DNAPL Characterization and Removal with Bioremediation Recirculation System and Electrical Resistance Heating
Melinda Montano, Shaw Environmental, Inc., Concord , CA
Vincent Chan, Shaw Environmental, Inc., Concord , CA
Wayne Akiyama, Shaw Environmental, Inc., Concord , CA
Dan Leigh, Shaw Environmental, Inc., Concord , CA 
Scott Anderson, U.S. Navy, San Diego, CA

Acetate as the Sole Electron Donor, Concurrent Fe(III) Rreduction, and the Prospect of Complete TCE Dechlorination without Dehalococcoides – New Concepts and Novel Data in Chlorinated Solvent Bioremediation
Kevin T. Finneran, PhD, University of Illinois - Urbana Champaign, Urbana, IL

A Comparison of Laboratory and Field Data for Nutrient Amended Carbon Substrates
Michael R. Sieczkowski, CHMM, JRW Bioremediation L.L.C., Lenexa, KS

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)Mechanisms and Kinetics of Extracellular Electron Shuttle Mediated Cyclic Nitramine (Biodegradation by a Novel Bacterial Isolate)
Student Presenter
Man Jae Kwon, University of Illinois - Urbana Champaign, Urbana, IL
Kevin T. Finneran, PhD, University of Illinois - Urbana Champaign, Urbana, IL  

Full-Scale Implementation of Bioremediation for PCE and TCE
Christopher Sullivan, Geosyntec Consultants, Acton , MA
Carl Elder, Geosyntec Consultants, Acton , MA
Douglas Larson, Geosyntec Consultants, Acton , MA

Biodegradation of an MEK Ground Water Plume over a Nine-Year Period
Richard D. Britton, The Whitman Companies, Inc., East  Brunswick, NJ
Cheryl Coffee, Congoleum Corporation, Trenton, NJ
Michael Percelay , The Whitman Companies, Inc., East  Brunswick , NJ
Keith McDermott, The Whitman Companies, Inc., East  Brunswick , NJ

DNAPL Characterization and Removal with Bioremediation Recirculation System and Electrical Resistance Heating
Melinda Montano, Shaw Environmental, Inc., 4005 Port Chicago Hwy, Concord , CA 94520 , USA , Tel: 925-288-2216, Fax: 925-827-2029, Email: melinda.montano@shawgrp.com
Vincent Chan, Shaw Environmental, Inc., 4005 Port Chicago Hwy, Concord , CA 94520 , USA , Tel: 925-288-2372, Fax: 925-288-0888, Email: vincent.chan@shawgrp.com
Wayne Akiyama, Shaw Environmental, Inc., 4005 Port Chicago Hwy, Concord , CA 94520 , USA , Tel: 925-288-2003, Fax: 925-288-0888, Email: wayne.akiyama@shawgrp.com
Dan Leigh, Shaw Environmental, Inc., 4005 Port Chicago Hwy, Concord, CA 94520, USA, Tel: 925-288-2193, Fax: 925-288-0888, Email: daniel.leigh@shawgrp.com
Scott Anderson, U.S. Navy, 1455 Frazee Road, Suite 900, San Diego, CA 92108, USA, Tel: 619-532-0700, Email: scott.d.anderson@navy.mil

A source area of PCE has recently been identified as a possible DNAPL plume at a site at Naval Station Treasure Island.  The plume will be delineated using Membrane Interface Probe (MIP) and FLUTe.  Then it will be treated using Electrical Resistance Heating, or alternatively, using Enhanced Solubility and Bioremediation.

Identification of the DNAPL is based on groundwater sampling results for an in situ bioremediation (ISB) system to treat dissolved chlorinated ethenes.  The system consisted of injection and extraction wells to distribute substrate and microbes through recirculated groundwater.  The source area is at the tip of the plume, which has not been comprehensively investigated.  Prior to treatment, the area had very high concentrations, as determined by groundwater sampling at well EW4.  During ISB, sampling showed substantially complete dechlorination.  After treatment, sampling showed that concentrations had rebounded to levels greater than 17,000 µg/L for CVOCs.  There was no rebound in other areas, and the rebound contaminant was mainly PCE, indicating that the PCE in this area exists as DNAPL.

MIP locations will be determined in a step-out pattern, starting in the vicinity of EW4.  Depth of borings will be up to 35 ft bgs, or the depth of the Bay Mud, which may be a confining layer.  The results will be used to determine the desired locations for FLUTe liners, and soil and groundwater sampling.  If the presence of DNAPL is confirmed, it is expected to take one of the following forms: 1) The form of pools, which have low surface area to mass.  2) The form of small residual globules disseminated through the aquifer, which have high surface area to mass.  Treatment will be by Electrical Resistance Heating or Enhanced Solubility and Bioremediation depending on the form of the DNAPL.

Acetate as the Sole Electron Donor, Concurrent Fe(III) Rreduction, and the Prospect of Complete TCE Dechlorination without Dehalococcoides – New Concepts and Novel Data in Chlorinated Solvent Bioremediation
Kevin T. Finneran, PhD, University of Illinois - Urbana Champaign, Dept of Civil and Environmental Engineering, NCEL 205 N. Mathews, Urbana, IL, 61801, Tel: 217-333-1514, Fax: 217-333-6967, Email: finneran@illinois.edu

Trichloroethylene (TCE) bioremediation has focused on reductive dechlorination in most applications because of prevalent anoxic, subsurface conditions that make anaerobic metabolism favorable as opposed to aerobic microbial metabolism.  There are several chlorinated solvent bioremediation dogmas that in large part have excellent data supporting them, but which at times have had anecdotal data against them.  We began investigating two such remediation tenets in detail.  The first is that acetate is a poor electron donor for complete dechlorination.  Complete dechlorinators require H2 as the primary electron donor, and acetate has to date only been identified as a good carbon source for these organisms.  The second widely held concept is that Fe(III) reduction is a strictly competitive process, and that Fe(III) must be fully reduced prior to the onset of cis-DCE or VC reduction to generate ethene.  While investigating hypotheses related to these concepts, molecular and physiological suggested complete dechlorination without the presence of Dehalococcoides microorganisms.  To date, these organisms are an absolute requirement, and many engineered remediation strategies are built on their presence or addition.

We obtained TCE-contaminated aquifer material from a confidential site in Connecticut .  We began two series of incubations using acetate as the sole electron donor.  The first series had acetate present at 1:1 stoichiometric concentration with respect to all electron acceptors (stoichiometric batch).  The second series had acetate present at 10X the necessary stoichiometry (10X batch), which is common practice in engineered strategies as a “factor of safety”.  TCE in the stoichiometric batch was quickly (<10 days) reduced to ethene without cis-DCE or VC accumulation at near TCE:ethene stoichiometry.  VC was also reduced to ethene in the stoichiometric batch when added as the primary chlorinated solvent.  However, in the 10X batch, TCE was slowly converted to ethene and reduction was incomplete (VC accumulated and TCE:ethene was not stoichiometric).  These data suggest that lower, metered electron donor addition, with acetate as the primary donor, can stimulate efficient dechlorination.

Fe(III) was concurrently reduced with ethene production in the stoichiometric batch, where Fe(II) quickly spiked then flattened in the 10X batch.  This demonstrates that stoichiometric electron donor can stimulate overlapping respiratory processes, most likely by increasing competition amongst the populations present.  An added positive benefit was the lack of methane in the stoichiometric batch, where methane was produced up to 30µM in the 10X batch.  Methane is a potent greenhouse gas, and its production needs to be limited in all remediation applications.  

Molecular analysis using Dehalococcoides specific PCR primers 728F and 1172R demonstrated that DHE-like organisms were not present in the aquifer material in any incubation from which DNA was extracted (repeated several times).  Nested PCR with universal Eubacterial primers and the DHE-specific primers as the internal (nested) pair also demonstrated lack of DHE-like molecular signatures.  Dehalococcoides ethenogenes strain FL2 was used as a positive control in all molecular analyses, and was repeatedly amplified using our techniques.  Amplified ribosomal 16S rDNA analysis (ARDRA) using universal Eubacterial primers 338F and 907R did not identify any DHE-like phylotypes in any of 900 clones tested.  The dominant genus identified in all sediment incubations was within the genus Desulfosporosinus.  Preliminary data with liquid enrichment cultures developed from this sediment indicate that cultures are presented both with and without Dehalococcoides.  This would be the first demonstration of complete dechlorination (TCE à ethene) in the absence of Dehalococcoides, with acetate as the sole electron donor.  Alternatively, it may also be the first evidence of Dehalococcoides using acetate as the sole electron donor.

A Comparison of Laboratory and Field Data for Nutrient Amended Carbon Substrates
Michael R. Sieczkowski, CHMM, JRW Bioremediation L.L.C., 14321 W 96^th Terrace, Lenexa , Kansas 66215 , USA , Office (913) 438-5544 extension 122, Fax (913) 438-5554, msieczkowski@jrwbiorem.com

The use of enhanced reductive dechlorination through the addition of carbon substrates has become a common remedial option for chlorinated solvents since the mid-1990s.  Since that time, many types of carbon substrates have been developed and tested with varied, but mostly positive results.  The use of a wide variety of substrates was driven predominantly by both substrate and application cost.  This led to the use of less expensive, non-engineered substrates.  This paradigm shift resulted in a shift in the relative cost of the substrate when compared to the total site cost.  Highly engineered substrates could account for more than 60% of the total cost of site work whereas non-engineered substrates generally account for a much lower percentage, in some cases as low as 25% of the total cost of site work.

In their efforts to further reduce total project costs, the industry looked at increasing degradation efficiency by augmenting systems with microbes specifically designed to degrade target contaminants or through the addition of nutrients.  Although microbial bioaugmentation can be successful, in many cases it does not produce a reduction in total project costs significant enough to warrant wide-spread use.  Nutrient addition likewise was not generally seen to dramatically impact overall costs on most sites. 

Similar to the move to develop more cost effective substrates, work to develop more cost effective nutrients has been a common goal.  Recent breakthroughs in the development of less costly nutrients (in some cases by more than two orders of magnitude) have made nutrient addition more economically viable.  This presentation reviews laboratory microcosm studies on a nutrient designed to increase anaerobic microbial efficiency and kinetics while reducing overall costs.  These results are then compared to in situ pilot field trails using a variety of carbon substrates including whey powder, ethyl lactate, and emulsified vegetable oil.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)Mechanisms and Kinetics of Extracellular Electron Shuttle Mediated Cyclic Nitramine (Biodegradation by a Novel Bacterial Isolate)
Student Presenter
Man Jae Kwon, University of Illinois - Urbana Champaign, Dept of Civil and Environmental Engineering, NCEL 205 N. Mathews, Urbana, IL, 61801, Tel: 217-333-6851, Fax: 217-333-6967, Email: mankwon@uiuc.edu
Kevin T. Finneran, PhD, University of Illinois - Urbana Champaign, Dept of Civil and Environmental Engineering, NCEL 205 N. Mathews, Urbana, IL, 61801, Tel: 217-333-1514, Fax: 217-333-6967, Email: finneran@uiuc.edu

A cyclic nitramine explosive, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), is a contaminant of concern at many military sites and live-fire training installations(Spalding and Fulton, 1988; Haas et al., 1990).  Groundwater contaminated by RDX is of growing environmental concern because of human health effects of RDX. The current study investigated the potential of utilizing indigenous microorganisms for in situ RDX bioremediation.

Microbial enrichments were generated using RDX-contaminated aquifer material that was incubated with a variety of electron donors and acceptors. Electron donors tested included H2, benzoate, formate, lactate, or acetate; electron acceptors included poorly crystalline Fe(III) oxide, or RDX. Approximately 60% of RDX was mineralized to CO2 in the cell suspension of the enrichment cultures incubated with lactate or acetate. 

A new bacterial species, MJ1, was enriched from the same aquifer material and isolated by streaking on an anaerobic agar slant with RDX as the sole electron acceptor.  The MJ1 colony was translucent, viscous, and of convex and circular shape with a size range of 1-2mm. The full 16S rRNA gene sequence of MJ1 showed that it is a novel species in the genus Desulfotomaculum. 16S rRNA phylogenetic cluster analysis demonstrated that MJ1 is closely related to species in Geobacteraceae, Shewanella, and Anaeromyxobacter, which have been shown to reduce RDX as well as Fe(III). MJ1 showed gram-negative staining behavior. However, cell walls of Desulfotomaculum, in general, contain the typical ultrastructure of gram-positive bacteria. MJ1 is rod-shaped. The optimal growth temperature and pH of MJ1 with Fe(III) and acetate were 30°C and 7, respectively. Electron donors utilized with Fe(III) were lactate, acetate, formate, ethanol, H2, benzoate. Electron acceptors utilized with acetate were Fe(III), fumarate, and AQDS. A preliminary experiment demonstrated that MJ1 under growth condition reduced RDX below the detection limit within 7 days. This isolate will be characterized further with respect to RDX transformation in detail.

Full-Scale Implementation of Bioremediation for PCE and TCE
Christopher Sullivan, Geosyntec Consultants, 289 Great Rd, Suite 105 , Acton , MA 01720 , Tel: 978-263-9588, Fax: 978-263-9594, Email: csullivan@geosyntec.com
Carl Elder, Geosyntec Consultants, 289 Great Rd, Suite 105 , Acton , MA 01720 , Tel: 978-263-9588, Fax: 978-263-9594, Email: celder@geosyntec.com
Douglas Larson, Geosyntec Consultants, 289 Great Rd, Suite 105 , Acton , MA 01720 , Tel: 978-263-9588, Fax: 978-263-9594, Email: dlarson@geosyntec.com  

This presentation describes the design, implementation, and operation of a full-scale bioaugmentation remedy over the 3.5 acre site that is the source to a 3500 foot plume.  Particular focus is given to the accomplishments achieved during the last five years and lessons learned from this project 

The site is a former manufacturing plant in Kansas .  Site geology consists of approximately 35 feet of fine sand underlain by limestone bedrock.  Groundwater is located approximately 15 feet below the ground surface.  Prior to implementing bioremediation, overburden groundwater was slightly oxidizing and aerobic (ORP = +200 mV, DO = 5 mg/l) with average nitrate and sulfate concentrations of approximately 7 and 25 mg/L, respectively.  Concentrations of PCE and TCE in site groundwater were initially as high as 2 mg/L and 11 mg/L, respectively. 

Bioremediation was proposed in 2003 as an alternative to pump and treat for the source area.  A bioremediation system consisting of two groundwater extraction wells, a treatment trailer and four injection wells was designed, permitted and installed in the Spring of 2004.  Full-scale operation of the bioremediation system began in July 2004.

Groundwater was initially amended with acetate to achieve iron reducing conditions.  After four months of operation, the electron donor was changed to lactate to maintain strongly reducing conditions.  The site was bioaugmented with KB-1™ in January and November 2005 (approximately six and sixteen months after system start-up).  The system has operated continuously since July 2004 at a pumping rate of approximately 12 gpm and lactate dose of about 130 mg/L (since November 2004).   

Bioremediation has destroyed approximately 99% of the mass within the treatment zone and more than 80% of the contaminant mass site-wide.  Nearly all of the remaining contaminant mass is located in a zone up gradient of the bioremediation system that was discovered after 2004.  PCE and TCE concentrations within the treatment zone on the western half of the site have decreased from approximately 2 mg/L to drinking water levels.  Concentrations on the eastern half of the site have decreased from as much as 10 mg/L to drinking water levels with the exception of two localized hotspots where TCE concentrations are approximately 100 ug/L.  Bioremediation has resulted in sufficient mass removal for the state regulators to approve reactivation of a municipal supply well that is located at the down gradient boundary of the site.  This well was deactivated in 1985 as a result of site contamination.  In August 2008, 500 gallons of emulsified soybean oil was added at select locations throughout the site to provide a persistent source of electron donor for bioremediation of residual contamination and the active treatment system was shutdown.

Biodegradation of an MEK Ground Water Plume over a Nine-Year Period
Richard D. Britton, The Whitman Companies, Inc.  116 Tices Lane , Unit B-1, East Brunswick , NJ 08816 , Tel: 732-390-5858, Fax: 732-390-9496, Email: rbritton@whitmanco.com
Cheryl Coffee , Congoleum Corporation.  1945 East State Street , Trenton , New Jersey 08619 , Tel: 609-584-3000, Fax: 609-584-3300, Email: ccoffee@congoleum.com
Michael Percelay , The Whitman Companies, Inc.  116 Tices Lane , Unit B-1, East Brunswick , New Jersey , 08816 , Tel: Fax: 732-390-5858, 732-390-9496, Email: mpercelay@whitmanco.com
Keith McDermott , The Whitman Companies, Inc.  116 Tices Lane, Unit B-1, East Brunswick, New Jersey, 08816, Tel: 732-390-5858, Fax: 732-390-9496, Email: kmcdermott@whitmanco.com

Methyl Ethyl Ketone (MEK) concentrations at an industrial facility located in Central, New Jersey (NJ) naturally degraded from concentrations of 354,000 ppm to ND ( < 10 ppb) between 1998 and 2007. 

In 1999, the maximum area of MEK concentrations that exceeded the NJ MEK ground water quality criterion of 300 ppb reached approximately 100,000 square feet.

Saturated subsurface materials (DTW=3.5 feet bgs) at the site are comprised primarily of fine to medium sand with a hydraulic conductivity of 4.7 x 10-3 cm/sec. 

Between 1998 and 2005, dissolved oxygen (DO) concentrations at the source area and other nearby monitoring wells were 0.0 mg/l, indicating that microorganisms were actively degrading MEK.  DO concentrations in source area wells began to rise in 2005 and rebounded to approximately 5 mg/l by 2007.

The timeframe for MEK to concentrations to degrade below the NJ ground water quality criterion of 300 ppb would have been predicted to be much shorter than nine years given the literature half-life values of 28 days and 7 days for anaerobic and aerobic biodegradation of MEK.  The persistence of elevated MEK concentrations suggested the presence of unremediated residual source material. 

A 2004 investigation in the vicinity of the source area monitoring wells employing a hydrophobic dye (Sudan IV) and FLUTe liners identified a 1500 square feet area of free-phase residual product near the initial discharge area. 

It is believed that the rapid decrease in MEK concentrations observed during late 2005 is correlated with the final dissolution of the identified pockets of residual product. 

Given the absence of any sensitive receptors, and a stable then shrinking ground water plume, natural attenuation was the most appropriate and cost effective remedial action to reach the NJ MEK ground water quality criteria of 300 ppb.