A Kinetic Model of Reductive TNT Transformation by Escherichia coli Resting Cells
Hong Yin, University of Connecticut, Storrs, CT
Thomas K. Wood, University of Connecticut, Storrs, CT  
Barth F. Smets, University of Connecticut, Storrs, CT

Combined Anaerobic Oxidation and Reductive Dechlorination for Optimizing Bioremediation of CVOCs: Theoretical and Practical Considerations
Eric C. Hince, Geovation Technologies, Inc., Florida, NY

Evaluation of Biological Treatment Options for N-Nitrosodimethylamine (NDMA) in Groundwater 
Paul B. Hatzinger, Shaw Environmental, Inc., Lawrenceville, NJ
Sheryl Streger, Shaw Environmental, Inc., Lawrenceville, NJ
Kevin McClay, Shaw Environmental, Inc., Lawrenceville, NJ
Robert J. Steffan, Shaw Environmental, Inc., Lawrenceville, NJ  

Treatment of Vinyl Chloride Contaminated Groundwater at a Landfill Site: Comparison of Biosparging and iSOC™ Bioremediation Systems for Plume Cut-off
Christine R. LeBlanc, East Coast Engineering, Inc., Marion, MA
James F. Begley, MT Environmental Restoration, Plymouth, MA

Treatment of PCP-Contaminated Soil Using an Engineered Ex Situ Biopile Process at a Superfund Site
Carl Rodzewich, Biogenie Corporation, Quakertown, PA 
Nile Fellows, Minnesota Pollution Control Agency, St. Paul, MN
Nicolas Moreau, Biogenie Corporation, Quakertown, PA
Michel Pouliot, Biogenie Corporation, Quakertown, PA 

A Comprehensive Performance Analysis of Hydrogen Release Compound (HRC®):  Lessons Learned and Future Directions
Anna Willett, Regenesis, San Clemente, CA 
Stephen S. Koenigsberg, San Clemente, CA 

Bioremediation of Dual Contaminant Mixtures: TCE and RDX

Travis S.M. Young, Department of Civil Engineering, University of Nebraska Lincoln, Lincoln, NE 68588-0531, Tel: 402-472-5026, Fax: 402-472-8934, Email: travisy74@yahoo.com
Matthew C. Morley, Department of Civil Engineering, University of Nebraska Lincoln, Lincoln, NE 68588-0531, Tel: 402-472-2057, Fax: 402-472-8934, Email: mmorley2@unl.edu
Daniel D. Snow, Water Sciences Laboratory, 103 Natural Resources Hall, University of Nebraska-Lincoln, Lincoln, NE 68583-0844, Tel: 402-472-7539, Fax: 402-472-9599, Email: dsnow1@unl.edu

High explosives and chlorinated solvents are common soil and groundwater contaminants at numerous military facilities worldwide. One such site is the former Nebraska Ordnance Plant (Mead, NE), which has areas of contaminated groundwater with both trichloroethylene (TCE) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazocine). Contaminated groundwater is currently extracted and treated using ex-situ adsorption to granular activated carbon (GAC). Bioremediation of this contaminant mixture is a potential alternative for treating groundwater at the site, as numerous studies have found that these contaminants are amenable to biological transformation under reducing conditions. In the present study, two cultures are being tested for biodegradation potential: anaerobic digester sludge from a municipal wastewater treatment plant, and a facultative culture enriched from solids backwashed from GAC contactors exposed to TCE- and RDX-contaminated groundwater. With no prior exposure to RDX, both cultures biodegraded RDX in single-component tests under anaerobic conditions. RDX concentrations decreased rapidly with evidence for transient production of nitroso-RDX metabolites. The culture enriched from the backwash solids degraded RDX at a much faster rate than the anaerobic sewage sludge.  Unacclimated anaerobic sludge did not transform TCE during initial 10-day batch tests. Both cultures are currently being grown in the presence of TCE, and additional tests will determine if the acclimated cultures can biodegrade TCE and mixtures of TCE and RDX. Successful biodegradation of this contaminant mixture will yield a new remedial option that can reduce the required time and costs for remediating this contaminant mixture.

A Kinetic Model of Reductive TNT Transformation by Escherichia coli Resting Cells

Hong Yin, Environmental Engineering Program, University of Connecticut, 261 Glenbrook Road, Storrs, CT 06269-2037, Tel: 860-486-0583, Fax: 860-486-2298, Email: yinh@engr.uconn.edu
Thomas K. Wood, Department of Chemical Engineering & Molecular and Cell Biology, University of Connecticut, 191 Auditorium Road, Storrs, CT 06269-3222, Tel: 860-486-2483 , Fax: 860-486-2959, Email: twood@ngr.uconn.edu
Barth F. Smets, Dept of Civil and Environmental Engineering & Molecular and Cell Biology, University of Connecticut, 261 Glenbrook Road, Storrs, CT 06269-2037,Tel: 860-486-2270, Fax: 860-486-2298, Email: barth.smets@uconn.edu

TNT biotransformation via nitro-reduction may be harnessed for bioremediation of hazardous waste sites. However, these transformations have been poorly understood or modeled. Traditional Michaelis-Menten model does not describe the nitro-reduction kinetics due to product toxicity and reducing power limitation experienced by cells. In this paper, TNT transformation by Escherichia coli was monitored and a kinetic model for TNT depletion was developed and experimentally calibrated. Using resting cells of aerobically pregrown Escherichia coli, 2,4,6-trinitrotoluene (TNT) was transformed via hydroxylaminodinitrotoluenes (2HADNT, 4HADNT, with 4HADNT as the dominant isomer) to 2,4-diamino-6-nitrotoluene (24DA6NT) as final product. Factors controlling the TNT transformation were quantified, including enzyme concentration, substrate concentration, and exogenous reducing power concentration. At higher cell densities, or by supplying glycerol as exogenous reducing power source, the rate and extent of TNT reduction increased revealing the importance of enzyme concentration and reducing power. Product toxicity (resulting from toxicity of TNT transformation intermediates) was quantified. A comprehensive model incorporating reducing power limitation, product toxicity, substrate inhibition, as well as substrate limitation adequately described the experimental observations.

Combined Anaerobic Oxidation and Reductive Dechlorination for Optimizing Bioremediation of CVOCs: Theoretical and Practical Considerations

Eric C. Hince, P.G., Geovation Technologies, Inc., 468 Route 17A, Florida, NY, 10921, Tel:  845-651-4141, Fax: 845-651-0040, Email: echince@geovation.com

In-situ anaerobic bioremediation has become a preferred remedy for ground-water contaminated with chlorinated solvents (CVOCs).  Most anaerobic bioremediation programs for CVOCs have focused on driving classical reductive dechlorination via the addition of carbon substrates (electron donors), bioaugmentation with Dehalococcoides cultures or both.  Relatively few examples of anaerobic oxidation processes have been demonstrated for CVOCs, either alone or in combination with reductive dechlorination.  Anaerobic oxidation processes coupled to the microbial reduction of electron acceptors such as nitrate, Mn(IV), Fe(III), and sulfate under anaerobic conditions may offer alternate catabolic pathways for CVOCs that may compliment reductive dechlorination.  For example, reductive dechlorination may proceed rapidly for highly chlorinated compounds such as PCE, TCE and 1,1,1-TCA and help activate these compounds for further degradation, whereas anaerobic oxidation processes may be more effective for less-chlorinated intermediates such as cDCE and vinyl chloride.  In addition, combined anaerobic oxidation-reduction treatment may help accelerate bioremediation by (a) improving electron flow and regenerating oxidants and reductants; (b) enhancing the biological removal of intermediates and byproducts of substrate-enhanced anaerobic treatment; and (c) promoting a broader array of catabolic processes than can directly or indirectly attack CVOCs and their daughter products.  Accordingly, the combination of anaerobic reduction and oxidation processes should provide means for improving and optimizing the anaerobic bioremediation of CVOCs, particularly for source areas of contamination.  Sites with large amounts of non-aqueous-phase contamination, (i.e., those that may require long-term treatment), may particularly benefit from this approach. Practical considerations for the assessment and implementation of combined anaerobic oxidation-reduction programs will be discussed, and data from ongoing anaerobic bioremediation programs will be presented to illustrate the efficacy and potential of this approach for CVOC source-area remediation.

Evaluation of Biological Treatment Options for N-Nitrosodimethylamine (NDMA) in Groundwater 

Paul B. Hatzinger, Ph.D., Princeton Research Center, Shaw Environmental, Inc., 4100 Quakerbridge Road, Lawrenceville, NJ 08648, Tel: 609-936-9300, Fax: 609-936-9221, Email: paul.hatzinger@shawgrp.com
Sheryl Streger, Princeton Research Center, Shaw Environmental, Inc., 4100 Quakerbridge Road, Lawrenceville, NJ 08648, Tel: 609-936-9300, Fax: 609-936-9221, Email: sheryl.streger@shawgrp.com
Kevin McClay, Princeton Research Center, Shaw Environmental, Inc., 4100 Quakerbridge Road, Lawrenceville, NJ 08648, Tel: 609-936-9300, Fax: 609-936-9221, Email: kevin.mcclay@shawgrp.com
Robert J. Steffan, Ph.D., Princeton Research Center, Shaw Environmental, Inc., 4100 Quakerbridge Road, Lawrenceville, NJ 08648, Tel: 609-936-9300, Fax: 609-936-9221, Email: robert.steffan@shawgrp.com

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging groundwater contaminant.  The origin of NDMA in groundwater and drinking water includes industrial, agricultural, water treatment, and military sources.  The objective of this project was to evaluate possible bioremediation strategies for treating NDMA-contaminated groundwater.  Our initial studies revealed that the propane-oxidizing strain Rhodococcus ruber ENV425 cometabolically degraded NDMA. The toluene-oxidizing strains, Pseudomonas mendocina KR1 and Pseudomonas sp. ENVPC5, each of which contains toluene-4-monoxygenase (T4MO) were also observed to degrade NDMA.  In addition, rapid removal of NDMA was achieved using a P. putida strain containing cloned T4MO, further demonstrating the role of T4MO in NDMA degradation. To evaluate the potential for ex situ biological treatment of NDMA, a chemostat was seeded with P. mendocina KR1 and continuously fed toluene (~ 2 g/day) as a primary substrate.  After establishing the culture at a dilution rate of 0.02 hr^-1,  NDMA was fed to the reactor at concentrations ranging from 25 to 250 mg/L, to represent typical groundwater concentrations.  Strain KR1 consistently degraded NDMA to below 0.2 mg/L during the 4-month study.  In addition to the reactor studies, the potential for in situ treatment of NDMA was examined using groundwater samples from a contaminated site in California. The addition of toluene, propane, and methane to microcosms prepared with these samples was not effective for stimulating indigenous organisms to cometabolize NDMA. However, appreciable losses of NDMA was observed in groundwater augmented with R. ruber ENV425 in the presence or absence of propane as a cosubstrate. The results from this research provide a firm basis for the development of in situ and ex situ biological approaches for NDMA remediation.

Treatment of Vinyl Chloride Contaminated Groundwater at a Landfill Site: Comparison of Biosparging and iSOC™ Bioremediation Systems for Plume Cut-off

Christine R. LeBlanc, East Coast Engineering, Inc., 156A Front Street, PO Box 745, Marion, MA 02738, Tel: 508-748-2460, Fax: 508-748-2553, Email: cleblanc@eastcoastengineering.com
James F. Begley, MT Environmental Restoration, 24 Bay View Avenue, Plymouth, MA 02360, Tel: 508-732-0121, Fax: 508-732-0122, Email: jbegley@cape.com

Persistent vinyl chloride plumes resulting from incomplete reductive dechlorination of chlorinated solvents in groundwater downgradient of landfill and dry cleaner sites often present significant risk to drinking water resources and potentially to indoor air quality.  A groundwater remediation program designed to treat a large deep vinyl chloride plume (85 ft below the water table) was implemented in June 2003 downgradient of a former landfill site in Massachusetts.  Response actions selected for the site include a system for vinyl chloride hot spot treatment by direct aerobic biostimulation using biosparging and  iSOC™ (in-situ Submerged Oxygen Curtain) technology.  iSOC™ is an in situ bioremediation/natural attenuation enhancement substrate delivery system.  The iSOC™ unit provides a large surface area for delivery of oxygen by mass transfer from the gas phase to the dissolved phase in groundwater without sparging.

Initial performance monitoring results have indicated that oxygen delivery is effectively stimulating aerobic bacteria and reducing vinyl chloride concentrations.  Dissolved oxygen has increased from less than 1 mg/L to 11 mg/L in the biosparging treatment zone.  Dissolved oxygen concentrations as high as 130 mg/L have been achieved in iSOC™ treatment wells and the concentration of vinyl chloride within the active iSOC™ treatment zone has been reduced to 3 to 4 ug/L from a baseline concentration of 10 to 15 ug/L.  The presentation will include an evaluation of treatment zones developed by the biosparging and iSOC™ systems, system performance, and the design, development, and optimization of aerobic treatment zones for plume cut-off.

Treatment of PCP-Contaminated Soil Using an Engineered Ex Situ Biopile Process at a Superfund Site

Carl Rodzewich, Biogenie Corporation, 2085 Quaker Pointe Drive, Quakertown, PA  18951, Tel: 215-538-1729, Fax: 215-538-8287, Email: crodzewich@biogenie-env.com
Nile Fellows, Minnesota Pollution Control Agency, 520 Lafayette Road, St. Paul, MN, 55155, Tel: 651-296-7299, Fax: 651-296-9707, Email: nile.fellows@pca.state.mn.us
Nicolas Moreau, Biogenie Corporation, 2085 Quaker Pointe Drive, Quakertown, PA  18951, Tel: 215-538-1729, Fax: 215-538-8287, Email: nmoreau@biogenie-env.com
Michel Pouliot, Biogenie Corporation, 2085 Quaker Pointe Drive, Quakertown, PA  18951, Tel: 215-538-1729, Fax: 215-538-8287, Email: mpouliot@biogenie-env.com

Releases of creosote and pentachlorophenol (PCP) at a New Brighton, Minnesota wood treatment facility, resulted in widespread soil and groundwater contamination.  Site investigations lead to the facility’s inclusion on the National Priorities List (NPL), and the Minnesota Pollution Control Agency was designated lead regulatory agency.  Biological treatment was the preferred remedy for 18,000 yd³ of contaminated soil, and Biogenie was contracted to design, construct and operate an ex situ Biopile to achieve the remedial objectives.  Most project owners and remedial managers perceive the Biopile process as a passive remediation technology, not fully appreciating the scientific and engineering expertise required to design and operate an effective Biopile.  This paper highlights the “behind the scenes” efforts of the scientific and engineering team responsible for the Biopile project, and specifically how those efforts achieved difficult remedial objectives within a treatment performance guarantee contract.

The New Brighton project required that an ex situ Biopile be used to achieve site reuse criteria of 10 mg/kg PCP and 5 mg/kg cPAHs, with a schedule dictating operation during winter months reaching -22°C. Biopile operating parameters were developed during laboratory Treatability Studies (TS) designed to optimize the biodegradation capacity of indigenous microorganisms. Specific operational parameters were also devised to manage soil containing a fair proportion of highly impacted woodchips. An engineering team applied the TS results during scale-up Biopile design and the project team mobilized to the site for construction.  Once operational the cleanup objectives were met within 18 to 38 weeks, depending on initial PCP concentrations which were up to 350 mg/kg.  Average PCP reduction rates achieved 95% for soils and 76% for soils containing wood chips.

With appropriate design and operating parameters, the engineered ex situ Biopile process enabled more than 96% of the total volume of soil to be successfully treated and backfilled on site, thus significantly reducing overall project costs.

A Comprehensive Performance Analysis of Hydrogen Release Compound (HRC®):  Lessons Learned and Future Directions 

Anna Willett, Regenesis, 1011 Calle Sombra, San Clemente, CA  92673, Tel: 949-366-8000, Fax: 949-366-8090, Email: awillett@regenesis.com
Stephen S. Koenigsberg, 1011 Calle Sombra, San Clemente, CA  92673, Tel: 949-366-8000, Fax: 949-366-8090, Email: skoenigsberg@regenesis.com

Since 1999, Hydrogen Release Compound (HRC^®) has been a commercially-available product for engineered bioremediation of anaerobically biodegradable contaminants.  HRC is a polylactate ester that, upon hydration or microbial cleavage of its ester bonds, slowly releases lactate.  Lactate serves as an electron donor and carbon source for microbial reductive biodegradation.  HRC is a viscous amber-colored liquid that is typically injected into a contaminated aquifer using direct push technology or backfill injection via a hollow stem auger.  Once in place, HRC creates a plume of lactate and its fermentation products (dissolved hydrogen and other organic acids) downgradient of the injection area and serves to accelerate anaerobic bioremediation processes.

In this review of HRC field application results, we summarize application types, contaminants treated, site types, application locations, injection methods, site lithology and hydrology, and concentration ranges of geochemical species.  The source of this information is a database of 474 HRC field applications, a series of 66 HRC publications (many by independent authors), and 10 detailed site case histories that are available electronically.  We will also address the overarching issue of incomplete dechlorination and discuss how the cause of incomplete dechlorination can be “diagnosed” and a remedy implemented. 

First and foremost, the hidden mass input from desorption of soil-phase contaminants and dissolution of residual DNAPL should be considered significant factors in an apparent slow down of dechlorination.  Additionally, competing electron acceptors such as ferric iron can have a significant effect on the rate of dechlorination.  For sites with these behaviors, we recommend, “more time and more electrons.”  On the other hand, if sufficient electron donor is present, but phylogenetic DNA tests show that the appropriate microbial community is not present in sufficient numbers, bioaugmentation with a dechlorinating inoculum is an appropriate remedy.  Site examples with monitoring data will be used to support these conclusions.

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