A Molecular Diagnostics "Smoking Gun" for Natural Attenuation
Aaron Peacock, Site Logic, Oak Ridge, TN

Extracellular Electron Shuttling in Bioremediation and Biotechnology
Kevin Finneran, University of Illinois, Urbana, IL

Bio-Trap™ Samplers Document In-Situ Microbial Oxidation and Assimilation of ¹³C-labeled Benzene under Denitrifying Conditions
Eric Hince, Geovation Technologies, Inc., Florida, NY

Improved Site Assessment of a Landfill-Leachate Plume using Microbial Community Profiles
Paula Mouser, University of Vermont, Burlington, VT

Obtaining Direct Evidence of ongoing InSitu Reductive Dechlorination using TCFE and RNA Analyses

Glenn M. White, Haley & Aldrich, Inc., 200 Town Centre Drive, Rochester, NY  14623, Tel: 585-321-4239, Fax: 585-486-4239, Email: gwhite@haleyaldrich.com
Greg Davis, Microbial Insights, Inc., 2340 Stock Creek Blvd., Rockford, TN 37853, Tel: 865-573-8188, Fax: 865-573-8133, Email: gdavis@microbe.com
Dora Ogles, Insights, Inc., 2340 Stock Creek Blvd., Rockford, TN 37853, Tel: 865-573-8188, Fax: 865-573-8133, Email: dogles@microbe.com
Aaron Peacock, Center for Biomarker Analysis, 10515 Research Drive, Suite 300, Knoxville, TN 37932,  Tel: 865-974-8014, Fax: 865-974-8027, Email: apeacock@UTK.edu
Jennifer Busch-Harris, Center for Applied Biogeosciences, University of Tulsa,  600 South College Ave, Tulsa, OK 74104, Tel: 918-631-3422, Fax: 918-631-3268, Email: Jennifer-busch@utulsa.edu
Eleanor Jennings, Center for Applied Biogeosciences, University of Tulsa, 600 South College Ave, Tulsa, OK 74104, Tel: 918-631-3028, Fax: 918-631-3268, Email: Eleanor-jennings@utulsa.edu
Kerry Sublette, Center for Applied Biogeosciences, University of Tulsa,  600 South College Ave, Tulsa, OK 74104, Tel: 918-631-3085, Fax: 918-631-3268, Email: kerry-sublette@utulsa.edu
Robert Pirkle, Microseeps Inc., 220 William Pit Way, Pittsburgh, PA 15238, Tel: 412-826-5245, Fax: 412-826-3433, Email: pmcloughlin@microseeps.com
Pat McLoughlin, Microseeps Inc., 220 William Pit Way, Pittsburgh, PA 15238, Tel: 412-826-5245, Fax: 412-826-3433, Email: rpirkle@microseeps.com

Ideally we would like to know the following three bits of direct information in order to evaluate anaerobic biodegradation of chlorinated solvents in groundwater: 1) is biodegradation occurring, 2) is it progressing to completion, and 3) how fast is it happening? However, with current methods we can only obtain insight enough to predict the answers to these questions.  This work is an effort to refine existing molecular biological tools (MBTs) to the point where they can provide us with direct, unambiguous information including proof of ongoing reductive dechlorination.  Bio-Traps impregnated with trichlorofluoroethene (TCFE) were deployed at a chlorinated solvent biostimulation site where ethene is routinely detected.  It was previously determined in the laboratory that the fluorinated daughter products will adhere to the Bio-Sep material in the Bio-Traps.  Bio-Traps are to be removed after 30, 60, and 120 days.  The 60-day traps contained 4.0 ug/bead of cis-1,2-dichlorofluoroethene (which constitutes proof of ongoing biodegradation) and significantly more RNA compared to the 30-day traps (1.0 X 10²  to 1.0 X 10⁶ gene copies per bead), which suggests that the amount of TCFE metabolic activity overtime may have increased.  The 60-day analysis also indicated TCE RDase at 1.0 X 10² gene copies per bead and VC RDase 1.0 X 10³ gene copies per bead.  This data will be compiled to track the rate of TCFE degradation on the Bio-Traps and correlate it to the expression of the functional genes involved in the degradation processes.  The long term goal of this work is to determine the significance of the amount of degraders in the subsurface with respect to degradation rate.  The 120-day results are pending.

Monitoring the Metabolic State of Geobacteraceae during in situ U(VI) Bioremediation

Dawn E. Holmes, University of Massachusetts Amherst, Dept of Microbiology, Morrill IV North Science Center, Amherst, MA, 01002, Tel: 413-577-0447, Fax: 413-545-1578, Email: dholmes@microbio.umass.edu
Regina A. O’Neil, University of Massachusetts Amherst, Dept of Microbiology, Morrill IV North Science Center, Amherst, MA, 01002, Tel: 413-577-0447, Fax: 413-545-1578, Email: rtarallo@microbio.umass.edu
Derek R. Lovley, University of Massachusetts Amherst, Dept of Microbiology, Morrill IV North Science Center, Amherst, MA, 01002, Tel: 413-545-9651, Fax: 413-545-1578, Email: dlovley@microbio.umass.edu

Stimulation of metal reducing microorganisms such as Geobacter species is a promising strategy for the reductive immobilization of U(VI) in contaminated aquifers. An understanding of the rates of microbial metal reduction will help develop strategies for optimization of the bioremediation process. However, it is difficult to monitor in situ metabolic rates using geochemical techniques. Therefore, a strategy for monitoring expression of genes that are indicative of metal reduction rates and the associated metabolic states of microorganisms participating in U(VI) reduction was developed. Relevant Geobacteraceae genes were identified from microarray experiments conducted under a variety of environmentally relevant conditions. Field experiments were conducted at a uranium-contaminated site in Rifle, CO.   Acetate was injected into the subsurface to stimulate the growth of metal-reducing microorganisms. During the active phase of U(VI) reduction, Geobacteraceae accounted for 90% and 57% of the bacterial sequences in the groundwater and sediments, respectively.  The in situ metabolic state of Geobacteraceae was determined by tracking the expression of stress response, nutrient limitation, and chemotaxis and motility genes.  Results show that the oxidative stress genes, cydA and sodA, as well as the gene heavy metal efflux gene, cusA, were expressed during bioremediation. Moreover, genes indicative of nitrogen (nifD), phosphorous (phoU), and Fe(II) (feoB and ideR) limitations were also expressed.  Interestingly, expression of the chemotaxis and motility gene, pilT, was inversely correlated with Fe(II) concentrations in the groundwater. As was expected, the number of mRNA transcripts from genes involved in central metabolism (gltA, mdh, and korA) and electron transport (mcpA and ompB) was correlated with acetate concentrations in the groundwater. These results demonstrate that monitoring the in situ transcript levels of key genes can provide insight into the rates of metabolism and nutrient requirements of Geobacteraceae during in situ bioremediation of uranium.

A Molecular Diagnostics “Smoking Gun” for Natural Attenuation

Aaron D. Peacock, Site Logic Consulting, Oak Ridge, TN, 37830, Tel: 865-385-1944, Email: apeacock@utk.edu
Jack Istok, Groundwater Research Laboratory, Oregon State University, Corvallis, OR, 97331, Tel: 541-737-8547, Fax: 541-737-9090, Email: jack.istok@oregonstate.edu
Greg A. Davis, Microbial Insights Inc., 2340 Stock Creek Blvd., Rockford, TN, 37853, Tel: 865-573-8188, Fax: 865-573-8133, Email gdavis@microbe.com
Dora Ogles, Microbial Insights Inc., 2340 Stock Creek Blvd., Rockford, TN, 37853, Tel: 865-573-8188, Fax: 865-573-8133, Email dogles@microbe.com
Eric Reas, Engineering and Land Planning, Clinton, NJ, 08809, Tel: 908-238-0544, Email: ereas@elp-inc.com

Demonstrating whether biodegradation of contaminants is occurring, or is likely to occur under a specific set of environmental conditions is a key factor in managing any site where biological processes are relied upon for remediation.  We have developed a combination of molecular tests and reactive tracers that provide “smoking gun” evidence of in-situ degradation of contaminants.  Field sampling and testing were used to investigate the relationship between baseline geochemical and microbial community data with in situ reductive dechlorination rates at a site contaminated with trichloroethene (TCE) and carbon tetrachloride (CTET).  Ten monitoring wells were selected to represent conditions along a groundwater flow path from the contaminant source zone to a wetlands groundwater discharge zone.  Background samples were analyzed for a suite of geochemical and microbial parameters; push-pull tests with fluorinated reactive tracers were used to measure in situ reductive dechlorination rates.  A principal component analysis identified three groups of wells with similar geochemical and microbial characteristics.  Push-pull tests were conducted using trichlorofluoroethene (TCFE) as a reactive tracer for TCE and trichlorofluoromethane (TCFM) as a reactive tracer for tetrachloromethane (CTET).  Injected TCFE was transformed in situ to cis- and trans-dichlorofluoroethene, chlorofluoroethene and, in one test, completely dechlorinated to fluoroethene.  Injected TCFM was transformed in situ to dichlorofluoromethane and chlorofluoromethane.  Zero-order TCFE transformation rates ranged from < 0.05 to 1.00 nM/hr (< 0.44 to 8.76 μM/yr).  A single TCFM transformation rate was estimated as < 0.05 nM/hr (0.44 μM/yr).  The results indicate that it is possible to use push-pull tests with reactive tracers to detect and quantify reductive dechlorination of chlorinated ethenes and ethanes under monitored natural attenuation conditions.  TCFE reduction rates were different for the three groups of wells identified by principal component analysis providing preliminary evidence that geochemical, microbiological, and in situ reductive dechlorination rates  may provide complimentary information. 

Extracellular Electron Shuttling in Bioremediation and Biotechnology 

Kevin T. Finneran, Environmental Engineering and Sciences, Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, 3221 Newmark Civil Engineering Laboratory, Urbana, IL 61801, Tel: 217-244-7956, Fax: 217-333-6968, Email: finneran@uiuc.edu

Extracellular electron shuttling compounds are molecules that transfer electrons between microbial biomass (as electron acceptors) to electronegative molecules in natural or engineered environments.  They are particularly useful to bioremediation applications in which the contaminant cannot be directly accessed by individual cells or in situations where Fe(III) reduction must be stimulated.  The molecules that have been studied most intensively to date are extracellular quinones and humic substances, which are natural organic compounds with multiple quinone functional groups.  Electron shuttling has been reported to promote biodegradation of numerous organic and inorganic contaminants.  Our work has focused on extracellular electron shuttling by prokaryotes and eukaryotes, and its effects on electronegative contaminants in subsurface environments.  In addition, our work has explored the novel biotechnology application of extracellular electron shuttling – increasing fermentative hydrogen production for hydrogen fuel.  The results presented in this seminar will include work conducted with prokaryotic transformation of the cyclic nitramine explosives RDX and HMX, and transformation of chlorinated organic molecules in contaminated sediment.  The talk will also address eukaryotic electron shuttling in aerobic environments, particularly amongst the white and brown rot fungi.  Finally, it will discuss data for increasing the molar hydrogen yield in fermentative pure cultures, and the role that reduced electron transfer molecules play in the hydrogen fuel economy.

Bio-Trap^TM Samplers Document In-Situ Microbial Oxidation and Assimilation of ¹³C-labeled Benzene under Denitrifying Conditions

Eric C. Hince, Geovation Consultants, Inc., 468 Route 17A, Florida, NY 10921, Tel: 845-651-4141, Fax: 845-651-0040, e-mail: echince@geovation.com
Greg Davis, Dora Ogles and Aaron Peacock, Microbial Insights, Inc., 2340 Stock Creek Blvd., Rockford, TN  37853-3044, Tel: 865-573-8188, Fax: 865- 573-8133, e-mail: gdavis@microbe.com, dogles@microbe.com,  apeacock@microbe.com
Kerry Sublette, Jennifer Busch-Harris and Eleanor Jennings; University of Tulsa, Center for Applied Biogeosciences, 600 S. College Ave, Tulsa, OK  74104, Tel: 918-631-3085, Fax: 865- 573-8133, e-mail:  kerry-sublette@utulsa.edu, jennifer-busch@utulsa.edu, eleanor-jennings@utulsa.edu

An in-situ denitrification-based bioremediation (“DBB”) program was implemented in the fall of 2004 to treat a gasoline-contaminated shallow aquifer at DoD-owned gasoline service station site in Maine.  Sampling of “smear zone” aquifer media documented a sharp decline in the sorbed-phase gasoline hydrocarbons in response to DBB treatment (Liyang Chu, et al., in submission).  Several molecular, culture-independent methods were used to characterize the denitrifying, gasoline-degrading microbial consortia stimulated by the DBB treatments, including denaturing gradient gel electrophoresis (DGGE), real-time polymerase chain reaction (qPCR) and multi-color fluorescence in-situ hybridization (“mFISH”) (Hince, 2005, U. Mass Soils Conf.; Hince and Ogles, 2005, IPEC).

A Bio-trap^TM study (http://www.microbe.com/biotrap.htm) was conducted from November to December 2005 to evaluate the intrinsic ability of the DBB-stimulated microbial consortia to degrade the aromatic hydrocarbons present in the aquifer.  Two Bio-traps^TM containing Bio-sep^TM beads loaded with ¹³C-labeled benzene were installed in two key wells:  DP-13 located in the source area of the historical gasoline release and DB-04 located mid-plume and in the main DBB treatment area.  The Bio-traps^TM were suspended in these wells for approximately one month using floats to maintain the Bio-traps^TM at a fixed distance beneath the water table and within the zone of highest hydrocarbon contamination.  Subsequently, the Bio-traps^TM were retrieved and shipped to Microbial Insights, Inc. (Rockford, TN) for molecular microbiological assays and determination of the mass of remaining ¹³C-labeled benzene.  MII also sequenced the eubacterial 16s rDNA separated via DGGE and analyzed the stable carbon isotope profiles (¹³C/¹²C) of the phospholipid fatty acid (PLFA) biomarkers recovered from the microbial biomass in the Bio-traps^TM.  The isotopic abundance of dominant fatty acids (those comprising greater then 1-2% of the total PLFA profile) was determined by gas chromatography-isotope ratio monitoring mass spectrometry (GC-IRMS).

The analytical results are among the most conclusive observed to date for the anaerobic oxidation and assimilation of benzene using ¹³C-labeled benzene in Bio-traps^TM.  Approximately 78% and 43%, respectively, of the ¹³C-labeled benzene was degraded in the Bio-traps^TM installed in DP-13 and DB-04 over a one-month period.  Biomarkers associated with the Proteobacteria dominated the PLFA profiles from both wells, consistent with the dominance of Gammaproteobacteria (Pseudomonadaceae) and Betaproteobacteria (Comamonadaceae) in the DGGE profiles and mFISH assays.  Whereas the DGGE profiles were dominated by 16s rDNA sequences related mostly to the family Pseudomonadaceae, quantitative mFISH analyses indicated that Betaproteobacteria were actually more abundant than the Gammaproteobacteria in site groundwater.

In DP-13, several lipid biomarkers exhibited δ-¹³C enrichment values that approached the theoretical maximum values (based on the initial loading of ¹³C-labeled benzene):  e.g., +5,992 (biomarker 16:1w7c) and +6,627 (biomarker 18:1w7c).  ¹³C enrichment was also observed in two fungal biomarkers, 18:2w6 (+4,738 δ-¹³C) and 20:4w6 (+5,106 δ-¹³C).  An unusual finding in DP-13 was the eukaryotic biomarker 20:5w3 (+5,133 δ-¹³C) often associated with microeukaryotic “grazers.” The Bio-sep^TM beads used in the Bio-traps^TM have small pore spaces that should limit access by the larger eukaryotic grazers.  Given the presence of other fungal biomarkers, the presence of the 20:5w3 biomarker may also be associated with fungal growth.  In DB-04, δ-¹³C enrichment values of lipid biomarkers were lower but still quite high:  e.g., +1,272 (biomarker 16:1w7c) and +2,996 (biomarker 18:1w7c).  ¹³C-labeled benzene degradation and δ-¹³C enrichment of lipid biomarkers correlated closely with the PLFA biomass and total microbial cell counts (determined via epi-fluorescent microscopy, DAPI staining), which were proportionately higher in DP-13 than DB-04.  

Improved Site Assessment of a Landfill-Leachate Plume using Microbial Community Profiles

Paula J. Mouser, Doctoral Student, Department of Civil and Environmental Engineering, University of Vermont, 213 Votey Building, Burlington, VT  05405; Tel: 802-656-1937, E-mail: Paula.Mouser@uvm.edu
Donna M. Rizzo, Assistant Professor, Department of Civil and Environmental Engineering, University of Vermont, 213 Votey Building, Burlington, VT  05405; Tel: 802-656-1495, E-mail: drizzo@cems.uvm.edu
Patrick O’Grady, Assistant Professor, Department of Environmental Science, Policy, and Management, University of California, Berkeley, 125A Hilgard, Berkeley, CA 94720; Tel: 510-643-7430; E-mail: ogrady@nature.berkeley.edu
Greg Druschel, Assistant Professor, Department of Geology, University of Vermont, 321 Delehanty Hall, Burlington, VT  05405; Tel: 802-656-3481; E-mail: gregory.druschel@uvm.edu
Lori Stevens, Associate Professor, Department of Biology, Ecology, and Molecular Systematics, University of Vermont, 318 Marsh Life Sciences Building, Burlington, VT 05405; Tel: 802-656-0445; E-mail: lori.stevens@uvm.edu

Many municipal landfills are unlined and have released leachate, or liquids that have come in contact with waste products, to the underlying soils and groundwater. Leachate-contaminated groundwater is rich in organic matter, nutrients, and metals, which distinctly changes the subsurface environment. Microbial communities are linked to changes in the subsurface hydrochemistry, and gaining quantitative information about the microorganisms to improve our knowledge of the movement and degradation of the leachate-contamination is not well researched. We sampled groundwater monitoring wells over time and space at a leachate-contaminated aquifer in northeastern New York using the 16S rRNA gene for Archaea, Bacteria, and Geobacteraceae. Community profiles were generated for each group of organisms using terminal restriction fragment length polymorphism (T-RFLP), and the community shifts over time were quantified using the Jaccard Index. Shifts in communities for three groundwater zones; unimpacted, plume fringes, and contaminated, followed distinct temporal trends and were significantly correlated to changes in groundwater hydrochemistry. Our results show how microbial community dynamics, monitored using molecular methods, can be used to improve our knowledge of the groundwater system at a leachate-contaminated site. 

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