Bioremediation of Relcalcitrant Compounds - Chapter 4 ppt

51 chapter four The federal integrated biotreatment research consortium (flask to field) Jeffrey W. Talley Contents 4.1 Introduction 51 4.1.1 Chlorinated solvents 52 4.1.2 PAHs 53 4.1.3 PCBs 53 4.2 Technical approach 54 4.3 Thrust area project results 55 4.3.1 Chlorinated solvents 55 4.3.2 PAHs 55 4.3.3 PCBs 56 References 56 4.1 Introduction The Department of Defense (DOD) has thousands of sites that have been contaminated with organic compounds that pose a serious threat to the environment. The remediation of these sites using existing technologies was problematic from an economic, technical, and political point of view. The Environmental Protection Agency (EPA) and DOD funded development and application of innovative remediation technologies to solve these problems. Of all of the innovative technologies, bioremediation was considered the most promising. L1656_C004.fm Page 51 Friday, June 17, 2005 9:12 AM © 2006 by Taylor & Francis Group, LLC 52 Bioremediation of Recalcitrant Compounds Biotreatment processes had been successfully demonstrated for treat- ment of a wide variety of easily degraded compounds, such as low-molec- ular-weight fuels and phenols. Strong potential existed for development of biotreatment processes directed toward contaminant groups traditionally more difficult to degrade, such as explosives, chlorinated solvents, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs). All of these compounds represented a major contaminant problem to the DOD. This project’s objectives enhanced existing funded efforts in DOD pro- grams, complemented both the EPA and DOD research strategies, and addressed problems experienced by environmental engineers involved in Superfund, Resource Conservation and Recovery Act (RCRA) activities, and international technology exchange programs. Dr. Jeffrey Talley, P.E., was the project director and was assisted by Deborah K. Belt. 4.1.1 Chlorinated solvents Chlorinated solvents entered the environment in massive amounts during the 1950s, 1960s, and 1970s. These contaminants have migrated through the subsurface and impacted groundwater at more than 1000 DOD sites. Con- taminated aquifers can be remediated by removing the solvents in the porous media of the subsurface. Laboratory and pilot-scale experiments have dem- onstrated the potential of cosolvent-enhanced in situ extraction to remove dense nonaqueous phase liquids (DNAPLs) in porous media. Although this method is effective for mass removal, residual amounts of cosolvents and contaminants are expected to remain at levels that could preclude meeting regulatory requirements. However, with the bulk of the DNAPLs extracted in situ , biotreatment becomes a viable polishing procedure. This was the emphasis of the work that was conducted by Dr. Guy Sewell, EPA. In situ biotreatment may transform the remaining contaminants to non- hazardous compounds at a rate in excess of the rate of dissolution or dis- placement. The efficacy of in situ bioremediation of chlorinated solvents is usually limited by transport and mixing considerations, i.e., supplying excess electron donors in conjunction to the chlorinated solvents at appro- priate concentrations. The delivery-and-extraction process facilitated the cosolvency effect and supplied electron donors (cosolvent, ethanol) and elec- tron acceptors (chlorinated solvent, tetrachloroethylene (PCE)) to the inher- ent bacteria. The synergism between these abiotic and biotic processes could minimize problems associated with the individual approaches and lead to the development of a treatment train approach, which could attenuate or eliminate the risks posed to human health and the environment by DNAPL sites. L1656_C004.fm Page 52 Friday, June 17, 2005 9:12 AM © 2006 by Taylor & Francis Group, LLC Chapter four: The federal integrated biotreatment research consortium 53 4.1.2 PAHs PAHs include industrial wastes such as petroleum and fuel residues, tars, and creosote that contaminate soils and sediments. Land farming is a com- mon treatment option for PAH-contaminated soils, but the removal of the high-molecular-weight (HMW) PAHs by this method is often problematic. The goal of this project, coordinated by Dr. Hap Pritchard Navel Research Laboratory (NRL), was to modify land farming by using bioaugmentation to improve degradation of PAHs. Bioaugmentation involved the addition of a biosurfactant-producing bacterium (strain Pa 64), a bulking agent (rice husks), and a carbon/nitrogen source (dried-blood fertilizer) (Pritchard et al., 1999). Microcosm studies conducted at NRL validated the method and determined the degradation kinetics. Lance Hansen conducted a pilot-scale study at the U.S. Army Engineer Research and Development Center (ERDC) in Vicksburg, MS, implementing bioaugmentation technology for PAH remediation. The study consisted of three metal pans (10 feet long × 3 feet wide × 2 feet deep), each filled with approximately 1 cubic yard of PAH-contaminated soil. One pan was untreated, one received bulking agent and dried blood, and the third received bulking agent, dried blood, and the bacteria. The pan study was designed to define the sampling strategy required to measure the effective- ness of bioaugmentation and to provide a realistic cost estimate for the bioaugmentation treatment (U.S. Army Corps of Engineers, 1996). Methods were developed and refined to monitor the progress and effectiveness of bioremediation. These included molecular biological techniques to monitor the presence of the inoculated organisms and their in situ activity (White and Ringelberg, 1998; Balkwill et al., 1988), respirometric techniques that monitor relative microbial activity based on CO 2 production, and genetic techniques that monitor the presence or absence of enzymes involved in nitrogen use and PAH degradation (Perkins et al., 2001). These techniques were correlated to standard contaminant analytical chemistry methods and were applied to the cost optimization of land-farming PAHs. 4.1.3 PCBs Research on microbial degradation of PCBs has been ongoing for more than 25 years and has shown that bioremediation requires a more sophisticated technology than the simplistic attempts that have been tried so far. The project conducted by Dr. Jim Tiedje at Michigan State University addressed key barriers to bioremediating PCBs: • Developing microorganisms that will grow on the major congeners produced by anaerobic dechlorination of PCBs •Improving bioavailability of PCBs through the use of surfactants • Optimizing field delivery of anaerobic or aerobic PCB bioremediation technologies L1656_C004.fm Page 53 Friday, June 17, 2005 9:12 AM © 2006 by Taylor & Francis Group, LLC 54 Bioremediation of Recalcitrant Compounds Genetically engineered microorganisms (GEMs) were developed that were capable of using PCB congeners as growth substrate under aerobic conditions. GEMs were modified to exhibit dechlorination genes that enabled the removal of chlorine before chlorocatechols were formed, avoid- ing toxicity (Tsoi et al., 1999). This approach avoids the need to manage cometabolism, which can be difficult in situ . These organisms can be used to remove products of anaerobic reductive PCB dechlorination, predomi- nantly ortho-chlorinated and ortho- + para-chlorinated congeners (Hrywna et al., 1999). Verniculite, as a carrier for the bacterial inoculum, improved survival of the GEMs in Picatinny soil. 4.2 Technical approach Figure 4.1 illustrates the technical approach used to develop new biotreat- ment technologies during the Flask to Field project. The technical approach within the consortium was to develop the most promising biotreatment processes at the bench scale and then validate the technology at the pilot and field scales. Engineering groups worked closely with scientists in eval- uating the potential of the resulting technologies and in the transfer of technologies from bench scale to field. The Technical Advisory Committee (TAC) periodically reviewed projects for technical merit. The recommenda- tions of these biotechnology experts to the thrust area coordinators served to further enhance the projects. This approach ensured that effective reme- diation technologies were developed within a reasonable time frame. Figure 4.1 Biotreatment process development in Federal Integrated Biotreatment Research Consortium (FIBRC). Chlorinated Solvents Explosives hPAHs PCBs Process Engineering CANDIDATES FOR DEMONSTRATION AND VALIDATION L1656_C004.fm Page 54 Friday, June 17, 2005 9:12 AM © 2006 by Taylor & Francis Group, LLC Chapter four: The federal integrated biotreatment research consortium 55 4.3 Thrust area project results A brief summary describing a major project from each thrust area is given below. 4.3.1 Chlorinated solvents The chlorinated solvents project, Solvent Extraction Residual Biotreatment (SERB), concentrated on the remediation of tetrachloroethylene. SERB tech- nology was validated in a field-scale study at Sage’s Dry Cleaner site, Jack- sonville, FL (Mravik et al., 1999; Sewell et al., 2000). PCE concentration was reduced by 70% in the aquifer using this technology. Significant levels (4 mg/l) of the dechlorination product, cis-1,2-dichloroethene (cis-DCE), were detected in groundwater samples in the area exposed to residual ethanol after 4 months and increased to 16 mg/l after 10 months. Maximum and minimum observed rates of dechlorination (based on cis-DCE production) were 43.6 and 4.2 µ g/l/day, respectively. These results indicated that over time, biotransformation had been enhanced. Microbial ecology studies using site materials indicated that the site remained biologically active. Microcosm studies indicated that anaerobic microbial populations generated a reducing equivalent balance by oxidation of the cosolvent (ethanol) that was linked to the reductive dechlorination of PCE. Molecular methods indicated the presence of known groups of dechlo- rinators. Overall, the project was successful. SERB research is still ongoing at the Jacksonville site and represents an attractive alternative for chlorinated solvents’ remediation. 4.3.2 PAHs Results from the PAH pilot-scale study implementing bioaugmentation tech- nology indicated that bioaugmentation did enhance PAH degradation (Hansen et al., 2000). Degradation of HMW PAHs into four-ring compounds (including BaP toxic equivalent compounds) was achieved. Low-molecu- lar-weight PAHs were extensively degraded in the first 2 to 3 months, and degradation of the HMW PAHs commenced in the fourth month in micro- cosms bioaugmented and treated with dried-blood fertilizer. A reduction of total PAHs (86 to 87%) was realized after 16 months in the pans that had been bulked, amended with dried-blood fertilizer, and bioaugmented. Com- parison of the two methods indicated that the time required to achieve 50% degradation of PAHs was decreased by half through bioaugmented land farming over traditional land farming methods. The soil used for this research was heavily contaminated with PAHs (7200 ppm) and would tra- ditionally not be considered a candidate for bioremediation. This research indicates that land farming that incorporates bioaugmentation technology may be an alternative to incineration for remediation of heavily PAH-contaminated sites. Bioaugmentation would be more cost effective than L1656_C004.fm Page 55 Friday, June 17, 2005 9:12 AM © 2006 by Taylor & Francis Group, LLC 56 Bioremediation of Recalcitrant Compounds incineration because of minimal soil excavation, material handling, and energy costs. 4.3.3 PCBs The PCB project has been very successful in cloning genes in bacteria that combine cometabolism of PCBs to chlorobenzoates, and the dechlorination and mineralization of chlorobenzoates, as a growth substrate. Microbiolog- ical, biochemical, and physiological characterization of the selected biphenyl degraders was completed. Based on the growth on PCB mixtures, toxicity testing, and survival in soil microcosms, a combination of two GEMs, Rhodo- coccus RHA1 tfcb and Burkholdena K W, were the most effective for achieving PCB degradation. The use of surfactants increased the solubility and reme- diation rates of the contaminant. Molecular probes were developed and used to track the bacteria in Picatinny arsenal soils and river sediments, using both genetic and polymerase chain reaction (PCR)–based techniques. The recombinant organisms survived in nonsterile sediment from Red Cedar River contaminated with Aroclor 1242 and maintained degradative activity, evidenced by reducing PCB levels by 78%. A pilot-scale study involving three different soil loadings (low, medium, and high solids reactors) is ongoing at ERDC. This study will evaluate the effects of different moisture contents on PCB bioremediation and application of GEMs, as well as determine the maximum soil loading rate optimum for GEMs’ activity to avoid or offset the subsequent costs of disposing of the stabilized soil. Bioremediation of PCBs is effective and offers lower energy and opera- tions costs than other technologies, but it may take longer to remediate the soil, and desorption kinetics may limit degradation rates. References Balkwill, D.L., Leach, F.R., Wilson, J.T., McNabb, J.F., and White, D.C. 1988. Equiva- lence or microbial biomass measures based on membrane lipid and cell wall components adenosine triphosphate, and direct counts in subsurface aquifer sediments. Microb. Ecol. 16: 73–84. Hansen, L.D., Nestler, C., and Ringelberg, D. 2000. Bioremediation of PAH/PCP contaminated soils from POPILE wood treatment facility. In Proceedings of the Second International Conference on Remediation of Chlorinated and Recalcitrant Compounds , G.B. Wickramanayake, A.R. Gavaskar, J.T. Gibbs, and J.L. Means, Eds. Battelle Press, Columbus, OH, pp. 145–152. Hrywna, Y., Tsoi, T.V., Maltseva, J.F., Quensen, J.F., III, and Tiedje, J.M. 1999. Con- struction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls. Appl. Environ. Microbiol . 65: 2163–2169. Mravik, S.C., Sewell, G.W., and Wood, A.L. 1999. Field evaluation of the Solvent Extraction Residual Biotreatment Technology in Abstracts of the 4th Interna- tional Symposium on Subsurface Microbiology . ISSM, Vail, CO. L1656_C004.fm Page 56 Friday, June 17, 2005 9:12 AM © 2006 by Taylor & Francis Group, LLC Chapter four: The federal integrated biotreatment research consortium 57 Perkins, E., Hansen, L.D., Nestler, C.C., and Bymes, J. 2001. Changes in Abundance of In-Situ Aromatic Degrading Bacteria during a Pilot Scale Landfarming of a Polycyclic Aromatic Hydrocarbon Contaminated Soil. Paper presented at Proceedings of the Ninth International Symposium on Microbial Ecology, Amsterdam. Pritchard, P.H., Jones-Meehan, J., Mueller, J.G., and Straube, W. 1999. Bioremediation of high molecular PAHs: application of techniques in bioaugmentation and bioavailability enhancement. In Novel Approaches for Bioremediation of Organic Pollution , R. Fass, Y. Flashner, and S. Reuveny, Eds. Kluwer Academic/Plenum Publishers, New York, pp. 157–169. Sewell, G.W., Mravik, S.C., and Wood, A.L. 2000. Field Evaluation of Solvent Extrac- tion Residual Biotreatment (SERB). Paper presented at 7th International FZK/ TW Conference on Contaminated Soil (ConSoil 2000), Leipzig, Germany, September 18–22. Tsoi, T.V., Plotaikova, E.G., Cole, J.R., Guerin, W.F., Bagdasarian, M., and Tiedie, J.M. 1999. Cloning, expression, and nucleotide sequence of the Pseudomonas aerug- inosa strain 142 ohb genes coding for oxygenolytic ortho-dehalogenation of halobenzoates. Appl. Environ. Microbiol. 65: 2151–2162. U.S. Army Corps of Engineers. 1996. Bioremediation Using Landfarming Systems, Engi- neering and Design , ETL 1110-1-176. USACE, Washington, DC. White, D.C. and Ringelberg, D.B. 1998. Signature lipid biomarker analysis. In Tech- niques in Microbe Ecology , R.S. Burlage, R. Atlas, D. Stahl, G. Geesey, and G. Sayler, Eds. Oxford University Press, New York, pp. 255–272. L1656_C004.fm Page 57 Friday, June 17, 2005 9:12 AM © 2006 by Taylor & Francis Group, LLC . 53 4. 1.3 PCBs 53 4. 2 Technical approach 54 4.3 Thrust area project results 55 4. 3.1 Chlorinated solvents 55 4. 3.2 PAHs 55 4. 3.3 PCBs 56 References 56 4. 1 Introduction The Department of Defense. demonstrated for treat- ment of a wide variety of easily degraded compounds, such as low-molec- ular-weight fuels and phenols. Strong potential existed for development of biotreatment processes. Land farming is a com- mon treatment option for PAH-contaminated soils, but the removal of the high-molecular-weight (HMW) PAHs by this method is often problematic. The goal of this project, coordinated