Product Citations and Abstracts

Biodegradation of the polycyclic aromatic hydrocarbons of creosote by undefined bacterial cultures was shown to be accompanied by the accumulation of neutral and acidic oxidation products. Formation of a number of identified neutral products is accounted for by demonstration of anomalous actions of an arene dioxygenase on the benzylic methylene and methylene carbons of napthenoaromatic hydrocarbons. Both neutral and acidic water-soluble fractions are also formed when various mixed bacterial cultures degrade weathered crude oil. While constituents of these fractions are not yet identified, the neutral materials have been shown to be toxic to developing embryos of invertebrates. These observations are discussed in relation to chemical and toxicological assessments of biodegradation of the complex chemical mixtures of fossil fuels.

Chapman, P.J. 1988. Constructing Microbial Strains for Degradation of Halogenated Aromatic Hydrocarbons. In: Environmental Biotechnology: Reducing Risks from Environmental Chemicals Through Biotechnology. Gilbert S. Omenn et al., Editor. Plenum Press, New York, NY. Pp. 81-95. (ERL,GB X568).

This paper examines methods that have been used to isolate and to construct bacteria capable of growing aerobically with chlorinated aromatic compounds, including chlorinated hydrocarbons. It also describes some recent work in this area of research.

[1-13C]acenaphthene, a tracer compound with a nuclear magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled 13C-NMR spectroscopy technique to study the biodegradation of acenaphthene by various bacterial cultures degrading aromatic hydrocarbons of creosote. Site-specific labeling at the benzylic position of acenaphthene allows 13C-NMR detection of chemical changes due to initial oxidations catalyzed by bacterial enzymes of aromatic hydrocarbon catabolism. Biodegradation of [1 -13 C]acenaphthene in the presence of naphthalene or creosote polycyclic aromatic compounds (PACs) was examined with an undefined mixed bacterial culture (established by enrichment on creosote PACs), and with isolates of individual naphthalene- and phenanthrene-degrading strains from this culture. From 13C-NMR spectra of extractable materials obtained in time course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as 1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of acenaphthene and subsequent reactions. Limited degradation of acenaphthene could be attributed to its oxidation by napthalene 1,2-dioxygenase or related dioxygenases, indicative of certain limitations of the undefined mixed culture with respect to acenaphthene catabolism. Coinoculation of the mixed culture with cells of acenaphthene-grown strain Pseudomonas sp. A2279 mitigated accumulation of partial transformation products and resulted in more complete degradation of acenaphthene. This study demonstrates the value of the stable isotope labeling approach and its ability to reveal incomplete mineralization even when as little as 2 to 3% of substrate is incompletely oxidized, yielding products of partial transformation. The approach outlined may prove useful in assessing bioremediation performance.

Pinkart, Holly C., Richard Devereux and Peter J. Chapman. 1998. Rapid Separation of Microbial Lipids Using Solid Phase Extraction Columns. J. Microbiol. Methods. 34(1):9-15. (ERL,GB 1038).

A method was developed to rapidly separate lipid classes commonly found in microbial communities. The method is based on the use of aminopropyl solid phase extraction columns to separate polyhydroxyalkoanates (PHA), phospholipids, sterols, triglycerides, diglycerides, monoglycerides, and steryl esters. Recoveries of all lipid classes, with the exception of PHA and sterols, ranged from 91% to greater than 99%. PHA were recovered at 69% of the standard, and sterols from 82-84% of the standard. When applied to the analysis of lipids extracted from the cyanobacterium Spirulina platensis, the method afforded excellent recovery and separation of phospholipids and diglycerides including saturated, monounsaturated and polyunsaturated fatty acids. The S. platensis lipids also contained hydrocarbons and phytol recovered in the steryl ester and diglyceride fractions, respectively. This method provided a high yield, specific and rapid separation of microbial lipids with little contamination from other lipid groups, and will be useful for the characterization of microbial communities in environmental samples.

Leblond, Jeffrey D. and Peter J. Chapman. 2000. Lipid Class Distribution of Highly Unsaturated Long Chain Fatty Acids in Marine Dinoflagellates. J. Phycol. 26(6):1103-1108. (ERL,GB 1094).

The very long chain highly unsaturated C28 fatty acids, octacosaheptaenoic [28:7(n-6)] and octacosaoctaenoic acid [28:8(n-3)], were found to be associated with phospholipids, obtained by fractionation of total lipid extracts into distinct lipid classes, in 4 and 6, respectively, of 16 examined dinoflagellates. An interfraction comparison of fatty acids associated with phospholipids and glycolipids has also shown that the phospholipid fractions contained the majority (over 75% in 12 of 16 strains) of docosahexaenoic acid [22:6(n-3)] and traces of tetracosanoic acid (24:0). By contrast, the highly unsaturated C18 fatty acids octadecatetraenoic [18:4(n-3)] and octadecapentaenoic acid [18:5(n-3)] were primarily recovered from a chloroplast-associated glycolipid fraction comprised of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol. In 12 of 16 strains, an interfraction comparison showed that over 90% of 18:5(n-3) was found to be associated with glycolipids.These findings indicate that the C28 fatty acids are located and probably synthesized in the cytoplasm or in an organelle other than the chloroplast, possibly with 22:6(n-3) and 24:0 as precursors, whereas the C18 fatty acids 18:4(n-3) and 18:5(n-3) are glycolipid constituents apparently synthesized within the chloroplast. The function(s) of these C28 fatty acids as components of phospholipids in cellular membranes is currently unknown.

Leblond, Jeffrey D. and Peter J. Chapman. 2002. Survey of the Sterol Composition of the Marine Dinoflagellates Karenia brevis, Karenia mikimotoi, and Karlodinium micrum: Distribution of Sterols Within Other Members of the Class Dinophyceae. EPA/600/J-01/420. J. Phycol. 38(8):670-682. (ERL,GB 1149).

The sterol composition of different marine microalgae was examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4-methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevisHansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two majors sterols, (24S)-4a-methyl-5a -ergosta-8(14),22-dien-3b-ol (ED) and its 27-nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen,two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi,and K. micrum,and and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.

Leblond, Jeffrey D., Terence J. Evens and Peter J. Chapman. 2003. Biochemistry of Dinoflagellate Lipids, with Particular Reference to the Fatty Acid and Sterol Composition of a Karenia brevis Bloom. EPA/600/J-03/491. Phycologia. 42(4):324-331. (ERL,GB 1160).

The harmful marine dinoflagellate, Karenia brevis (Dinophyceae), frequently forms large toxic blooms in the waters off of the west coast of Florida (USA) and is responsible for massive fish kills and public health concerns. Despite decades of field studies on this organism, no investigation has yet characterized the lipid composition of a K. brevis bloom. To address this lack of information, samples from a 1999 K. brevis bloom from the northwest Florida coast were analyzed for their fatty acid and sterol composition. Fatty acids found in lipid fractions containing membrane phospholipids, chloroplast-associated glycolipids, and storage triglycerides differed significantly. The glycolipid fraction was found to contain octadecapentaenoic acid [18:5(n-3)], a fatty acid commonly associated with dinoflagellates. The phospholipid fraction was found to contain small amounts of two recently described, highly unsaturated fatty acids, octacosaoctaenoic acid [28:8(n-3)] and octacosaheptaenoic acid [28:7(n-6)]. Fatty acids from the triglyceride fraction were more abundant than those associated with glycolipid or phospholipids. Sterols were found mainly as free sterols and were dominated by two compounds (24S)-4a-methyl-5a-ergosta-8(14),22-dien-3b-ol (ED) and its 27-nor derivative (NED). The lipid composition of these samples very closely resembles laboratory-grown cultures of K. brevis and serves to provide an in situ field validation of past laboratory examinations of this organism. The implications of our data are discussed in the context of the physiological autecology of K. brevis, in the form of a minireview on the biochemistry of dinoflagellate lipids, as studied in both the laboratory and the environment.

Leblond, Jeffrey D. and Peter J. Chapman. Unpublished. Polyunsaturated C27 Hydrocarbons in the Marine Dinoflagellate, Pyrocystis lunula (Dinophyceae): Preliminary Characterization. J. Phycol. 33 p. (ERL,GB 1179).

The lipids of different algal species have revealed a diversity of fatty acids, sterols, and hydrocarbons, several of which are considered of chemotaxonomic value and candidates for useful chemical biomarkers with the potential for characterizing phytoplankton community composition. Neutral fractions obtained from the lipids of two strains of Pyrocystis lunula, in the course of characterizing over forty dinoflagellates, were found to contain an abundant quantity of long-chain polyunsaturated hydrocarbons, along with previously reported keto-steranes. Gas chromatographic examination showed a retention time of the predominant hydrocarbon, relative to standards, which together with its molecular weight (m/z = 364) and mass spectrum suggested a multiply unsaturated C27 compound (C27H40) with eight double bonds. Reduction (Adams catalyst) gave a product identical to authentic n-heptacosane indicating a straight chain hydrocarbon. While the positions and stereochemistry of double bonds have not been established, the carbon number of this hydrocarbon and the number of double bonds strongly suggest a relationship to, and formation by decarboxylation of, the recently described, long-chain polyunsaturated C28 fatty acid, [28:8(n-3)], shown to be a constituent of dinoflagellate phospholipids. This hydrocarbon was not found in any other genus of the over forty examined dinoflagellates, nor was it found in isolates of two other species of Pyrocystis, P. fusiformis and P. noctiluca. The function(s) of this compound in P. lunula is currently unclear.

Leblond, Jeffrey D. and Peter J. Chapman. 2004. Sterols of the Heterotrophic Dinoflagellate, Pfiesteria piscicida (Dinophyceae): Is There a Lipid Biomarker?. J. Phycol. 40(1):104-111. (ERL,GB 1191).

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid-Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health, and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC-MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that, like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4a-methyl-substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.

Leblond, Jeffrey D., Jeremy L. Dahmen, Rebecca L. Seipelt, Matthew J. Elrod-Erickson, A. Bruce Cahoon, Rodney Kincaid, James C. Howard, Terence J. Evens and Peter J. Chapman. 2005. Lipid Composition of Chlorarachniophytes (Chlorarachniophyceae) from the Genera Bigelowiella, Gymnochlora, and Lotharella. J. Phycol. 41:311-321. (ERL,GB 1218).

The Chlorarachniophyceae are unicellular eukaryotic algae characterized by an amoeboid morphology that may be the result of secondary endosymbiosis of a green alga by a nonphotosynthetic amoeba or amoeboflagellate. Whereas much is known about the phylogeny of chlorarachniophytes, little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, four organisms from three genera were examined for their fatty acid and sterol composition. Fatty acids from lipid fractions containing chloroplast-associated glycolipids, storage triglycerides, and cytoplasmic membrane-associated polar lipids were characterized. Glycolipid-associated fatty acids were of limited composition, principally eicosapentaenoic acid [20:5(n-3)] and hexadecanoic acid (16:0). Triglyceride-associated fatty acids, although minor, were found to be similar in composition. The polar lipid fraction was dominated by lipids that did not contain phosphorus, and had a more variable fatty acid composition with 16:0 and docosapentaenoic acid [22:5(n-3)] dominant along with a number of minor C18 and C20 fatty acids. Crinosterol and one of the epimeric pair poriferasterol/stigmasterol were the sole sterols. Several genes required for synthesis of these sterols were computationally identified in Bigelowiella natans. One sterol biosynthesis gene showed the greatest similarity to SMT1 of the green alga, Chlamydomonas reinhardtii. However, homologues to other species, mostly green plant species, were also found. Further, the method used for identification suggested that the sequences have been transferred to a genetic compartment other than the likely original location, the nucleomorph nucleus.

Shields, Malcolm S., Stacy O. Montgomery, Peter J. Chapman, Stephen M. Cuskey and P.H. Pritchard. 1989. Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4. EPA/600/J-89/168. Appl. Environ. Microbiol. 55(6):1624-1629. (ERL,GB 668). (Avail. from NTIS, Springfield, VA: PB90-129537)

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of 18O- and 16O- labeled 3-methylcatechol from toluene in an atmosphere of 18O2 and 16O 2 established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.

Mueller, James G., Peter J. Chapman and P. Hap Pritchard. 1989. Creosote-Contaminated Sites: Their Potential for Bioremediation. EPA/600/J-89/170. Environ. Sci. Technol. 23(10):1197-1201. (ERL,GB 671). (Avail. from NTIS, Springfield, VA: PB90-129552)

The demonstrated ability to biologically transform toxic organic chemicals into innocuous compounds under laboratory conditions has led to the view that microbial systems may be employed advantageously to remediate hazardous waste sites polluted with similar materials. Hence, bioremediation is a rapidly developing technology with the potential to provide efficient and economic means of removing organic pollutants from contaminated materials. However, bioremediation is currently restricted by a variety of factors. One of the most significant of these is the difficulty of effectively degrading complex mixtures of chemicals as often found at hazardous waste sites. Although preliminary evidence shows that such treatments are potentially effective in situ, additional data on the performance of bioremediation efforts in large scale field trials is also needed. Bioremediation of creosote-contaminated materials is reviewed here by characterizing coal-tar creosote, identifying techniques for assessing the biodegradability of its many chemical constitutents, examining known routes of microbial transformation of these chemicals, and reviewing the performance of previous bioremediation efforts. This approach is developed as a model system to project the potential application of bioremediation to ameliorate environments contaminated by complex mixtures of structurally diverse hazardous chemicals.

Mueller, James G., Peter J. Chapman and P. Hap Pritchard. 1989. Action of a Fluoranthene-Utilizing Bacterial Community on Polycyclic Aromatic Hydrocarbon Components of Creosote. EPA/600/J-89/425. Appl. Environ. Microbiol. 55(12):3085-3090. (ERL,GB 674). (Avail. from NTIS, Springfield, VA: PB90-245721)

Cultures enriched by serial transfer through a mineral salts medium containing fluoranthene were used to establish a stable, 7-membered bacterial community from a sandy soil highly contaminated with coal-tar creosote. This community exhibited an ability to utilize fluoranthene as sole carbon source for growth as demonstrated by increases in protein concentration and changes in absorption spectra when grown on fluoranthene in liquid culture. Biotransformation of other polycyclic aromatic hydrocarbons (PAHs) was verified by demonstrating their disappearance from an artificial PAH mixture using capillary gas chromatography. When grown on fluoranthene as sole carbon source and subsequently exposed to fluoranthene plus 16 additional PAHs typical of those found in creosote, this community transformed all PAHs present in this defined mixture. After 3 days of incubation, 13 of the original 17 PAH components were degraded to levels below the limit of detection (10 ng/L). Continued incubation resulted in extensive degradation of the remaining 4 compounds. The ability of this community to utilize a high molecular weight PAH as sole carbon source, in conjunction with its ability to transform a diverse array of PAHs, suggests that it may be of value in the bioremediation of environments contaminated with PAHs such as those impacted by creosote.

Mueller, James G., Peter J. Chapman, Beat O. Blattmann and P. Hap Pritchard. 1990. Utilization of Fluoranthene by Pseudomonas paucimobilis Strain EPA505. In: Gas, Oil, Coal, and Environmental Biotechnology II. EPA/600/D-89/254. Cavit Akin and Jared Smith, Editors. Institute of Gas Technology, Chicago, IL. Pp. 243-253. (ERL,GB 690).

Pseudomonas paucimobilis strain EPA505 was previously purified from a 7-membered bacterial community originally isolated from a creosote-contaminated soil for its ability to degrade polycyclic aromatic hydrocarbon (PAH) components of creosote. The unique ability of this organism to utilized fluoranthene as sole source of carbon and energy for growth in pure culture was demonstrated by increase in bacterial biomass, changes in UV-absorption, decrease in aqueous fluoranthene concentration, and the production of metabolites when fluoranthene was supplied as sole carbon source in liquid culture. Compounds accumulating in fluoranthene culture medium during growth of EPA505 have been distinguished by HPLC and UV-absorption properties. Based on precedents established for bacterial degradation of similar compounds, speculative pathways are proposed to illustrate the novel biochemistry employed by strain EPA505 in the utilization of fluoranthene. Whereas utilization of fluoranthene appears to involve previously undefined variations on established oxygenation and ring cleavage processes, these findings suggest the potential of this and other organisms for accelerating the biotransformation of other environmental pollutants currently considered recalcitrant to microbiological attack. Hence, strain EPA505 and other organisms similarly isolated for their ability to degrade fluoranthene and related compounds may prove useful to remediation efforts employing biological processes.

Mueller, James G., Peter J. Chapman, Beat O. Blattmann and P. Hap Pritchard. 1990. Isolation and Characterization of a Fluoranthene-Utilizing Strain of Pseudomonas paucimobilis. EPA/600/J-90/109. Appl. Environ. Microbiol. 56(4):1079-1086. (ERL,GB 691). (Avail. from NTIS, Springfield, VA: PB90-264698)

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compounds as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.

Mueller, James G., Suzanne E. Lantz, Beat O. Blattmann and Peter J. Chapman. 1991. Bench-Scale Evaluation of Alternative Biological Treatment Processes for the Remediation of Pentachlorophenol- and Creosote-Contaminated Materials: Slurry-Phase Bioremediation. EPA/600/J-91/331. Environ. Sci. Technol. 25(6):1055-1061. (ERL,GB 721). (Avail. from NTIS, Springfield, VA: PB92-129683)

Performance data on slurry-phase bioremediation of pentachlorophenol (PCP)- and creosote-contaminated sediment and surface soil were generated at the bench-scale level. Aqueous slurries, containing 0.05% Triton X-100 to facilitate the soil washing process and to help stabilize the suspensions, were prepared from sediment and surface soil freshly obtained from the American Creosote Works Superfund site at Pensacola, Florida. Slurries (1.1 L) were incubated for 30 days in separate, 1.5 L bioreactors operated in the batch mode at 28.5°C with continuous mixing (300 rpm). Dissolved oxygen and pH were maintained at 90% and pH=7.0. Samples removed with time from each reactor were extracted and analyzed by gas chromatography for PCP and 42 monitored creosote constitutents to delineate the activity of indigenous microorganisms. Changes in microbial biomass were also recorded. Excluding PCP, benzo[b]fluoranthene, benzo[k]fluoranthene and indeno[123-cd]pyrene, slurry-phase bioremediation of highly contaminated sediment (pH adjusted) resulted in rapid and extensive biodegradation (3-5 days to biodegrade >50% of targeted compounds) of monitored constituents. Conversely, microbial activity in surface soil slurries was slower (14-21 days required to biodegrade greater than 50% of targeted compounds) and generally confined to the more readily biodegradable, lower-molecular-weight compounds. These data suggest that slurry-phase bioremediation strategies can be effectively employed to remediate creosote-contaminated materials.

Mueller, James G., Suzanne E. Lantz, Beat O. Blattmann and Peter J. Chapman. 1991. Bench-Scale Evaluation of Alternative Biological Treatment Processes for the Remediation of Pentachlorophenol- and Creosote-Contaminated Materials: Solid-Phase Bioremediation. EPA/600/J-91/335. Environ. Sci. Technol. 25(6):1045-1055. (ERL,GB 722). (Avail. from NTIS, Springfield, VA: PB92-129725)

Bench-scale biotreatability studies were performed to evaluate the potential for using a solid-phase bioremediation process to ameliorate pentachlorophenol (PCP)- and creosote-contaminated sediment and surface soil present at the American Creosote Works Superfund site, Pensacola, Florida. Surface soil and sediment were contaminated with approximately 1 and 7 % (weight basis) organic pollutants, respectively, but the more recalcitrant creosote constitutents (i.e., high-molecular-weight polycyclic aromatic hydrocarbons) were proportionately higher in the surface soil indicative of creosote weathering. The effects of tilling and fertilization on the rate and extent of biodegradation of PCP and 42 targeted creosote constituents by indigenous microflora were monitored by gas chromatographic analysis of organic extracts of soil and sediment. Changes in microbial populations (total heterotrophs and the number of phenanthrene-degraders) were also recorded. Specially designed "landfarming chambers" allowed for the quantitative analysis of targeted pollutants lost through abiotic processes (i.e., leaching, adsorption, volatilization). Data corrected for these losses represented an accurate assessment of biological activity towards creosote and PCP. In general, solid-phase bioremediation resulted in slow and predictable losses of targeted pollutants (i.e., low-molecular-weight creosote constituents were more readily biodegraded than higher-molecular-weight contaminants), and the more recalcitrant pollutants (e.g., PCP) tended to persist. Biodegradation in surface soils was stimulated by the addition of inorganic soluble nutrients, but this treatment had little effect with the more highly contaminated, alkaline sediments. Performance data from these studies suggest that full-scale site remediation employing solid-phase bioremediation strategies may not effectively meet acceptable treatment standards in the time defining these studies.

Mueller, James G., Douglas P. Middaugh, Suzanne E. Lantz and Peter J. Chapman. 1991. Biodegradation of Creosote and Pentachlorophenol in Contaminated Groundwater: Chemical and Biological Assessment. EPA/600/J-91/328. Appl. Environ. Microbiol. 57(5):1277-1285. (ERL,GB 728). (Avail. from NTIS, Springfield, VA: PB92-129659)

Shake flask studies examined the rate and extent of biodegradation of pentachlorophenol (PCP) and 42 components of coal-tar creosote present in contaminated groundwater recovered from the American Creosote Works (ACW) Superfund site, Pensacola, Florida. The ability of indigenous soil microorganisms to remove these contaminants from aqueous solutions was determined by gas chromatographic analysis of organic extracts of biotreated groundwater. Changes in potential environmental and human health hazards associated with the biodegradation of this material were determined at intervals by Microtox® assays and fish toxicity and teratogenicity tests. After 14 days incubation at 30°C, indigenous microorganisms effectively removed 100, 99, 94, 88 and 87% of measured phenolic, lower-molecular-weight polycyclic aromatic hydrocarbons (PAH), S-heterocycle, N-heterocycle and O-heterocycle constituents of creosote, respectively. However, only 53% of the higher-molecular-weight PAHs were degraded; PCP was not removed. Despite the removal of a majority of the organic contaminants through biotreatment, only a slight decrease in toxicity and teratogenicity of biotreated groundwater was observed. These data suggest that toxicity and teratogenicity are associated with compounds difficult to treat biologically, and that one may not necessarily rely on indigenous microorganisms to effectively remove these compounds in a reasonable time span; to this end, alternative or supplemental approaches may be necessary. Similar measures of toxicity and teratogenicity of treated material may offer a simple, yet important, guide to bioremediation effectiveness.

Shields, Malcolm S., Stacy O. Montgomery, Stephen M. Cuskey, Peter J. Chapman and P.H. Pritchard. 1991. Mutants of Pseudomonas cepacia G4 Defective in Catabolism of Aromatic Compounds and Trichloroethylene. EPA/600/J-91/337. Appl. Environ. Microbiol. 57(7):1935-1941. (ERL,GB 730). (Avail. from NTIS, Springfield, VA: PB92-129741)

Pseudomonas cepacia strain G4 possesses a novel pathway of toluene catabolism that is shown to be responsible for the degradation of trichloroethylene (TCE). This pathway involves conversion of toluene via o-cresol to 3-methylcatechol. In order to determine the enzyme of toluene degradation that is responsible for TCE degradation, chemically induced mutants, blocked in the toluene ortho -monooxygenase (TOM) pathway of G4, were examined. Mutants of the phenotypic class designated TOM A- were all defective in their ability to oxidize toluene, o-cresol, m-cresol, and phenol, suggesting that a single enzyme is responsible for conversion of these compounds to their hydroxylated products (3-methylcatechol from toluene, o-cresol, and m-cresol and catechol from phenol) in the wild type. Mutants of this class did not degrade TCE. Two other mutant classes blocked in toluene catabolism, TOM B-, which lacked catechol-2,3-dioxygenase or TOM C-, which lacked 2-hydroxy-6-oxoheptadienoic acid hydrolase activity, were fully capable of TCE degradation. Therefore, TCE degradation is directly associated with the monooxygenation capability responsible for toluene, cresol and phenol hydroxylation.

Resnick, Sol M. and Peter J. Chapman. 1994. Physiological Properties and Substrate Specificity of a Pentachlorophenol-Degrading Pseudomonas Species. EPA/600/J-94/444. Biodegradation. 5(1):47-54. (ERL,GB 750). (Avail. from NTIS, Springfield, VA: PB95-112157)

A bacterial strain capable of utilizing pentachlorophenol (PCP) as sole source of carbon and energy for growth was isolated from enrichment cultures containing 100 mg/1 PCP in a mineral salts medium inoculated with contaminated soil from a lumber treatment waste site. The isolate, designated strain SR3, was identified as a species of Pseudomonas by virtue of its physiological and biochemical characteristics. Mineralization of PCP by Pseudomonas sp. strain SR3 was demonstrated by loss of detectable PCP from growth medium, stoichiometry of chloride release (5 equivalents of chloride per mole of PCP), and formation of biomass consistent with the concentration of PCP mineralized. PCP-induced cells of strain SR3 showed elevated rates of oxygen consumption in the presence of PCP, and with different chlorinated phenols, with complete degradation of 2,3,5,6-, 2,3,6-, 2,4,6-, 2,4, and 2,6-chloro-substituted phenols. Concentration of PCP up to 175 mg/liter supported growth of this organism, but maximal rates of PCP removal were observed at a PCP concentration of100 mg/liter. Based on its degradative properties, Pseudomonas sp. strain SR3 appears to have utility in bioremediation of soil and water contaminated with PCP.

Jeffrey, Wade H., Stephen M. Cuskey, Peter J. Chapman, Sol Resnick and Ronald H. Olsen. 1992. Characterization of Pseudomonas putida Mutants Unable to Catabolize Benzoate: Cloning and Characterization of Pseudomonas Genes Involved in Benzoate Catabolism and Isolation of a Chromosomal DNA Fragment Able to Substitute for xylS in Activation of the TOL Lower-Pathway Promoter. EPA/600/J-92/381. J. Bacteriol. 174(15):4986-4996. (ERL,GB 766). (Avail. from NTIS, Springfield, VA: PB93-121135)

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represented by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression frm OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.

Eaton, Richard W. and Peter J. Chapman. 1992. Bacterial Metabolism of Naphthalene: Construction and Use of Recombinant Bacteria to Study the Ring Cleavage of 1,2-Dihydroxynaphthalene and Subsequent Reactions. EPA/600/J-93/059. J. Bacteriol. 174(23):7542-7554. (ERL,GB 795). (Avail. from NTIS, Springfield, VA: PB93-168938)

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated using recombinant bacteria carrying genes cloned from the NAH7 plasmid. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by the NAH7 plasmid. The gene encoding this enzyme, nahD, was cloned on a 1.95 kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibrium of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1 kb MluI-StuI restriction fragment. Mechanisms for both the isomerase- and hydratase/aldolase- catalyzed reactions are proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75 kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. By identifying the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.

Grifoll, Magdalena, Sergey A. Selifonov and Peter J. Chapman. 1995. Transformation of Substituted Fluorenes and Fluorene Analogs by Pseudomonas sp. Strain F274. EPA/600/J-02/250. Appl. Environ. Microbiol. 61(9):3490-3493. (ERL,GB 908).

Pseudomonas sp. F274, previously shown to catabolize fluorene via fluorenone and its angular dioxygenation, 2',3'-dihydroxy-2-carboxybiphenyl, phthalate and protocatechuate, was examined for its abililty to transform substituted fluorenes and S- and N-heterocyclic analogues. Halogen- and methyl-substituted fluorenes were metabolized to the correspondingly substituted phthalates via attack on the unsubstituted ring. In the case of 1-methylfluorene, initial oxidation of the methyl group to carboxyl prevented all other transformations but 9-monooxygenation. The strain also oxidized S-heteroatoms and benzylic methylenic groups of fluorene analogues. No angular dioxygenation of S- and N-heterocycles was observed.

Grifoll, Magdalena, Sergey A. Selifonov, Charylene V. Gatlin and Peter J. Chapman. 1995. Actions of a Versatile Fluorene-Degrading Bacterial Isolate on Polycyclic Aromatic Compounds. Appl. Environ. Microbiol. 61(10):3711-3723. (ERL,GB 909).

Pseudomonas cepacia F297 showed the ability to grow with fluorene as a sole source of carbon and energy; its growth yield corresponded to assimulation of about 40% of fluorene carbon. Accumulation of a ring meta-cleavage product during growth and identification of 1-indanone in growth media and washed cell suspensions suggest that strain F297 metabolizes fluorene by mechanisms analogous to those of naphthalene degradation. In addition to fluorene, strain F297 utilized for growth a wide variety of polycyclic aromatic compounds (PACs), including naphthalene, 2,3-dimethylnaphthalene, phenanthrene, anthracene, and dibenzothiophene. Fluorene-induced cells of the strain also transformed 2,6-dimethylnaphthalene, biphenyl, dibenzofuran, acenaphthene and acenaphthylene. Identification of products formed from those substrates (GC-MS) in washed cell suspensions, indicates that Pseudomonas cepacia F297 carries out the following reactions: (i) aromatic ring oxidation and cleavage, apparently using the pyruvate released for growth, (ii) methyl group oxidations, (iii) methylenic oxidations, and (iv) S-oxidations of aromatic sulfur heterocycles. Strain F297 grew with a creosote-PAC mixture, producing almost complete removal of all aromatic compounds containing 2-3 rings in 14 days, as demonstrated by GC analysis of the remaining PACs recovered from cultures. Identification of key chemicals confirmed that not only are certain compounds depleted, but also the anticipated reaction products are found.

Eaton, Richard W. and Peter J. Chapman. 1995. Formation of Indigo and Related Compounds from Indolecarboxylic Acids by Aromatic Acid-Degrading Bacteria: Chromogenic Reactions for Cloning Genes Encoding Dioxygenases That Act on Aromatic Acids. EPA/600/J-96/165. J. Bacteriol. 177(23):6983-6988. (ERL,GB 920).

The p-cumate-degrading strain Pseudomonas putida F1 and the m- and p -toluate-degrading strain P. putida mt-2 transform indole-2-carboxylate and indole-3-carboxylate to colored products identified here as indigo, indirubin, and isatin. A mechanism by which these products could be formed spontaneously following dioxy-genase-catalyzed dihydroxylation of the indolecarboxylates is proposed. Indolecarboxylates were employed as chromogenic substrates for identifying recombinant bacteria carrying genes encoding p-cumate dioxygenase and toluate dioxygenase. Dioxygenase gene carrying bacteria could be readily distinguished as dark green-blue colonies among other colorless recombinant Escherichia coli colonies on selective agar plates containing either indole-2-carboxylate or indole-3-carboxylate.

Selifonov, Sergey A., Magdalena Grifoll, Richard W. Eaton and Peter J. Chapman. 1996. Oxidation of Naphthenoaromatic and Methyl-Substituted Aromatic Compounds by Naphthalene 1,2-Dioxygenase. EPA/600/J-96/161. Appl. Environ. Microbiol. 62(2):507-514. (ERL,GB 930).

Oxidation of acenaphthene, acenaphthylene and fluorene was examined with recombinant strain Pseudomonas aeruginosa PA01 (pRE695), expressing naphthalene dioxygenase genes cloned from plasmid NAH7. Acenaphthene underwent monooxygenation to 1-acenaphthenol with subsequent conversion to 1-acenaphthenone, cis- and trans- acenaphthene-1,2-diols, while acenaphthylene was dioxygenated to give cis-acenaphthene-1,2-diol. Non-specific dehydrogenase activities present in the host strain led to the conversion of both acenaphthene-1,2-diols to 1,2-acenaphthoquinone. The latter was spontaneously oxidized to naphthalene-1,8-dicarboxylic acid. No aromatic ring dioxygenation products were detected from acenaphthene and acenaphthylene. Mixed monooxygenase and dioxygenase actions of naphthalene dioxygenase on fluorene yielded products of benzylic 9-monooxygenation, aromatic ring dioxygenation, or both. Action of naphthalene dioxygenase on a variety of methyl-substituted aromatic compounds, including 1,2,4-trimethylbenzene and isomers of dimethylnaphthalene, resulted in formation of benzylic alcohols, methyl group monooxygenation products, which were subsequently converted to the corresponding carboxylic acids by dehydrogenase(s) in the host strain. Benzylic monooxygenation of methyl groups was strongly predominant over aromatic ring dioxygenation and essentially non-specific with respect to the substitution pattern of the aromatic substrates. In addition to monooxygenating benzylic methyl and methylene groups, naphthalene dioxygenase behaved as a sulfoxygenase, catalyzing monooxygenation of the sulfur heteroatom of 3-methylbenzothiophene.

Mueller, James G., Suzanne E. Lantz, Beat O. Blattmann, Douglas P. Middaugh and Peter J. Chapman. 1990. Alternative Biological Treatment Processes for Remediation of Creosote-Contaminated Materials: Bench-Scale Treatability Studies. EPA/600/9-90/049. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 74 p. (Avail. from NTIS, Springfield, VA: PB91-179085)

Bench-scale biotreatability studies were performed to determine the most effective of two bioremediation application strategies to ameliorate creosote- and pentachlorophenol (PCP)-contaminated soils present at the American Creosote Works Superfund site, Pensacola, Florida: solid-phase bioremediation or slurry-phase bioremediation. When indigenous microorganisms were employed as biocatalysts, solid-phase bioremediation was slow and ineffective (8-12 weeks required to biodegrade greater than 50% of resident organics). Biodegradation was limited to lower-molecular-weight constituents rather than the more hazardous, higher-molecular-weight (HMW) compounds); PCP and HWM polycyclic aromatic hydrocarbons (PAHs) containing 4 or more fused rings resisted biological attach. Moreover, supplementation with aqueous solution of inorganic nutrients had little effect on the overall effectiveness of the treatment strategy. Alternatively, slurry-phase bioremediation was much more effective: greater that 50% of targeted organics were biodegraded in 14 days. Again, however, more persistent contaminants, such as PCP and HMW PAHs, were not extensively degraded when subjected to the action of indigenous microorganisms.

Shields, Malcolm S., Stacy O. Montgomery, Peter J. Chapman, Stephen M. Cuskey and Parmely H. Pritchard. 1990. Involvement of a Toluene Degradative Pathway in the Biodegradation of Trichloroethylene by Pseudomonas cepacia Strain G4. In: Bioremediation of Hazardous Wastes. EPA/600/9-90/041. U.S. Environmental Protection Agency, Office of Research and Development, Biosystems Technology Development Program, Washington, DC. Pp. 49-52. (ERL,GB X721).

The oxidation of trichloroethylene (TCE) by bacteria possessing enzymes that act on toluene is now established for three species: P. cepacia, P. putida, and P. mendocina. In all cases an oxygenase acting directly on the aromatic ring of toluene is responsible for an attack on the alternate substrate TCE. P. cepacia, strain G4, was shown to catabolize toluene by a novel pathway. HPLC and GC/MC analysis indicated toluene hydroxylations at first the ortho and then meta positions to sequentially form o-cresol and 3-methylcatechol. Analysis of pathway intermediates (by GC/MS) formed in a defined atmosphere of 18O2 and 16O2 confirmed the sequential nature of this monooxygenation activity and the source of the oxygen as O2. The generation of several mutants unable to metabolize a variety of related aromatic compounds--toluene, phenol, cresol, catechol and hydroxymuconic semialdehyde (HMS)--revealed that the only mutants that also suffered loss of TCE-metabolizing ability were those that coincidentally lost the ability to hydroxylate toluene, phenol, o- or m-cresol. The principle of toluene ring oxygenation has led to the identification of strains of Nocardia, Alcaligenes, and Acinetobacter as capable of TCE degradation.

Eaton, Richard W., Peter J. Chapman and P. Hap Pritchard. 1990. Degradation of Heterocyclics. In: Bioremediation of Hazardous Wastes. EPA/600/9-90/041. U.S. Environmental Protection Agency, Office of Research and Development, Biosystems Technology Development Program, Washington, DC. Pp. 46-48. (ERL,GB X722).

Many contaminants of ground water are heterocyclic compounds that are potentially hazardous to human health and to the environment. These classes of compounds along with many others are formed as combustion products in the generation of synthetic fuels from fossil fuels. Groundwater contamination by these chemicals has been shown in the vicinity of coal gasification plants, oil shale-retorting facilities, and at wood treatment facilities where coal-tar derived creosote has been used. To develop biological approaches to treat sites contaminated with these and other chemicals, the microbiology and biochemistry of their biodegradation must be understood. This research is designed to provide such insights.

Mueller, James G., Peter J. Chapman, Parmely H. Pritchard, Ron Thomas, Ellis L. Kline and Suzanne E. Lantz. 1990. Development of a Sequential Treatment System for Creosote-Contaminated Soil and Water: Bench Studies. In: Bioremediation of Hazardous Wastes. EPA/600/9-90/041. U.S. Environmental Protection Agency, Office of Research and Development, Biosystems Technology Development Program, Washington, DC. Pp. 42-43. (ERL,GB X723).

A triphasic, sequential treatment system for the potential remediation of environments contaminated by mixtures of hazardous wastes has been co-developed by Southern BioProducts (SBP), Incorporated, Atlanta, GA and the Gulf Breeze Environmental Research Laboratory, Gulf Breeze, FL. This approach integrates established soil-washing technology (phase I) with dewatering and chemical fractionation through a patented filtration process (phase II). Together, these steps reduce the volume of material to be subsequently treated from 100 to 100,000 fold. Volume reduction of this magnitude facilitates phase III of this system: biodegradation of susceptible pollutants employing EPA/SBP-patented bacteria capable of utilizing high-molecular-weight creosote constituents as growth substrates. The efficacy of this treatment system on creosote-contaminated soil and water is currently being evaluated at the bench-scale level.

Mueller, James G., Peter J. Chapman and Parmely H. Pritchard. 1990. Aerobic Biodegradation of PAHs. In: Bioremediation of Hazardous Wastes. EPA/600/9-90/041. U.S. Environmental Protection Agency, Office of Research and Development, Biosystems Technology Development Program, Washington, DC. Pp. 23-24. (ERL,GB X725).

Biological degradation represents the major route through which polycyclic aromatic hydrocarbons (PAHs) and other organic chemicals are removed from contaminated environments. In most cases, lower molecular weight PAHs containing two or three rings are readily degraded biologically. Conversely, higher-molecular-weight PAHs are considered resistant to biological action and tend to persist in contaminated environments. Unfortunately, higher molecular weight PAHs represent the greatest risk to public and environmental health. For bioremediation to be considered as an acceptable remedial action alternative for PAH-contaminated sites (i.e., creosote waste sites, coal gasification sites, petroleum refineries), biotreatment processes must be capable of destroying these chemicals. The isolation of microbial systems capable of utilizing high-molecular-weight PAHs (e.g., fluoranthene) as sole sources of carbon and energy for growth partially addresses this challenge.

Folsom, Brian R., Peter J. Chapman and Parmely H. Pritchard. 1990. Performance of a Recirculating Bioreactor for the Degradation of TCE. In: Bioremediation of Hazardous Wastes. EPA/600/9-90/041. U.S. Environmental Protection Agency, Office of Research and Development, Biosystems Technology Development Program, Washington, DC. Pp. 6-8. (ERL,GB X726).

Of the volatile organic chemicals found as common ground-water contaminants, trichloroethylene (TCE) has received significant attention; and as a result, a number of bacterial systems with the ability to degrade TCE by cometabolism are now recognized. In these systems, TCE is degraded by bacterial enzymes that are typically expressed following induction with other chemicals. Previously, one of these TCE-degrading organisms was isolated by investigators at the Gulf Breeze Environmental Research Laboratory. This organism has subsequently been indentified as Pseudomonas cepacia strain G4 and requires either toluene, o-cresol, m-cresol, or phenol for induction of TCE degradative enzymes. A novel toluene degradative pathway involving sequential hydroxylation of toluene at ortho and meta positions to form 3-methylcatechol has been characterized. TCE is completely degraded to CO2, Cl, and unidentified, nonvolatile products by this organism.

Morris, Pamela J., Michael E. Shelton and Peter J. Chapman. 1995. Co-Contaminated Sites: Biodegradation of Fossil Fuels in the Presence of PCBs. In: Bioremediation of Recalcitrant Organics. Robert E. Hinchee, Daniel B. Anderson and Ronald E. Hoeppel, Editors. Battelle Press, Columbus, OH. Pp. 123-130. (ERL,GB X823).

Polychlorinated biphenyl (PCB)-contaminated sites are often co-contaminated with fossil fuels making biodegration more difficult. Our current studies examine biodegradation of the fossil fuel components of two PCB-contaminated sites: (1) a former racing Drag Strip soil contaminated with Aroclor 1242 and (2) a sediment from Silver Lake contaminated with Aroclor 1260. The sandy surface soil at the Drag Strip site contains 1.9% organic carbon and 1.5% fossil fuel component. Analysis of the solvent-extractable organic fraction, by alumina column chromatography, shows the distribution of organics to be 91.2% hydrocarbons, 7.8% polars, and 1.1% asphaltenes. This oil is extremely weathered and contains few readily biodegradable components. Enrichments have yielded undefined mixed cultures of bacteria capable of extensive degradation of components of both the Drag Strip and Silver Lake site materials. One culture, enriched from a creosote-contaminated soil adjacent to a utility pole, transformed approximately 28% and 37% (by weight) of the Drag Strip and Silver Lake oils, respectively. While the presence of fossil fuels has been shown to inhibit aerobic PCB degradation, our studies show that the presence of PCBs negatively impacts fossil fuel biodegradation. continuing studies will examine the nature of PCB inhibition of fossil fuel biodegradation.