Aaron D. Peacock

Stimulating the In Situ Activity of Geobacter Species To Remove Uranium from the Groundwater of a Uranium-Contaminated Aquifer

ABSTRACT The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 μM in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species.

Plant diversity, soil microbial communities, and ecosystem function: Are there any links?

A current debate in ecology centers on the extent to which ecosystem function depends on biodiversity. Here, we provide evidence from a long-term field manipulation of plant diversity that soil microbial communities, and the key ecosystem processes that they mediate, are significantly altered by plant species richness. After seven years of plant growth, we determined the composition and function of soil microbial communities beneath experimental plant diversity treatments containing 1-16 species. Microbial community bio- mass, respiration, and fungal abundance significantly increased with greater plant diversity, as did N mineralization rates. However, changes in microbial community biomass, activity, and composition largely resulted from the higher levels of plant production associated with greater diversity, rather than from plant diversity per se. Nonetheless, greater plant pro- duction could not explain more rapid N mineralization, indicating that plant diversity affected this microbial process, which controls rates of ecosystem N cycling. Greater N availability probably contributed to the positive relationship between plant diversity and productivity in the N-limited soils of our experiment, suggesting that plant-microbe in- teractions in soil are an integral component of plant diversitys influence on ecosystem

Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

ABSTRACT The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.

Diversity and Characterization of Sulfate-Reducing Bacteria in Groundwater at a Uranium Mill Tailings Site

ABSTRACT Microbially mediated reduction and immobilization of U(VI) to U(IV) plays a role in both natural attenuation and accelerated bioremediation of uranium-contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex., was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from δ-Proteobacteria, gram-positive organisms, and theNitrospira division. PLFA analysis detected at least 52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0. Desulfotomaculum andDesulfotomaculum-like sequences were the most dominant DSR genes detected. Those belonging to SRB within δ-Proteobacteria were mainly recovered from low-uranium (≤302 ppb) samples. OneDesulfotomaculum-like sequence cluster overwhelmingly dominated high-U (>1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences (P = 0.0001). This strong association indicates that Desulfotomaculum has remarkable tolerance and adaptation to high levels of uranium and suggests the organisms possible involvement in natural attenuation of uranium. The in situ activity level of Desulfotomaculum in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research.

Utilization of Microbial Biofilms as Monitors of Bioremediation

A down-well aquifer microbial sampling system was developed using glass wool or Bio-Sep beads as a solid-phase support matrix. Here we describe the use of these devices to monitor the groundwater microbial community dynamics during field bioremediation experiments at the U.S. Department of Energy Natural and Accelerated Bioremediation Research Program’s Field Research Center at the Oak Ridge National Laboratory. During the 6-week deployment, microbial biofilms colonized glass wool and bead internal surfaces. Changes in viable biomass, community composition, metabolic status, and respiratory state were reflected in sampler composition, type of donor, and groundwater pH. Biofilms that formed on Bio-Sep beads had 2–13 times greater viable biomass; however, the bead communities were less metabolically active [higher cyclopropane/monoenoic phospholipid fatty acid (PLFA) ratios] and had a lower aerobic respiratory state (lower total respiratory quinone/PLFA ratio and ubiquinone/menaquinone ratio) than the biofilms formed on glass wool. Anaerobic growth in these systems was characterized by plasmalogen phospholipids and was greater in the wells that received electron donor additions. Partial 16S rDNA sequences indicated that Geobacter and nitrate-reducing organisms were induced by the acetate, ethanol, or glucose additions. DNA and lipid biomarkers were extracted and recovered without the complications that commonly plague sediment samples due to the presence of clay or dissolved organic matter. Although microbial community composition in the groundwater or adjacent sediments may differ from those formed on down-well biofilm samplers, the metabolic activity responses of the biofilms to modifications in groundwater geochemistry record the responses of the microbial community to biostimulation while providing integrative sampling and ease of recovery for biomarker analysis.

Lipid and carbon isotopic evidence of methane-oxidizing and sulfate-reducing bacteria in association with gas hydrates from the Gulf of Mexico

An integrated lipid biomarker–carbon isotope approach reveals new insight to microbial methane oxidation in the Gulf of Mexico gas-hydrate system. Hydrate-bearing and hydrate-free sediments were collected from the Gulf of Mexico slope using a research submersible. Phospholipid fatty acids consist mainly of C16–C18 compounds, which are largely derived from bacteria. The phospholipid fatty acids suggest that total biomass is enhanced 11–30-fold in gas-hydrate–bearing sediment compared to hydrate-free sediment. Lipid biomarkers indicative of sulfate-reducing bacteria are strongly depleted in 13C (δ13C = −48‰ to −70‰) in the hydrate-bearing samples, suggesting that they are involved in the oxidation of methane (δ13C = −47‰ for thermogenic methane and −70‰ for biogenic methane). Isotopic properties of other biomarkers suggest that sulfur-oxidizing bacteria ( Beggiatoa ) may also contribute to the lipid pool in hydrate-bearing samples, which are characterized by less negative δ13C values (to −11.2‰). In the hydrate-free sample, fatty acid biomarkers have δ13C values of −27.6‰ to −39.6‰, indicating that crude oil (average ∼−27‰) or terrestrial organic carbon (average ∼−20‰) are the likely carbon sources. Our results provide the first lipid biomarker–stable isotope evidence that sulfate- reducing bacteria play an important role in anaerobic methane oxidation in the Gulf of Mexico gas hydrates. The coupled activities of methane-oxidizing and sulfate-reducing organisms contribute to the development of ecosystems in deep-sea environments and result in sequestration of carbon as buried organic carbon and authigenic carbonates. These have implications for studying climate change based on carbon budgets.

Kineococcus radiotolerans sp. nov., a radiation-resistant, gram-positive bacterium.

A gram-type positive, motile, coccus-shaped organism was isolated from a radioactive work area. Strain SRS30216T is an orange-pigmented bacterium that is catalase-positive, oxidase-negative and urease-negative. The orange pigment is most likely a carotenoid with absorption peaks at approximately 444, 471 and 501 nm. Cells normally grew in clusters, but individual, motile, flagellated cells were also observed. Growth of strain SRS30216T occurred at temperatures between 11 and 41 degrees C, between pH 5 and 9 and at NaCl concentrations up to and including 5%. Fatty acid composition was limited, with >90% of the fatty acids being anteiso 15:0. Alkenes of 19-24 carbons in length were detected during examination of the neutral lipids. Strain SRS30216T demonstrated high levels of resistance to gamma-radiation and desiccation. The most closely related recognized species is Kineococcus aurantiacus RA 333T, which is 93% similar in 16S rDNA sequence. DNA-DNA hybridization revealed only 31% similarity between these two organisms. It is proposed that SRS30216T (= ATCC BAA-149T = DSM 14245T) represents the type strain of a novel species in the genus Kineococcus, Kineococcus radiotolerans sp. nov..

Physiological and taxonomic description of the novel autotrophic, metal oxidizing bacterium, Pseudogulbenkiania sp. strain 2002

A lithoautotrophic, Fe(II) oxidizing, nitrate-reducing bacterium, strain 2002 (ATCC BAA-1479; =DSM 18807), was isolated as part of a study on nitrate-dependent Fe(II) oxidation in freshwater lake sediments. Here we provide an in-depth phenotypic and phylogenetic description of the isolate. Strain 2002 is a gram-negative, non-spore forming, motile, rod-shaped bacterium which tested positive for oxidase, catalase, and urease. Analysis of the complete 16S rRNA gene sequence placed strain 2002 in a clade within the family Neisseriaceae in the order Nessieriales of the Betaproteobacteria 99.3% similar to Pseudogulbenkiania subflava. Similar to P. sublfava, predominant whole cell fatty acids were identified as 16:17c, 42.4%, and 16:0, 34.1%. Whole cell difference spectra of the Fe(II) reduced minus nitrate oxidized cyctochrome content revealed a possible role of c-type cytochromes in nitrate-dependent Fe(II) oxidation. Strain 2002 was unable to oxidize aqueous or solid-phase Mn(II) with nitrate as the electron acceptor. In addition to lithotrophic growth with Fe(II), strain 2002 could alternatively grow heterotrophically with long-chain fatty acids, simple organic acids, carbohydrates, yeast extract, or casamino acids. Nitrate, nitrite, nitrous oxide, and oxygen also served as terminal electron acceptors with acetate as the electron donor.

Measuring soil microbial community diversity using polar lipid fatty acid and denaturing gradient gel electrophoresis data.

The possibility of calculating useful microbial community diversity indices from environmental polar lipid fatty acid and 16S rDNA PCR-DGGE data was investigated. First, the behavior of the species richness, Shannons, and Simpsons diversity indices were determined on polar lipid fatty acid profiles of 115 pure cultures, communities constructed from those profiles with different numbers of species, and constructed communities with different distributions of species. Differences in the species richness of these artificial communities was detected by all three diversity indices, but they were insensitive to the evenness of the distribution of species. Second, data from a field experiment with substrate addition to soil was used to compare the methods developed for lipid- and DNA-based diversity indices. Very good agreement was found between indices calculated from environmental polar lipid fatty acid profiles and denaturing gradient gel electrophoresis profiles from matched samples (Pearsons correlation coefficient r=0.95-0.96). A method for data pre-treatment for diversity calculations is described.

Carbon isotope signatures of fatty acids in Geobacter metallireducens and Shewanella algae

The goal of this study was to examine carbon isotope fractionations associated with lipid biomarkers of different ironreducing bacteria. Experiments were conducted using a Geobacter metallireducens strain GS-15 (grown on acetate) and a Shewanella algae strain BrY (grown on lactate). Both organisms were grown anaerobically using ferric citrate as the electron acceptor. The d 13 C of total biomass of GS-15 was � 34.3x, which was about 7.2xlower than the substrate acetate (d 13 C=� 27.1x). The d 13 C of total biomass of BrY was � 30.0x, which was about 7.1xlower than the substrate lactate (d 13 C=� 22.9x). Isotopic fractionations (e) between fatty acid sand biomass within the same strain were consistently greater for BrY (� 10.9xto � 15.5x) than for GS-15 (� 4.5x to � 8.6x). The lipid biomarker 10Me16:0, which was only found in GS-15 cultures, had an e value of � 5.39F0.73x(n=2). The differences in fractionation may reflect the carbon assimilation pathways by which the fatty acids were synthesized: the smaller fractionations in GS-15 may be associated with the tricarboxylic cycle, whereas the greater fractionations in BrY may be associated with the serine pathway. The difference in isotope fractionations between lipid biomarkers and total biomass of different iron-reducing bacteria may be used to enhance our understanding of the microbial community structure in an iron-reducing environment. D 2002 Published by Elsevier Science B.V.