecting samples from hydrothermal vents, a new bidirectional high pressure-resistant sample valve with balanced poppet was designed. The sample valve utilizes a soft high performance plastic ＂PEEK＂ as poppet. The poppet with inapposite dimension is prone to occur to plastic deformation or rupture for high working pressure in experiments. To address this issue, based on the fmite element model, simulated results on stress distribution of the poppet with different structure parameters and preload spring force were obtained. The static axial deformations on top of the poppet were experimented. The simulated results agree with the experimental results. The new sample valve seals well and it can withstand high working pressure.

Distribution of large-scale detachment faults on mid-ocean ridges in relation to spreading rates

Large-scale detachment faults on mid-ocean ridges （MORs） provide a window into the deeper earth. They have megamullion on their corrugated surfaces, with exposed lower crustal and upper mantle rocks, rela- tively high residual Bouguer gravity anomaly and P-wave velocity, and are commonly associated with ocean- ic core complex. According to 30 detachment faults identified on MORs, we found that their distances to the axis mostly range from 5 to 50 km, half-spreading rates range from 6.8 to 17 mm/a, and activity time ranges from recent to 3 Ma. Most of the detachment faults are developed on the slow spreading Mid-Atlantic Ridge （MAR） and ultra-slow spreading Southwest Indian Ridge （SWIRl, with the dominant half-spreading rates of 7-13 mm/a, especially 10-13 mm/a. Furthermore, they mostly occur at the inside corner of one segment end and result in an asymmetric seafloor spreading. The detachment faults on MORs are mainly controlled by the tectonism and influenced by the magmatism. Long-lived detachment faults tend to be formed where the ridge magma supply is at a moderate level, although the tectonism is a first-order controlling factor. At the slow spreading ridges, detachment faults tend to occur where local magma supply is relatively low, whilst at the ultra-slow spreading ridges, they normally occur where local magma supply is relatively high. These faults are accompanied by hydrothermal activities, with their relationships being useful in the study of hydrothermal polymetallic sulfides and their origin.

Mitochondrial phylogenomics reveal the origin and adaptive evolution of the deep-sea caridean shrimps(Decapoda:Caridea)

The deep-sea is considered as the most extensive ecosystem on the Earth.It is meaningful for elucidating the life origins by exploring the origin and adaptive genetic mechanisms of the large deepsea organisms.Caridean shrimps have colonized and successfully adapted to deep-sea environments.They provide an ideal model to analyze the origin and adaptive evolution of modern deep-sea fauna.Here,we conducted the phylogenetic analyses of mitocho ndrial genomes(mitogenomes)from carideans,including 11 newly sequences reported in this investigation to explore the habitat origins,divergence times,and adaptive evolution of deep-sea(seamounts and hydrothermal vents)caridean shrimps.The results showed that the species of deep-sea Caridea formed a monophyletic group.Phylogenetic analysis supported that the deepsea caridean shrimps may originated from shallow sea.The hydrothermal vents alvinocaridid shrimps and Lebbeus shinkaiae from Thoridae underwent a second range expansion from seamounts to vent ecosystems.Estimates of divergence time showed that the caridean shrimps invaded into deep-sea at 147.75 Ma.The divergence of most of the modern seamount and hydrothermal vent species are in the late Cretaceous/early Tertiary.This may associate with the geological events of the Western Pacific,the climate change,and the global deep-water anoxic/dysoxic events during this period.Twenty-two potentially important adaptive residues were detected in the deep-sea shrimp lineage,which were located in atp6,atp8,cox1,cox3,cytb,nad2,nad4 l,and nad5.This investigation adds our understanding of the evolutionary history of deep-sea caridean shrimps,and provides insights into the mitochondrial genetic basis of deep-sea adaptation in this group.

Phylogenetic diversity and bioactivity of culturable deepsea-derived fungi from Okinawa Trough

Deep-sea sediments are now recognized as a home for rich and largely microbial community.Recently,it has been believed in an increasing number of studies that bacteria could be abundant in deepsea sediments of many types;however,fungi in deep-sea sediments remain relatively unknown.The phylogenetic diversity and bioactivity of culturable deep-sea-derived fungi from Okinawa Trough sediments were investigated in traditional method combined with fungal identification of molecular biology in this study.A total of 76 isolates belonged to 15 fungal taxa were recovered in a harsh condition of low nutrient and low temperature,indicating that the fungal communities in deep-sea sediments from Okinawa Trough were relatively abundant and diversified.Aspergillus,Cladosporium,and Penicillium were the dominant fungal genera,while Mycosphaerella,Purpureocillium,and Schizophyllum were relatively rare in the deep-sea sediments from Okinawa Trough.Among the six genera recovered,Mycosphaerella was firstly recovered from deep-sea sediments in this study.Moreover,about 75%of the extracts from the 15 fungal representative isolates displayed distinct bioactivity against at least one indicator bacterium or marine macrofouler,emphasizing the potentials of these deep-sea-derived fungi from Okinawa Trough as producers of bioactive metabolites.Notably,isolates Cladosporium oxysporum SCSIO z001 and Penicillium citrinum SCSIO z049 displayed a wide spectrum of bioactivities,isolates Cladosporium cladosporioides SCSIO z015,Cladosporium sphaerospermum SCSIO z030,and Penicillium verruculosum SCSIO z007 exhibited a strong anti-bacterial-growth activity,and isolate Penicillium chrysogenum SCSIO z062 displayed a strong anti-larval-settlement activity.These results suggest that these isolates deserved further study as potential sources of novel bioactive metabolites.

Eyes of diff ering colors in Alvinocaris longirostris from deep-sea chemosynthetic ecosystems: genetic and molecular evidence of its formation mechanism

Coloration is an important phenotypic trait for multiple adaptive functions.It is interesting to fi nd white-eye(AW)and orange-eye(AO)phenotypes in the shrimp Alvinocaris longirostris inhabiting the deep-sea cold seep and hydrothermal vent areas of the northwestern Pacifi c.By comparative transcriptome analyses,1491 diff erentially expressed genes(DEGs)were identified between AW and AO.Among them,many DEGs were associated with immunity,antioxidation,and detoxifi cation.Two signifi cant enzyme encoding genes,xanthine dehydrogenase,and tryptophan oxidase involved in pigment biosynthesis pathways were up-regulated in AW and AO,respectively,which might be related to the diff erences of white and orange eye phenotypes.Moreover,single nucleotide polymorphism(SNP)calling detected that genotypes of 28 SNP distributing in 14 unigenes were completely diff erent between AW and AO.Particularly,there were three and two non-synonymous mutations in immune genes crustin Pm5 and antimicrobial peptide,respectively.Results indicate that the diff erence in eye color is probably resulted from immune response to variable micro-environmental stressors encountered in the dispersal process of the shrimps,such as symbiotic microbes,pathogens,and toxic substances,and might be genetically fi xed at last.The suggested pathway preliminarily explained the formation mechanism of diff erent eye phenotypes in Alvinocaridid shrimps,providing a basis for further study on adaptive evolution of eyes in deep-sea chemosynthetic faunas.