Relating Microbial Community Structure and Geochemistry in Deep Regolith Developed on Volcaniclastic Rock in the Luquillo Mountains,Puerto Rico

Fe oxidation is often the first chemical reaction that initiates weathering and disaggregation of intact bedrock into regolith. Here we explore the use of pyrosequencing tools to test for evidence that bacteria participate in these reactions in deep regolith. We analyze regolith developed on volcaniclastic rocks of the Fajardo formation in a ridgetop within the rainforest of the Luquillo Mountains of Puerto Rico. In the 9-m-deep regolith profile, the primary minerals chlorite, feldspar, and pyroxene are detected near 8.3 m but weather to kaolinite and Fe oxides found at shallower depths. Over the regolith profile, both total and heterotrophic bacterial cell counts generally increase from the bedrock to the surface. Like other soil microbial studies, the dominant phyla detected are Proteobacteria, Acidobacteria, Planctomycetes, and Actinobacteria. Proteobacteria (α, β, γ and δ) were the most abundant at depth (6.8–9 m, 41–44%), while Acidobacteria were the most abundant at the surface (1.4–4.4 m, 37–43%). Despite the fact that Acidobacteria dominated surficial communities while Proteobacteria dominated near bedrock, the near-surface and near-bedrock communities were not statistically different in structure but were statistically different from mid-depth communities. Approximately 21% of all sequences analyzed did not match known sequences: the highest fraction of unmatched sequences was greatest at mid-depth (45% at 4.4 m). At the regolith-bedrock interface where weathering begins, several lines of evidence are consistent with biotic Fe oxidation. At that interface, iron-related bacterial activity tests and culturing indicate the presence of iron-related bacteria, and phylogenetic analyses identified sub-phyla containing known iron-oxidizing microorganisms. Cell densities of iron-oxidizers in the deep saprolite were estimated to be on the order of 10⁵ cells g^?1. Overall Fe loss was also observed at the regolith-bedrock interface, consistent with bacterial production of organic acids and leaching of Fe-organic complexes. Fe-organic species were also detected to be enriched near the bedrock-regolith interface. In this and other deep weathering profiles, chemolithoautotrophic bacteria that use Fe for energy and nitrate or oxygen as an electron acceptor may play an important role in initiating disaggregation of bedrock.     

Solar-driven chemical energy source for a Martian biota

Microorganisms deep in the Martian soil could derive energy indirectly from the sun via chemical reactions involving atmospheric photolysis products of the solar ultraviolet flux. The Viking discovery of a chemically uniform regolith which, though poor in organics, is rich in sulfur-containing compounds suggests reaction sequences in which sulfur is recycled through reduced and oxidized states by biologically catalyzed reactions with photochemically-produced atmospheric constitutents. One candidate reaction, reduction of soil sulfate minerals by molecular hydrogen, is already exploited on earth by bacteria of the ubiquitous and tenaciousDesulfovibrio genus.     

Drought deaths in Eucalyptus globulus (Labill.) plantations in relation to soils, geomorphology and climate

The occurrence of tree deaths in young, 3 to 6 year old Eucalyptus globulus plantations established on farmland in south-western Australia was found to be strongly related to factors indicative of poor soil water storage capacity. Seven years after planting tree survival was significantly less on soils <2 m deep compared to >2 m deep (22% vs 70%). This is due to the limited ability of some soils to store a sufficient proportion of the annual rainfall within the root-zone to meet the plant water demand in a region with a recurrent annual summer drought. There are practical difficulties in routinely surveying soils to depths in excess of 2 m over broad areas, to predict the likelihood of tree death. On the granitic basement rocks of south-west Western Australia, the occurrence of ferricrete gravels provides a useful surrogate indicator for the presence of deeper soils. In this region the distribution of soil depth and soil fertility has a geomorphic basis, being related to previous patterns of deep weathering and regolith stripping. Soils have developed on various horizons of deeply weathered profiles, formed from granites and gneisses. These materials have been stripped to a variable extent by erosion, leading to a range of soil depths. The original weathered profiles, which correspond to the soils with ferricrete gravels, comprise the deepest soil/regolith materials (~30-50 m deep); whereas along drainage lines the regolith has been completely stripped, the soils are shallow and plantations are most susceptible to drought. Knowledge of the relationship between soil depth and plantation performance allows regional indications of drought risk to be developed from regional soil mapping and the production of more efficient sampling designs for site assessment.     

The coupling of biological iron cycling and mineral weathering during saprolite formation, Luquillo Mountains, Puerto Rico

Corestones of quartz diorite bedrock in the Rio Icacos watershed in Puerto Rico weather spheroidally to form concentric sets of partially weathered rock layers (referred to here as rindlets) that slowly transform to saprolite. The rindlet zone (0.2–2 m thick) is overlain by saprolite (2–8 m) topped by soil (0.5–1 m). With the objective of understanding interactions between weathering, substrate availability, and resident micro‐organisms, we made geochemical and microbiological measurements as a function of depth in 5 m of regolith (soil + saprolite). We employed direct microscopic counting of total cell densities; enumeration of culturable aerobic heterotrophs; extraction of microbial DNA for yield calculations; and biochemical tests for iron‐oxidizing bacteria. Total cell densities, which ranged from 2.5 × 10⁶ to 1.6 × 10¹⁰ g^?1 regolith, were higher than 10⁸ g^?1 at three depths: in the upper 1 m, at 2.1 m, and between 3.7 and 4.9 m, just above the rindlet zone. High proportions of inactive or unculturable cells were indicated throughout the profile by very low percentages of culturable heterotrophs (0.0004% to 0.02% of total cell densities). The observed increases in total and culturable cells and DNA yields at lower depths were not correlated with organic carbon or total iron but were correlated with moisture and HCl‐extractable iron. Biochemical tests for aerobic iron‐oxidizers were also positive at 0.15–0.6 m, at 2.1–2.4 m, and at 4.9 m depths. To interpret microbial populations within the context of weathering reactions, we developed a model for estimating growth rates of lithoautotrophs and heterotrophs based on measured substrate fluxes. The calculations and observations are consistent with a model wherein electron donor flux driving bacterial growth at the saprolite–bedrock interface is dominated by Fe(II) and where autotrophic iron‐oxidizing bacteria support the heterotrophic population and contribute to bedrock disaggregation and saprolite formation.     

Microbial Communities in Subpermafrost Saline Fracture Water at the Lupin Au Mine, Nunavut, Canada

We report the first investigation of a deep subpermafrost microbial ecosystem, a terrestrial analog for the Martian subsurface. Our multidisciplinary team analyzed fracture water collected at 890 and 1,130 m depths beneath a 540-m-thick permafrost layer at the Lupin Au mine (Nunavut, Canada). ¹⁴C, ³H, and noble gas isotope analyses suggest that the Na–Ca–Cl, suboxic, fracture water represents a mixture of geologically ancient brine, ~25-kyr-old, meteoric water and a minor modern talik-water component. Microbial planktonic concentrations were ~10³ cells mL^?1. Analysis of the 16S rRNA gene from extracted DNA and enrichment cultures revealed 42 unique operational taxonomic units in 11 genera with Desulfosporosinus, Halothiobacillus, and Pseudomonas representing the most prominent phylotypes and failed to detect Archaea. The abundance of terminally branched and midchain-branched saturated fatty acids (5 to 15 mol%) was consistent with the abundance of Gram-positive bacteria in the clone libraries. Geochemical data, the ubiquinone (UQ) abundance (3 to 11 mol%), and the presence of both aerobic and anaerobic bacteria indicated that the environment was suboxic, not anoxic. Stable sulfur isotope analyses of the fracture water detected the presence of microbial sulfate reduction, and analyses of the vein-filling pyrite indicated that it was in isotopic equilibrium with the dissolved sulfide. Free energy calculations revealed that sulfate reduction and sulfide oxidation via denitrification and not methanogenesis were the most thermodynamically viable consistent with the principal metabolisms inferred from the 16S rRNA community composition and with CH₄ isotopic compositions. The sulfate-reducing bacteria most likely colonized the subsurface during the Pleistocene or earlier, whereas aerobic bacteria may have entered the fracture water networks either during deglaciation prior to permafrost formation 9,000 years ago or from the nearby talik through the hydrologic gradient created during mine dewatering. Although the absence of methanogens from this subsurface ecosystem is somewhat surprising, it may be attributable to an energy bottleneck that restricts their migration from surface permafrost deposits where they are frequently reported. These results have implications for the biological origin of CH₄ on Mars.     

Karyotype analysis and DNA content in some species of Chrysolaena (Vernonieae,Asteraceae)

Cytogenetic characterization by karyotyping and determination of DNA content by flow cytometry of five species of Chrysolaena (Vernonieae, Asteraceae) was performed. This is the first study of nuclear DNA content realized in the genus. The 2C-values were compared with the ploidy level and the total karyotype length (TKL) of each species. Mitotic analysis revealed a base chromosome number x = 10 for all entities and different ploidy levels, from diploid (2n = 2x = 20) to octoploid (2n = 8x = 80). All species showed bimodal karyotypes composed of metacentric and submetacentric chromosomes. The average chromosome size (ML) varied from 1.86 μm to 2.70 μm, while the TKL ranged from 18.65 μm to 80.55 μm. The intrachromosomal asymmetry index (A₁) varied from 0.27 to 0.38, while the interchromosomal asymmetry index (A₂) ranged from 0.19 to 0.25. A new cytotype is reported for the first time for C. propinqua. Accessory chromosomes found in C. verbascifolia, C. cognata, C. flexuosa, and C. propinqua are also reported as new.     

Conditioning film and initial biofilm formation on electrochemical CaCO3 deposition on a metallic net in the marine environment

Electrochemical deposition of minerals is a unique technology for artificial reef constructions, relying on calcium carbonate (CaCO₃) build-up over metallic structures through electrolysis of seawater. The present study traces the first 72 h following electric current termination on bacterial biofilm build-up on a metallic net covered with CaCO₃. 16S rRNA clone libraries indicated a dynamic succession. Proteobacteria and Bacteroidetes were evident at all sampling times while Cyanobacteria appeared only within the first 8 h. A significant increase in total organic carbon (TOC) and total protein was observed after 48 h with a significant correlation (R ² = 0.74), indicating TOC is a good tool for characterizing initial biofilm formation. 18S rRNA gene sequences obtained 72 h following current termination indicated a significant presence of Cnidarians (51%). Understanding the dynamics among primary bacterial settlers is important because they play a crucial role in driving the colonization of sessile invertebrate communities on artificial, as well as natural surfaces.     

Solar-driven chemical energy source for a Martian biota.

Microorganisms deep in the Martian soil could derive energy indirectly from the sun via chemical reactions involving atmospheric products of the solar ultraviolet flux. The Viking discovery of a chemically uniform regolith which, though poor in organics, is rich in sulfur-containing compounds suggests reaction sequences in which sulfur is recycled through reduced and oxidized states by biologically catalyzed reactions with photochemically-produced atmospheric constituents. One candidate reaction, reduction of soil ssufate minerals by molecular hydrogen, is already exploited on earth by bacteria of the ubiquitous and tenacious Desulfovibrio genus.     

Design of a beneficial product for newborn calves by combining Lactobacilli,minerals, and vitamins

Diarrhea is one of the most frequent diseases affecting newborn calves in intensive systems. Several strategies were proposed to protect and improve health, such as probiotics. This work was directed to design a product containing freeze-dried bacteria, vitamins, and minerals, as well as to optimize conditions with lyoprotectors, combine strains and add vitamins, minerals, and inulin to the product. The lyoprotectors were milk, milk-whey, and actose, and products were stored for 6 months at 4°C. Combined bacteria were freeze-dried in milk and the final products were added with minerals, vitamins, and insulin. The viable cells were determined by the plate count assay and antibiotic profiles to differentiate strains. Lactobacillus johnsonii CRL1693, L. murinus CRL1695, L. mucosae CRL1696, L. salivarius CRL1702, L. amylovorus CRL1697, and Enterococcus faecium CRL1703 were evaluated. The optimal conditions were different for each strain. Milk and milk whey maintained the viability during the process and storage after 6 months for most of the strains, except for L. johnsonii. Lactose did not improve cell’s recovery. L. murinus was viable for 6 months in all the conditions, with similar results in enterococci. In strains combined before freeze-dried, the viability decreased deeply, showing that one-step process with bacteria mixtures, vitamins, and minerals were not adequate. Freeze-dried resistance depends on each strain and must be lyophilized individually.     

Bacterial composition of soils of the Lake Wellman area, Darwin Mountains, Antarctica

The aim of this study was to examine the bacterial composition of high latitude soils from the Darwin–Hatherton glacier region of Antarctica. Four soil pits on each of four glacial drift sheets were sampled for chemical and microbial analyses. The four drifts—Hatherton, Britannia, Danum, and Isca—ranged, respectively, from early Holocene (10 ky) to mid-Quaternary (ca 900 ky). Numbers of culturable bacteria were low, with highest levels detected in soils from the younger Hatherton drift. DNA was extracted and 16S rRNA gene clone libraries prepared from samples below the desert pavement for each of the four drift sheets. Between 31 and 262 clones were analysed from each of the Hatherton, Britannia, and Danum drifts. Bacterial sequences were dominated by members of the phyla Deinococcus-Thermus, Actinobacteria, and Bacteroidetes. Culturable bacteria, including some that clustered with soil clones (e.g., members of the genera Arthrobacter, Adhaeribacter, and Pontibacter), belonged to Actinobacteria and Bacteroidetes. The isolated bacteria are ideal model organisms for genomic and phenotypic investigations of those attributes that allow bacteria to survive and/or grow in Antarctic soils because they have close relatives that are not tolerant of these conditions.     

The Black Water Chemocline of Meromictic Lower Mystic Lake,Massachusetts, U.S.A.

Lower Mystic Lake, Massachusetts, USA, has an anoxic black water layer just below the top of the chemocline (15.5–16.0 m). Bacterial concentrations averaged 10.4 × 10⁶ cells/ml in the black water layer and 4.0 × 10⁶ cells/ml below 17 m. Below the chemocline, microbial concentrations were linearly correlated to the vertical light absorption coefficient, r = 0.82. Phototrophic bacteria were not detected below the top of the chemocline, due to a low PAR that never exceeded 0.0001% surface illumination. Sulfate‐reducing bacteria and methanogens were enriched from the monimolimnion in selective media. Below the chemocline, H₂S concentrations were in excess of 11 mmoles/l and Fe, Mn, CH₄ and CO₂ concentrations were elevated compared to the mixolimnion. Nuisance releases of H₂S occurred from the lake in 1965. Although the monimolimnion remains a highly reduced environment rich in H₂S, the potential of further nuisance releases is small due to the diminished volume of the monimolimnion and the relatively deep chemocline.     

Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp.

The microbial community associated with a marine sponge (Haliclona sp.) collected from Tateyama city, Japan was studied using 16S rRNA gene clone libraries. Two DNA templates were prepared using methods recommended for Gram-positive and Gram-negative bacteria in the Qiagen kit manual. From each DNA template, two 16S rRNA genes were PCR amplified, using the combination of universal bacterial primer 27f and primers 1385r and 1492r, respectively. A total of 347 clones were sequenced and compared with those available in DNA data banks. These sequences were members of ten bacterial phyla. Interestingly, more than 30 % of the clones represent novel sequences. A comparison of these sequences with sequences in a library prepared from DNA extracted from the surrounding water shows minimum DNA contamination. Taxonomically, the highest diversity was detected in the clone library prepared using a combination of primers 27f and 1492r and DNA isolated using the Gram-positive bacteria protocol. The potential of Haliclona sp.-associated bacteria to produce secondary metabolites was studied by cloning and sequencing the polyketide synthase (PKS, type 1) gene using the same DNA samples. Analysis of partial sequences derived from the sponge metagenome revealed 27 unique ketosynthase domains of PKS type I. This study suggests strongly that this Haliclona sp. plays host to diverse novel bacteria with a potential to produce novel polyketides.     

UV-absorbing bacteria in coral mucus and their response to simulated temperature elevations

Reef-building corals encompass various strategies to defend against harmful ultraviolet (UV) radiation. Coral mucus contains UV-absorbing compounds and has rich prokaryotic diversity associated with it. In this study, we isolated and characterized the UV-absorbing bacteria from the mucus of the corals Porites lutea and Acropora hyacinthus during the pre-summer and summer seasons. A total of 17 UV-absorbing bacteria were isolated and sequenced. The UV-absorbing bacteria showed UV absorption at wavelengths ranging from λ _max = 333 nm to λ _min = 208 nm. Analysis of the DNA sequences revealed that the majority of the UV-absorbing bacteria belonged to the family Firmicutes and the remaining belonged to the family Proteobacteria (class Gammaproteobacteria). Comparison of the sequences with the curated database yielded four distinct bacterial groups belonging to the genus Bacillus, Staphylococcus, Salinicoccus and Vibrio. The absorption peaks for the UV-absorbing bacteria shifted to the UV-A range (320–400 nm) when they were incubated at higher temperatures. Deciphering the complex relationship between corals and their associated bacteria will help us to understand their adaptive strategies to various stresses.     

Microbial biogeochemistry of Boiling Springs Lake: a physically dynamic,oligotrophic, low‐pH geothermal ecosystem

Boiling Springs Lake (BSL) in Lassen Volcanic National Park, California, is North America's largest hot spring, but little is known about the physical, chemical, and biological features of the system. Using a remotely operated vessel, we characterized the bathymetry and near‐surface temperatures at sub‐meter resolution. The majority of the 1.2 ha, pH 2.2 lake is 10 m deep and 50–52 °C, but temperatures reach 93 °C locally. We extracted DNA from water and sediments collected from warm (52 °C) and hot (73–83 °C) sites separated by 180 m. Gene clone libraries and functional gene microarray (GeoChip 3.0) were used to investigate the BSL community, and uptake of radiolabeled carbon sources was used to assess the relative importance of heterotrophic vs. autotrophic production. Microbial assemblages are similar in both sites despite the strong temperature differential, supporting observations of a dynamic, convectively mixed system. Bacteria in the Actinobacteria and Aquificales phyla are abundant in the water column, and Archaea distantly related to known taxa are abundant in sediments. The functional potential appears similar across a 5‐year time span, indicating a stable community with little inter‐annual variation, despite the documented seasonal temperature cycle. BSL water‐derived DNA contains genes for complete C, N, and S cycles, and low hybridization to probes for N and S oxidation suggests that reductive processes dominate. Many of the detected genes for these processes were from uncultivated bacteria, suggesting novel organisms are responsible for key ecosystem services. Selection imposed by low nutrients, low pH, and high temperature appear to result in low diversity and evenness of genes for key functions involved in C, N, and S cycling. Conversely, organic degradation genes appear to be functionally redundant, and the rapid assimilation of radiolabeled organic carbon into BSL cells suggests the importance of allochthonous C fueling heterotrophic production in the BSL C cycle.     

Authigenic mineralization and detrital clay binding by freshwater biofilms: The Brahmani river,India

This study sought to understand the origin and fate of one of the bitumen mounds found on the bottom of Lake Baikal. These mounds are located at a depth of 900 m beneath oil spots detected on the surface of Lake Baikal (53° 18′24^″, 108° 23′20^″). The two mounds were sampled with a manipulator from a “MIR” deep-water manned submersible. Mature mound No. 8 was subjected to chemical and microbiological studies. Mound No. 3 was subjected only to chemical studies; we failed to perform microbiological analyses of this mound for logistic reasons. Oil spots collected from the water surface, samples of mound No. 3 and No. 8, were subjected to GC/MS analysis. The water contained aliphatic hydrocarbons with chains between C₈ and C₂₃, with the most abundant chain length being C₁₈. Mound No. 3 with the most abundant chain length being C₁₈ actively released oil droplets into the water. It contained 770 mg/g of C₁₃-C₃₂ n-alkanes, with a maximum at C₂₃ (160 mg/g). Mound No. 8 was inactive and contained 148 mg/g of aliphatic C₂₂-C₃₄ n-alkanes, with a maximum at C₂₅. Mound No. 8 also consisted of 3% inorganic matter, 48% unresolved complex mixture (UCM) and less than 1% other compounds (polyaromatic hydrocarbons, isoprenoids, carotenoids, and hopanes). The core of this sample used as inoculate, yielded Rhodococci when cultivated on oil as the only source of carbon. Cultivation of the sample on agar-containing Raymond inorganic medium with crude West Siberian oil as the only source of carbon revealed colonies of these bacteria, which all appeared identical. PCR was performed with DNA isolated from 5 colonies, using primers for 16S rRNA genes. Comparison of the sequences of the 5 PCR products over a length of 714 bp revealed that they were almost identical. Phylogenetic analysis of these homologous sequences showed that they were similar to the corresponding sequences of the genus Rhodococcus. Substrate demands, the morphology of the colonies, and SEM and TEM data confirmed that the isolates obtained could indeed be Rhodococci. All of the isolates could grow in bulk cultures with inorganic medium supplemented with crude oil. Moreover, all of the isolates degraded aliphatic hydrocarbons with lengths between C₁₁ and C₂₉. C₂₃-C₂₉ hydrocarbons were degraded completely. The isolates could grow at 4–37°C. The most unexpected finding was that of the many microorganisms capable of consuming oil, only Rhodococci exhibited this ability in the inactive bitumen mound. The possible mechanisms of how crude oil is transformed into bitumen mounds and mature bitumen are discussed.     

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly 62% of all DNA sequences analyzed. Other phylogenetic groups identified included Proteobacteria (20%), Actinobacteria (9%), Cyanobacteria (4%), and Bacteroidetes (2%). The composition of RNA-based libraries (1122 sequences) was similar to the DNA-based libraries with a few notable exceptions: Proteobacteria were more dominant in the RNA clone libraries (i.e., 35% RNA; 20% DNA). Differences in the Proteobacteria composition were also observed; alpha-Proteobacteria was 22 times more abundant in the RNA-based clones while beta-Proteobacteria was eight times more abundant in the DNA libraries. Nearly twice as many DNA operational taxonomic units (OTUs) than RNA OTUs were observed at distance 0.03 (101 DNA; 53 RNA). Twenty-four OTUs were shared between all RNA- and DNA-based libraries (OTU_0.03) representing only 18% of the total OTUs, but 81% (1527/1883) of all sequences. Such differences between clone libraries demonstrate the necessity of generating both RNA- and DNA-derived clone libraries to compare these two different molecular approaches for community analyses.     

Methanogenic and Sulphate-Reducing Microbial Communities in Deep Groundwater of Crystalline Rock Fractures in Olkiluoto,Finland

The long-term safety of final disposal of spent nuclear fuel in the deep geosphere is dependent on stability of biogeochemical conditions at the disposal site. Microbial processes, such as sulphate reduction and methanogenesis, may have profound effects on site biogeochemistry. In this study, sulphate-reducing bacteria and methane-producing archaea were investigated at depths ranging from 68 to 545 m in crystalline rock fractures at an intended spent nuclear fuel disposal site in Olkiluoto, Finland. Denaturing gradient gel electrophoresis detected diverse sulphate-reducing bacterial communities in all samples. Although the number of dsrB gene copies was below 10³ copies ml^?1 in all analyzed samples according to real-time quantitative PCR, their abundance was highest in samples that had the highest sulphate concentrations. Several distinct mcrA gene fragments were also recovered from most of the analyzed samples by cloning, although the number of methanogens was lower than that of sulphate-reducing bacteria when measured by mcrA-targeted quantitative PCR. The detected gene fragments were most closely related to sequences obtained from aquatic and deep subsurface environments. Results imply that sulphate reduction, methanogenesis, and anaerobic methane oxidation may all take place in the Olkiluoto deep geobiosphere.     

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.     

Phylogenetic diversity of transition and anoxic zone bacterial communities within a near‐shore anoxic basin: Nitinat Lake

At two stations surveyed in Nitinat Lake, a ～200‐m‐deep anoxic tidal fjord, sulfide was detected as close as 15 m from the surface. Biological characterization, determined from small subunit ribosomal RNA gene sequencing, of the chemocline and anaerobic zone revealed many sequences related to sulfur‐oxidizing bacteria, suggesting that sulfur cycling is a dominant process. γ‐ and ε‐Proteobacteria related to thiotrophic symbionts, as well as Chlorobium sp., dominated the transition zone. These are expected to play a role in dark and phototrophic CO₂ fixation, respectively. ε‐Proteobacteria phylotype abundance increased with depth, eventually comprising 69–97% of all sequences recovered from the anoxic zone. The vast majority (74%) of these phylotypes were affiliated with a novel Acrobacter sp. group (NITEP5). Quantification of NITEP5 revealed that up to 2.8 × 10⁵ cells ml^?1 were present in the anoxic zone. Surprisingly, although sequences related to known sulfate‐reducing bacteria were recovered from the transition zone, quantification of the dsr gene and ³⁵SO₄^2? uptake tests suggest that sulfate‐reduction within the water column is negligible. Overall, sequence diversity between different vertical zones was high, although the spatial segregation of γ‐Proteobacteria, Chlorobi, and ε‐Proteobacteria did not appear to vary significantly between seasons.     

Anammox bacterial populations in deep marine hypersaline gradient systems

To extend the knowledge of anaerobic ammonium oxidation (anammox) habitats, bacterial communities were examined in two hypersaline sulphidic basins in Eastern Mediterranean Sea. The 2 m thick seawater–brine haloclines of the deep anoxic hypersaline basins Bannock and L’Atalante were sampled in intervals of 10 cm with increasing salinity. ¹⁵N isotope pairing incubation experiments showed the production of ²⁹N₂ and ³⁰N₂ gases in the chemoclines, ranging from 6.0 to 9.2 % salinity of the L’Atalante basin. Potential anammox rates ranged from 2.52 to 49.65 nmol N₂ L^?1 day^?1 while denitrification was a major N₂ production pathway, accounting for more than 85.5 % of total N₂ production. Anammox-related 16S rRNA genes were detected along the L’Atalante and Bannock haloclines up to 24 % salinity, and the amplification of the hydrazine synthase genes (hzsA) further confirmed the presence of anammox bacteria in Bannock. Fluorescence in situ hybridisation and sequence analysis of 16S rRNA genes identified representatives of the marine anammox genus ‘Candidatus Scalindua’ and putatively new operational taxonomic units closely affiliated to sequences retrieved in marine environments that have documented anammox activity. ‘Scalindua brodae’ like sequences constituted up to 84.4 % of the sequences retrieved from Bannock. The anammox community in L’Atalante was different than in Bannock and was stratified according to salinity increase. This study putatively extends anammox bacterial habitats to extremely saline sulphidic ecosystems.