Bioremediation of Relcalcitrant Compounds - Chapter 6 docx

147 chapter six Enhancing PCB bioremediation James M. Tiedje, Tamara V. Tsoi, Kurt D. Pennell, Lance D. Hansen, Altaf Wani, and Desirée P. Howell Contents 6.1 Project background and rationale 148 6.2 Objectives 151 6.2.1 Overall objectives 151 6.2.2 Research objectives to design PCB-growing GEMs 152 6.2.3 Research objectives to enhance PCB remediation 153 6.2.4 Field-Test Phase Objectives 154 6.3 Technical approach 155 6.3.1 Summary 155 6.3.2 FeSO 4 amendment 157 6.3.3 Sequential inoculations 157 6.3.4 Surfactant Amendments 161 6.4 Accomplishments of the flask evaluation 162 6.4.1 Designing and testing PCB-growing GEMs 162 6.4.1.1 Characterization of aerobic PCB metabolism by biphenyl-degrading organisms 162 6.4.1.2 Conceptual proof of designing PCB growth pathway 167 6.4.1.3 Developing gene transfer system for G+/G– PCB-degrading bacteria 170 6.4.1.4 Degradative capabilities of the recombinant RHA1( fcb ) 171 6.4.1.5 Survival and activity of GEM RHA1( fcb ) in nonsterile soil 173 L1656_C006.fm Page 147 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC 148 Bioremediation of Recalcitrant Compounds 6.4.1.6 Developing and testing molecular tracking recombinant organisms in situ 175 6.4.1.7 Construction of multiple ortho-PCB dechlorinator LB400( ohb ) 177 6.4.1.8 Growth on defined PCB mixtures 182 6.4.1.9 Validation of PCB remediation strategy in soil (microcosm studies) 182 6.4.1.10 Developing protocol for inoculum delivery 184 6.4.1.11 Recommendations for inoculum delivery during pilot test 187 6.4.1.12 Compatibility of anaerobic and aerobic phases in remediation process 187 6.4.2 Microbial-surfactant compatibility experiments 188 6.4.3 Plasmid stability studies 189 6.4.4 PCB-surfactant solubilization experiments 191 6.4.5 Mathematical modeling 192 6.4.6 PCB transformation experiments 193 6.5 Accomplishments of the pilot evaluation 196 6.5.1 Site consideration for field test 196 6.5.2 Site description 200 6.5.3 Pilot-scale demonstration 201 6.5.4 Sampling schedule 204 6.5.5 Analytical methods 206 6.6 Conclusions 208 6.7 Recommendations for further transitional research 208 References 209 6.1 Project background and rationale PCBs remain among the most expensive hazardous waste cleanup problems facing the country. Through the use of existing technology, principally incin- eration, the cleanup cost is estimated to exceed $20 billion. If PCB concen- trations could be reduced in situ using bioremediation approaches, these costs could be substantially reduced. The research on microbial degradation of PCBs has a 20-year history (Ahmed and Focht, 1973), and many field trials of PCB bioremediation have taken place. This research has shown that biore- mediation requires a more sophisticated technology than the simplistic attempts that have been tried thus far. The 20 years of PCB research, however, defined the barriers that must be overcome to achieve successful bioreme- diation, and the discoveries in basic biochemistry and molecular biology have now provided feasible approaches to overcome these barriers. The fundamental barriers to bioremediation of PCBs are: • The absence from nature of organisms that will grow on PCBs L1656_C006.fm Page 148 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC Chapter six: Enhancing PCB bioremediation 149 • The slow rate of reductive dechlorination and the fact that it is usually incomplete • The low solubility of PCBs, and hence their poor bioavailability • Practical barriers, including a microbial delivery technology that en- sures high survivability of introduced microorganisms in soils • Appropriate field-scale remediation technologies. Polychlorinated biphenyls represent a class of chlorinated compounds, the general structure for which is given in Figure 6.1. Each of the numbered positions may or may not be chlorinated, resulting in 209 different congeners. For industrial purposes, PCBs were manufactured as complex mixtures con- taining from 60 to 90 congeners by the catalytic chlorination of biphenyl (Shulz et al., 1989). Depending on the amount of chlorine added, these mixtures were mobile oils, viscous liquids, or sticky resins, but all were nonflammable, thermally stabile, chemically inert, and excellent electrical insulators. Because of these properties, they were widely used as dielectric fluids in electrical capacitors and transformers and as plasticizers. Smaller but still significant amounts were used as lubricants, hydraulic fluids, heat transfer fluids, cutting oils, extenders in waxes, pesticides, and inks, and in carbonless copy paper (Hutzinger et al., 1974). In the United States and Great Britain, nearly all PCBs were manufac- tured by Monsanto under the trade name Aroclor and given a four-digit numerical designation. The first two digits in the numerical designation indicate either PCBs (12), polychlorinated terphenyls (PCTs) (54), or mixtures of PCBs and PCTs (25 or 44), and the last two digits indicated the percent chlorine by weight. Thus, Aroclor 1242, for example, is a PCB mixture that is 42% chlorine by weight and averages 3.1 chlorines per molecule. Aroclor 1260 contains 60% chlorine and averages about six chlorines per molecule. Aroclor 1016 is an exception to this scheme; it contains 41% chlorine by weight and appears to be a fractional distillation product from Aroclor 1242, with a marked reduction in the amount of congeners with five or more chlorines. In other countries, PCB mixtures were manufactured under the trade names Fenclor, Pheneclor, Pyralene, Clophen, and Kanechlor, to name a few (Hutzinger et al., 1974). Figure 6.1 The general structure of polychlorinated biphenyl compounds. 4 3 5 2′ 3′ 6 Biphenyl 26′ 5′ 11′ 4′ L1656_C006.fm Page 149 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC 150 Bioremediation of Recalcitrant Compounds As the result of manufacturing processes and spills, several hundred million pounds of PCBs have been released into the environment (Hutzinger and Veerkamp, 1981), and the same properties that made them so industrially useful make them environmentally persistent. Because they are sparingly soluble in water, they have a limited potential for migration through soil, and even the bulk of PCBs deposited in sediments may remain in place for decades. They are also lipid soluble and therefore bioaccumulate, increasing risks associated with exposure through the food chain. A variety of adverse biological effects have been ascribed to them. Perhaps the most notable ecotoxicological effect of PCBs concerns poor reproductive success and deformities in some fish and fish-eating birds (Ludwig et al., 1993). Also, PCBs are suspected carcinogens, and there is epidemiological evidence that they can cause abnormal neurological development in infants and children and alter immunological responses (ATSDR, 2000). Thus, they are recognized as one of the most problematic and persistent environmental contaminants. The remediation of PCB-contaminated soils and sediments typically involves excavation of the contaminated material followed by landfill dis- posal or incineration. The high costs, long-term liability, and regulatory issues associated with this approach have reduced the attractiveness of exca- vation as an ultimate remediation option. In addition, excavation and off-site transport of PCB-contaminated wastes may actually increase the potential for human exposure. Recognition of the potential economic and health impli- cations associated with traditional PCB treatment methods has led to a renewed interest in the development of in situ and on-site treatment tech- nologies, including enhanced bioremediation processes. Due to their low solubility in water (or hydrophobicity) and low vapor pressures, PCB con- geners are not effectively removed from soil/sediment systems by conven- tional abiotic remediation technologies such as soil vapor extraction or sol- vent flushing. Thus, the current state-of-the-art for PCB remediation typically involves the excavation of PCB-contaminated soil/sediment, followed by incineration. Estimated costs for incineration are on the order of $300 to $600/ton of soil, including transportation and excavation costs. As noted above, this remediation method frequently involves increased risk of human exposure, due to the excavation and transport of PCB-contaminated soils. The primary routes of exposure for this scenario are inhalation and dermal contact with soil particles containing sorbed-phase PCBs. A competing in situ technology that is currently under development involves thermal desorption and oxidation of PCB-contaminated surface soils (Iben et al., 1996). The technique involves the use of a thermal blanket containing resistive tubular heaters spaced at 8-cm intervals. The thermal blanket is placed over the soil surface and covered with a layer of insulation (vermiculite or ceramic fiber) and an impermeable sheet of fiberglass-rein- forced silicon rubber. Off-gases are extracted through a central tube and passed through a thermal oxidizer operated at about 900˚C. A pilot-scale study has been conducted at an abandoned racetrack where PCB-containing oil was applied to the soil surface to reduce dust. PCB concentrations L1656_C006.fm Page 150 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC Chapter six: Enhancing PCB bioremediation 151 averaged approximately 680 mg/kg from 0 to 7.5 cm (0 to 3 in.) depth and 100 mg/kg for 7.5 to 15.0 cm (3 to 6 in.) depth. Below 15 cm (6 in.), PCB concentrations were below the 2 mg/kg target level. The thermal blanket was heated to about 900˚C for 21 h, with temperatures at the 15 cm depth maintained at 250˚C for about 29 h after the heaters were turned off. In most cases, soil PCB concentrations were reduced to well below the 2 mg/kg target. The costs for thermal blanket remediation were estimated at $150 to $200/ton for a larger site (>6 ha) for a treatment depth of 15 cm (6 in.). These costs may be viewed as rather optimistic because they were based on a scaled-up application (not the actual test case) and the depth of treatment was only 6 in. Limitations and concerns of this technology include: •Small depth of treatment • Potential for downward migration of mobilized PCBs • Potential formation of undesirable low-temperature thermal prod- ucts near the edge of the treated zone 6.2 Objectives 6.2.1 Overall objectives The primary objectives of the research described herein was to: • Develop genetically engineered organisms that will grow on PCBs • Evaluate surfactants and FeSO 4 to enhance PCB dechlorination •Implement and test PCB bioremediation in pilot-scale reactors The goal of the first objective was to construct pathways for PCB deg- radation that would result in bacteria capable of using PCB congeners as a growth substrate, and to use these organisms to remove products of anaer- obic reductive PCB dechlorination (i.e., the less chlorinated mono-, di-, and trichlorobiphenyls, predominantly ortho- and ortho+para-chlorinated con- geners). To achieve this goal, two metabolic capabilities were combined in the same organism, (a) cometabolism of PCBs to chlorobenzoates and dechlo- rination and (b) mineralization of chlorobenzoates as a growth substrate. Our research activities have focused on several biphenyl-degrading, PCB-cometabolic bacterial strains studied in our laboratory and the dechlo- rination genes found, isolated, and studied as part of the Great Lakes and Mid-Atlantic Hazardous Suibstance Research Center (GLMAC HSRC) project. Once those strains were constructed, the ability of the designed organisms to enhance PCB degradation in soils was evaluated. The practical effectiveness of constructed bacteria was tested at the U.S. Army Engineer Waterways Experiment Station (WES). The second objective of the project was designed to identify and evaluate surfactants capable of enhancing the bioremediation of PCBs in soils and sediments. Research activities specifically focused on the selection of L1656_C006.fm Page 151 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC 152 Bioremediation of Recalcitrant Compounds surfactants that are compatible with the engineered bacteria discussed below, including Rhodococcus erythreus NY05, Rhodococcus RHA1, and Comamonas testosteroni VP44. The experimental approach involved a systematic screen- ing of selected surfactants in microbial batch systems for toxicity or inhibi- tory effects, prior to the addition of engineered bacteria and surfactant to a contaminated soil or sediment system. The ideal surfactant candidate would not be readily utilized as a growth substrate by the bacteria, possibly serving as a preferential growth substrate over PCBs or reducing the selective pres- sure for PCB growth genes. In addition to biological compatibility, the selected surfactants were also tested for sorptive losses to several natural soils, capacity to solubilize PCB congeners, coupled solubilization and micro- bial transformation, and effects on plasmid stability. The third objective focused on pilot-scale implementation of PCB biore- mediation using soil collected from Lake Ontario Ordinance Works (LOOW) Picatinny Arsenal and General Electric at a site located in Rome, GA. The practical effectiveness of a two-phase anaerobic–aerobic bioremediation sys- tem was evaluated in cooperation with engineers in the Strategic Environ- mental Research and Development Program (SERDP) bioconsortium at Georgia Tech and the Waterways Experiment Station (WES). 6.2.2 Research objectives to design PCB-growing GEMs • Develop gene cloning and chromosomal integration technique in Rhodococcus strains. • Design genetically enhanced para- and ortho-PCB-growing gram-negative and Rhodococcus strains. • Evaluate the fate of the designed organisms and their effect on PCBs in soils. • Develop methods allowing the tracking of introduced organisms and genes in situ . • Evaluate effect of anaerobic-aerobic shift and FeSO 4 and FeS on sur- vivability and PCB degradative activity of the designed organisms in soils. • Develop suitable protocol for soil inoculation with the engineered microorganisms. • Enhance anaerobic reductive PCB dechlorination in PCB-contaminat- ed soil. • Evaluate the recombinant PCB remediation two-phase technology on the pilot scale using PCB-contaminated soils. • Evaluate the feasibility of anaerobic PCB dechlorination in contami- nated soils to enrich the congeners that would be accessible for deg- radation by aerobic genetically enhanced microorganisms. • Establish methods allowing rapid and quantitative detection of ge- netically engineered PCB-growing bacteria in situ . L1656_C006.fm Page 152 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC Chapter six: Enhancing PCB bioremediation 153 • Characterize the PCB-growing variants of strains Rhodococcus sp. RHA1 and Burkholderia xenovorans LB400 possessing hydrolytic para-dechlorination ( fcb ) and oxygenolytic ortho-chlorobenzoate ( ohb ) dechlorination genes, respectively, for their substrate range. • Evaluate the survivability of the recombinant PCB-growing organ- isms RHA1( fcb ) and LB400( ohb ) and their impact on PCBs in soil microcosms. • Evaluate the effect of FeSO 4 and FeS on the fate and activity of the recombinant strains LB400( ohb ) and RHA1( fcb ) in soil microcosms. • Evaluate the survivability of introduced biphenyl-degrading bacteria in soils. • Isolate and evaluate the feasibility of genetic enhancement of indig- enous biphenyl-degrading organisms from the PCB-contaminated soil to increase chances for successful PCB remediation in this envi- ronment, which has a complex contamination profile. 6.2.3 Research objectives to enhance PCB remediation • Investigate the growth of Comamonas testosteroni VP44 and Rhodococ- cus erythreus NY05 on biphenyl and 4-chlorobiphenyl (4-CBP) in the presence of selected nonionic surfactants (Tween 80, Tergitol NP-15, and Witconol SN-120). • Develop analytical methods (high-performance liquid chromatogra- phy (HPLC) and gas chromatography (GC)) to measure the concen- tration of both surfactant and PCB congeners in the aqueous phase. •Measure the rates of micellar solubilization and equilibrium solubi- lization capacity of the selected surfactant for specific PCB congeners. •Measure the sorption and desorption of surfactants on natural soils possessing a range of organic carbon (OC) contents (Wurtsmith aqui- fer material, 0.02% OC; Appling soil, 0.7% OC; and Webster soil, 3.5% OC) and the influence of surfactant sorption on PCB sorption by the solid phase. • Quantify the aerobic degradation of both surfactant and PCB conge- ners in aqueous systems by recombinant variants of Comamonas tes- tosteroni VP44 and Rhodococcus erythreus NY05. • Conduct microcosm experiments (500-ml reactors) to assess the de- sorption and aerobic dechlorination of sorbed-phase PCB congeners in the presence of Tergitol NP-15 and Tween 80. • Develop and test a mathematical model to describe the coupled sorption/desorption, micellar solubilization, and transformation of PCB congeners under sequential anaerobic and aerobic conditions. • Investigate the growth of Comamonas testosteroni VP44 and Rhodococ- cus erythreus NY05 on biphenyl and 4-CBP in the presence of selected nonionic surfactants (Tween 80, Tergitol NP-15, and Witconol SN-120). L1656_C006.fm Page 153 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC 154 Bioremediation of Recalcitrant Compounds • Design and test mixing capacity of low-water-content bioreactor (~1/ 5 scale) for the treatment of PCB-contaminated soils. • Perform economic analysis of bioreactor system and compare costs to competing technologies based on site-specific information. • Optimize anaerobic–aerobic reactor treatment scheme, including ma- terials handling; the addition of bulking agents, biocarriers, and nu- trients; and disposal procedures. • Conduct surfactant performance tests using PCB-contaminated soils to test the potential for surfactant sorption losses and PCB phase distribution and PCB desorption rates. • Assist in scale-up of the bioreactor and implementation of pilot-scale tests. • Assist in regulatory compliance with regard to PCBs and genetically engineered microorganisms (GEMs) handling and disposal procedures. • Coordinate interaction among PCB thrust area PIs (J. Tiedje at MSU, K. Pennel at Georgia Tech, and L. Hansen at WES) and incorporate laboratory results into field-scale reactor design and operation. • Evaluate mixing performance and materials addition of full-scale reactor during operation. 6.2.4 Field-Test Phase Objectives • Evaluate the effects of vermiculite and Fe(II) on survival and activity of GEMs in soil. • Evaluate survivability and PCB degradative activity of GEMs in con- taminated soils. • Develop protocol for preparation and delivery of GEMs in pilot-scale reactors. • Conduct laboratory-scale experiments to evaluate the effects of sur- factant additions on PCB desorption and biodegradation under mix- ing regimes similar to those utilized in the pilot-scale reactors. • Develop and evaluate mathematical models designed to simulate PCB desorption and biodegradation in the presence and absence of surfactants. •Validate the design and strategy for two-phase anaerobic–aerobic bioremediation of PCB-contaminated soils using laboratory-scale soil microcosms. • Evaluate the use of PCB-growing GEMs in combination with en- hanced anaerobic dechlorination and surfactants for bioremediating PCB-contaminated soils. • Design pilot-scale treatment systems, including slurry and land-farming reactors, to test PCB remediation technologies. • Develop cost estimates for pilot-scale treatment systems and perform economic analysis of full-scale implementation with comparisons to conventional PCB treatment technologies. L1656_C006.fm Page 154 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC Chapter six: Enhancing PCB bioremediation 155 • Evaluate the effects of different solids’ loading rates (i.e., water con- tents) on the application of amendments and GEMs and the biore- mediation of PCBs. • Determine the maximum solids’ loading rate for optimum activity of GEMs in order to offset subsequent dewatering costs for the dis- posal/reuse of stabilized soils. • Identify PCB-contaminated sites available for the field test and eval- uate feasibility of their PCB treatment. • Conduct ex situ two-phase anaerobic–aerobic PCB remediation pi- lot-scale tests and evaluate efficiency of the PCB removal and per- formance of GEMs. 6.3 Technical approach 6.3.1 Summary This project addresses key barriers to bioremediating PCBs, which are to: • Develop microorganisms that will grow on the major congeners pro- duced by anaerobic dechlorination of PCBs •Improve bioavailability of PCBs through use of surfactants • Optimize field delivery of anaerobic–aerobic PCB bioremediation technology •Validate the new two-phase anaerobic–aerobic bioremediation strat- egy in a pilot-scale test Three central components of the project were: the use of a combination of genetically engineered organisms that will grow on PCBs, the use of surfactants to enhance the bioavailability, and bioslurry experiments as a first stage in a flask-to-field transfer technology (Figure 6.2). The lack of organisms that grow on PCBs results from the fact that organisms with PCB (biphenyl moiety)-cometabolizing activity do not seem to have the ability to use the chlorobenzoate product for growth. Moreover, those few organisms that have this ability would attempt to metabolize it via the chlorocatechol pathway, forming an acyl halide, which is immediately toxic. Hence, if any organism in nature does have the capacity to grow on PCBs, it would be suicidal. Through the use of dechlorination genes, we have devised a scheme to remove chlorines before chlorocatechols are formed, thereby providing an energy (growth) product and avoiding toxicity. This approach should be a more desired solution for PCB remediation because it would avoid the need to manage cometabolism, which can be difficult to achieve in situ . One of the biological barriers to effective PCB remediation is slow rates of intrinsic anaerobic dechlorination. The first step in optimizing a sequential anaerobic-aerobic biotreatment process for PCBs is to maximize the extent of dechlorination. Several dechlorination processes, generally believed to be L1656_C006.fm Page 155 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC 156 Bioremediation of Recalcitrant Compounds due to the actions of different species of microorganisms, have been recog- nized on the basis of their congener specificities (Table 6.1). In Table 6.1, dechlorination process M, for example, removes both flanked and unflanked meta-chlorines. A flanked meta-chlorine is one that is adjacent to another (ortho- or para-) chlorine. An unflanked chlorine has no other chlorine next to it. Not fully captured in Table 6.1 is the fact that these processes also vary in their abilities to attack more heavily chlorinated Figure 6.2 Sequential anaerobic–aerobic PCB remediation strategy. Table 6.1 Regiospecificities of the Various Described PCB Dechlorination Processes Dechlorination Activity Susceptible Chlorines M Flanked and unflanked meta Q Flanked and unflanked para H Flanked para Doubly flanked meta H’ Flanked para Meta of 2,3- and 2,3,4- groups P Flanked para N Flanked meta T Meta of hepta- and octa-CBs LP Unflanked para Aerobic PCB Dechlorination Genes Organisms - Burkholderia LB400 (G−) - Rhodococcus RHA1 (G+) Inocula Technology - Vermiculite Low, Medium and High Solids Reactors Anaerobic Reductive Dechlorination FeSO 4 - Rates/Conc. Seed Inoculum - Hudson river General Scheme Twe en 80 - Sorption/Availability ohb, fcb G+/− Cassettes L1656_C006.fm Page 156 Tuesday, July 12, 2005 7:47 AM © 2006 by Taylor & Francis Group, LLC [...]... 12 16 20 24 Time, h 2-CBA 4-CBA 2, 4-CBA 100 Degradation of Mix M by the resting cells of Rhodococcus sp RHA1 (+fcb) (A) and accumulation of a chlorobenzoates (B) 50 0 2-CB 4-CB 2-2 ′/2, 6- CB 2, 4-CB 2-4 ′ -CB 2, 4-2 ′ -CB Figure 6. 10 Degradation of mix M by RHA1(fcb) © 20 06 by Taylor & Francis Group, LLC 2, 4-4 ′ -CB L 165 6_C0 06. fm Page 173 Tuesday, July 12, 2005 7:47 AM Chapter six: Enhancing PCB bioremediation. .. each peak number © 20 06 by Taylor & Francis Group, LLC L 165 6_C0 06. fm Page 163 Tuesday, July 12, 2005 7:47 AM Chapter six: Enhancing PCB bioremediation 163 RHA1 was indicated by nonstoichiometric yield of 2-chlorobenzoic acid (2-CBA) and the appearance of yellow color with a maximum absorbance at a wavelength of 394 nm, 2-hydroxy -6 - oxo -6 - phenylhexa-2,4-dienoic acid (HOPDA) Some fading of the yellow color... © 20 06 by Taylor & Francis Group, LLC L 165 6_C0 06. fm Page 162 Tuesday, July 12, 2005 7:47 AM 162 Bioremediation of Recalcitrant Compounds 6. 4 Accomplishments of the flask evaluation 6. 4.1 Designing and testing PCB-growing GEMs 6. 4.1.1 Characterization of aerobic PCB metabolism by biphenyl-degrading organisms Analysis of the dechlorination patterns in anaerobic sediments resulted in identification of eight... biphenyl-grown resting cell assays We observed complete degradation of 2-, 4-, and 2,4-CB after 24 h of incubation Accumulation of equimolar amounts of 2- and 2,4-CBA was observed; however, no 4-CBA was detected, whereas the parent RHA1 accumulated stoichiometric quantities of 4-CBA from 4-CB This suggested activity of the enzymes encoded by the fcb operon in the recombinant strain Because the wild-type... only NY05 produced traces of 2-CBA from 2,4′-CB as transformation product No significant decrease of these HOPDAs, nor a © 20 06 by Taylor & Francis Group, LLC L 165 6_C0 06. fm Page 164 Tuesday, July 12, 2005 7:47 AM 164 Bioremediation of Recalcitrant Compounds corresponding increase of the chlorobenzoic acids, was detected during another 72 h of incubation, suggesting that meta-cleavage products formed... strain RHA1 using the phylogeny of these strain and close relatives All candidate primers included more than one species of the genus Rhodococcus because of the inherent difficulty in designing a 16S rRNA-based strain-specific probe The © 20 06 by Taylor & Francis Group, LLC L 165 6_C0 06. fm Page 1 76 Tuesday, July 12, 2005 7:47 AM 1 76 Bioremediation of Recalcitrant Compounds RHA1 16S rRNA gene sequence corresponding... toward para-chlorinated ring We have identified several intermediate products of aerobic PCB oxidation with potential biotoxic effects, among them meta-cleavage products; chlorinated HOPDAs and dihydrodiols and monoand dihydroxybiphenyls The amount of (Cl)HOPDA produced by © 20 06 by Taylor & Francis Group, LLC L 165 6_C0 06. fm Page 166 Tuesday, July 12, 2005 7:47 AM 166 Bioremediation of Recalcitrant Compounds. .. costs and increase chances of success © 20 06 by Taylor & Francis Group, LLC L 165 6_C0 06. fm Page 161 Tuesday, July 12, 2005 7:47 AM Chapter six: Enhancing PCB bioremediation 161 H Figure 6. 5 Engineering PCB pathways 6. 3.4 Surfactant Amendments Although microbial transformation of PCBs has been the subject of intense study over the past 25 years, it is now apparent that the use of simplistic remediation... stable Recovery of about 5% of 2,4′-CB as 4-CBA by both strains showed that they could also oxidize the ortho-chlorinated ring Advantageously to NY05 and VP44, strains LB400 and RHA1 efficiently degraded 80 to 100% of 2,2 - and 2,4,2′-CB (chlorine in ortho-position on each ring of the biphenyl moiety) Both strains transformed 2,4,2′-CB into equimolar amounts of 2,4-CBA, and there was a 60 to 80% transformation... and indicate that dechlorination was limited to the meta-positions In other words, process Q or para-dechlorination activity was lost, and even N activity was diminished The profile for Silver Lake microorganisms alone indicates that high levels of ortho- and para-substituted congeners, notably 2 4-2 6- CB (peak 21), 2 4-2 4-CB (peak 26) , and 24 6- 2 4-CB (peak 34), were formed These congeners could serve as . a wavelength of 394 nm, 2-hydroxy -6 - oxo -6 - phenylhexa-2,4-dienoic acid (HOPDA). Some fading of the yellow color and a shift toward shorter wave- lengths were detected during the next two days of incubation,. microorganisms alone indicates that high levels of ortho- and para-substituted congeners, notably 2 4-2 6- CB (peak 21), 2 4-2 4-CB (peak 26) , and 24 6- 2 4-CB (peak 34), were formed. These congeners could. evaluation 1 96 6.5.1 Site consideration for field test 1 96 6.5.2 Site description 200 6. 5.3 Pilot-scale demonstration 201 6. 5.4 Sampling schedule 204 6. 5.5 Analytical methods 2 06 6 .6 Conclusions 208 6. 7