1998 RESEARCH PROJECTS Program Element 2 Community Dynamics and Microbial Ecology PROJECT: Vadose Zone Microbial Community Structure and Activity in Metal/Radionuclide-Contaminated Sediments PRINCIPAL INVESTIGATOR: Fred K. Brockman PROGRAM ELEMENT 2 Community Dynamics and Microbial Ecology OBJECTIVE: The structure and diversity of a microbial community determines its function, which in turn, affects the success of bioremediation. The objective of the project is to determine the affect of unsaturated flow rate, exogenous nutrients, and metal concentrations on microbial community structure, activity, diversity, and dynamics in contaminated vadose zones. We will test three specific hypotheses in field studies (representing long-term metal/radionuclide exposure) and in microcosm studies (short-term exposure, < 1 yr). Field studies will be performed at sites historically contaminated with metals/radionuclides and/or at NABIR Field Research Centers. Short-term effects will be studied in microcosms containing vadose zone sediments. Two types of microcosms will be used: batch and unsaturated flow columns. Data from batch microcosm experiments will be used to select appropriate conditions and critical treatments to be tested in unsaturated flow column experiments. Unsaturated flow column experiments will be performed using intact sediment cores; experiments will simulate the dynamic effects of a contaminant plume as it penetrates deep vadose zone sediments and will determine the effects of treatments that may be used during accelerated bioremediation in the field. Community structure will be assessed by three methods: culturing, nucleic acid based techniques (rRNA analysis and in situ hybridization of flotation films), and phospholipid fatty acid (PLFA) biomarker profiles. Microbial activities will be measured by radiotracer methods. Initial laboratory microcosm studies will focus on chromate and lead; year-3 experiments will analyze contaminated (likely metal and radionuclide) field samples. PROJECT: Determination of the Structure of Metal- and Humics-Reducing Microbial Communities in Subsurface Environments Contaminated with Uranium and Other Metals PRINCIPAL INVESTIGATOR: Derek Lovley PROGRAM ELEMENT 2 Community Dynamics and Microbial Ecology OBJECTIVE: Microorganisms that can use metals and/or humic substances as terminal electron acceptors have the potential to aid in the remediation of metal-contaminated environments via several mechanisms. The objectives of this research are: 1) to determine what metal- and humics-reducing bacteria predominate at contaminated DOE sites; 2) to determine the ability of these organisms to reduce toxic metals, humic substances, and various forms of Fe(III) and Mn(IV) oxides; 3) to develop molecular techniques that will provide a rapid method for assessing the metal- and humics-reducing communities in subsurface environments; 4) to determine how the activity of metal- and humics-reducing organisms might best be stimulated in the subsurface through a study of their physiology; and 5) to test whether strategies for stimulating metal and/or humics reduction suggested by the physiological studies will in fact stimulate the activity of metal- and humics-reducing bacteria in subsurface sediment samples. In order to meet these objectives, metal- and humics-reducing bacteria will be recovered from subsurface environments with a variety of culturing techniques that we have recently developed for the study of such organisms in other sedimentary environments. The physiology of these isolates will be evaluated in detail with emphasis on: 1) the capacity of these organisms to reduce toxic metals and humics; 2) the potential influence that alternative electron acceptors might have on metal reduction; and 3) the suitability of various electron donors to promote metals and humics reduction. The 16S rRNA sequences of the isolates will be determined and this data will be used to develop 16S rRNA probes and 16S rDNA PCR primers. These molecular tools will be used for determining the distribution of metal- and humics-reducing bacteria naturally present in contaminated subsurface environments and for monitoring the effect of strategies designed to enhance the growth and activity of metal and humics reducers. Microorganisms that can use metals and/or humic substances as terminal electron acceptors have the potential to aid in the remediation of metal-contaminated environments via several mechanisms. APPROACH: These studies will: 1) advance the basic understanding of the metal- and humics-reducing microbial community in subsurface environments; 2) provide cultures of subsurface metal- and humics-reducing bacteria that are needed for further investigation into the biochemistry and regulation of this process in the subsurface; 3) provide insights into the natural attenuation of metals by metal- and humics-reducing bacteria; 4) suggest mechanisms for accelerating the bioremediation of metal-contaminated subsurface environments; and 5) provide tools for evaluating the structure of the metal- and humics-reducing microbial community in order to better predict natural attenuation and to better monitor the effectiveness of treatment strategies designed to enhance the activity of metal- and humics-reducing bacteria. PROJECT: Systematic Analysis of Microbial Communities in a Chromium Contaminated Super Fund Site PRINCIPAL INVESTIGATOR: Terence L. Marsh PROGRAM ELEMENT 2 Community Dynamics and Microbial Ecology OBJECTIVE: The objectives are to: 1) Survey the microbial community in up to 200 soil samples from a site contaminated with heavy metals using new rapid molecular techniques that are culture-independent; 2) Identify phylogenetic signatures of microbial populations that correlate with metal ion contamination; 3) Cultivate these diagnostic strains using traditional as well as novel cultivation techniques in order to identify organisms that may be of value in site evaluation/management or bioremediation. APPROACH: Analysis of metal concentration profiles will be performed at a site contaminated by chromium and other metals. Total community DNA will be extracted, and amplified, and the PCR product digested with 3-6 restriction enzymes to provide RFLPs to create a community profile. Profiles will be compared between communities from pristine environments versus. contaminated environments. PROJECT: Horizontal Gene Transfer as Adaptive Response to Heavy Metal Stress in Subsurface Microbial Communities PRINCIPAL INVESTIGATOR: Barth F. Smets PROGRAM ELEMENT 2 Community Dynamics and Microbial Ecology OBJECTIVE: Analysis of metal concentration profiles will be performed at a site contaminated by chromium and other metals. total community DNA will be extracted, and amplified, and the PCR product digested with 3-6 restriction enzymes to provide RFLPs to create a community profile. Profiles will be compared between communities from pristine environments versus contaminated environments. APPROACH: Two different scenarios of plasmid transfer will be studied, both at different heavy metal stress intensities. The first scenario will examine the transfer of a naturally occurring TOL plasmid harboring transposon-located xyl genes. A variant of this plasmid, TOL::Tn401, carrying a stable transposon-located antibiotic resistance marker will be used. Transposition of the xyl genes from the plasmid to the chromosome and stability of the plasmid in both donor strain and the resident community will be monitored. Quantification of plasmid transfer to the indigenous community will be performed after a selective elimination of the donor strain. The donor strain (in casu Pseudomonas putida) will carry a suicide gene circuit that consist of a lethal gene (gef gene). The entire suicide gene circuit is firmly inserted in the chromosome such that horizontal transfer of this gene circuit can not occur. The TOL plasmid does not encode functions directly related to bacterium-heavy-metal interactions; however it serves as a well studied model system for a naturally occurring catabolic conjugal transfer. The second scenario will examine transfer of pMOL155 an IncQ non-conjugal mobilizable plasmid harboring czc genes encoding resistances against Cd2+, Zn2+, and Co2+. Its transfer from the host strain (in casu Alcaligenes eutrophus) to the resident microbial community can only occur in a two-step process via a process called retro-transfer. The initial transfer of a conjugal plasmid, such as RP4 plasmid, to the pMOL155 Alcaligenes eutrophus bearing strain is required to allow direct pMOL155 transfer. Thus in absence of the RP4 plasmid, pMOL155 will only be transferred to the indigenous community via its own DNA mobilizing abilities. Furthermore, a P. putida strain with a chromosomal conditional suicide gene will be used to deliver RP4 in the resident community. The ability of the resident community to transfer the acquired RP4 will also be confirmed by using Rifr P. fluorescens strains as recipient. This set of experiments will allow one to quantify the ability of the microcosm community to mobilize plasmid DNA by means of naturally resident conjugal plasmids or the introduced conjugal plasmid RP4. The above mentioned conjugal transfer experiments will also be examined in liquid batch media with strains isolated from the analyzed microcosms. The isolated strains will be typed for their metabolic abilities as well as by their 16S rRNA gene sequence. A potential difficulty in examining gene transfer in natural ecosystems, concerns the establishment of a specific phenotype in the recipient upon gene transfer. To address such concern, we will insert PrlacZ (that is expressed in wide array of bacteria) in TOL::Tn401 and pMOL155 and use these constructions in donor strains in which the expression of the PrlacZ is strongly repressed. The transfer rates based on the expression of the PrlacZ will be compared to those obtained on the basis of the characters that are linked to TOL::Tn401 and pMOL155 plasmids (growth on m-toluate and Cd2+ and Zn2+ resistance respectively). We will also construct an element linking Pr to the green fluorescent protein gene (gfp) by using available gfp containing vectors. By placing such construct on a mobile DNA element, a very facile evaluation of gene transfer will be based on expression of the green fluorescent protein in the recipient strain. The effect of the applied stresses on the microcosm microbial communities will be monitored using an ARDRA (Amplified Ribosomal DNA Restriction Analysis) analysis. DNA will be extracted from the soil microcosms samples and the 16S rRNA genes amplified by PCR. The amplified genes will be digested by restriction enzymes and the digestion patterns at different experimental conditions compared. PROJECT: Noncompetitive Microbial Diversity Patterns in Soils: Their Causes and Implications for Bioremediation PRINCIPAL INVESTIGATOR: James M. Tiedje PROGRAM ELEMENT 2 Community Dynamics and Microbial Ecology OBJECTIVE: The goal of this study is to establish a scientific foundation for in situ bioremediation of DOE contaminated sites through understanding the mechanisms that control structure, composition, function and dynamics of microbial communities. Towards this goal, research will be focused on the following objectives: (1) to determine the structure and composition of microbial communities as influenced by radioactive and mixed waste contaminants; (2) to determine the mechanisms responsible for high soil microbial diversity. APPROACH: To achieve these objectives, the following hypotheses will be tested: (1) species richness is reduced by toxicity in both competitive and noncompetitive communities, but dominance is increased only in the competitive community and unaffected in the noncompetitive community; (2) phylogenetic diversity of microbial communities at toxic sites is lower compared to adjacent nontoxic sites, and the proportion of gram positive bacteria is increased; (3) the noncompetitive diversity pattern is primarily a result of spatial isolation of soil populations; (4) carbon resource heterogeneity is an additional factor contributing to the noncompetitive diversity pattern. We will test these hypotheses by (1) comparing microbial community structure and composition at DOE radioactive and mixed waste sites and the adjacent noncontaminated sites using a 16S rRNA gene cloning approach followed by restriction pattern analyses and sequence analyses; (2) examining the existing structure of microbial communities from soils with different textures and moisture; and (3) experimental manipulation of the degree of spatial isolation and resource heterogeneity in laboratory. Also quantitative indices will be developed for assessing the differentiation of microbial community diversity patterns. This research will be conducted as a collaborative project at Michigan State University and Oak Ridge National Laboratory. [Back to Award Recipients Page]