Title: Introduction Text: The biodegradation of chlorinated ethene contaminants at the DNAPL: water interface may be achieved or enhanced through the process of bioaugmentation (shown here). In this process, non-native microbial cultures that have demonstrated the ability to completely reductively dechlorinate these contaminants are injected into groundwater near the DNAPL:water interface. Dechlorination is then achieved by supplying the added microorganisms with appropriate electron donor(s) and nutrients. The dechlorination process may occur via several metabolic pathways, but dehalorespiration is the primary pathway currently exploited for bioaugmentation. NAVFAC is currently working on laboratory and field demonstrations of this DNAPL treatment approach. This Web Tool summarizes the concepts behind the use of bioaugmentation for DNAPL treatment and provides an overview of on-going NAVFAC efforts in technology development.

Title: DNAPL Text: DNAPL is the abbreviation for Dense, Nonaqueous-Phase Liquid. DNAPL forms when chemicals that are hydrophobic, immiscible with water, and have densities greater than that of water exceed their solubility limit and remain in liquid state. Chlorinated ethene compounds including trichloroethene (TCE) and tetrachloroethene (or perchloroethene; PCE) are commonly detected groundwater contaminants that form DNAPL in groundwater environments. These compounds were often used as degreasing solvents in manufacturing processes.

Title: Dechlorination Text: Dechlorination is the process by which chlorine atoms are removed from an organic molecule.

Title: Background (1 of 2) Text: There is a critical need for remedial technologies that can effectively treat, reduce, or contain DNAPL in the saturated zone at reduced treatment times and lower costs. The difficulty in physically removing DNAPL from contaminated aquifers has emphasized the need for effective in situ treatment and/or containment technologies. Left untreated or uncontained, DNAPL can serve as a continuous source of chlorinated ethene contamination to groundwater above maximum contaminant levels (MCLs).

Title: Background (2 of 2) Text: There are several remedial technologies available to address DNAPL contamination. One technology showing promise for treating DNAPL source zones is bioremediation. At many sites, chlorinated solvents are biodegraded by naturally occurring (indigenous) subsurface microorganisms. This process is referred to as intrinsic bioremediation. If engineering steps are required to promote or enhance chlorinated solvent biodegradation by the native microorganisms, then the process is referred to as enhanced bioremediation or biostimulation. Adding non-naturally occurring microorganism cultured in the laboratory to enhance biodegradation is termed bioaugmentation and is the focus of this Web Tool.

Title: Reactive Barrier Text: Minimizes the migration of contaminated groundwater by intercepting and degrading the dissolved phase contaminants.

Title: Containment Text: Minimizes the migration of contaminated groundwater by either preventing groundwater flow or hydraulically containing the contaminated groundwater.

Title: MNA Text: Monitored natural attenuation is appropriate at sites where site-specific remedial action objectives can be achieved within a reasonable time frame when compared to more active remedial methods. Natural attenuation processes include "biodegradation; dispersion; dilution; sorption; volatilization; radioactive decay; and chemical or biological stabilization, transformation, or destruction of contaminants" (EPA, OSWER Directive 9200.4-17P).

Title: Flushing-Alcohol Text: Removes DNAPL by either mobilizing pure phase contaminant or increasing the solubility of the contaminant.

Title: Oxidant Text: Removes DNAPL via chemical mechanisms by rapidly degrading the dissolved phase contaminant.

Title: Volatilization Text: Removes vapor phase contaminant from either the vadose or saturated zones by enhancing partitioning into the vapor phase.

Title: Thermal Text: Removes DNAPL by heating the subsurface and enhancing volatilization and/or mobilization of the DNAPL pure phase.

Title: Biostimulation Text: Removes DNAPL mass by enhancing the rate of biodegradation within the source zone.

Title: Bioaugmentation Text: Minimizes migration of contaminated groundwater (increases degradation rate and promotes complete dechlorination to ethene) by increasing the activity of dechlorinating microorganisms.

Title: Bioremediation Text: Bioremediation refers to a group of technologies that exploit the metabolic properties of microorganisms to degrade hazardous substances into less toxic or nontoxic compounds. Bioremediation technologies have been used to treat a wide range of contaminants including chlorinated solvents.

Title: Objectives Text: As part of the Department of Defense (DoD) Environmental Security Technology Certification Program (ESTCP), NAVFAC is currently working on technology development for the Biodegradation of DNAPL through Bioaugmentation of Source Areas. The objectives for this project are: 1. to enhance the dissolution rate of a DNAPL via supplemented biological activity (bioaugmentation) near or at the DNAPL:water interface, leading to accelerated source cleanup; and 2. to contain the source area by rapidly degrading the high concentrations of dissolved phase contaminants that emanate from the DNAPL source area. This animation shows the potential effect of bioaugmentation on accelerated DNAPL mass reduction due to an enhanced dissolution rate.

Title: Technology Description (1 of 4) Text: Reductive dechlorination occurs under anaerobic conditions and is a well-understood pathway for degradation of chlorinated ethene and ethane compounds as well as other chlorinated contaminants. Play this video to view the reductive dechlorination of PCE (the parent compound) through the succession of daughter products to the final end product of ethene. Reductive dechlorination involves the step-wise replacement of individual chlorine atoms with hydrogen atoms. Bioremediation of DNAPL actually proceeds in the aqueous phase adjacent to the DNAPL:water interface. Here, dechlorination of the parent compound maintains a sharper concentration gradient between the phases. This accelerates the mass transfer rate of the parent compound from the non-aqueous phase into the solution, which in turn maximizes the rate of biodegradation.

Title: Technology Description (2 of 4) Text: Several bacterial species have been isolated and identified, including Dehalococcoides ethenogenes, that use compounds such as PCE and TCE as terminal electron acceptors (i.e., respiration). While dehalorespiring bacteria have been detected at a number of chlorinated solvent contaminated sites, the common occurrence of PCE or TCE dechlorination stalling at the formation of 1,2-cis-dichloroethene (cDCE) and vinyl chloride (VC) suggests that these specific microorganisms (Dehalococcoides species) may not be ubiquitous and/or present in sufficient numbers in groundwater systems. However, the stall may simply be due to conditions that are not sufficiently reducing. Bioaugmentation may prove useful under such situations and/or when delivery of suitable electron donors and nutrients fails to stimulate satisfactory levels of dehalo-respiration by the indigenous populations.

Title: Electron Acceptor Text: Microorganisms obtain energy for growth by transferring electrons from an electron donor to an electron acceptor. An electron acceptor is a compound that receives or accepts electrons during cellular respiration. The microorganism collects the energy from the reaction for its use in cell growth and maintenance. Under dehalorespiration, chlorinated ethene compounds such as PCE, TCE, cDCE, and VC serve as the electron acceptors.

Title: Dehalorespiration Text: Dehalorespiration is an anaerobic respiratory pathway where electrons are transferred from an electron donor (hydrogen) to a halogenated molecule resulting in the displacement of the halogen atom with replacement by a hydrogen atom. The transfer of electrons produces a net energy gain for the dehalorespiring organism. For example, chlorine is a type of halogen atom and is replaced with a hydrogen atom during dehalorespiration of PCE to TCE.

Title: Technology Description (3 of 4) Text: In dehalorespiration, the chlorinated ethene acts as an electron acceptor while an electron donor is required to provide energy for this process (McCarty, 1994). Hydrogen is generally considered the direct electron donor for reductive dechlorination, and is typically produced from the anaerobic oxidation of other carbon substrates, such as organic acids or alcohols (Maymo-Gatell et al., 1997).

Title: Electron Donor Text: An electron donor is a compound that gives up or “donates” electrons during cellular respiration. Hydrogen is generally considered the direct electron donor for dehalorespiration. Hydrogen is produced from the fermentation of any number of simple organic substrates.

Title: Technology Description (4 of 4) Text: This figure represents an overall schematic of the addition of a dehalorespiring culture and/or electron donor to a source area to promote enhanced DNAPL dissolution. To the extent possible, the site should be characterized so that the vertical and horizontal extent of the source area is well defined. If the extent of the source area is not well defined, this can lead to incomplete remediation and/or additional cleanup expenditures.

Title: Advantages of Bioaugmentation Text: Enhancing the dissolution rate of a DNAPL will decrease cleanup times. A source zone with a faster dissolution rate will cost less to contain from a long-term operation and maintenance (O&M) perspective. Mass will be destroyed and not simply transferred to another medium. Remediation of the contaminants is performed in situ with minimal aboveground impact, minimal waste generation, and a small footprint at a given site. Relatively low capital costs compared to other ex situ remedial options such as pump-and-treat because the primary capital expenditure is related to injection well installation or direct-push points.

Title: Limitations of Bioaugmentation Text: Like any source remediation technology there is a need to understand and identify the source extent and mass in order to minimize the zone of treatment. Such an effort would require capital cost expenditures. A limitation of all source remediation technologies involves contacting the treatment with the DNAPL/source material. This could include limitations related to delivering nutrients and/or microorganisms to the source area. Certain geochemical conditions (e.g., low pH or high sulfate) may be inhibitory to biodegradation. Some co-contaminants may inhibit dechlorination such as chloroform and hydrogen sulfide.

Title: Comparison: Biostimulation vs. Bioaugmentation Text: It is often difficult to determine whether or not a site has biological limitations. For this reason, it is recommended that biostimulation be tested first for a few months at any site where bioaugmentation is under consideration. There are several important considerations in comparing the cost effectiveness of bioaugmentation over biostimulation alone. Business models vary considerably for the use of the bioaugmentation approach and costs can be high compared to performance benefits. Cultures can be sold by the liter or in an unlimited supply for the site based on a license. Handling of these cultures is particularly important as most are strict anaerobes with low tolerance of dissolved oxygen. There is no common standard to infer the quality (i.e., the dechlorinating potential) and consistency of the cultures received from the vendor. The cultures can change over time and in some cases lose some of the function for which they were acquired. There may be competition with native microorganisms and competitive exclusion of the introduced culture may create conditions in which there is a low survivability. Also, the effective distribution of the cultured microbes is not trivial and a cost effective distribution method may be a significant technical challenge. Monitoring of bioaugmentation plots is also a technical challenge because the mere presence of the introduced organism may not be as helpful as determining whether it is generating the necessary enzymes.

Title: Technical Approach Text: The ESTCP demonstration project for the Biodegradation of DNAPL through Bioaugmentation of Source Areas was completed at the Dover National Test Site in Delaware. There were six phases of the project: Pre-Demonstration Laboratory Testing Initial Operation Baseline Operation Electron Donor Addition Bioaugmentation Post Bioaugmentation

Title: Pre-Demonstration Activities (1 of 3) Text: Prior to initiating the bioaugmentation demonstration, a number of pre-demonstration activities were completed to provide essential data required to effectively implement and interpret the performance of the bioaugmentation demonstration. These activities included pre-design laboratory testing and site instrumentation and characterization. The demonstration required the selection of an appropriate dehalogenating microbial consortium. The three cultures tested were: The Pinellas culture KB-1™ Toronto Main (TM) KB-1 was chosen as the appropriate dehalogenating microbial consortium.

Title: Pre-Demonstration Activities (2 of 3) Text: Two identical parallel-plate model aquifers (boxes) were constructed to characterize spatial trends in dechlorination, assess microbial activity around a PCE DNAPL zone, and identify any inhibitory conditions limiting dechlorination of volatile organic compounds (VOCs). One of the boxes is shown in the photograph. Both boxes were operated at the University of Toronto for 2.5 years. Both boxes received soil and groundwater from the site. Sampling ports were used to monitor for VOCs and several other parameters. Groundwater was fed from the left and flowed from left to right in this view.

Title: Pre-Demonstration Activities (3 of 3) Text: Both boxes received electron donor, but only Box 2 was inoculated with KB-1. Comparison of PCE removal rates in the model aquifers showed that the dissolution rate increased due to bioaugmentation. Biostimulation alone did not result in enhanced PCE dissolution and reductive dechlorination of PCE was not observed. However, bioaugmentation with KB-1 resulted in enhanced PCE dissolution by ~2.2 times (see Phase 3 in the figure).

Title: Demonstration Testing and Evaluation (1 of 2) Text: The next phase of the project involved field implementation of the concepts demonstrated in the laboratory. As shown here, a groundwater recirculation system was installed to allow for the injection of electron donor and cultured microbes into the demonstration plot. During the Initial Operation Phase from March 2002 to May 2002, groundwater was recirculated to establish steady state geochemical conditions and optimize flowrates. Field parameters such as VOCs, inorganics, volatile fatty acids (VFAs), and others were monitored. In the Baseline Operation Phase from May 2002 to February 2003, groundwater was recirculated under optimized pumping conditions in order to attain a baseline PCE dissolution rate. The Electron Donor Addition Phase was completed in March 2003 to July 2003. During this phase, ethanol and sodium lactate were injected in order to evaluate the ability of the indigenous microbial population to enhance dissolution rate above observed baseline. During this period, biodegradation was NOT observed with biostimulation alone.

Title: Demonstration Testing and Evaluation (2 of 2) Text: For the Bioaugmentation Phase from July 2003 to February 2005, groundwater was recirculated with electron donor (ethanol and sodium lactate) and the selected microbial consortium (KB-1) to evaluate the ability of the consortium to enhance the dissolution rate above the previous two phases. The Post Bioaugmentation Phase which was started in March 2005 is the final phase and these activities are still on-going. Electron donor addition was ceased in order to evaluate the ability of the established microbial population to enhance the DNAPL dissolution rate above observed baseline without the constant electron donor supply.

Title: Results Text: Bioaugmentation with KB-1™ resulted in the generation of TCE, cis-1,2-DCE, vinyl chloride, and ethene in many locations in the test plot. As shown in the graph, it has resulted in ethene being the dominant product (as % of total ethenes) in the groundwater sampled at the extraction wells. Only through bioaugmentation has the distribution of daughter products changed significantly. Bioaugmentation has increased the mean mass discharge when compared to the Biostimulation and Baseline stages. A final tracer test to assess changes in patterns over the experimental period is planned. This study has demonstrated that enhanced dissolution of PCE DNAPL using biological means is feasible at the field scale. The effective delivery of electron donor to the source area is a critical factor in the performance of this technology.

Title: References Text: Maymo-Gatell, X., J.M. Gossett and S.H. Zinder. 1997. "Dehalococcus Ethenogenes Strain 195: Ethene production from halogenated aliphatics." In: In Situ and On-Site Bioremediation: Volume 3. Alleman, B.C. And Leeson, A. (Eds). Battelle Press, Columbus, OH. McCarty, P.L. 1994. "An Overview of Anaerobic Transformation of Chlorinated Solvents." Symposium on Intrinsic Bioremediation of Ground Water 135 - 142. NAVFAC ESTCP Project Profile. Biodegradation of Dense Non-Aqueous Phase Liquids (DNAPL) Through Bioaugmentation of Source Areas.

Title: Contact Text: For more information about this project, please contact: T2 NFESC POC (805) 982-1656 or NFESC POC (805) 982-1616 PRTH_NFESCT2@navy.mil