2001 RESEARCH PROJECTS Program Element 2 Community Dynamics and Microbial Ecology

PROJECT:	Impact of Biostimulation Conditions on Diversity and Dynamics of Key Genes Involved in Metal Reduction
PRINCIPAL INVESTIGATOR:	Fred J. Brockman
PROGRAM ELEMENT 2	Community Dynamics and Microbial Ecology

The relationships between diversity, ecosystem function, and stability of community structure over time remains a major challenge in microbial ecology. However, this knowledge is needed to anticipate the effects of perturbations (e.g., bioremediation operating parameters) on the performance of the system. For example, different types, rates, or timing of nutrient additions may impact intra-species competition and generate different community structure end-states, which may profoundly impact the performance of a bioremediative process such as uranium reduction. We propose to collaborate with and assist four currently-funded NABIR investigators (names and institutions on the title page) in the analysis of select Field Research Center (FRC) samples from both laboratory and field studies investigating biological uranium reduction. Samples will be from batch and dynamic column experiments, and from groundwater and core samples before and during in situ biostimulation to promote uranium reduction. Conserved functional genes for metal reduction and sulfite reduction will be amplified from the samples and approximately 10,000 clones sequenced, without a prior restriction enzyme screening step, at the DOE Joint Genome Institute. Three hypotheses (in short form here) will address diversity in three key metal and uranium reducing genes, how diversity in these genes changes in response to different conditions imposed in laboratory studies and in response to field bioremediation phases, and the utility of the diversity information for micrarray analysis at the population- and expression-levels. The resulting robust data sets will (a) characterize macrodiversity and microdiversity within the functional genes at the FRC site, (b) describe how nutritional and hydrogeochemical experimental factors impact the diversity and dynamics of the functional genes, and (c) be exploited to construct and utilize a high resolution DNA array at the FRC site.

PROJECT:	Integrated Particle Handling Methods for Multiplexed Microbial Identification and Characterization in Sediments
PRINCIPAL INVESTIGATOR:	Darrell Chandler
PROGRAM ELEMENT 2	Community Dynamics and Microbial Ecology

Molecular analysis of subsurface microbial communities requires some combination of sample collection, concentration, cell lysis, nucleic acid purification, PCR amplification and specific detection in order to address fundamental questions of microbial community dynamics, activity and function in the environment. Consequently, molecular methods are still relatively ineffectual for monitoring community dynamics during bioremediation, due primarily to the cost, technical difficulty and retrospective nature of the analyses. For ucleic acid analyses to meaningfully contribute to bioremediation efforts they must not only contribute to the fundamental understanding of microbial ecology, but also be formatted in such a manner that in-field analysis can be achieved. The objective of the proposed work is therefore to develop an integrated microbial and nucleic acid detection method and prototype system for the characterization and analysis of subsurface sediments, focusing on the molecular detection of metal- and sulfate-reducingbacteria and activity in sediments obtained from the Oak Ridge Field Research Center. We will meet this objective by combining environmental molecular microbiology with novel renewable surface techniques, microfluidic systems and microparticle analytical chemistry. The fluidic systems will be used to evaluate hypotheses on the integrated biochemistry that is necessary to directly detect 16S rRNA from metal-reducing microbial communities on a suspension microarray, without using the polymerase chain reaction (PCR). These investigations will include the use of peptide nucleic acid capture and detection probes and “tunable surface” concepts to increase nucleic acid capture and detection efficiency and/or mitigate interferences due to co-extracted humic acids. The unified microparticle sample preparation method and suspension array will then be used to characterize the 16S rRNA metal- and sulfate-reducing microbial community in FRC sediment microcosms before and after biostimulation.

Bioremediation of dissolved metals exploits the biological transformation of soluble toxic species to insoluble, less toxic ones. This grant is aimed at developing a quantitative, high-throughput molecular assessment tool that will enable field monitoring of radionuclide-transforming microorganisms via detection of 16S rDNA, RNA, intergenic spacer region sequences, metal-resistance genes, and genes coding for enzymes that carry out metal-immobilizing and metal-detoxifying reactions. The tool is based upon a new multiplex technology that involves hybridization of nucleic acids on the surface of microscopic, fluorescent, polystyrene beads in order to identify specific target sequences in complex mixtures of DNA or RNA. Following capture of target sequences obtained from environmental samples, the fluorescent beads are analyzed by flow cytometry.

We have established a multiplex method for measuring the abundance of target sequences in PCR products generated from community DNA. Experiments were conducted on 16S rRNA genes amplified from contaminated groundwater. Now we need to test a greater variety of groundwater and sediment samples, develop better sets of molecular probes, increase the rapidity of the data analysis, improve the lower detection limit in PCR products, and apply the method towards understanding microbial communities. With collaborators, we will initially analyze field samples from the UMTRA site and microcosms constructed with uranium-contaminated material from the Aberdeen Proving Grounds in Maryland. Furthermore, we will study the impact of various electron donors on in situ kinetics of U reduction using FRC samples generated in push-pull experiments by the Istok/Krumholz team. Later this work will be expanded to chromate-contaminated sites. After improvements in the detection limit are made, we will continue to investigate the detection of mRNA in total RNA.

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