Acid mine drainage biogeochemistry at Iron Mountain,California

The Richmond Mine at Iron Mountain, Shasta County, California, USA provides an excellent opportunity to study the chemical and biological controls on acid mine drainage (AMD) generation in situ, and to identify key factors controlling solution chemistry. Here we integrate four years of field-based geochemical data with 16S rRNA gene clone libraries and rRNA probe-based studies of microbial population structure, cultivation-based metabolic experiments, arsenopyrite surface colonization experiments, and results of intermediate sulfur species kinetics experiments to describe the Richmond Mine AMD system. Extremely acidic effluent (pH between 0.5 and 0.9) resulting from oxidation of approximately 1 × 10⁵ to 2 × 10⁵ moles pyrite/day contains up to 24 g/1 Fe, several g/1 Zn and hundreds of mg/l Cu. Geochemical conditions change markedly over time, and are reflected in changes in microbial populations. Molecular analyses of 232 small subunit ribosomal RNA (16S rRNA) gene sequences from six sites during a sampling time when lower temperature (<32°C), higher pH (>0.8) conditions predominated show the dominance of Fe-oxidizing prokaryotes such as Ferroplasma and Leptospirillum in the primary drainage communities. Leptospirillum group III accounts for the majority of Leptospirillum sequences, which we attribute to anomalous physical and geochemical regimes at that time. A couple of sites peripheral to the main drainage, "Red Pool" and a pyrite "Slump," were even higher in pH (>1) and the community compositions reflected this change in geochemical conditions. Several novel lineages were identified within the archaeal Thermoplasmatales order associated with the pyrite slump, and the Red Pool (pH 1.4) contained the only population of Acidithiobacillus. Relatively small populations of Sulfobacillus spp. and Acidithiobacillus caldus may metabolize elemental sulfur as an intermediate species in the oxidation of pyritic sulfide to sulfate. Experiments show that elemental sulfur which forms on pyrite surfaces is resistant to most oxidants; its solublization by unattached cells may indicate involvement of a microbially derived electron shuttle. The detachment of thiosulfate (S₂O₃^2-) as a leaving group in pyrite oxidation should result in the formation and persistence of tetrathionate in low pH ferric iron-rich AMD solutions. However, tetrathionate is not observed. Although a S₂O₃^2--like species may form as a surface-bound intermediate, data suggest that Fe³⁺ oxidizes the majority of sulfur to sulfate on the surface of pyrite. This may explain why microorganisms that can utilize intermediate sulfur species are scarce compared to Fe-oxidizing taxa at the Richmond Mine site.

     

Biogeochemical processes in a clay formation in situ experiment: Part D - Microbial analyses - Synthesis of results

The purpose of the Porewater Chemistry (PC) experiment at the Mont Terri (MT) Underground Rock Laboratory (URL) was to measure geochemical parameters, such as pH, Eh and pCO₂, in the porewater of the Opalinus Clay formation. Although the PC experiment was designed and implemented carefully from a geochemical perspective, conditions were not sterile and some microbial and nutrient contamination likely occurred. Microbial activity in the added synthetic porewater in the borehole was apparent shortly after initiation of the experiment and affected the geochemical parameters observed in the porewater. This paper summarizes the results from microbial analyses of post-termination PC water and overcore clay samples, conducted to attempt to elucidate the role of microbial activity in the evolution of the geochemical conditions in the PC experiment. Microbial analyses of the PC borehole water, and of clay overcore samples from around the borehole, were carried out at three laboratories and included both molecular biology and culturing methods.Results indicated the presence of heterotrophic aerobic and anaerobic organisms that resulted likely from the initial, non-sterile conditions, sustained by suspected contamination with organic matter (glycerol, acetone). The results also indicated the presence of NO₃-reducers, Fe-reducers, SO₄-reducers and methanogens (i.e., Bacteria as well as Archaea), suggesting a reducing environment with Fe(III)- and SO₄ reduction, and methanogenesis occurring in the PC water and adjacent clay. A black precipitate containing pyrite (identified by XRD and SEM) and a strong H₂S smell in the porewater confirmed the occurrence of SO₄ reduction. Microorganisms identified in the porewater included Pseudomonas stutzeri, Bacillus licheniformis, Desulfosporosinus spp. and Hyphomonas spp. Species identified in enrichment cultures from the overcore samples included Pseudomonas stutzeri, three species of Trichococcus spp., Caldanaerocella colombiensis, Geosporobacter subterrenus and Desulfosporosinus lacus. Overall the results indicated a thriving microbial community in the PC water and adjacent clay in contrast to “undisturbed” Opalinus Clay for which limited evidence for a small viable microbial community has been given in a previous study.     

Carbon isotope fractionation in phospholipid fatty acid biomarkers of bacteria and fungi native to an acid mine drainage lake

This study identifies isotope signatures associated with autotrophic and heterotrophic microbial communities that may provide a means to determine carbon cycling relationships in situ for acid mine drainage (AMD) sites. Stable carbon isotope ratios (δ¹³C) of carbon sources, bulk cells, and membrane phospholipids (PLFA) were measured for autotrophic and heterotrophic microbial enrichment cultures from a mine tailings impoundment in northern Ontario, Canada, and for pure strains of the sulfur oxidizing bacteria Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The autotrophic enrichments had indistinguishable PLFA distributions from the pure cultures, and the PLFA cyc-C_19:0 was determined to be a unique biomarker in this system for these sulfur oxidizing bacteria. The PLFA distributions produced by the heterotrophic enrichments were distinct from the autotrophic distributions and the C_18:2 PLFA was identified as a biomarker for these heterotrophic enrichments. Genetic analysis (16S, 18S rRNA) of the heterotrophic cultures indicated that these communities were primarily composed of Acremonium fungi.Stable carbon isotope analysis revealed that bulk cellular material in all autotrophic cultures was depleted in δ¹³C by 5.6–10.9‰ relative to their atmospheric CO₂ derived carbon source, suggesting that inorganic carbon fixation in these cultures is carbon limited. Individual PLFA from these autotrophs were further depleted by 8.2–14.6‰ compared to the bulk cell δ¹³C, which are among the largest biosynthetic isotope fractionation factors between bulk cell and PLFA reported in the literature. In contrast, the heterotrophic bulk cells were not significantly fractionated in δ¹³C relative to their carbon source and heterotrophic PLFA ranged from 3‰ enriched to 4‰ depleted relative to the isotopic composition of their total biomass. These distinct PLFA biomarkers and isotopic fractionations associated with autotrophic and heterotrophic activity in this laboratory study provide potential biomarkers for delineating autotrophic and heterotrophic carbon cycling in AMD environments.     

A microbial pathway for the formation of gold-anomalous calcrete

The formation of pedogenic carbonate (calcrete) in terrestrial environments is commonly mediated by microorganisms. In Australia, Au-anomalous calcrete is an important sampling medium for geochemical exploration, but current models describing its formation do not include a confirmed microbial component. This study demonstrates that bacterial communities in calcareous sands from dunes overlying the Barns Gold Deposit in semi-arid South Australia, are capable of mediating the biomineralisation of Au-anomalous carbonates. Bacterial enrichment cultures obtained from calcareous sands at three depths (0.1, 0.64 and 2.1 m, plus abiotic control) were incubated in urea and Ca²⁺-containing growth media (pH 8), unamended and amended with Au (100 parts-per-billion, ppb) as Au–aspartic-acid complex. During the incubation of the enrichment cultures urea was turned over to NH₄⁺ within 96 h to 220 h. The solution pH increased concurrently by approximately 1.2 units, and Au-anomalous Ca-carbonate crystallites were precipitated on cells, which functioned as nucleation sites; no carbonate precipitation was observed in abiotic controls. Compared to the medium, Au was strongly enriched in these carbonates and appeared to be uniformly dispersed in the individual crystallites, as shown using LA-ICP-MS; a similar distribution is present in naturally occurring Au-anomalous calcrete. Phylogenetic 16S rRNA PCR DGGE analyses, shotgun cloning and functional microbial analyses (BioLog, ureC quantitative PCR) demonstrated that naturally occurring and culture-enriched bacterial communities were dominated by alkaliphylic, halotolerant Bacillus spp. The indigenous bacterial communities were capable of utilising amino acids (including l-aspartic acid) and urea, which appears to lead to the destabilisation of the Au–amino acid complexes and concomitant co-precipitation of Au in the Ca-carbonates. In conclusion, a model combining geomicrobial– with evapotranspiration– and plant-based components is likely to best describe the formation of (Au-anomalous) calcrete in semi-arid and arid zones.     

Bioleaching potential of bacterial communities in historic mine waste areas

Microbial activity has the potential to alter all cultural heritage in mining and metallurgy, due to metal mobilization by leaching. This communication shows the consequences of the bioleaching ability of two natural enrichments on copper slag samples from a historic ore smelting site in Sangerhausen (Mansfeld, Südharz, Saxony-Anhalt, Germany). Enrichment cultures gained from mine drainage were dominated by either the iron and sulfur-oxidizing Acidithiobacillus ferrivorans, or by the iron-oxidizing Leptospirillum. During 35 days of bioleaching in media containing copper slag pulp, inoculated with these enrichments, the change in pH and solubilized metal concentrations of the systems were monitored. Both bacterial strains were completely different from each other in their pattern of pH variation and rates of metal solubilization. The maximum removal of Cu (1725 mg/l) and Zn (715 mg/l) from copper slag substrate was achieved with enrichment culture of A. ferrivorans SCUT-1. However, maximum Mn (207 mg/l), Pb (86 mg/l), and Ni (75 mg/l) removal was observed with enrichment culture of Leptospirillum strain YQP-1. Implications for metal mobilization along with alteration of artifacts from not only historic mining areas but also aspects of decontamination and remediation are discussed.     

Geochemical and mineralogical aspects of sulfide mine tailings

Tailings generated during processing of sulfide ores represent a substantial risk to water resources. The oxidation of sulfide minerals within tailings deposits can generate low-quality water containing elevated concentrations of SO₄, Fe, and associated metal(loid)s. Acid generated during the oxidation of pyrite [FeS₂], pyrrhotite [Fe_(1−x)S] and other sulfide minerals is neutralized to varying degrees by the dissolution of carbonate, (oxy)hydroxide, and silicate minerals. The extent of acid neutralization and, therefore, pore-water pH is a principal control on the mobility of sulfide-oxidation products within tailings deposits. Metals including Fe(III), Cu, Zn, and Ni often occur at high concentrations and exhibit greater mobility at low pH characteristic of acid mine drainage (AMD). In contrast, (hydr)oxyanion-forming elements including As, Sb, Se, and Mo commonly exhibit greater mobility at circumneutral pH associated with neutral mine drainage (NMD). These differences in mobility largely result from the pH-dependence of mineral precipitation–dissolution and sorption–desorption reactions. Cemented layers of secondary (oxy)hydroxide and (hydroxy)sulfate minerals, referred to as hardpans, may promote attenuation of sulfide-mineral oxidation products within and below the oxidation zone. Hardpans may also limit oxygen ingress and pore-water migration within sulfide tailings deposits. Reduction–oxidation (redox) processes are another important control on metal(loid) mobility within sulfide tailings deposits. Reductive dissolution or transformation of secondary (oxy)hydroxide phases can enhance Fe, Mn, and As mobility within sulfide tailings. Production of H₂S via microbial sulfate reduction may promote attenuation of sulfide-oxidation products, including Fe, Zn, Ni, and Tl, via metal-sulfide precipitation. Understanding the dynamics of these interrelated geochemical and mineralogical processes is critical for anticipating and managing water quality associated with sulfide mine tailings.     

Reactive iron(III) in sediments: Chemical versus microbial extractions

The availability of particulate Fe(III) to iron reducing microbial communities in sediments and soils is generally inferred indirectly by performing chemical extractions. In this study, the bioavailability of mineral-bound Fe(III) in intertidal sediments of a eutrophic estuary is assessed directly by measuring the kinetics and extent of Fe(III) utilization by the iron reducing microorganism Shewanella putrefaciens, in the presence of excess electron donor. Microbial Fe(III) reduction is compared to chemical dissolution of iron from the same sediments in buffered ascorbate-citrate solution (pH 7.5), ascorbic acid (pH 2), and 1 M HCl. The results confirm that ascorbate at near-neutral pH selectively reduces the reactive Fe(III) pool, while the acid extractants mobilize additional Fe(II) and less reactive Fe(III) mineral phases. Furthermore, the maximum concentrations of Fe(III) reducible by S. putrefaciens correlate linearly with the iron concentrations extracted by buffered ascorbate-citrate solution, but not with those of the acid extractions. However, on average, only 65% of the Fe(III) reduced in buffered ascorbate-citrate solution can be utilized by S. putrefaciens, probably due to physical inaccessibility of the remaining fraction of reactive Fe(III) to the cells. While the microbial and abiotic reaction kinetics further indicate that reduction by ascorbate at near-neutral pH most closely resembles microbial reduction of the sediment Fe(III) pool by S. putrefaciens, the results also highlight fundamental differences between chemical reductive dissolution and microbial utilization of mineral-bound ferric iron.     

Pyrite oxidation by Acidithiobacillus ferrooxidans at various concentrations of dissolved oxygen

Pyrite oxidation rates were examined at various concentrations of dissolved oxygen (DO) in the presence of the sulfur and iron oxidizer Acidithiobacillus ferrooxidans. Five different batch experiments were performed at room temperature for 75 days under various DO levels (273, 129, 64.8, 13.2, and ≤ 0.006 μM), containing pyrite grains (particle size 63–250 μm) and a modified 9K nutrient medium at pH 3. The reactors were inoculated with A. ferrooxidans. In all experiments, pH decreased with time and sulfur and iron were released to the solution, indicating pyrite oxidation at all DO levels. Pyrite oxidation rates (ca. 5 × 10^− 10 mol m^− 2 s^− 1 at 273 μM DO) from all experiments showed positive correlation with DO, Fe(III), and bacterial concentration. These rates were significantly slower than rates presented in other published studies, but this is probably due to the significantly greater Fe(III) concentration at lower pH in these previous studies. The results obtained in this study suggest that ferric iron reduction at the pyrite surface is the primarily mechanism for microbial pyrite oxidation in the presence of DO. The results from our study support the indirect mechanism of sulfide oxidation, where A. ferrooxidans oxidizes ferrous iron in the presence of DO, which then oxidizes pyrite.     

Isolation,biodegradation ability and molecular detection of hydrocarbon degrading bacteria in petroleum samples from a Brazilian offshore basin

The detection of microorganisms with potential for biodeterioration and biodegradation in petroleum fields is of great relevance, since these organisms may be related to a decrease in petroleum quality in the reservoirs or damage in the production facilities. In this sense, petroleum formation water and oil samples were collected from the Campos Basin, Brazil, with the aim of isolating microorganisms and evaluating their ability to degrade distinct classes of hydrocarbon biomarkers (9,10-dihydrophenanthrene, phytane, nonadecanoic acid and 5α-cholestane). Twenty eight bacterial isolates were recovered and identified by sequencing their 16S rRNA genes. Biodegradation assays revealed that bacterial metabolism of hydrocarbons occurred through reactions based on oxidation, carbon–carbon bond cleavage and generation of new bonds or by the physical incorporation of hydrocarbons into microbial cell walls. Based on the biodegradation results, selective PCR-based systems were developed for direct detection in petroleum samples of bacterial groups of interest, namely Bacillus spp., Micrococcus spp., Achromobacter xylosoxidans, Dietzia spp. and Bacillus pumilus. Primer sets targeting 16S rRNA genes were designed and their specificity was confirmed in silico (i.e. computational analysis) and in PCR reactions using DNA from reference strains as positive and negative controls. Total DNA from oil was purified and the amplification tests revealed the presence of the target bacteria in the samples, unraveling a significant potential for petroleum deterioration in the reservoirs sampled, once proper conditions are present for hydrocarbon degradation. The application of molecular methods for rapid detection of specific microorganisms in environmental samples would be valuable as a supporting tool for the evaluation of oil quality in production reservoirs.     

Reactive modelling of CO2 intrusion into freshwater aquifers: current requirements, approaches and limitations to account for temperature and pressure effects

The modelling of CO₂ intrusion into virtual freshwater aquifers after a leakage from CO₂ storage formations is a well-established approach for the identification of monitoring parameters and for the risk assessment. At presence, standard or close-to-standard conditions in terms of temperature (T), i.e. 25?°C and pressure (P), i.e. 1?C5?bar, are assumed. This approach neglects the fact that temperature and pressure conditions change with the depth of the freshwater aquifer. This study tests the accuracy of T?CP corrections of the geochemical constants in the system gaseous CO₂?Cwater?Cmineral which are performed by the simulators PhreeqC (Parkhurst and Appelo in User??s guide to phreeqc (version 2)??a computer program for speciation, batch reaction, one-dimensional transport, and inverse geochemical calculations. Technical report, US Department of the Interior, 1999) and TOUGHREACT (Xu et?al. in Toughreact user??s guide: a simulation program for non-isothermal multiphase reactive geochemical transport in variably saturated geologic media. Technical report, Lawrence Berkeley National Laboratory, 2004). It further identifies the impact of T and P variations on the predicted concentrations of the monitoring parameters pH and total inorganic carbon (TIC) and on the predicted concentration of the trace metal lead (Pb) in 3D multiphase-multicomponent simulations of virtual aquifers. The results reveal a strong imprecision in the correction of kinetic rates of mineral dissolution and a lack of corrections of sorption equilibrium states. The predicted pH and concentrations of TIC and lead depend strongly on the assumed T and P conditions. It is concluded that a neglect of T and P effects results in inaccurate predictions of groundwater chemistry. The impact assessment and monitoring strategies based on currently available modelling results consequently require strong improvements.     

The distribution, speciation and geochemical cycling of selenium in a sedimentary environment, Kesterson Reservoir, California, U.S.A.

The well-defined and intensively studied episode of Se contamination at Kesterson Reservoir (Merced County, California, U.S.A.) provided a unique opportunity to describe the distribution, speciation and geochemical transformations of Se in a variety of geochemical and ecological settings, ranging from permanent ponds to semi-arid grasslands and salt flats. Kesterson Reservoir comprises 500 ha of land contaminated with Se from agricultural drain water. In most places. Se was concentrated in surficial organic detritus and the surficial decimeter of mineral soil. At dry sites, selenate ion predominated below 20 cm depth. Elemental selenium (Se⁰) also was prominent. The amount of zero-valent Se increased slowly with time. Although selenate is thermodynamically stable in the vadose zone in the presence of oxygen, Se⁰ is an additional, metastable product of the mineralization of organic selenium. Thiols and inorganic sulfides dramatically increase the solubility of Se⁰. Decreasing pH inhibits the reaction, explaining the observed decrease in solubility and biological availability of Se in soil and aquatic systems at low pH. Adding thiols or methionine to soil increases the emission of volatile Se compounds several-fold, suggesting that thiols play a major role in the microbial cycling of Se in soil.     

Controls on the geochemistry of rare earth elements along a groundwater flow path in the Carrizo Sand aquifer,Texas, USA

Groundwater samples were collected along a groundwater flow path in the Carrizo Sand aquifer in south Texas, USA. Field measurements that included pH, specific conductivity, temperature, dissolved oxygen (DO), oxidation–reduction potentials (Eh in mV), alkalinity, iron speciation, and H₂S concentrations were also conducted on site. The geochemistry (i.e., concentrations, shale-normalized patterns, and speciation) of dissolved rare element elements (REEs) in the Carrizo groundwaters are described as a function of distance along a flow path. Eh and other redox indicators (i.e., DO, Fe speciation, H₂S, U, and Re) indicate that redox conditions change along the flow path in the Carrizo Sand aquifer. Within the region of the aquifer proximal to the recharge zone, groundwaters exhibit both highly oxidizing and localized mildly reducing conditions. However, from roughly 10 km to the discharge zone, groundwaters are reducing and exhibit a progressive decrease in redox conditions. Dissolved REE geochemical behavior exhibits regular variations along the groundwater flow path in the Carrizo Sand aquifer. The changes in REE concentrations, shale-normalized patterns, and speciation indicate that REEs are not conservative tracers. With flow down-gradient, redox conditions, pH and solution composite, and adsorption modify groundwater REE concentrations, fractionation patterns, and speciation.     

Effects of sediment iron mineral composition on microbially mediated changes in divalent metal speciation: Importance of ferrihydrite

Dissimilatory metal reducing bacteria (DMRB) can influence geochemical processes that affect the speciation and mobility of metallic contaminants within natural environments. Most investigations into the effect of DMRB on sediment geochemistry utilize various synthetic oxides as the Fe^III source (e.g., ferrihydrite, goethite, hematite). These synthetic materials do not represent the mineralogical composition of natural systems, and do not account for the effect of sediment mineral composition on microbially mediated processes. Our experiments with a DMRB (Shewanella putrefaciens 200) and a divalent metal (Zn^II) indicate that, while complexity in sediment mineral composition may not strongly impact the degree of “microbial iron reducibility,” it does alter the geochemical consequences of such microbial activity. The ferrihydrite and clay mineral content are key factors. Microbial reduction of a synthetic blend of goethite and ferrihydrite (VHSA-G) carrying previously adsorbed Zn^II increased both [Zn^II-aq] and the proportion of adsorbed Zn^II that is insoluble in 0.5 M HCl. Microbial reduction of Fe^III in similarly treated iron-bearing clayey sediment (Fe-K-Q) and hematite sand, which contained minimal amounts of ferrihydrite, had no similar effect. Addition of ferrihydrite increased the effect of microbial Fe^III reduction on Zn^II association with a 0.5 M HCl insoluble phase in all sediment treatments, but the effect was inconsequential in the Fe-K-Q. Zinc k-edge X-ray absorption spectroscopy (XAS) data indicate that microbial Fe^III reduction altered Zn^II bonding in fundamentally different ways for VHSA-G and Fe-K-Q. In VHSA-G, ZnO₆ octahedra were present in both sterile and reduced samples; with a slightly increased average Zn-O coordination number and a slightly higher degree of long-range order in the reduced sample. This result may be consistent with enhanced Zn^II substitution within goethite in the microbially reduced sample, though these data do not show the large increase in the degree of Zn-O-metal interactions expected to accompany this change. In Fe-K-Q, microbial Fe^III reduction transforms Zn-O polyhedra from octahedral to tetrahedral coordination and leads to the formation of a ZnCl₂ moiety and an increased degree of multiple scattering. This study indicates that, while many sedimentary iron minerals are easily reduced by DMRB, the effects of microbial Fe^III reduction on trace metal geochemistry are dependent on sediment mineral composition.     

Biomineralization of As(V)-hydrous ferric oxyhydroxide in microbial mats of an acid-sulfate-chloride geothermal spring, Yellowstone National Park

Acid-sulfate-chloride (pH∼3) geothermal springs in Yellowstone National Park (YNP) often contain Fe(II), As(III), and S(-II) at discharge, providing several electron donors for chemolithotrophic metabolism. The microbial populations inhabiting these environments are inextricably linked with geochemical processes controlling the behavior of As and Fe. Consequently, the objectives of the current study were to (i) characterize Fe-rich microbial mats of an ASC thermal spring, (ii) evaluate the composition and structure of As-rich hydrous ferric oxides (HFO) associated with these mats, and (iii) identify microorganisms that are potentially responsible for mat formation via the oxidation of Fe(II) and or As(III). Aqueous and solid phase mat samples obtained from a spring in Norris Basin, YNP (YNP Thermal Inventory NHSP35) were analyzed using a complement of chemical, microscopic and spectroscopic techniques. In addition, molecular analysis (16S rDNA) was used to identify potentially dominant microbial populations within different mat locations. The biomineralization of As-rich HFO occurs in the presence of nearly equimolar aqueous As(III) and As(V) (∼12 μM), and ∼ 48 μM Fe(II), forming sheaths external to microbial cell walls. These solid phases were found to be poorly ordered nanocrystalline HFO containing mole ratios of As(V):Fe(III) of 0.62 ± 0.02. The bonding environment of As(V) and Fe(III) is consistent with adsorption of arsenate on edge and corner positions of Fe(III)-OH octahedra. Numerous archaeal and bacterial sequences were identified (with no closely related cultured relatives), along with several 16S sequences that are closely related to Acidimicrobium, Thiomonas, Metallosphaera and Marinithermus isolates. Several of these cultured relatives have been implicated in Fe(II) and or As(III) oxidation in other low pH, high Fe, and high As environments (e.g. acid-mine drainage). The unique composition and morphologies of the biomineralized phases may be useful as modern-day analogs for identifying microbial life in past Fe-As rich environments.     

Distributions and assemblages of microbial communities along a sediment core retrieved from a potential hydrate-bearing region offshore southwestern Taiwan

Assessing the impacts of methane released from hydrate-bearing environments on global carbon cycling would require detailed insights into the distributions and capacities of microbial communities at different horizons of sediment column. In this study, we conducted geochemical, gene abundance and diversity analyses for a sediment core retrieved from a potential hydrate-bearing region off southwestern Taiwan. Geochemical profiles were characterized by a sulfate-to-methane transition with decreasing total organic carbon and nitrogen in sediments, and increasing dissolved inorganic carbon, ammonium and total sulfur in sediments. Bacterial and archaeal 16S rRNA and amoA gene abundances decreased with depth. In contrast, ANME-1 and -2 16S rRNA gene abundances increased significantly across the sulfate-to-methane transition and peaked at different horizons below this interface. A total of 124,379 bacterial and 130,351 archaeal reads were recovered through tag-pyrosequencing of 16S rRNA genes and categorized into 9014 bacterial and 6394 archaeal operational taxonomic units on the basis of 97% sequence similarity, respectively. Major bacterial phyla/divisions and archaeal groups (>5% of the total reads) detected included Chloroflexi, Planctomycetes, OP9, Deltaproteobacteria, BHI80-139, MBG-B, Halobacteria, MCG, Thermoplasmata, ANME-1 and MG-I. The abundance variations of most major OTUs (>0.5% of the total reads) were statistically correlated with those of geochemical parameters. These lines of evidence suggest that the populations represented by the major OTUs or detected by group-specific primers were compartmentalized into different horizons and involved directly or indirectly in the cycling of methane, sulfate, organic carbon and nitrogen. Overall, this study demonstrates that the deep sequencing coverage combined with the quantification of gene abundance and geochemical characterization would enable to uncover the detailed distributions and potential metabolic capabilities of specific groups from complexly structured microbial communities in methane-rich marine sediments.     

Quantitative study of geochemical processes in the Dogger aquifer,Paris Basin,France

The quantification of geochemical reactions in hydrothermal aquifers requires an exhaustive approach because of their interdependence. All chemical elements likely to have a quantitative influence on dissolution or precipitation reactions have to be taken into account. Geochemical constraints the number of which equals the number of the chemical elements, were determined with the help of chemical analyses of the solution. In the case study of the Dogger aquifer (Paris Basin, France), 12 elements are taken into account (Al, C, Cl, Ca, F, H, K, Mg, Na, O, S, Si). Three constraints apply to water-activity, neutrality and conservation of chloride in aqueous solutions. The determination of the remaining constraints was based on saturation indices, stability diagrams of minerals and partial pressure of carbon dioxide (pCO₂). The present-day solutions are at equilibrium with respect to nine minerals (albite, anhydrite, chalcedony, calcite, dolomite, fluorite, kaolinite, K-feldspar and illite or chlorite). Thus, it was demonstrated that the composition of these solutions (including computed pCO₂) is only a function of temperature and chloride content. Moreover, it was possible to test the validity of the geochemical system by computing its speciation and comparing these theoretical results with actual chemical analyses (pH and concentrations). Finally, geochemical simulations were used in predicting what quantities would be dissolved or precipitated, as either the temperature or the chlorinity varied. Although the rock is predominantly calcareous, these quantities could not be determined if the influence of the aluminosilicates were neglected. This chemical component approach with which one can pose and solve rigorously the chemical equilibrium problem constitutes a prerequisite for the quantitative study of geochemical processes related to fluid flow.     

Effect of electron donors on the fractionation of sulfur isotopes by a marine Desulfovibrio sp.

Sulfur isotope effects produced by microbial dissimilatory sulfate reduction are used to reconstruct the coupled cycling of carbon and sulfur through geologic time, to constrain the evolution of sulfur-based metabolisms, and to track the oxygenation of Earth’s surface. In this study, we investigate how the coupling of carbon and sulfur metabolisms in batch and continuous cultures of a recently isolated marine sulfate reducing bacterium DMSS-1, a Desulfovibrio sp., influences the fractionation of sulfur isotopes.DMSS-1 grown in batch culture on seven different electron donors (ethanol, glycerol, fructose, glucose, lactate, malate and pyruvate) fractionates ³⁴S/³²S ratio from 6‰ to 44‰, demonstrating that the fractionations by an actively growing culture of a single incomplete oxidizing sulfate reducing microbe can span almost the entire range of previously reported values in defined cultures. The magnitude of isotope effect correlates well with cell specific sulfate reduction rates (from 0.7 to 26.1 fmol/cell/day). DMSS-1 grown on lactate in continuous culture produces a larger isotope effect (21-37‰) than the lactate-grown batch culture (6‰), indicating that the isotope effect also depends on the supply rate of the electron donor and microbial growth rate. The largest isotope effect in continuous culture is accompanied by measurable changes in cell length and cellular yield that suggest starvation. The use of multiple sulfur isotopes in the model of metabolic fluxes of sulfur shows that the loss of sulfate from the cell and the intracellular reoxidation of reduced sulfur species contribute to the increase in isotope effects in a correlated manner. Isotope fractionations produced during sulfate reduction in the pure culture of DMSS-1 expand the previously reported range of triple sulfur isotope effects (³²S, ³³S, and ³⁴S) by marine sulfate reducing bacteria, implying that microbial sulfur disproportionation may have a smaller ³³S isotopic fingerprint than previously thought.     

Electron flow in acidic subsurface sediments co-contaminated with nitrate and uranium

The combination of low pH and high concentrations of nitrate and radionuclides in the subsurface is representative of many sites within the U.S. nuclear weapons complex managed by the Department of Energy (DOE), including the DOE’s Environmental Remediation Sciences Program Field Research Center (ORFRC), in Oak Ridge, Tennessee. In order to provide a further understanding of the coupled microbiological and geochemical processes limiting radionuclide bioremediation, we determined the rates and pathways of terminal-electron accepting processes (TEAPs) in microcosm experiments using close to in situ conditions with ORFRC subsurface materials. At the in situ pH range of 4-5, carbon substrate utilization and TEAP rates were diminished, such that nitrate was not depleted and metal reduction was prevented. Upon biostimulation by pH neutralization and carbon substrate addition, TEAPs were stimulated to rates that rival those measured in organic-rich surficial sediments of aquatic environments, and extremely high nitrate concentrations (0.4-0.5 M) were not found to be toxic to microbial metabolism. Metal reduction under neutral pH conditions started once nitrate was depleted to low levels in response to biostimulation. Acidity controlled not only the rates but also the pathways of microbial activity. Denitrification predominated in sediments originating from neutral pH zones, while dissimilatory nitrate reduction to ammonium occurred in neutralized acidic microcosms amended with glucose. Electron donors were determined to stimulate microbial metabolism leading to metal reduction in the following order: glucose > ethanol > lactate > hydrogen. In microcosms of neutralized acidic sediments, 80-90% of C equivalents were recovered as fermentation products, mainly as acetate. Due to the stress imposed by low pH on microbial metabolism, our results indicate that the TEAPs of acidic subsurface sediment are inherently different from those of neutral pH environments and neutralization will be necessary to achieve sufficient metabolic rates for radionuclide remediation.     

Uranyl adsorption and surface speciation at the imogolite-water interface: Self-consistent spectroscopic and surface complexation models

Macro- and molecular-scale knowledge of uranyl (U(VI)) partitioning reactions with soil/sediment mineral components is important in predicting U(VI) transport processes in the vadose zone and aquifers. In this study, U(VI) reactivity and surface speciation on a poorly crystalline aluminosilicate mineral, synthetic imogolite, were investigated using batch adsorption experiments, X-ray absorption spectroscopy (XAS), and surface complexation modeling. U(VI) uptake on imogolite surfaces was greatest at pH ∼7-8 (I = 0.1 M NaNO₃ solution, suspension density = 0.4 g/L [U(VI)]_i = 0.01-30 μM, equilibration with air). Uranyl uptake decreased with increasing sodium nitrate concentration in the range from 0.02 to 0.5 M. XAS analyses show that two U(VI) inner-sphere (bidentate mononuclear coordination on outer-wall aluminol groups) and one outer-sphere surface species are present on the imogolite surface, and the distribution of the surface species is pH dependent. At pH 8.8, bis-carbonato inner-sphere and tris-carbonato outer-sphere surface species are present. At pH 7, bis- and non-carbonato inner-sphere surface species co-exist, and the fraction of bis-carbonato species increases slightly with increasing I (0.1-0.5 M). At pH 5.3, U(VI) non-carbonato bidentate mononuclear surface species predominate (69%). A triple layer surface complexation model was developed with surface species that are consistent with the XAS analyses and macroscopic adsorption data. The proton stoichiometry of surface reactions was determined from both the pH dependence of U(VI) adsorption data in pH regions of surface species predominance and from bond-valence calculations. The bis-carbonato species required a distribution of surface charge between the surface and β charge planes in order to be consistent with both the spectroscopic and macroscopic adsorption data. This research indicates that U(VI)-carbonato ternary species on poorly crystalline aluminosilicate mineral surfaces may be important in controlling U(VI) mobility in low-temperature geochemical environments over a wide pH range (∼5-9), even at the partial pressure of carbon dioxide of ambient air (p_CO2 = 10^−3.45 atm).     

Elucidating microbial processes in nitrate- and sulfate-reducing systems using sulfur and oxygen isotope ratios: The example of oil reservoir souring control

Sulfate-reducing bacteria (SRB) are ubiquitous in anoxic environments where they couple the oxidation of organic compounds to the production of hydrogen sulfide. This can be problematic for various industries including oil production where reservoir “souring” (the generation of H₂S) requires corrective actions. Nitrate or nitrite injection into sour oil fields can promote SRB control by stimulating organotrophic nitrate- or nitrite-reducing bacteria (O-NRB) that out-compete SRB for electron donors (biocompetitive exclusion), and/or by lithotrophic nitrate- or nitrite-reducing sulfide oxidizing bacteria (NR-SOB) that remove H₂S directly. Sulfur and oxygen isotope ratios of sulfide and sulfate were monitored in batch cultures and sulfidic bioreactors to evaluate mitigation of SRB activities by nitrate or nitrite injection. Sulfate reduction in batch cultures of Desulfovibrio sp. strain Lac15 indicated typical Rayleigh-type fractionation of sulfur isotopes during bacterial sulfate reduction (BSR) with lactate, whereas oxygen isotope ratios in unreacted sulfate remained constant. Sulfur isotope fractionation in batch cultures of the NR-SOB Thiomicrospira sp. strain CVO was minimal during the oxidation of sulfide to sulfate, which had δ¹⁸O_SO4 values similar to that of the water-oxygen. Treating an up-flow bioreactor with increasing doses of nitrate to eliminate sulfide resulted in changes in sulfur isotope ratios of sulfate and sulfide but very little variation in oxygen isotope ratios of sulfate. These observations were similar to results obtained from SRB-only, but different from those of NR-SOB-only pure culture control experiments. This suggests that biocompetitive exclusion of SRB took place in the nitrate-injected bioreactor. In two replicate bioreactors treated with nitrite, less pronounced sulfur isotope fractionation and a slight decrease in δ¹⁸O_SO4 were observed. This indicated that NR-SOB played a minor role during dosing with low nitrite and that biocompetitive exclusion was the major process. The results demonstrate that stable isotope data can contribute unique information for understanding complex microbial processes in nitrate- and sulfate-reducing systems, and offer important information for the management of H₂S problems in oil reservoirs and elsewhere.