Sponsored by Geovation Engineering, P.C.

Advanced Diagnostic Tools and Applications to Site Design, Management and Closure
Stephen S. Koenigsberg, Ph.D., WSP Environmental Strategies, Irvine, CA

Applying Environmental Biotechnology to a VCUP Program for the Remediation of a Mixed Chlorinated Solvent Plume            
William R. Mahaffey, Ph.D., Pelorus EnBiotech Corp, Evergreen, CO

Comparison of Sediment, Groundwater, and Bio-Trap Microbial Samples from a Biostimulation Study at Rifle, Colorado
Aaron Peacock, Ph.D., Haley & Aldrich, Rockford, TN

Combining Transcriptomic and Proteomic Methods to Develop Bioindicators of Chlorinated Solvent Bioremediation
Ruth Richardson, Cornell University, Ithaca, NY

In-Situ Stable Isotope and Fluorinated Analog Probing to Evaluate Fate of cDCE via both Reductive Dechlorination and Anaerobic Oxidation
Eric Hince, Geovation Engineering, P.C., Florida, NY 

Reductive Dechlorination of Chlorinated Solvents with Electrodes as the Electron Donor
Derek R. Lovley, University of Massachusetts, Amherst, MA

Round Table Discussion:  "Current Practices and Future Directions in the Application of Molecular Biological Tools and Emerging Biotechnologies"

Advanced Diagnostic Tools and Applications to Site Design, Management and Closure 

Stephen Koenigsberg, WSP Environmental Strategies, 4199 Campus Drive, Suite 550, Irvine, CA  92612
John Simon, WSP Environmental Strategies, 11911 Freedom Drive , 9th Floor, Reston , VA 20190
Matt Burns, WSP Environmental Strategies, 1740 Massachusetts Avenue , Boxborough , MA   01719
David Sarr, WSP Environmental Strategies, 11911 Freedom Drive , 9th Floor, Reston , VA 20190
Scott Haitz, WSP Environmental Strategies, 11911 Freedom Drive , 9th Floor, Reston , VA 20190

Site remediation has evolved from energy intensive, mechanically driven remediation processes to more effective and cost efficient in situ processes.  In many cases, in-situ remediation is best served if integrated with advanced diagnostics that employ molecular biological tools (MBTs) and compound specific isotope analysis (CSIA).  Diagnostic protocols, taken alone or in combination, can help with site assessment and subsequently assure the best in-situ remedy from among the available options.  As an extension of this, the diagnostics can further be used for optimization of on-going remedial operations. 

While advanced diagnostics can assist in site assessment and the design and management of in situ remediation options, there are also important extensions of this work to site closure, with an emphasis on monitored natural attenuation (MNA).  We have applied advanced diagnostics in ten remedial operation s s ince March, 2006.  In eight cases the diagnostics were used to help site assessment and/or remedy selection and these results will be summarized. In two cases, which will be discussed in detail, there was movement directly to an MNA status based on the use of MBTs.

The larger of the two sites that received and MNA ruling had soil and groundwater concentrations of chlorinated solvents greater than the Georgia Risk Reduction Standards (RRS).  Applications of phospholipid fatty acid analysis (PLFA) and nucleic acid diagnostics were employed to assess the status of natural attenuation prepare a petition for MNA.  With the results of these analyses the site was put into an MNA status.  This ruling was in concert with excavation of the source and an ozone treatment of source groundwater.  The MBT data was used to successfully establish that downgradient bioremediation barriers could be held in abeyance subject to the impacts of the source treatments and if permanently avoided an average cost savings of $1.2 MM will be realized.

Applying Environmental Biotechnology to a VCUP Program for the Remediation of a Mixed Chlorinated Solvent Plume

William R. Mahaffey, Pelorus EnBiotech Corp, 3528 Evergreen Parkway, Evergreen, CO 80439, Tel: 303-670-2875, Fax: 303-670-5139
Mark Miller, DOMANI Sustainability, LLC, Denver, CO, 80202, Tel: 303-232-0193, Fax: 303-232-0394/865-974-8027
Duane Wanty, Invensys, Inc., 33 Commercial Street C41-TE, Foxboro, MA  02035, Tel: 508-549-6004, Fax: 508-549-6152
Aaron Peacock, Center for Biomarker Analysis, 10515 Research Drive, Suite 300, Knoxville, TN 37932-2575, Tel: 865-974-8030

Molecular diagnostics inconjunction with standard bioindicator parameters are becoming valuable tools for site evaluation, remedy selection and post remedial site evaluation.  A former solvent recovery process at an industrial facility released chlorinated volatile organic compounds (CVOCs) to the groundwater.  The major constituents exceeding Illinois Tier 1, Class 1 groundwater standards are; 1,1,1-Trichloroethane [TCA], 1,1-Dichloroethene (1,1-DCE), 1,1-Dichloroethane (1,1-DCA), Tetrachloroethene [PCE] and Trichloroethene [TCE].  Under a voluntary cleanup program a source area removal action was performed to remove soil impacts and a groundwater remediation program implemented.  A supplemental site characterization was implemented to collect groundwater data on organic and inorganic bioindicator parameters.  In addition microbial traps were emplaced in a series of wells throughout the plume to collect samples of the microbial community and analyze the community structure using molecular genomic tools. Specifically, the presence and response of dechlorinating microbes to stimulation with anaerobic aquifer conditioners was monitored.  The objective was to use the molecular genomic tools and bioindicator parameter data to define and guide the remedial system design, and monitor remedial performance.  Over a period eighteen months, six organic acid nutrient injection events were performed to stimulate anaerobic reductive dechlorination of the target contaminants.  Transect mass flux analysis and spatial moments analyses were used for analyzing performance monitoring data and demonstrated complete remediation and retraction of the dissolved phase plume to the source area. Only two of eleven monitoring wells had impacts exceeding the regulatory limits.  In the nine down gradient monitoring wells choroethenes were completely degraded to ethene and 1,1,1-TCA was transformed to ethane with transient accumulation of 1,1-DCA, and chloroethane.  Analyses of microbial traps showed up to three orders of magnitude increases in the Dehalococcoides ethenogenes and Dehalobactor sp. subsequent to stimulation with organic nutrient supplementaton.  In addition key reductive dehalogenase gene copies (TCE rd, BAV-1 VCrd and VCrd) were observed to increase during the treatment program.  

Comparison of Sediment, Groundwater, and Bio-trap Microbial Samples from a Biostimulation Study at Rifle, Colorado

Aaron D. Peacock, Haley & Aldrich, Inc. 103 Newhaven Rd, Oak Ridge, TN 37830,  Tel: 913-787-4172, Fax: 913-599-5822, Email: apeacock@haleyaldrich.com
David B. Hedrick, Microbial Insights, Inc. 2340 Stock Creek Blvd. , Rockford , TN 37853 , Tel: 865-573-8188 Email: dhedrick@microbe.com

In bioremediation, it is crucial to be able to monitor the subsurface microbial community in terms of its biomass, community composition, and activities.  However, the subsurface is difficult to access and has large sample-to-sample variation.  Sediment samples are the gold standard of subsurface conditions.  However, the great expense of obtaining quality samples of subsurface sediments limits the number available.  Also, due to technical or legal restrictions, the locations that can be sampled may be further limited.  The high variation in the subsurface environment means that any one sample is not a very good indicator of subsurface conditions - several samples must be taken before reliable estimates can be made.  Bioremedial treatments (for example, addition of carbon or oxygen) will affect a portion of the microbial community, and there may be another portion which is still present but unaffected.  Groundwater samples are typically much more readily available.  Since the goal is groundwater quality, wells have usually already been installed for groundwater sampling.  These groundwater samples must either be filtered on-site, or chilled and shipped to a laboratory for analysis.  Bio-traps are small perforated plastic cylinders packed with Bio-Sep beads, which are composed of the plastic Nomex and powdered activated carbon.  The internal structure of the Bio-Sep provides a large surface area for bacterial colonization.  Bio-traps are suspended in existing wells, for a period of time, and then retrieved and analyzed by methods appropriate for sediment samples including molecular, lipid, and microscopic techniques.  In this study we compared sediment, groundwater and Bio-trap samples taken during a biostimulation event designed to reduce the amount of soluble Uranium in site groundwater.  Results show the groundwater community reacts more strongly to subsurface treatments than the total subsurface community, while Bio-traps appear to capture just the active portion of the microbial community from the groundwater.

Combining Transcriptomic and Proteomic Methods to Develop Bioindicators of Chlorinated Solvent Bioremediation

Robert M. Morris, UC Santa Barbara
Brian G. Rahm, Cornell University , Civil & Environmental Engineering, Ithaca , NY 14853
Stephen H. Zinder, Cornell University , Microbiology, Ithaca , NY 14853
Ruth E. Richardson, Cornell University , Civil & Environmental Engineering, Ithaca , NY 14853 , Email: rer26@cornell.edu

Molecular bioindicators (DNA, RNA, and protein) show promise for aiding bioremediation efforts at chloroethene-contaminated field sites. While DNA can be analyzed to show the presence of Dehalococcoides (DHC) populations, RNAs and enzymatic proteins serve as more appropriate bioindicators of instantaneous activity of those populations. In this study, we elucidate several RNA and protein bioindicators that are highly expressed during chloroethene dehalorespiration by DHC.

The genome of Dehalococcoides ethenogenes strain 195 was first screened for candidate bioindicator genes including a housekeeping gene (RNA polymerase), reductive dehalogenases (RDases), and other genes with potential roles in the dehalorespiration process. Expression of these genes was determined at the level of RNA via reverse transcription quantitative polymerase chain reaction (qRTPCR). To validate the RNA results and to develop protein-based bioindicators we also ran shotgun proteomics (via tandem mass spectrometry of digested proteins -GeLC/MS/MS) on proteins from these cultures. The most highly expressed RDases were TceA, PceA and two other putative RDases (DET gene numbers 1545 and 1559). Outside of the RDases, the most highly expressed candidates were the hydrogenase Hup and a gene annotated as formate dehydrogenase (“Fdh”). 

The proteomic analyses were then extended to three other cultures containing DHC strains: pure culture strain CBDB1 (grown on trichlorophenol), the bioaugmentation culture KBlTM, and an uncharacterized PCE-to-ethene enrichment. Through these comparative proteomic studies, we detected several proteins (including "Fdh") in all cultures and on all substrates. As they are not strain-specific, such proteins might serve as good bioindicators of general DHC activity. In contrast, the suite of RDases detected varied considerably. Most notably, only cultures which respired vinyl chloride (VC) to ethene contained peptides matching VC RDases - suggesting that this protein is a specific bioindicator of VC-dechlorination activity. Our results have generated a suite of RNA and protein bioindicators that, with further development, should aid in situ bioremediation efforts.

In-Situ Stable Isotope and Fluorinated Analog Probing to Evaluate Fate of cDCE via both Reductive Dechlorination and Anaerobic Oxidation

Eric C. Hince, Geovation Engineering, P.C., 468 Route 17A, Florida, NY, 10921, Tel: 845-651-4141, Fax: 845-651-0040, Email: echince@geovation.com
Edward Sullivan, The Whitman Companies, Inc., 116 Tices Lane, Unit B-1, East Brunswick, New Jersey  08816, Tel: 732-390-5858 Ext 236, Fax: 732-390-9496, Email: ESullivan@whitmanco.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, Email: gdavis@microbe.com, Email: dogles@microbe.com, Email: 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, Email:  kerry-sublette@utulsa.edu, Email: jennifer-busch@utulsa.edu, Email: eleanor-jennings@utulsa.edu

Anaerobic biostimulation and bioaugmentation techniques are now among the most widely used remedies for chloroethene-contaminated groundwater aquifers.  Most of the literature and reports on field applications have focused on either biostimulation of reductive dechlorination or bioaugmentation with commercial cultures containing Dehalococcoides spp. whereas relatively little has been published concerning the anaerobic oxidation of chloroethenes.  As cDCE is a potentially persistent daughter of PCE and TCE, bioremediation strategies that can simultaneously promote both reductive dechlorination and anaerobic oxidation processes may be more rapid and effective than reductive dechlorination alone.  As reported by Paul Bradley (USGS) and his colleagues, anaerobic oxidation processes can be the dominant biodegradation pathways for partially reduced chloroethenes such as cDCE and vinyl chloride at some sites.  Nitrate and Mn(IV) appears to provide sufficient energy as electron acceptors for the anaerobic oxidation of both cDCE and vinyl chloride, whereas Fe(III) reduction likely provides sufficient energy for oxidation of vinyl chloride but perhaps not cDCE.

This paper will provide a progress report on the use of Bio-trapTM samplers to investigate both in-situ reductive dechlorination and anaerobic oxidation processes at two different chloroethene contamination sites in the greater New York metropolitan area.  Pairs of Bio-trapTM samplers have been designed and installed as follows:  one sampler with Bio-sepTM beads loaded with 13C-labeled cDCE will be used to investigate the anaerobic oxidation pathway whereas the second sampler with Bio-sepTM beads loaded with DCFE (a fluorinated analog of cDCE) will be used as a tracer for reductive dechlorination.  At one site in southern New York, complete reductive dechlorination is already occurring (prior to remediation) and a biostimulation demonstration program designed to promote simultaneous reductive dechlorination and anaerobic oxidation is underway.  At a second site in northern New Jersey, nano-scale iron was injected into a TCE source area that resulted in a large decrease in TCE and large increases in the biological daughter cDCE and total inorganic carbon without appreciable increases in vinyl chloride.  The available data from the NJ site suggests that the nano iron injection stimulated anaerobic oxidation and mineralization of cDCE to inorganic carbon.

Bio-trapsTM loaded with “normal” (12C) cDCE were installed and analyzed at both sites from June 2006 through January 2007 to investigate the relative rates of microbial biomass growth and cDCE degradation prior to the deployment of the 13C-labeled cDCE and DCFE loaded Bio-trapsTM.  The initial results have shown considerable losses of cDCE and the collection of PLFA biomarkers mostly associated with the Proteobacteria, consistent with potential heterotrophic anaerobic oxidation of cDCE.  The first 13C-labeled cDCE and DCFE loaded Bio-trapsTM were deployed at the NY demonstration site in January 2007.  The Bio-trapsTM will periodically be retrieved for analysis and replaced with new sets of Bio-trapsTM.  Bio-trapsTM will be analyzed for residual 13C-labeled cDCE, DCFE and the fluorinated daughters of DCFE to quantify cDCE degradation rates and for analysis via gas chromatography/isotope ratio-mass spectrometry (GC/IR-MS) to assess the incorporation of 13C into PLFA biomarkers.  Analysis of inorganic carbon and the 13C/12C isotopic signature thereof will help quantify the respiratory (versus biomass) fraction of cDCE oxidized anaerobically.  Quantitative PCR will be used to enumerate Dehalococcoides bacteria and the functional Rdase genes involved in reductive dechlorination and multi-color fluorescence in-situ hybridization (“FISH”) will be used to conduct co-localization studies of Dehalococcoides and other bacteria and to enumerate other important groups of anaerobic bacteria including Deltaproteobacteria and Firmicutes.  A battery of biogeochemical species will be analyzed to evaluate and correlate changes in biogeochemistry with the SIP and molecular microbiological data collected.  

Reductive Dechlorination of Chlorinated Solvents with Electrodes as the Electron Donor

Sarah M. Strycharz, Kelly P. Nevin, and Derek R. Lovley, Department of Microbiology, University of Massachusetts-Amherst, Amherst, MA 01003

The addition of various electron donors to groundwater to promote reductive dechlorination of chlorinated solvents has proven to be an effective bioremediation strategy in many instances.  However, adding the appropriate levels of electron donor that will stimulate reductive dechlorination, without unduly stimulating other forms of anaerobic respiration, can be problematic.  Furthermore, it can be difficult to add organic electron donors exactly to the required site for the most strategic application, especially when bioremediating source zone contamination.  Previous studies in our laboratory have demonstrated that Geobacter species can electrically interact with electrodes.  For example, Geobacter sulfurreducens and Geobacter metallireducens can generate electricity by oxidizing a variety of organic compounds to carbon dioxide with electron transfer to the anodes of microbial fuel cells.  When electrodes are poised at negative potentials Geobacter species can accept electrons, reducing contaminants such as nitrate and uranium.  Previous studies in the laboratory of Frank Loeffler have demonstrated that Geobacter lovleyi can reduce the chlorinated solvents PCE and TCE to DCE with acetate serving as the electron donor.  To determine whether an electrode might also serve as an electron donor for reductive dechlorination, G. lovleyi was pregrown on the graphite electrode surfaces with acetate as the electron donor and PCE as the electron acceptor.  Once dechlorination of PCE to DCE was observed the medium was replaced with a medium that did not contain acetate and the electrode was poised at –500 mV versus a silver/silver chloride reference electrode.  PCE was rapidly dechlorinated to DCE with the electrode serving as the sole electron donor.  Repeated additions of PCE continued to be rapidly removed.  Control electrodes, poised at –500 mV, but without a G. lovleyi biofilm, did not dechlorinate PCE.  As in previous studies in which an electrode served as an electron donor for the reduction of various other electron acceptors, hydrogen produced as the result of proton reduction at the electrode surface could be ruled out as an important electron donor for reductive dechlorination.  This is the first demonstration that electrodes can serve as an electron donor for microbially catalyzed reductive dechlorination.  The possibility that other types of dechlorinating microorganisms, such has Dehalococcoides species, might also accept electrons from electrodes is currently under investigation. Using electrodes to supply electrons to the subsurface for reductive dechlorination may prove to be a beneficial bioremediation approach, especially for the treatment of source zones.

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