Yoshiko Fujita

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.

The Effect of the CO32- to Ca2+ Ion activity ratio on calcite precipitation kinetics and Sr2+partitioning

BackgroundA proposed strategy for immobilizing trace metals in the subsurface is to stimulate calcium carbonate precipitation and incorporate contaminants by co-precipitation. Such an approach will require injecting chemical amendments into the subsurface to generate supersaturated conditions that promote mineral precipitation. However, the formation of reactant mixing zones will create gradients in both the saturation state and ion activity ratios (i.e., aCO32-/aCa2+). To better understand the effect of ion activity ratios on CaCO3 precipitation kinetics and Sr2+ co-precipitation, experiments were conducted under constant composition conditions where the supersaturation state (Ω) for calcite was held constant at 9.4, but the ion activity ratio (r=aCO32-/aCa2+) was varied between 0.0032 and 4.15.ResultsCalcite was the only phase observed, by XRD, at the end of the experiments. Precipitation rates increased from 41.3 ± 3.4 μmol m-2 min-1 at r = 0.0315 to a maximum rate of 74.5 ± 4.8 μmol m-2 min-1 at r = 0.306 followed by a decrease to 46.3 ± 9.6 μmol m-2 min-1 at r = 1.822. The trend was simulated using a simple mass transfer model for solute uptake at the calcite surface. However, precipitation rates at fixed saturation states also evolved with time. Precipitation rates accelerated for low r values but slowed for high r values. These trends may be related to changes in effective reactive surface area. The aCO32-/aCa2+ ratios did not affect the distribution coefficient for Sr in calcite (DPSr2+), apart from the indirect effect associated with the established positive correlation between DPSr2+ and calcite precipitation rate.ConclusionAt a constant supersaturation state (Ω = 9.4), varying the ion activity ratio affects the calcite precipitation rate. This behavior is not predicted by affinity-based rate models. Furthermore, at the highest ion ratio tested, no precipitation was observed, while at the lowest ion ratio precipitation occurred immediately and valid rate measurements could not be made. The maximum measured precipitation rate was 2-fold greater than the minima, and occurred at a carbonate to calcium ion activity ratio of 0.306. These findings have implications for predicting the progress and cost of remediation operations involving enhanced calcite precipitation where mineral precipitation rates, and the spatial/temporal distribution of those rates, can have significant impacts on the mobility of contaminants.

Geophysical monitoring and reactive transport modeling of ureolytically-driven calcium carbonate precipitation

Ureolytically-driven calcium carbonate precipitation is the basis for a promising in-situ remediation method for sequestration of divalent radionuclide and trace metal ions. It has also been proposed for use in geotechnical engineering for soil strengthening applications. Monitoring the occurrence, spatial distribution, and temporal evolution of calcium carbonate precipitation in the subsurface is critical for evaluating the performance of this technology and for developing the predictive models needed for engineering application. In this study, we conducted laboratory column experiments using natural sediment and groundwater to evaluate the utility of geophysical (complex resistivity and seismic) sensing methods, dynamic synchrotron x-ray computed tomography (micro-CT), and reactive transport modeling for tracking ureolytically-driven calcium carbonate precipitation processes under site relevant conditions. Reactive transport modeling with TOUGHREACT successfully simulated the changes of the major chemical components during urea hydrolysis. Even at the relatively low level of urea hydrolysis observed in the experiments, the simulations predicted an enhanced calcium carbonate precipitation rate that was 3-4 times greater than the baseline level. Reactive transport modeling results, geophysical monitoring data and micro-CT imaging correlated well with reaction processes validated by geochemical data. In particular, increases in ionic strength of the pore fluid during urea hydrolysis predicted by geochemical modeling were successfully captured by electrical conductivity measurements and confirmed by geochemical data. The low level of urea hydrolysis and calcium carbonate precipitation suggested by the model and geochemical data was corroborated by minor changes in seismic P-wave velocity measurements and micro-CT imaging; the latter provided direct evidence of sparsely distributed calcium carbonate precipitation. Ion exchange processes promoted through NH4+ production during urea hydrolysis were incorporated in the model and captured critical changes in the major metal species. The electrical phase increases were potentially due to ion exchange processes that modified charge structure at mineral/water interfaces. Our study revealed the potential of geophysical monitoring for geochemical changes during urea hydrolysis and the advantages of combining multiple approaches to understand complex biogeochemical processes in the subsurface.

Responses of Ammonia-Oxidizing Bacterial and Archaeal Populations to Organic Nitrogen Amendments in Low-Nutrient Groundwater

ABSTRACT To evaluate the potential for organic nitrogen addition to stimulate the in situ growth of ammonia oxidizers during a field scale bioremediation trial, samples collected from the Eastern Snake River Plain Aquifer in Idaho before, during, and after the addition of molasses and urea were subjected to PCR analysis of ammonia monooxygenase subunit A (amoA) genes. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were present in all of the samples tested, with AOA amoA genes outnumbering AOB amoA genes in all of the samples. Following urea addition, nitrate levels rose and bacterial amoA copy numbers increased dramatically, suggesting that urea hydrolysis stimulated nitrification. Bacterial amoA diversity was limited to two Nitrosomonas phylotypes, whereas archaeal amoA analyses revealed 20 distinct operational taxonomic units, including several that were markedly different from all previously reported sequences. Results from this study demonstrate the likelihood of stimulating ammonia-oxidizing communities during field scale manipulation of groundwater conditions to promote urea hydrolysis.

Design and Validation of ureC-based Primers for Groundwater Detection of Urea-Hydrolyzing Bacteria

Polymerase chain reaction primers based on the ureC gene are described for use in detecting diverse groundwater urea-hydrolyzing bacteria. Six degenerate primers were designed and evaluated for their ability to detect the gene encoding the large catalytic subunit of urease, ureC. Five combinations of these primers were tested pair-wise and displayed an overlapping detection range for bacterial isolates. Pair L2F/L2R exhibited the greatest detection range for described bacterial species and for bacterial isolates from groundwater samples belonging to the bacterial divisions Firmicutes, Actinobacteria, and the α , β , and γ subdivisions of Proteobacteria. Primers L2F/L2R exhibited a greater detection range than previously described ureC-specific primers, and amplified novel ureC sequences from groundwater isolates in the genera Hydrogenophaga, Acidovorax, Janthinobacterium, and Arthrobacter. A comparative phylogenetic analysis of ureC and 16S rRNA genes was performed to determine the utility of groundwater ureC sequence information as a phylogenetic marker for ureolytic species. Our results were consistent with previous analyses of urease genes which demonstrated that the ureC gene has undergone lateral transfer and is not a robust phylogenetic marker. However, the ureC-specific primers, L2F/L2R, demonstrate a broad detection range for ureolytic species, and can serve to enhance functional diversity analyses of ureolytic bacteria.

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100‐bp insertion in helix 6

Two different versions of the 16S rRNA gene, one of which contained an unusual 100-bp insertion in helix 6, were detected in isolate UFO1 acquired from the Oak Ridge Integrated Field-Research Challenge (ORIFRC) site in Tennessee. rRNA was extracted from UFO1 and analyzed by reverse transcriptase-quantitative PCR with insert- and non-insert-specific primers; only the noninsert 16S rRNA gene sequence was detected. Similarly, PCR-based screening of a cDNA library (190 clones) constructed from reverse-transcribed rRNA from UFO1 did not detect any clones containing the 100-bp insert. Examination of cDNA with primers specific to the insert-bearing 16S rRNA gene, but downstream of the insert, suggests that the insert was excised from rRNA. Inspection of other 16S rRNA genes in the GenBank database revealed that a homologous insert sequence, also found in helix 6, has been reported in other environmental clones, including those acquired from ORIFRC enrichments. These findings demonstrate the existence of widely divergent copies of the 16S rRNA gene within the same organism, which may confound 16S rRNA gene-based methods of estimating microbial diversity in environmental samples.

Inducing mineral precipitation in groundwater by addition of phosphate

BackgroundInduced precipitation of phosphate minerals to scavenge trace elements from groundwater is a potential remediation approach for contaminated aquifers. The success of engineered precipitation schemes depends on the particular phases generated, their rates of formation, and their long term stability. The purpose of this study was to examine the precipitation of calcium phosphate minerals under conditions representative of a natural groundwater. Because microorganisms are present in groundwater, and because some proposed schemes for phosphate mineral precipitation rely on stimulation of native microbial populations, we also tested the effect of bacterial cells (initial densities of 105 and 107 mL-1) added to the precipitation medium. In addition, we tested the effect of a trace mixture of propionic, isovaleric, formic and butyric acids (total concentration 0.035 mM).ResultsThe general progression of mineral precipitation was similar under all of the study conditions, with initial formation of amorphous calcium phosphate, and transformation to poorly crystalline hydroxylapatite (HAP) within one week. The presence of the bacterial cells appeared to delay precipitation, although by the end of the experiments the overall extent of precipitation was similar for all treatments. The stoichiometry of the final precipitates as well as Rietveld structure refinement using x-ray diffraction data indicated that the presence of organic acids and bacterial cells resulted in an increasing a and decreasing c lattice parameter, with the higher concentration of cells resulting in the greatest distortion. Uptake of Sr into the solids was decreased in the treatments with cells and organic acids, compared to the control.ConclusionsOur results suggest that the minerals formed initially during an engineered precipitation application for trace element sequestration may not be the ones that control long-term immobilization of the contaminants. In addition, the presence of bacterial cells appears to be associated with delayed HAP precipitation, changes in the lattice parameters, and reduced incorporation of trace elements as compared to cell-free systems. Schemes to remediate groundwater contaminated with trace metals that are based on enhanced phosphate mineral precipitation may need to account for these phenomena, particularly if the remediation approach relies on enhancement of in situ microbial populations.

Experimental and Numerical Analysis of Parallel Reactant Flow and Transverse Mixing with Mineral Precipitation in Homogeneous and Heterogeneous Porous Media

Formation of mineral precipitates in the mixing interface between two reactant solutions flowing in parallel in porous media is governed by reactant mixing by diffusion and dispersion and is coupled to changes in porosity/permeability due to precipitation. The spatial and temporal distribution of mixing-dependent precipitation of barium sulfate in porous media was investigated with side-by-side injection of barium chloride and sodium sulfate solutions in thin rectangular flow cells packed with quartz sand. The results for homogeneous sand beds were compared to beds with higher or lower permeability inclusions positioned in the path of the mixing zone. In the homogeneous and high permeability inclusion experiments, BaSO