Microbial Diversity in Ultra-High-Pressure Rocks and Fluids from the Chinese Continental Scientific Drilling Project in China

Microbial communities in ultra-high-pressure (UHP) rocks and drilling fluids from the Chinese Continental Scientific Drilling Project were characterized. The rocks had a porosity of 1 to 3.5% and a permeability of ~0.5 mDarcy. Abundant fluid and gas inclusions were present in the minerals. The rocks contained significant amounts of Fe₂O₃, FeO, P₂O₅, and nitrate (3 to 16 ppm). Acridine orange direct counting and phospholipid fatty acid analysis indicated that the total counts in the rocks and the fluids were 5.2 × 10³ to 2.4 × 10⁴ cells/g and 3.5 × 10⁸ to 4.2 × 10⁹ cells/g, respectively. Enrichment assays resulted in successful growth of thermophilic and alkaliphilic bacteria from the fluids, and some of these bacteria reduced Fe(III) to magnetite. 16S rRNA gene analyses indicated that the rocks were dominated by sequences similar to sequences of Proteobacteria and that most organisms were related to nitrate reducers from a saline, alkaline, cold habitat; however, some phylotypes were either members of a novel lineage or closely related to uncultured clones. The bacterial communities in the fluids were more diverse and included Proteobacteria, Bacteroidetes, gram-positive bacteria, Planctomycetes, and Candidatus taxa. The archaeal diversity was lower, and most sequences were not related to any known cultivated species. Some archaeal sequences were 90 to 95% similar to sequences recovered from ocean sediments or other subsurface environments. Some archaeal sequences from the drilling fluids were >93% similar to sequences of Sulfolobus solfataricus, and the thermophilic nature was consistent with the in situ temperature. We inferred that the microbes in the UHP rocks reside in fluid and gas inclusions, whereas those in the drilling fluids may be derived from subsurface fluids.     

Microbial diversity in ultra-high-pressure rocks and fluids from the Chinese Continental Scientific Drilling Project in China

Microbial communities in ultra-high-pressure (UHP) rocks and drilling fluids from the Chinese Continental Scientific Drilling Project were characterized. The rocks had a porosity of 1 to 3.5% and a permeability of approximately 0.5 mDarcy. Abundant fluid and gas inclusions were present in the minerals. The rocks contained significant amounts of Fe2O3, FeO, P2O5, and nitrate (3 to 16 ppm). Acridine orange direct counting and phospholipid fatty acid analysis indicated that the total counts in the rocks and the fluids were 5.2 x 10(3) to 2.4 x 10(4) cells/g and 3.5 x 10(8) to 4.2 x 10(9) cells/g, respectively. Enrichment assays resulted in successful growth of thermophilic and alkaliphilic bacteria from the fluids, and some of these bacteria reduced Fe(III) to magnetite. 16S rRNA gene analyses indicated that the rocks were dominated by sequences similar to sequences of Proteobacteria and that most organisms were related to nitrate reducers from a saline, alkaline, cold habitat; however, some phylotypes were either members of a novel lineage or closely related to uncultured clones. The bacterial communities in the fluids were more diverse and included Proteobacteria, Bacteroidetes, gram-positive bacteria, Planctomycetes, and Candidatus taxa. The archaeal diversity was lower, and most sequences were not related to any known cultivated species. Some archaeal sequences were 90 to 95% similar to sequences recovered from ocean sediments or other subsurface environments. Some archaeal sequences from the drilling fluids were >93% similar to sequences of Sulfolobus solfataricus, and the thermophilic nature was consistent with the in situ temperature. We inferred that the microbes in the UHP rocks reside in fluid and gas inclusions, whereas those in the drilling fluids may be derived from subsurface fluids.     

Microbiological Assessment of Circulation Mud Fluids During the First Operation of Riser Drilling by the Deep-Earth Research Vessel Chikyu

Quality assurance and control (QA/QC) is significant for the scientific drilling in order to accurately characterize physical, geochemical, and biological properties in the cored deep subseafloor materials. To explore the deep subseafloor life and its biosphere, identification and control of microbial contamination in drilling cores is critical for highly sensitive molecular analyses as well as cultivations, especially for the evaluation of low biomass and/or extremely harsh deep environments. Here we report some microbiological characteristics of circulation mud fluids before and after the first riser drilling operation by the newly constructed deep-earth research vessel Chikyu. During the Chikyu shakedown expedition CK06-06 in 2006, we used the riser system for drilling 547 to 647 meter below the seafloor into the sediments offshore the Shimokita Peninsula of Japan. Cultivation experiments showed that no microbial growth was observed in the precirculation mud fluid, while 4 × 10⁵ colonies per 1 ml were observed in the postcirculation mud fluid; all cultured bacterial isolates were found to be Halomonas. Using culture-independent molecular analysis, 16S rRNA gene sequences of Xanthomonas, which is used for industrial production of the mud fluid viscosifier “xanthan gum”, were predominantly detected in the precirculation mud fluid, while Halomonas sequences consistently dominated the clone library constructed from the postcirculation mud fluid. Archaeal 16S rRNA genes were amplified only from the postcirculation mud fluid; these archaeal clone sequences were affiliated to the Marine Crenarchaeota Group I (MGI), Marine Euryarchaeota Group II (MGII), Miscellaneous Crenarchaeotic Group (MCG), South African Gold Mine Euryarchaeotic Group (SAGMEG), Soil Group, and Methanococcus aeolicus. These results suggest that Halomonas contaminated and grew in the tank of circulation mud fluids, and other indigenous deep subseafloor microbial components, especially deep subsurface archaea, were also mixed into the post-circulation mud fluid.     

深部生物圈古菌的研究进展与展望

古菌作为深部生物圈中常见的原核生物,广泛分布于各类海洋沉积生境中,在沉积物生物地球化学循环中发挥着重要作用。由于不同的古菌类群对环境条件存在生理适应性差异,它们分别在近岸沿海和开阔大洋沉积物中构成了厌氧微生物生态系统和好氧微生物生态系统。本文通过对近岸与远洋、沉积物与上覆水体两个不同维度的古菌群落结构进行比较,以及对出现在深部生物圈中的常见古菌(奇古菌门(Thaumarchaeota)、深古菌门(Bathyarchaeota)、底栖古菌目(Thermoprofundales)、Asgard古菌超级门、乌斯古菌门(Woesearchaeota))的分布、代谢和环境适应机制进行论述,总结了深部生物圈中古菌的研究进展,并在此基础上展望了几个未来研究的方向与重点。     

Mariana Forearc Serpentinite Mud Volcanoes Harbor Novel Communities of Extremophilic Archaea

Extremophilic archaeal communities living in serpentinized muds influenced by pH 12.5 deep-slab derived fluids were detected and their richness and relatedness assessed from across seven serpentinite mud volcanoes located along the Mariana forearc. In addition, samples from two near surface core sections (Holes D and E) at ODP Site 1200 from South Chamorro were subjected to SSU rDNA clone library and phylogenetic analysis resulting in the discovery of several novel operational taxonomic units (OTUs). Five dominant OTUs of Archaea from Hole 1200D and six dominant OTUs of Archaea from Hole 1200E were determined by groups having three or more clones. Terminal-restriction fragment length polymorphism (T-RFLP) analysis revealed all of the dominant OTUs were detected within both clone libraries. Cluster analysis of the T-RFLP data revealed archaeal community structures from sites on Big Blue and Blue Moon to be analogous to the South Chamorro Hole 1200E site. These unique archaeal community fingerprints resulted from an abundance of potential methane-oxidizing and sulfate-reducing phylotypes. This study used deep-sea sediment coring techniques across seven different mud volcanoes along the entire Mariana forearc system. The discovery and detection of both novel Euryarchaeota and Marine Benthic Group B Crenarcheaota phylotypes could be efficacious archaeal indicator populations involved with anaerobic methane oxidation (AMO) and sulfate reduction fueled by deep subsurface serpentinization reactions.     

Predominance of Methanolobus spp. and Methanoculleus spp. in the Archaeal Communities of Saline Gas Field Formation Fluids

The microbial communities in sulfate-rich, saline formation fluids of a natural gas reservoir in Lower Saxony, Germany were investigated to enhance the knowledge about microbial communities in potential carbon dioxide sequestration sites. This investigation of the initial state of the deep subsurface microbiota is necessary to predict their influence on the long-term stability and storage capacity of such sites. While the bacterial 16S rDNA gene library was comprised of sequences affiliating with the Firmicutes, the Alphaproteobacteria, the Gammaproteobacteria and the Thermotogales, the archaeal 16S rDNA libraries were simply dominated by two phylotypes related to the genera Methanolobus and Methanoculleus. The monitoring of the archaeal communities in different formation fluid samples by T-RFLP and Real-Time-PCR indicated that these two methanogenic genera dominated at all, whereas the proportion of the two groups varied. Thus, methylotrophic and autotrophic methanogenesis seems to be of importance in the reservoir fluids, dependent on the provided reduction equivalents and substrates and it also may influence the fate of CO₂ in the subsurface.     

Geochemical Influence on Diversity and Microbial Processes in High Temperature Oil Reservoirs

The diversity of thermophilic microbial assemblages detected within two neighboring high temperature petroleum formations was shown to closely parallel the different geochemical regimes existing in each. A high percentage of archaeal 16S rRNA gene sequences, related to thermophilic aceticlastic and hydrogenotrophic methanogens, were detected in the natural gas producing Rincon Formation. In contrast, rRNA gene libraries from the highly sulfidogenic Monterey Formation contained primarily sulfur-utilizing and fermentative archaea and bacteria. In addition to the variations in microbial community structure, microbial activities measured in microcosm experiments using high temperature production fluids from oil-bearing formations also demonstrated fundamental differences in the terminal respiratory and redox processes. Provided with the same suite of basic energy substrates, production fluids from the South Elwood Rincon Formation actively generated methane, while thermophilic microflora within the Monterey production fluids were dominated by hydrogen sulfide producing microorganisms. In both cases, molecular hydrogen appeared to play a central role in the stimulation of carbon and sulfur cycling in these systems. In methanogenic production fluids, the addition of sulfur compounds induced a rapid shift in the terminal electron accepting process, stimulating hydrogen sulfide formation and illustrating the metabolic versatility of the subsurface thermophilic assemblage. The high similarity between microbial community structure and activity corresponding with the prevalent geochemical conditions observed in deep subsurface petroleum reservoirs suggests that the resident microflora have adapted to the subsurface physicochemical conditions and may actively influence the geochemical environment in situ.     

Microbial diversity in degraded and non-degraded petroleum samples and comparison across oil reservoirs at local and global scales

Microorganisms have shown their ability to colonize extreme environments including deep subsurface petroleum reservoirs. Physicochemical parameters may vary greatly among petroleum reservoirs worldwide and so do the microbial communities inhabiting these different environments. The present work aimed at the characterization of the microbiota in biodegraded and non-degraded petroleum samples from three Brazilian reservoirs and the comparison of microbial community diversity across oil reservoirs at local and global scales using 16S rRNA clone libraries. The analysis of 620 16S rRNA bacterial and archaeal sequences obtained from Brazilian oil samples revealed 42 bacterial OTUs and 21 archaeal OTUs. The bacterial community from the degraded oil was more diverse than the non-degraded samples. Non-degraded oil samples were overwhelmingly dominated by gammaproteobacterial sequences with a predominance of the genera Marinobacter and Marinobacterium. Comparisons of microbial diversity among oil reservoirs worldwide suggested an apparent correlation of prokaryotic communities with reservoir temperature and depth and no influence of geographic distance among reservoirs. The detailed analysis of the phylogenetic diversity across reservoirs allowed us to define a core microbiome encompassing three bacterial classes (Gammaproteobacteria, Clostridia, and Bacteroidia) and one archaeal class (Methanomicrobia) ubiquitous in petroleum reservoirs and presumably owning the abilities to sustain life in these environments.

     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

Thermophilic prokaryotes from deep subterranean habitats

The deep continental biosphere consists of geologically isolated ecosystems differing in their physicochemical, geological, and trophic parameters. Most of the deep ecosystems exist at elevated temperatures (50–120°C), which favor the development of thermophilic microorganisms. In many cases, the indigenous nature of subsurface microorganisms is questionable due to problems of collecting representative and non-contaminated samples. In spite of the numerous studies on the deep biosphere microbial communities, the number of cultivated thermophiles isolated from subsurface environments not associated with petroleum deposits does not exceed 30 species. More than half of the thermophilic species isolated from deep subsurface belong to the Firmicutes. The majority of the underground thermophiles are represented by strict or facultative anaerobes, with capacity for sulfate and iron reduction notably widespread. Most thermophilic subsurface microorganisms are organotrophs, although chemolithoautotrophic thermophiles also have been reported. This review deals with the phylogenetic diversity and physiological properties of the cultivated thermophilic prokaryotes isolated from various deep subterranean habitats.     

Molecular characterization of microbial communities in fault-bordered aquifers in the Miocene formation of northernmost Japan

We investigated the diversity and distribution of archaeal and bacterial 16S rRNA gene sequences in deep aquifers of mid‐ to late Miocene hard shale located in the northernmost region of the Japanese archipelago. A major fault in the north‐west–south‐east (NW–SE) direction runs across the studied area. We collected three groundwater samples from boreholes on the south‐west (SW) side of the fault at depths of 296, 374 and 625 m below ground level (m.b.g.l.) and one sample from the north‐east (NE) side of the fault at a depth of 458 m.b.g.l. The groundwater samples were observed to be neutral and weakly saline. The total microbial counts after staining with acridine orange were in the order 10⁵?10⁶ cells mL^?1 and 10³ cells mL^?1 in the aquifers to the SW and to the NE of the fault, respectively. A total of 407 archaeal and bacterial 16S rRNA gene sequences (204 and 203 sequences, respectively) were determined for clone libraries constructed from all groundwater samples. Phylogenetic analyses showed that the libraries constructed from the SW aquifers were generally coherent but considerably different from those constructed from the NE aquifer. All of the archaeal clone libraries from the SW aquifers were predominated by a single sequence closely related to the archaeon Methanoculleus chikugoensis, and the corresponding bacterial libraries were mostly predominated by the sequences related to Bacteroidetes, Firmicutes and δ‐Proteobacteria. In contrast, the libraries from the NE aquifer were dominated by uncultured environmental archaeal clones with no methanogen sequences and by β‐proteobacterial clones with no sequences related to Bacteroidetes and δ‐Proteobacteria. Hence, the possible coexistence of methanogens and sulphate reducers in Horonobe deep borehole (HDB) on the SW side is suggested, particularly in HDB‐6 (374 m.b.g.l.). Moreover, these organisms might play an important geochemical role in the groundwater obtained from the aquifers.     

Culturable prokaryotic diversity of deep,gas hydrate sediments: first use of a continuous high‐pressure,anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG)

Deep subseafloor sediments may contain depressurization‐sensitive, anaerobic, piezophilic prokaryotes. To test this we developed the DeepIsoBUG system, which when coupled with the HYACINTH pressure‐retaining drilling and core storage system and the PRESS core cutting and processing system, enables deep sediments to be handled without depressurization (up to 25 MPa) and anaerobic prokaryotic enrichments and isolation to be conducted up to 100 MPa. Here, we describe the system and its first use with subsurface gas hydrate sediments from the Indian Continental Shelf, Cascadia Margin and Gulf of Mexico. Generally, highest cell concentrations in enrichments occurred close to in situ pressures (14 MPa) in a variety of media, although growth continued up to at least 80 MPa. Predominant sequences in enrichments were Carnobacterium, Clostridium, Marinilactibacillus and Pseudomonas, plus Acetobacterium and Bacteroidetes in Indian samples, largely independent of media and pressures. Related 16S rRNA gene sequences for all of these Bacteria have been detected in deep, subsurface environments, although isolated strains were piezotolerant, being able to grow at atmospheric pressure. Only the Clostridium and Acetobacterium were obligate anaerobes. No Archaea were enriched. It may be that these sediment samples were not deep enough (total depth 1126–1527 m) to obtain obligate piezophiles.     

Diversity and Community Structure of Archaea in Deep Subsurface Sediments from the Tropical Western Pacific

Archaeal 16S rRNA gene clone libraries using PCR amplicons from eight different layers of the MD06-3051 core were obtained from the tropical Western Pacific sediments. A total of 768 clones were randomly selected, and 264 representative clones were sequenced by restriction fragment length polymorphism. Finally, 719 valid clones and 104 operational taxonomic units were identified after chimera-check and ≥97% similarity analysis. The phylogenetic analysis of 16S rDNA sequences obtained from sediment samples were very diverse and showed stratification with depth. Majority of the members were most closely related to uncultivated groups and physiologically uncharacterized assemblages. All phylotypes were affiliated with Crenarchaeota (76%) and Euryarchaeota (24%), respectively. Deep-sea archaeal group (DSAG, 41% of total clones) and miscellaneous crenarchaeotic group (MCG, 29% of total clones) belonging to Crenarchaeota were the most predominant archaeal 16S rDNA phylotypes in clone libraries. Phylotypes in this study shared high similarity with those in subsurface sediments from Peru Margin sites, which indicated that different geographical zones might host similar members of archaeal populations based on similar sedimentary environments. In our study, members of DSAG and MCG seemed to dominate certain layers of the nonhydrate sediments, suggesting a wide ecophysiological adaptation than previously appreciated. The spatial distribution and community structure of these groups might vary with the different geochemical gradients of the environment.     

Characteristics of Naturally Grown Biofilms in Deep Groundwaters and Their Heavy Metal Sorption Property in a Deep Subsurface Environment

Two biofilm samples were collected from anaerobic groundwater in the depth range of 158.8–199.4 m in a borehole drilled in the Tono area, Japan, to understand their effects on the migration behavior of heavy metals in subsurface environments. The depth range is featured geologically by the lignite formation of sedimentary rocks that bear a uranium ore and the underlying granitic formation. Microbiomes of the derived biofilms, as well as of the ambient bacterioplankton, were characterized based on 16S rRNA gene sequences (clones) or phylotypes, and their heavy metal sorption properties were examined with reference to geochemical features of groundwaters. Phylotypic compositions of the four microbiomes, i.e., of biofilm vs. planktonic bacteria as well as in granitic vs. sedimentary rock groundwaters showed significant differences. In addition, each microbiome was dominated by one or two distinctive phylotypes. In bacterioplankton, the phylotype related to a betaproteobacterial environmental clone dominated 54% of the sequenced clones derived from sedimentary rock groundwater, whereas those related to Denitratisoma oestradiolicum and Clostridium sp. dominated 45% and 37%, respectively, of the clones derived from granitic groundwater. In biofilms, the phylotypes related to Methylobacillus flagellatus and Ignavibacterium album accounted for 77% and 78% of the clones of the biofilms derived from the sedimentary rock and granitic groundwaters, respectively. Chemical and mineralogical analyses demonstrated that high amounts of heavy metals such as Fe, Ni, Cu, Zn, As, Cd, Pb, Th and U accumulated in the biofilms; and their sorption properties varied between biofilms presumably with influences of co-occurring Fe-hydroxides and sulfide minerals under the redox conditions of approximately ?360 to 0 mV in subsurface environments. The biofilm-mineral interaction provides an implication for possible retardation of radionuclide migration in subsurface hydrology, which is of practical interest in geological disposal systems for high-level radioactive waste.     

Characterization of methanogenic and prokaryotic assemblages based on mcrA and 16S rRNA gene diversity in sediments of the Kazan mud volcano (Mediterranean Sea)

The diversity of the methyl‐coenzyme reductase A (mcrA) and 16S rRNA genes was investigated in gas hydrate containing sediment from the Kazan mud volcano, eastern Mediterranean Sea. mcrA was detected only at 15 and 20 cm below seafloor (cmbsf) from a 40‐cm long push core, while based on chemical profiles of methane, sulfate, and sulfide, possible anaerobic oxidation of methane (AOM) depth was inferred at 12–15 cmbsf. The phylogenetic relationships of the obtained mcrA, archaeal and bacterial 16S rRNA genes, showed that all the found sequences were found in both depths and at similar relative abundances. mcrA diversity was low. All sequences were related to the Methanosarcinales, with the most dominant (77.2%) sequences falling in group mcrA‐e. The 16S rRNA‐based archaeal diversity also revealed low diversity and clear dominance (72.8% of all archaeal phylotypes) of the Methanosarcinales and, in particular, ANME‐2c. Bacteria showed higher diversity but 83.2% of the retrieved phylotypes from both sediment layers belonged to the δ‐Proteobacteria. These phylotypes fell in the SEEP‐SRB1 putative AOM group. In addition, the rest of the less abundant phylotypes were related to yet‐uncultivated representatives of the Actinobacteria, Spirochaetales, and candidate divisions OP11 and WS3 from gas hydrate‐bearing habitats. These phylotype patterns indicate that AOM is occurring in the 15 and 20 cmbsf sediment layers.     

Microbial Community Stratification Controlled by the Subseafloor Fluid Flow and Geothermal Gradient at the Iheya North Hydrothermal Field in the Mid-Okinawa Trough (Integrated Ocean Drilling Program Expedition 331)

The impacts of lithologic structure and geothermal gradient on subseafloor microbial communities were investigated at a marginal site of the Iheya North hydrothermal field in the Mid-Okinawa Trough. Subsurface marine sediments composed of hemipelagic muds and volcaniclastic deposits were recovered through a depth of 151 m below the seafloor at site C0017 during Integrated Ocean Drilling Program Expedition 331. Microbial communities inferred from 16S rRNA gene clone sequencing in low-temperature hemipelagic sediments were mainly composed of members of the Chloroflexi and deep-sea archaeal group. In contrast, 16S rRNA gene sequences of marine group I Thaumarchaeota dominated the microbial phylotype communities in the coarse-grained pumiceous gravels interbedded between the hemipelagic sediments. Based on the physical properties of sediments such as temperature and permeability, the porewater chemistry, and the microbial phylotype compositions, the shift in the physical properties of the sediments is suggested to induce a potential subseafloor recharging flow of oxygenated seawater in the permeable zone, leading to the generation of variable chemical environments and microbial communities in the subseafloor habitats. In addition, the deepest section of sediments under high-temperature conditions (∼90°C) harbored the sequences of an uncultivated archaeal lineage of hot water crenarchaeotic group IV that may be associated with the high-temperature hydrothermal fluid flow. These results indicate that the subseafloor microbial community compositions and functions at the marginal site of the hydrothermal field are highly affected by the complex fluid flow structure, such as recharging seawater and underlying hydrothermal fluids, coupled with the lithologic transition of sediments.     

Phylogenetic Diversity of Archaea and Bacteria in a Deep Subsurface Paleosol

Abstract A low-biomass paleosol 188 m below the ground surface at the Department of Energy's Hanford Site in south-central Washington State was recovered and maintained at the in situ temperature (17°C) as an intact core or homogenized sediment for 0, 1, 3, 10, and 21 weeks post-sampling. Bacterial and archaeal 16S rRNA genes were amplified by PCR and cloned. Of 746 bacterial and 190 archaeal clones that were categorized by restriction fragment length polymorphism (RFLP), 242 bacterial and 16 archaeal clones were partially sequenced and compared against the small subunit ribosomal RNA database (RDP) and GenBank. Six bacterial and 16 archaeal clones sequences, with little similarity to those in public databases, were sequenced in their entirety, and subjected to more detained phylogenetic analysis. The most frequently occurring clones types were related to Pseudomonas, Bacillus, Micrococcus, Clavibacter, Nocardioides, Burkholderia, Comamonas, and Erythromicrobium. Clone sequences whose RDP similarity value was ≥0.6 consistently grouped with their nearest RDP neighbor during phylogenetic analysis. Six truly novel eubacterial sequences were identified; they consistently cluster with or near the Chloroflexaceae and sequences recovered from the Sargasso Sea. Sixteen unique archaeal RFLP groups were identified from 190 randomly-sampled clones. The novel archaeal rDNA clones formed a coherent clade along the major Crenarchaea branch containing all previously described mesophilic crenarchae clones, but remained firmly associated with 16S rDNA clones previously obtained from a thermal Fe/S spring in Yellowstone National Park. The wealth of group-specific genetic information identified during this study will now allow us to address specific hypotheses related to in situ stimulation of these deep subsurface microorganisms and changes in microbial community composition resulting from subsurface contamination or remediation processes at the Hanford Site. Revised: 21 October 1997; Accepted: 20 November 1997     

Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval

The diversity and structure of the archaeal community in the effluent leachate from a full-scale recirculating landfill was characterized by direct 16S rRNA gene (16S rDNA) retrieval. Total-community DNA was extracted from the microbial assemblages in the landfill leachate, and archaeal 16S rDNAs were amplified with a universally conserved primer and an Archaea-specific primer. The amplification product was then used to construct a 16S rDNA clone library, and 70 randomly selected archaeal clones in the library were grouped by restriction fragment length polymorphism (RFLP) analysis. Sequencing and phylogenetic analysis of representatives from each unique RFLP type showed that the archaeal library was dominated by methanogen-like rDNAs. Represented in the kingdom of Euryarchaeota were phylotypes highly similar to the methanogenic genera Methanoculleus, Methanosarcina, Methanocorpusculum, Methanospirillum and Methanogenium, where the clone distribution was 48, 11, 3, 1 and 1, respectively. No sequences related to known Methanosaeta spp. were retrieved. Four rDNA clones were not affiliated with the known methanogenic Archaea, but instead, they were clustered with the uncultured archaeal sequences recently recovered from anaerobic habitats. Two chimeric sequences were identified among the clones analyzed.     

Diversity of the Microeukaryotic Community in Sulfide-Rich Zodletone Spring (Oklahoma)

The microeukaryotic community in Zodletone Spring, a predominantly anaerobic sulfide and sulfur-rich spring, was examined using an 18S rRNA gene cloning and sequencing approach. The majority of the 288 clones sequenced from three different locations at Zodletone Spring belonged to the Stramenopiles, Alveolata, and Fungi, with members of the phylum Cercozoa, order Diplomonadida, and family Jakobidae representing a minor fraction of the clone library. No sequences suggesting the presence of novel kingdom level diversity were detected in any of the three libraries. A large fraction of stramenopile clones encountered were monophyletic with either members of the genus Cafeteria (order Bicosoecida) or members of the order Labyrinthulida (slime nets), both of which have so far been encountered mainly in marine habitats. The majority of the observed fungal clone sequences belonged to the ascomycetous yeasts (order Saccharomycetales), were closely related to yeast genera within the Hymenobasidiomycetes (phylum Basidiomycetes), or formed a novel fungal lineage with several previously published or database-deposited clones. To determine whether the unexpected abundance of fungal sequences in Zodletone Spring clone libraries represents a general pattern in anaerobic habitats, we generated three clone libraries from three different anaerobic settings (anaerobic sewage digester, pond sediment, and hydrocarbon-exposed aquifer sediments) and partially sequenced 210 of these clones. Phylogenetic analysis indicated that clone sequences belonging to the kingdom Fungi represent a significant fraction of all three clone libraries, an observation confirmed by phospholipid fatty acid and ergosterol analysis. Overall, this work reveals an unexpected abundance of Fungi in anaerobic habitats, describes a novel, yet-uncultured group of Fungi that appears to be widespread in anaerobic habitats, and indicates that several of the previously considered marine protists could also occur in nonmarine habitats.     

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

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