Frederick S. Colwell

Microbial Communities from Methane Hydrate-Bearing Deep Marine Sediments in a Forearc Basin

ABSTRACT Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35°C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.

Subscribed Content Calcium Carbonate Precipitation by Ureolytic Subsurface Bacteria

Coprecipitation in carbonate minerals offers a means of slowing the transport of divalent radionuclides and contaminant metals (e.g.,90Sr2+, UO2+, Co2+) in the subsurface. It may be possible to accelerate this process by stimulating the native microbial community to generate chemical conditions favoring carbonate precipitation. In a preliminary evaluation of this approach, we investigated the ability of ureolytic subsurface bacteria to produce alkaline conditions conducive to calcium carbonate precipitation. Groundwater samples from the Eastern Snake River Plain (ESRP) aquifer in Idaho were screened for urea-hydrolyzing microorganisms; three isolates were selected for further evaluation. Analysis of 16S rRNA gene sequences indicated that two of the ESRP isolates were of the genus Pseudomonas , and the other was a Variovorax sp. The specific urease activities of the ESRP isolates appeared to be similar to each other but less than that of Bacillus pasteurii , a known urease-positive organism. However, calcium carbonate was rapidly precipitated in all cultures that were supplied with urea and calcium, and X-ray diffraction analyses indicated that calcite was always the predominant carbonate polymorph produced. The correspondence between measured calcium concentrations and equilibrium predictions suggested that the rate of calcite precipitation was directly linked to the rate of urea hydrolysis. These results are promising with respect to the potential utility of this approach for in situ remediation and indicate that further evaluation of this approach under conditions more closely simulating environmental conditions is warranted.

Pore‐size constraints on the activity and survival of subsurface bacteria in a late cretaceous shale‐sandstone sequence, northwestern New Mexico

To investigate the distribution of microbial biomass and activities to gain insights into the physical controls on microbial activity and potential long‐term survival in the subsurface, 24 shale and sandstone cores were collected from a site in northwestern New Mexico. Bacterial biomass in the core samples ranged from below detection to 31.9 pmol total phospholipid fatty acid (PLFA) g‐1 of rock with no apparent relationship between lithology and PLFA abundance. No metabolic activities, as determined by anaerobic mineralization of [14C]acetate and [14C]glucose and 35SO4 2‐ reduction, were detected in core samples with pore throats <0.2 fan in diameter, smaller than the size of known bacteria. However, enrichments revealed the presence of sulfate‐re‐ducing bacteria, and 35SO4 2‐ reduction was detected upon extended (14 days) incubation in some small‐pore‐throat samples. In contrast, relatively rapid rates of metabolic activity were more common in core samples containing a significant fraction of pore throat...

Isolation of a Methanogen from Deep Marine Sediments That Contain Methane Hydrates, and Description of Methanoculleus submarinus sp. nov.

ABSTRACT We isolated a methanogen from deep in the sediments of the Nankai Trough off the eastern coast of Japan. At the sampling site, the water was 950 m deep and the sediment core was collected at 247 m below the sediment surface. The isolated methanogen was named Nankai-1. Cells of Nankai-1 were nonmotile and highly irregular coccoids (average diameter, 0.8 to 2 μm) and grew with hydrogen or formate as a catabolic substrate. Cells required acetate as a carbon source. Yeast extract and peptones were not required but increased the growth rate. The cells were mesophilic, growing most rapidly at 45°C (no growth at ≤10°C or ≥55°C). Cells grew with a maximum specific growth rate of 2.43 day−1 at 45°C. Cells grew at pH values between 5.0 and 8.7 but did not grow at pH 4.7 or 9.0. Strain Nankai-1 grew in a wide range of salinities, from 0.1 to 1.5 M Na+. The described phenotypic characteristics of this novel isolate were consistent with the in situ environment of the Nankai Trough. This is the first report of a methanogenic isolate from methane hydrate-bearing sediments. Phylogenetic analysis of its 16S rRNA gene sequence indicated that it is most closely related to Methanoculleus marisnigri (99.1% sequence similarity), but DNA hybridization experiments indicated a DNA sequence similarity of only 49%. Strain Nankai-1 was also found to be phenotypically similar to M. marisnigri, but two major phenotypic differences were found: strain Nankai-1 does not require peptones, and it grows fastest at a much higher temperature. We propose a new species, Methanoculleus submarinus, with strain Nankai-1 as the type strain.

Metagenomic profiling : microarray analysis of an environmental genomic library

ABSTRACT Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ∼1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.

Attached and unattached microbial communities in a simulated basalt aquifer under fracture- and porous-flow conditions.

ABSTRACT Bench scale column studies were used to examine the partitioning of microorganisms between groundwater and a geologic medium and to examine the effect of hydrogeology (i.e., porous- versus fracture-flow) on organism partitioning. Replicated columns were constructed with intact basalt core segments that contained natural fractures and with the same basalt crushed into particles. The columns were perfused with groundwater, and upon reaching a steady state, the columns were sacrificed and the attached and unattached communities were analyzed by multiple approaches. The analyses included the total number of cells, the phylogenetic affiliation of the cells (i.e., the α, β, and γ subclasses of the class Proteobacteria and gram positives with high G+C DNA content) by fluorescent in situ hybridization (FISH), number and taxonomic affiliation by fatty acid methyl ester profiles of culturable heterotrophs, most-probable-number estimates of methanotrophs and phenol oxidizers, and whole-community sole carbon source utilization patterns from Biolog GN microplates. In the packed columns, about 99% of the total biomass (per cubic centimeter of porous medium) was attached to the geologic medium. Lack of equitable units precluded a comparison of attached and unattached biomasses in the fractured columns where the attached biomass was expressed per unit of surface area. Compositional differences in the attached and unattached communities were evidenced by (i) the recovery ofPseudomonas stutzeri, an Enterococcus sp., andBacillus psychrophilus from the groundwater and not from the basalt, (ii) differences between community carbon source utilization patterns, and (iii) the relative abundances of different phylogenetic groups estimated by FISH in both column types. In the packed columns, attached communities were depleted of members of the α- and β-Proteobacteria subclasses in comparison to those in the corresponding groundwater. In the fractured columns, attached communities were enriched in gram-positive Bacteriaand γ-Proteobacteria and depleted of β-Proteobacteria, in comparison to those in the corresponding groundwater. Segregation of populations and their activities, possibly modified by attachment to geologic media, may influence contaminant fate and transport in the subsurface and impact other in situ applications.

Ages and magnetic structures of the South China Sea constrained by deep tow magnetic surveys and IODP Expedition 349

Combined analyses of deep tow magnetic anomalies and International Ocean Discovery Program Expedition 349 cores show that initial seafloor spreading started around 33 Ma in the northeastern South China Sea (SCS), but varied slightly by 1-2 Myr along the northern continent-ocean boundary (COB). A southward ridge jump of approximate to 20 km occurred around 23.6 Ma in the East Subbasin; this timing also slightly varied along the ridge and was coeval to the onset of seafloor spreading in the Southwest Subbasin, which propagated for about 400 km southwestward from approximate to 23.6 to approximate to 21.5 Ma. The terminal age of seafloor spreading is approximate to 15 Ma in the East Subbasin and approximate to 16 Ma in the Southwest Subbasin. The full spreading rate in the East Subbasin varied largely from approximate to 20 to approximate to 80 km/Myr, but mostly decreased with time except for the period between approximate to 26.0 Ma and the ridge jump (approximate to 23.6 Ma), within which the rate was the fastest at approximate to 70 km/Myr on average. The spreading rates are not correlated, in most cases, to magnetic anomaly amplitudes that reflect basement magnetization contrasts. Shipboard magnetic measurements reveal at least one magnetic reversal in the top 100 m of basaltic layers, in addition to large vertical intensity variations. These complexities are caused by late-stage lava flows that are magnetized in a different polarity from the primary basaltic layer emplaced during the main phase of crustal accretion. Deep tow magnetic modeling also reveals this smearing in basement magnetizations by incorporating a contamination coefficient of 0.5, which partly alleviates the problem of assuming a magnetic blocking model of constant thickness and uniform magnetization. The primary contribution to magnetic anomalies of the SCS is not in the top 100 m of the igneous basement.

Combined microbial community-level analyses for quality assurance of terrestrial subsurface cores☆

Bacterial communities from surface soils, groundwater, drilling muds and deep subsurface cores were profiled by sole carbon source utilization and by phospholipid ester-linked fatty acid analysis. The combination of these functional and structural methods successfully distinguished communities from disparate origins. Multivariate analysis of the data showed good agreement between the results of the two methods. Subsurface communities tended to respire amino acids over carbohydrates and demonstrated preferential use of individual compounds such as acetate and Tween as sole carbon sources. PLFA profiles indicated that the groundwaters predominately contained gram negative aerobic heterotrophic populations, the drilling muds and cuttings were populated by gram negative anaerobes and the core communities were composed of anaerobic gram negative bacteria and gram positive bacteria. The utility of this approach as a component of quality assurance of core samples obtained for microbiological analysis during mud rotary coring was demonstrated. Monitoring of controlled bioprocesses, environmental remediation and detection of environmental disturbance are some of the numerous potential applications for these community-level characterization methods. Since combined analyses such as these can simultaneously provide specific information about individual community members and about community-level function, it is hoped that these methods will prove useful in answering fundamental questions in microbial ecology, such as the relationship between in situ community structure and its measurable function.

Observations pertaining to the origin and ecology of microorganisms recovered from the deep subsurface of Taylorsville Basin, Virginia

To understand the conditions under which microorganisms exist in deep hydrocarbon reservoirs, sidewall cores were collected from a natural gas‐bearing formation, 2800 m below the surface in Taylorsville Basin, Virginia. Data from chemical and microbial tracers and controls indicate that the interiors of some sidewall cores contained microorganisms indigenous to the rock formation. The cultured microorganisms were composed primarily of saline‐tolerant, thermophilic fermenting, Fe(III)‐reducing, and sulfate‐reducing bacteria (1 to 104 cells/g). The physiological capabilities of the cultured microorganisms are compatible with the temperature (76°C), pressure (32 MPa), and salinity (≈0.8 wt.% NaCl equivalent) in the sampled interval. The petrological data indicated that the strata contain intercrystalline pores of micrometer size, that occur between late diagenetic cement in siltstone and within cross‐cutting, mineralized fractures in shale. These pores made up only 0.04% of the rock volume, were mostly gas‐f...

Estimates of biogenic methane production rates in deep marine sediments at Hydrate Ridge, Cascadia Margin

ABSTRACT Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.