Author Correction: A distinct and active bacterial community in cold oxygenated fluids circulating beneath the western flank of the Mid-Atlantic ridge.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A distinct and active bacterial community in cold oxygenated fluids circulating beneath the western flank of the Mid-Atlantic ridge.

The rock-hosted, oceanic crustal aquifer is one of the largest ecosystems on Earth, yet little is known about its indigenous microorganisms. Here we provide the first phylogenetic and functional description of an active microbial community residing in the cold oxic crustal aquifer. Using subseafloor observatories, we recovered crustal fluids and found that the geochemical composition is similar to bottom seawater, as are cell abundances. However, based on relative abundances and functional potential of key bacterial groups, the crustal fluid microbial community is heterogeneous and markedly distinct from seawater. Potential rates of autotrophy and heterotrophy in the crust exceeded those of seawater, especially at elevated temperatures (25 °C) and deeper in the crust. Together, these results reveal an active, distinct, and diverse bacterial community engaged in both heterotrophy and autotrophy in the oxygenated crustal aquifer, providing key insight into the role of microbial communities in the ubiquitous cold dark subseafloor biosphere.

Novel microbial assemblages inhabiting crustal fluids within mid-ocean ridge flank subsurface basalt.

Although little is known regarding microbial life within our planet's rock-hosted deep subseafloor biosphere, boreholes drilled through deep ocean sediment and into the underlying basaltic crust provide invaluable windows of access that have been used previously to document the presence of microorganisms within fluids percolating through the deep ocean crust. In this study, the analysis of 1.7 million small subunit ribosomal RNA genes amplified and sequenced from marine sediment, bottom seawater and basalt-hosted deep subseafloor fluids that span multiple years and locations on the Juan de Fuca Ridge flank was used to quantitatively delineate a subseafloor microbiome comprised of distinct bacteria and archaea. Hot, anoxic crustal fluids tapped by newly installed seafloor sampling observatories at boreholes U1362A and U1362B contained abundant bacterial lineages of phylogenetically unique Nitrospirae, Aminicenantes, Calescamantes and Chloroflexi. Although less abundant, the domain Archaea was dominated by unique, uncultivated lineages of marine benthic group E, the Terrestrial Hot Spring Crenarchaeotic Group, the Bathyarchaeota and relatives of cultivated, sulfate-reducing Archaeoglobi. Consistent with recent geochemical measurements and bioenergetic predictions, the potential importance of methane cycling and sulfate reduction were imprinted within the basalt-hosted deep subseafloor crustal fluid microbial community. This unique window of access to the deep ocean subsurface basement reveals a microbial landscape that exhibits previously undetected spatial heterogeneity.

Phylogenetic diversity of microorganisms in subseafloor crustal fluids from Holes 1025C and 1026B along the Juan de Fuca Ridge flank.

To expand investigations into the phylogenetic diversity of microorganisms inhabiting the subseafloor biosphere, basalt-hosted crustal fluids were sampled from Circulation Obviation Retrofit Kits (CORKs) affixed to Holes 1025C and 1026B along the Juan de Fuca Ridge (JdFR) flank using a clean fluid pumping system. These boreholes penetrate the crustal aquifer of young ocean crust (1.24 and 3.51 million years old, respectively), but differ with respect to borehole depth and temperature at the sediment-basement interface (147 m and 39°C vs. 295 m and 64°C, respectively). Cloning and sequencing of PCR-amplified small subunit ribosomal RNA genes revealed that fluids retrieved from Hole 1025C were dominated by relatives of the genus Desulfobulbus of the Deltaproteobacteria (56% of clones) and Candidatus Desulforudis of the Firmicutes (17%). Fluids sampled from Hole 1026B also contained plausible deep subseafloor inhabitants amongst the most abundant clone lineages; however, both geochemical analysis and microbial community structure reveal the borehole to be compromised by bottom seawater intrusion. Regardless, this study provides independent support for previous observations seeking to identify phylogenetic groups of microorganisms common to the deep ocean crustal biosphere, and extends previous observations by identifying additional lineages that may be prevalent in this unique environment.

Activity and phylogenetic diversity of sulfate-reducing microorganisms in low-temperature subsurface fluids within the upper oceanic crust.

The basaltic ocean crust is the largest aquifer system on Earth, yet the rates of biological activity in this environment are unknown. Low-temperature (<100°C) fluid samples were investigated from two borehole observatories in the Juan de Fuca Ridge (JFR) flank, representing a range of upper oceanic basement thermal and geochemical properties. Microbial sulfate reduction rates (SRR) were measured in laboratory incubations with (35)S-sulfate over a range of temperatures and the identity of the corresponding sulfate-reducing microorganisms (SRM) was studied by analyzing the sequence diversity of the functional marker dissimilatory (bi)sulfite reductase (dsrAB) gene. We found that microbial sulfate reduction was limited by the decreasing availability of organic electron donors in higher temperature, more altered fluids. Thermodynamic calculations indicate energetic constraints for metabolism, which together with relatively higher cell-specific SRR reveal increased maintenance requirements, consistent with novel species-level dsrAB phylotypes of thermophilic SRM. Our estimates suggest that microbially-mediated sulfate reduction may account for the removal of organic matter in fluids within the upper oceanic crust and underscore the potential quantitative impact of microbial processes in deep subsurface marine crustal fluids on marine and global biogeochemical carbon cycling.

Microbial diversity within basement fluids of the sediment-buried Juan de Fuca Ridge flank.

Despite its immense size, logistical and methodological constraints have largely limited microbiological investigations of the subseafloor basement biosphere. In this study, a unique sampling system was used to collect fluids from the subseafloor basaltic crust via a Circulation Obviation Retrofit Kit (CORK) observatory at Integrated Ocean Drilling Program borehole 1301A, located at a depth of 2667 m in the Pacific Ocean on the eastern flank of the Juan de Fuca Ridge. Here, a fluid delivery line directly accesses a 3.5 million years old basalt-hosted basement aquifer, overlaid by 262 m of sediment, which serves as a barrier to direct exchange with bottom seawater. At an average of 1.2 × 10(4) cells ml(-1), microorganisms in borehole fluids were nearly an order of magnitude less abundant than in surrounding bottom seawater. Ribosomal RNA genes were characterized from basement fluids, providing the first snapshots of microbial community structure using a high-integrity fluid delivery line. Interestingly, microbial communities retrieved from different CORKs (1026B and 1301A) nearly a decade apart shared major community members, consistent with hydrogeological connectivity. However, over three sampling years, the dominant gene clone lineage changed from relatives of Candidatus Desulforudis audaxviator within the bacterial phylum Firmicutes in 2008 to the Miscellaneous Crenarchaeotic Group in 2009 and a lineage within the JTB35 group of Gammaproteobacteria in 2010, and statistically significant variation in microbial community structure was observed. The enumeration of different phylogenetic groups of cells within borehole 1301A fluids supported our observation that the deep subsurface microbial community was temporally dynamic.

Iron coated pottery granules for arsenic removal from drinking water.

A new media, iron coated pottery granules (ICPG) has been developed for As removal from drinking water. ICPG is a solid phase media that produces a stable Fe-Si surface complex for arsenic adsorption. Scanning electron microscopy (SEM) was used to document the physical attributes (grain size, pore size and distribution, surface roughness) of the ICPG media. Several advantages of the ICPG media such as (a) its granular structure, (b) its ability to absorb As via the F(0) coating on the granules' surface; (c) the inexpensive preparation process for the media from clay material make ICPG media a highly effective media for removing arsenic at normal pH. A column filtration test demonstrated that within the stability region (flow rate lower than 15L/h, EBCT >3 min), the concentration of As in the influent was always lower than 50 microg/L. The 2-week system ability test showed that the media consistently removed arsenic from test water to below the 5 microg/L level. The average removal efficiencies for total arsenic, As(III), and As(V) for a 2-week test period were 98%, 97%, and 99%, respectively, at an average flow rate of 4.1L/h and normal pH. Measurements of the Freundlich and Langmuir isotherms at normal pH show that the Freundlich constants of the ICPG are very close to those of ferric hydroxide, nanoscale zero-valent iron and much higher than those of nanocrystalline titanium dioxide. The parameter 1/n is smaller than 0.55 indicating a favorable adsorption process [K. Hristovski, A. Baumgardner, P. Westerhoff, Selecting metal oxide nanomaterials for arsenic removal in fixed bed columns: from nanopowders to aggregated nanoparticle media, J. Hazard. Mater. 147 (2007) 265-274]. The maximum adsorption capacity (q(e)) of the ICPG from the Langmuir isotherm is very close to that of nanoscale zero-valent indicating that zero-valent iron is involved in the process of the As removal from the water. The results of the toxicity characteristic leaching procedure (TCLP) analysis revealed that the media was non-hazardous, as shown by the ND (non-detectable) result for arsenic. The mechanism of As adsorption by ICPG has not been determined. Formation of Fe-Si complexes on the surface of the ICPG system may be responsible for the tight bonding of the As to the IGPC media.

Towards measuring particle-associated fecal indicator bacteria in tropical streams.

A protocol to enumerate particle-associated microbial indicator bacteria (heterotrophic plate count [HPC], enterococci [ENT] and Clostridium perfringens [CP]) by membrane filtration in a tropical stream is proposed that relies on high-speed homogenization and chemical treatment. Application of this protocol to stream samples suggest that ENT measurements are more biased by the presence of aggregates than HPC or CP. Whole sample treatment typically increased the colony forming units (CFU) count by 9-52%. Analysis of different settled fractions and examination of the number of indicators recoverable from particles retained on a 5 microm filter in relation to the number of particles containing target indicators both indicate that relatively more ENT form aggregates than HPC or CP. Although the bias is smallest for CP, this does not imply that CP is a better indicator as this depends on the unknown extent to which pathogens are themselves found associated with particles.

The microbial biosphere of sediment-buried oceanic basement.

Low-temperature hydrothermal fluids, circulating within the vast volume of sediment-buried upper oceanic basement on the flanks of the global mid-ocean ridge system, represent a largely unexplored habitat that could potentially host a significant and unique microbial biosphere. The present state of knowledge and many remaining challenges are discussed.

Processing deep-sea particle-rich water samples for fluorescence in situ hybridization: consideration of storage effects, preservation, and sonication.

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters.

Autotrophic ammonia oxidation in a deep-sea hydrothermal plume.

Direct evidence for autotrophic ammonia oxidation is documented for the first time in a deep-sea hydrothermal plume. Elevated NH(4) (+) concentrations of up to 341+/-136 nM were recorded in the plume core at Main Endeavour Field, Juan de Fuca Ridge. This fueled autotrophic ammonia oxidation rates as high as 91 nM day(-1), or 92% of the total net NH(4) (+) removal. High abundance of ammonia-oxidizing bacteria was detected using fluorescence in situ hybridization. Ammonia-oxidizing bacteria within the plume core (1.0-1.4x10(4) cells ml(-1)) accounted for 7.0-7.5% of the total microbial community, and were at least as abundant as methanotrophs. Ammonia-oxidizing bacteria were a substantial component of the particle-associated communities (up to 51%), with a predominance of the r-strategist Nitrosomonas-like cells. In situ chemolithoautotrophic organic carbon production via ammonia oxidation may yield 3.9-18 mg C m(-2) day(-1) within the plume directly over Main Endeavour Field. This rate was comparable to that determined for methane oxidation in a previous study, or at least four-fold greater than the flux of photosynthetic carbon reaching plume depths measured in another study. Hence, autotrophic ammonia oxidation in the neutrally buoyant hydrothermal plume is significant to both carbon and nitrogen cycling in the deep-sea water column at Endeavour, and represents another important link between seafloor hydrothermal systems and deep-sea biogeochemistry.

Branched aliphatic alkanes with quaternary substituted carbon atoms in modern and ancient geologic samples.

A pseudohomologous series of branched aliphatic alkanes with a quaternary substituted carbon atom (BAQCs, specifically 2,2-dimethylalkanes and 3,3- and 5,5-diethylalkanes) were identified in warm (65 degrees C) deep-sea hydrothermal waters and Late Cretaceous black shales. 5,5-Diethylalkanes were also observed in modern and Holocene marine shelf sediments and in shales spanning the last 800 million years of the geological record. The carbon number distribution of BAQCs indicates a biological origin. These compounds were observed but not identified in previous studies of 2.0 billion- to 2.2 billion-year-old metasediments and were commonly misidentified in other sediment samples, indicating that BAQCs are widespread in the geological record. The source organisms of BAQCs are unknown, but their paleobiogeographic distribution suggests that they have an affinity for sulfides and might be nonphotosynthetic sulfide oxidizers.

Fluids from aging ocean crust that support microbial life.

Little is known about the potential for life in the vast, low-temperature (<100 degrees C) reservoir of fluids within mid-ocean ridge flank and ocean basin crust. Recently, an overpressured 300-meter-deep borehole was fitted with an experimental seal (CORK) delivering crustal fluids to the sea floor for discrete and large-volume sampling and characterization. Results demonstrate that the 65 degrees C fluids from 3.5-million-year-old ocean crust support microbial growth. Ribosomal RNA gene sequence data indicate the presence of diverse Bacteria and Archaea, including gene clones of varying degrees of relatedness to known nitrate reducers (with ammonia production), thermophilic sulfate reducers, and thermophilic fermentative heterotrophs, all consistent with fluid chemistry.