B11C-0617
Microbial Populations Associated with Phosphate-Mediated Vadose Zone Sequestration of Strontium and Uranium
Significant quantities of metals and radionuclides are contained in thick unsaturated zones at several contaminated sites in the western US. In many cases, this contamination has migrated to underlying groundwater, sometimes decades after being released into the subsurface. Because of the prohibitive costs associated with physically removing the contamination, an attractive remedy to this problem is to develop methods for long-term in situ stabilization of the contamination in the vadose zone. Our research focuses on developing a method of introducing gaseous compounds to stimulate precipitation of stable phosphate mineral phases in the vadose zone to immobilize soluble contaminants thus minimizing further transport to groundwater. Preliminary studies have demonstrated that biological precipitation of phosphate minerals can be stimulated under unsaturated conditions by injection of triethyl phosphate (TEP) gas. Microorganisms hydrolyze TEP, releasing inorganic phosphate, catalyzing the precipitation of metals and radionuclide-containing phosphate minerals. Our initial results demonstrate that a mixed culture of aerobic microorganisms from vadose zone sediments, enriched with TEP, produce significantly higher concentrations of inorganic phosphate than the no TEP control. A high-density microarray (PhyloChip) capable of detecting up to 9,000 prokaryotic taxa will be used to identify the microbial community composition of the enriched culture. In addition, the metabolically active organisms will be investigated through extraction and hybridization of ribosomal RNA. Organisms capable of hydrolyzing TEP to inorganic phosphate will be further characterized to determine the requirements for aerobic microbially-mediated radionuclide immobilization. The chemical and isotopic compositions of the reactants and products will be measured to enable in situ monitoring of microbial TEP utilization. The result of these studies will be the basis for unsaturated column experiments designed to test different delivery methods for TEP and other nutrients, and to estimate potential rates of phosphate-mediated radionuclide immobilization in the vadose zone.
B11C-0618
Environmental Proteomic Analysis in a Contaminated Aquifer: From Column to Field- Scale
Column flow-through cells packed with sediment material from a uranium-contaminated site (Rifle, CO) were operated under environmentally relevant conditions. Iron-sulfide and calcite precipitation was observed in column effluent tubing whilst subsequent SEM analysis of the tubing revealed the presence of large numbers of microorganisms. Column effluent tubing was removed, flash-frozen and the recovered biomass analyzed using proteomic techniques. Following the successful extraction of protein from the tubing, 2D-LC-MS/MS results revealed that the gram-negative bacterium Dechloromonas aromatica was present in the tubing community. Out of 2710 unique peptides identified in the sample, 1814 matched this species, indicating that it may play an important role in these biofilms. Similar darkening of pump tubing has been observed in field scale bioremediation projects at the Rifle site, whilst 16S rRNA techniques were used to identify D. aromatica playing an important role in a selenium-bioremediation process occurring in the same tubing. This data demonstrates that the proteomic analysis of column-based material may be useful when attempting to scale-up to field-based proteomic studies.
B11C-0619
Generalized Logical Network Modeling of Interactions Among Bacteria in Aerosols Under Meteorological Factors Using High Density Phylogenetic Microarrays
The generalized logical network model utilizes temporal information in the 16S rRNA gene concentration time- course to examine interactions of bacteria within a microbial community under meteorological factors. The Biowatch aerosol bacterial community data set (Brodie et al., PNAS 104[1]:299-304, 2007) of 8,763 taxa intensities was generated using 237 16S rRNA oligonucleotide phylogenetic microarrays at 30 locations throughout the U.S. over time-courses of up to 20 weeks at each location. Each microarray contains about 9,000 probe sets, with an average of 24 probes per set. Seventy-two meteorological factors were measured at the time each microarray was analyzed. In a generalized logical network, a generalized truth table, associated with every node representing either a bacterial taxon or a meteorological factor, describes the represented bacterial behavior dictated by some environmental factors in addition to associations with other bacterial taxa. The optimal generalized logics at each bacterial node in the network will be searched so that they best explain the observed time-course data. Determination of an optimal logic will involve parent node selection and generalized truth-table generation. The maximum number of parents is set to a given number. If the current node shows consistent behavior during transition from one state to another given the parent nodes, then the parent nodes are kept. The actual goodness of the transition is calculated using the chi-square test. In addition to the dependency of the concentrations of bacteria on meteorological factors, with various time delays, the initial generalized logical network modeling results indicate that the concentrations of specific bacterial taxa are also associated with concentrations of other bacteria.
B11C-0620
Electrochemical Performance and Microbial Characterization of Thermophilic Microbial Fuel Cells
Significant research effort is currently focused on microbial fuel cells (MFC) as a source of renewable energy. To date, most of these efforts have concentrated on MFCs operating at mesophilic temperatures. However, many previous studies have reported on the superiority of thermophilic conditions in anaerobic digestion and demonstrated a net gain in energy yield, in terms of methane, relative to the increased energy requirements of operation. Because of this, our recent studies on MFCs focused on investigating the operation and microbiology associated with thermophilic MFCs operating at 55°C. Over 100-day operation, these MFCs were highly stable and achieved a maximum power density of 24mW/m2 and a columbic efficiency of 89 percent with acetate as the sole electron donor. In order to characterize the microbial community involved in thermophilic electricity generation, DNA and RNA were isolated from the electrode and PhyloChip analyses performed. Exploring the changes in the microbial community over time in electricity producing MFC revealed an increase in relative abundance of populations belonging to the Firmicutes, Chloroflexi, and alpha Proteobacteria by at least one order of magnitude. In contrast, these populations decreased in the open circuit and no electron donor amended controls. In order to better characterize the active microbial populations, we enriched and isolated a novel organism, strain JR, from samples collected from an operating MFC. Based on 16S rRNA sequence analysis strain JR was a member of the family Peptococcaceae, within the Phylum Firmicutes, clustering with Thermincola ferriacetica (98 percent similarity). Phenotypic characterization revealed that strain JR was capable of thermophilic dissimilatory reduction of insoluble electron acceptors such as amorphous Fe(III); as well as reduction of the model quinone 2,6-anthraquinone disulfonate. Thermincola strain JR was also capable of producing current by coupling acetate oxidation to anodic electron transfer. This represents the first organism isolated from a thermophilic microbial fuel cell and also the first representative of this genus capable of anodic electron transfer. The results of this study indicate the potential advantages for thermophilic MFCs and the novel microbiology associated with their operation.
B11C-0621
A Functional Genomic Approach to Chlorinated Ethenes Bioremediation
With the recent advances in genomic sciences, a knowledge-based approach can now be taken to optimize the bioremediation of trichloroethene (TCE). During the bioremediation of a heterogeneous subsurface, it is vital to identify and quantify the functionally important microorganisms present, characterize the microbial community and measure their physiological activity. In our field experiments, quantitative PCR (qPCR) was coupled with reverse-transcription (RT) to analyze both copy numbers and transcripts expressed by the 16S rRNA gene and three reductive dehalogenase (RDase) genes as biomarkers of Dehalococcoides spp. in the groundwater of a TCE-DNAPL site at Ft. Lewis (WA) that was serially subjected to biostimulation and bioaugmentation. Genes in the Dehalococcoides genus were targeted as they are the only known organisms that can completely dechlorinate TCE to the innocuous product ethene. Biomarker quantification revealed an overall increase of more than three orders of magnitude in the total Dehalococcoides population and quantification of the more liable and stringently regulated mRNAs confirmed that Dehalococcoides spp. were active. Parallel with our field experiments, laboratory studies were conducted to explore the physiology of Dehalococcoides isolates in order to develop relevant biomarkers that are indicative of the metabolic state of cells. Recently, we verified the function of the nitrogenase operon in Dehalococcoides sp. strain 195 and nitrogenase-encoding genes are ideal biomarker targets to assess cellular nitrogen requirement. To characterize the microbial community, we applied a high-density phylogenetic microarray (16S PhyloChip) that simultaneous monitors over 8,700 unique taxa to track the bacterial and archaeal populations through different phases of treatment. As a measure of species richness, 1,300 to 1,520 taxa were detected in groundwater samples extracted during different stages of treatment as well as in the bioaugmentation culture. We found that the community structure was sensitive to manipulation such as the injection of whey. In addition to Dehalococcoides spp., the PhyloChip also detected dechlorinating bacteria from other phyla such as Sulfurospirillum multivorans and Dehalobacter restrictus. Although these organisms only dechlorinate TCE to dichloroethene, their populations increase at the site over time suggested they also played an important role. Over 600 subfamilies were also found to be active in the microbial community with many of those being important players in geochemical processes. Overall, through the use of high throughput molecular techniques, a comprehensive view of the functionally important organisms and the microbial community was obtained, providing knowledge that can be used to guide the manipulation of the bioremediation processes to achieve the most efficient treatment.
B11C-0622
Binning of shallowly sampled metagenomic sequence fragments reveals that low abundance bacteria play important roles in sulfur cycling and degradation of complex organic polymers in an acid mine drainage community
Our understanding of environmental microbiology has been greatly enhanced by community genome sequencing of DNA recovered directly the environment. Community genomics provides insights into the diversity, community structure, metabolic function, and evolution of natural populations of uncultivated microbes, thereby revealing dynamics of how microorganisms interact with each other and their environment. Recent studies have demonstrated the potential for reconstructing near-complete genomes from natural environments while highlighting the challenges of analyzing community genomic sequence, especially from diverse environments. A major challenge of shotgun community genome sequencing is identification of DNA fragments from minor community members for which only low coverage of genomic sequence is present. We analyzed community genome sequence retrieved from biofilms in an acid mine drainage (AMD) system in the Richmond Mine at Iron Mountain, CA, with an emphasis on identification and assembly of DNA fragments from low-abundance community members. The Richmond mine hosts an extensive, relatively low diversity subterranean chemolithoautotrophic community that is sustained entirely by oxidative dissolution of pyrite. The activity of these microorganisms greatly accelerates the generation of AMD. Previous and ongoing work in our laboratory has focused on reconstrucing genomes of dominant community members, including several bacteria and archaea. We binned contigs from several samples (including one new sample and two that had been previously analyzed) by tetranucleotide frequency with clustering by Self-Organizing Maps (SOM). The binning, evaluated by comparison with information from the manually curated assembly of the dominant organisms, was found to be very effective: fragments were correctly assigned with 95% accuracy. Improperly assigned fragments often contained sequences that are either evolutionarily constrained (e.g. 16S rRNA genes) or mobile elements that are not expected to reflect the tetranucleotide frequency signature of the host genome. Four unknown tetranucleotide frequency clusters with significant sequence (6 Mb total) were noted and analyzed further. Based on phylogenetic markers and BLAST results, these clusters represent low abundance bacteria including Acintobacteria, Firmicutes, and Proteobacteria. Functional analysis of these clusters revealved that the low- abundance bacteria harbor genes that could potentially encode important ecosystem functions such as sulfur utilization (e.g. polysulfide reductase) and polymer degradation (e.g. chitinase and glycoside hydrolase). We conclude that ESOM clustering of tetranucleotide frequency patterns is an effective method for rapidly binning shotgun community genomic sequences and a valuable tool for analyzing minor community members, which despite their low abundance may play crucial ecological roles.
B11C-0623
Predicting Structure and Function for Novel Proteins of an Extremophilic Iron Oxidizing Bacterium
Proteins isolated from uncultivated microbial populations represent the functional components of microbial processes and contribute directly to community fitness under natural conditions. Investigations into proteins in the environment are hindered by the lack of genome data, or where available, the high proportion of proteins of unknown function. We have identified thousands of proteins from biofilms in the extremely acidic drainage outflow of an iron mine ecosystem (1). With an extensive genomic and proteomic foundation, we have focused directly on the problem of several hundred proteins of unknown function within this well-defined model system. Here we describe the geobiological insights gained by using a high throughput computational approach for predicting structure and function of 421 novel proteins from the biofilm community. We used a homology based modeling system to compare these proteins to those of known structure (AS2TS) (2). This approach has resulted in the assignment of structures to 360 proteins (85%) and provided functional information for up to 75% of the modeled proteins. Detailed examination of the modeling results enables confident, high-throughput prediction of the roles of many of the novel proteins within the microbial community. For instance, one prediction places a protein in the phosphoenolpyruvate/pyruvate domain superfamily as a carboxylase that fills in a gap in an otherwise complete carbon cycle. Particularly important for a community in such a metal rich environment is the evolution of over 25% of the novel proteins that contain a metal cofactor; of these, one third are likely Fe containing proteins. Two of the most abundant proteins in biofilm samples are unusual c-type cytochromes. Both of these proteins catalyze iron- oxidation, a key metabolic reaction supporting the energy requirements of this community. Structural models of these cytochromes verify our experimental results on heme binding and electron transfer reactivity, and provide details for a working hypothesis of electron flow within the biofilm's major bacterium. Nearly 7% of the novel proteins contain tetratrico peptide repeat (TPR) modules, a protein-protein interaction domain that participates in signal transduction and a wide variety of other cellular functions. Like many biofilms, the various organisms in this community use unknown mechanisms to communicate, relying upon each other for survival. Especially interesting is evidence that most of these novel TPR proteins are located in the extracellular or membrane fractions, suggesting their role in intracellular communication. (1) Ram et al, 2005, Science 308:1915-20, "Community Proteomics of a Natural Microbial Biofilm" (2) Zemla et al, 2005, Nucleic Acids Res 33 (Web Server issue):W111-5, "AS2TS system for protein structure modeling and analysis" This work was funded by the DOE Genomics: GTL Program and was performed under the auspices of the DOE by the University of California, Lawrence Livermore National Laboratory under contract W-7405-Eng-48.
B11C-0624
Influence of Electron Donor Type and Concentration on Microbial Population Structure During Uranium Reduction and Remobilization
Enhanced reductive precipitation of U(VI) through stimulation of indigenous microorganisms is an attractive, low- cost strategy for in-situ remediation of contaminated groundwaters and sediments. The rate of organic carbon (OC) supply determines not only the amount of electron donor available for bioreduction of U(VI), but also affects the resulting concentration of aqueous (bi)carbonate generated by microbial respiration. Increased (bi)carbonate concentrations drive aqueous U(VI) concentrations to higher levels and make U(IV) oxidation under reducing conditions favorable. We designed a long-term column study to investigate the effects of different OC forms and supply rates on the stability of bioreduced U and on the structure and dynamics of the microbial communities. OC was supplied as acetate or lactate at four different concentrations and columns were sampled at three time points. In the columns receiving high OC supply the time points correspond to a phases of net U-reduction, U(IV) reoxidation and U(VI) remobilization, and stable levels of U mobilization. DNA was extracted from column sediments, 16S rRNA genes were amplified and the communities analyzed using a high-density phylogenetic microarray (PhyloChip). Lactate and acetate supplied at equivalent rates had a similar impact on uranium mobility with higher OC resulting in re-oxidation of U(IV) after an initial period of U(VI) reduction. Similarly, organic carbon (OC) supply rate, not OC form, had the largest impact on microbial community structure. The diversity (richness) of bacterial and archaeal communities increased over time with those receiving lactate having higher initial richness. Known U-reducing bacteria were present in all columns and time points, however the dynamics of these organisms varied with both organic carbon supply rate and form. This data demonstrates that the initial rate of electron donor supply during heavy metal remediation strongly impacts microbial community development. Uranium re-mobilization occurred irrespective of electron donor form, and this occurred despite the presence of multiple species of U-reducing bacteria.
B11C-0625
Diverse anaerobic Cr(VI) tolerant bacteria from Cr(VI)-contaminated 100H site at Hanford
Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water. Cr(VI) is more soluble, toxic, carcinogenic, and mutagenic compared to its reduced form Cr(III). In order to stimulate microbially mediated reduction of Cr(VI), a poly-lactate compound HRC was injected into the chromium contaminated aquifers at site 100H at Hanford. Based on the results of the bacterial community composition using high-density DNA microarray analysis of 16S rRNA gene products, we recently investigated the diversity of the dominant anaerobic culturable microbial population present at this site and their role in Cr(VI) reduction. Positive enrichments set up at 30°C using specific defined anaerobic media resulted in the isolation of an iron reducing isolate strain HAF, a sulfate reducing isolate strain HBLS and a nitrate reducing isolate, strain HLN among several others. Preliminary 16S rDNA sequence analysis identifies strain HAF as Geobacter metallireducens, strain HLN as Pseudomonas stutzeri and strain HBLS as a member of Desulfovibrio species. Strain HAF isolated with acetate as the electron donor utilized propionate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Growth was optimal at 37°C, pH of 6.5 and 0% salinity. Strain HLN isolated with lactate as electron donor utilized acetate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Optimal growth was observed at 37°C, at a pH of 7.5 and 0.3% salinity. Anaerobic active washed cell suspension of strain HLN reduced almost 95 micromolar Cr(VI) within 4 hours relative to controls. Further, with 100 micromolar Cr(VI) as the sole electron acceptor, cells of strain HLN grew to cell numbers of 4.05X 107/ml over a period of 24hrs after an initial lag, demonstrating direct enzymatic Cr(VI) reduction by this species. 10mM lactate served as the sole electron donor. These results demonstrate that Cr(VI) immobilization at the Hanford 100H site could be mediated by direct microbial metabolism apart from indirect chemical reduction of Cr(VI) by end products of microbial activity.
B11C-0626
Biofilm function and variability in a hydrothermal ecosystem: insights from environmental genomes
The ability to adapt to variable environmental conditions is key to survival for all organisms, but may be especially crucial to microorganisms in extreme environments such as hydrothermal systems. Streamer biofilm communities (SBCs) made up of thermophilic chemotrophic microorganisms are common in alkaline-chloride geothermal environments worldwide, but the in situ physiochemical growth parameters and requirements of SBCs are largely unknown [1]. Hot springs in Yellowstone National Park's alkaline geyser basins support SBC growth. However, despite the relative geochemical homogeneity of source pools and widespread ecosystem suitability in these regions (as indicated by energetic profiling [2]), SBCs are not ubiquitous in these ecosystems. The ability of hydrothermal systems to support the growth of SBCs, the relationship between these geochemically driven environments and the microbes that live there, and the function of individuals in these communities are aspects that are adressed here by applying environmental genomics. Analysis of 16S rRNA and total membrane lipid extracts have revealed that community composition of SBCs in "Bison Pool" varies as a function of changing environmental conditions along the outflow channel. In addition, a significant crenarchaeal component was discovered in the "Bison Pool" SBCs. In general, the SBC bacterial diversity triples while the archaeal component varies little (from 3 to 2 genera) in a 5-10°C gradient with distance from the source. While these SBCs are low in overall diversity, the majority of the taxa identified represent uncultured groups of Bacteria and Archaea. As a result, the community function of these taxa and their role in the formation of the biofilms is unknown. However, recent genomic analysis from environmental DNA affords insight into the roles of specific organisms within SBCs at "Bison Pool," and integration of these data with an extensive corresponding geochemical dataset may indicate shifting community function with geochemical variability. For example, calculations of energy availability and genomic data indicate a myriad of potential heterotrophic and autotrophic metabolic functions present at "Bison Pool" (genes for all known autotrophic C- fixation pathways, H2, CO, and formate oxidation, cellulose degredation, and Fe and As redox), as well as oxygenic and anoxygenic photosynthesis. These microbial communities and their environments are ideal for coordination of geochemical and genomic data, enabling informed analysis of SBC function and growth criteria. [1] Jahnke, L. et al. (2001) AEM 67, 5179-5189 [2] Meyer-Dombard, D. et al. (2005) Geobiology 3, 211-227