Coping with polychlorinated biphenyl (PCB) toxicity: Physiological and genome-wide responses of Burkholderia xenovorans LB400 to PCB-mediated stress

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Temperature determines the pattern of anaerobic microbial dechlorination of Aroclor 1260 primed by 2,3,4,6-tetrachlorobiphenyl in Woods Pond sediment.

Reductive dechlorination of the Aroclor 1260 residue in Woods Pond (Lenox, Mass.) sediment samples was investigated for a year at incubation temperatures from 4 to 66 degrees C. Sediment slurries were incubated anaerobically with and without 2,3,4,6-tetrachlorobiphenyl (2346-CB; 350 microM) as a primer for dechlorination of the Aroclor 1260 residue. Dechlorination of the Aroclor residue occurred only in live samples primed with 2346-CB and only at 8 to 34 degrees C and 50 to 60 degrees C. The extent and pattern of polychlorinated biphenyl (PCB) dechlorination were temperature dependent. At 8 to 34 degrees C, the dechlorination resulted in 28 to 65% decreases of the hexathrough nonachlorobiphenyls and corresponding increases in the tri- and tetrachlorobiphenyls. At 12 to 30 degrees C, 30 to 40% of the hexa- through nonachlorobiphenyls were dechlorinated in just 3 months. The optimal temperature for overall chlorine removal was 20 to 27 degrees C. We observed four different microbial dechlorination processes with different but partially overlapping temperature ranges, i.e., Process N (flanked meta dechlorination) at 8 to 30 degrees C, Process P (flanked para dechlorination) at 12 to 34 degrees C, Process LP (unflanked para dechlorination) at 18 to 30 degrees C, and Process T (a very restricted meta dechlorination of specific hepta- and octachlorobiphenyls) at 50 to 60 degrees C. These temperature ranges should aid in the development of strategies for the enrichment and isolation of the microorganisms responsible for each dechlorination process. The incubation temperature determined the relative dominance of the four PCB dechlorination processes and the extent and products of dechlorination. Hence, understanding the effects of temperature on PCB dechlorination at contaminated sites should assist in predicting the environmental fate of PCBs or planning bioremediation strategies at those sites.     

Coping with Polychlorinated Biphenyl (PCB) Toxicity: Physiological and Genome-Wide Responses of Burkholderia xenovorans LB400 to PCB-Mediated Stress

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.     

Enhancement of PCB degradation by Burkholderia xenovorans LB400 in biphasic systems by manipulating culture conditions

Two-phase partitioning bioreactors (TPPBs) can be used to biodegrade environmental contaminants after their extraction from soil. TPPBs are typically stirred tank bioreactors containing an aqueous phase hosting the degrading microorganism and an immiscible, non-toxic and non-bioavailable organic phase functioning as a reservoir for hydrophobic compounds. Biodegradation of these compounds in the aqueous phase results in thermodynamic disequilibrium and partitioning of additional compounds from the organic phase into the aqueous phase. This self-regulated process can allow the delivery of large amounts of hydrophobic substances to degrading microorganisms. This paper explores the reactor conditions under which the polychlorinated biphenyl (PCB) degrader Burkholderia xenovorans LB400 can degrade significant amounts of the PCB mixture Aroclor(R) 1242. Aroclor(R) degradation was found to stall after approximately 40 h if no carbon source other than PCBs was available in the reactor. Sodium pyruvate was found to be a suitable carbon source to maintain microbial activity against PCBs and to function as a substrate for additional cell growth. Both biphenyl (while required during the inoculum preparation) and glucose had a negative effect during the Aroclor(R) degradation phase. Initial Aroclor(R) 1242 degradation rates in the presence of pyruvate were high (6.2 mg L(-1) h(-1)) and 85% of an equivalent concentration of 100 mg Aroclor(R) 1242 per L aqueous phase could be degraded in 48 h, which suggest that solvent extraction of PCBs from soil followed by their biodegradation in TPPBs might be a feasible remediation option.     

Isolation and Characterization of a Mixed Culture That Degrades Polychlorinated Biphenyls

A mixed culture composed of two Pseudomonas strains, designated as KKL101 and KKS102, was isolated from soil. This mixed culture had an enhanced ability to degrade various polychlorinated biphenyls (PCBs) which include highly chlorinated components. They did not grow individually on the mineral salts medium supplemented with a highly chlorinated PCB (PCB48, a mixture of mainly tetrachlorobiphenyl) and biphenyl. When the spent medium of KKL101 was added to the washed cell preparation of KKS102, however, the latter grew on these carbon sources, producing yellow compounds which were identified as metabolic intermediates of the carbon sources, biphenyl and PCBs. These results suggest that KKL101 produces a growth factor(s) essential for KKS102 to grow on PCBs and that the growth of KKL101 is supported by the metabolic intermediates produced by KKS102. It appears that these two bacterial strains have a symbiotic relationship. From the analysis of the degradation products of various PCB congeners, it was found that strain KKS102 degrades a wide range of PCBs which have been considered to be refractory to biological degradation.     

Reductive dechlorination of PCB-contaminated raisin river sediments by anaerobic microbial granules

A polychlorinated biphenyl (PCB)-dechlorinating anaerobic microbial consortium, developed in a granular form, demonstrated extensive dechlorination of PCBs present in Raisin River sediments at room (20 degrees to 22 degrees C) and at a relatively low (12 degrees C) temperature. Highly chlorinated PCB congeners were dechlorinated and less chlorinated compounds were produced. The homolog comparison showed that tri-, tetra-, penta-, hexa-, and heptachlorobiphenyl compounds decreased significantly, and mono- and dichlorobiphenyl compounds increased. After 32 weeks of incubation at 12 degrees C, the predominant less chlorinated products included 2-, 4-, 2-2/26-, 24-, 2-4-, 24-2-, 26-2-, and 26-4-CB. Among these, 24- and 24-2-CB did not accumulate at room temperature, suggesting a further dechlorination of these congeners. Predominantly meta dechlorination (i.e., pattern M) was catalyzed by the microbial consortium in the granules. Dechlorination in the control studies without granules was not extensive. This study is the first demonstration of enhanced reductive dechlorination of sediment PCBs by an exogenous anaerobic microbial consortium. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 182-190, 1997.     

Degradation of anaerobic reductive dechlorinationproducts of Aroclor 1242 by four aerobic bacteria

We studied the aerobic degradation of eight PCB congeners which comprise from 70 to 85% of the anaerobic dechlorination products from Aroclor 1242, including2-, 4-, 2,4-, 2,6-, 2,2'-, 2,4'-, 2,2',4-, and2,4,4'-chlorobiphenyl (CB), and the biodegradation of their mixtures designed to simulate anaerobic dechlorination profiles M and C. StrainsComamonas testosteroni VP44 and Rhodococcus erythreus NY05 preferentially oxidizeda para-substituted ring, while Rhodococcus sp. RHA1, similar to well known strain Burkholderia sp. LB400, preferably attackedan ortho-chlorinated ring. Strains with ortho-directed attack extensively degraded2,4'- and 2,4,4'-CB into 4-chlorobenzoate, while bacteria with para-directed attack transformed these congeners mostly into potentially problematicmeta-cleavage products. The strains that preferentiallyoxidized an ortho-substituted ring readily degradedseven of the eight congeners supplied individually; only 2,6-CB was poorly degraded. Degradationof 2,2'- and 2,4,4'-CB was reduced when present in mixtures M and C. Higher efficiencies of degradation of the individual congeners and defined PCB mixtures M and C and greater production of chlorobenzoates were observed with bacteria that preferentially attackan ortho-substituted ring. PCB congeners 2,4'-, 2,2',4-, and 2,4,4'-CB canbe used to easily identify bacteria with ortho-directed attack whichare advantageous for use in the aerobic stage of the two-phase (anaerobic/aerobic)PCB bioremediation scheme.     

Degradation of Polychlorinated Biphenyls by UV-Catalyzed Photolysis

Photolysis of five polychlorinated biphenyl (PCB) congeners [2,4,4′-trichlorobiphenyl (PCB 28), 2,2′,5,′5-tetrachlorobiphenyl (PCB 52), 2,2′,4,5,5′-pentachlorobiphenyl (PCB 101), 2,2′,4,4′,5,′5-hexachlorobiphenyl (PCB 153) and 2,2′,3,4,4′,5,′5-heptachlorobiphenyl (PCB 180)] individually and in combination were carried out in the solvents methanol, ethanol, and 2-propanol. The disappearance of parent congener generally increased with UV intensity. The solvents had significant or limited effect on the removal of PCBs depending on the congener used. Because 2-propanol was highly toxic and methoxylated products were formed when methanol was used, ethanol was selected as the optimum solvent. The results of photolysis of the PCB mixture showed that PCB 52 was formed and accumulated after 4 h of photolysis. The addition of sodium hydroxide increased the rate of photolysis of the PCB mixture. One hundred percent removal can be obtained of the PCB in mixture in 90 min under optimized conditions. Gas chromatography–mass spectrometry was used to determine the intermediates of the photolysis of PCBs under optimized conditions. For the PCB congeners and mixture studied, the major photolytic intermediates were less chlorinated congeners, and biphenyl was the major product with minor amounts of hydroxylated PCBs, ethylated, dimethylated, and methylated biphenyls. Biphenyl could be further degraded by a prolonged photolysis. Toxicity of the PCB mixture during photolysis was monitored by the Microtox^® test. It was found that the toxicity increased at the early stage of photolysis, and gradually decreased as the reaction proceeded. After 90 min, the EC₅₀ of the reaction mixture was similar to that of the untreated sample.     

Transformation of polychlorinated biphenyls by a novel BphA variant through the meta-cleavage pathway

The transformation of 20 polychlorinated biphenyls (PCBs) through the meta-cleavage pathway by recombinant Escherichia coli cells expressing the bphEFGBC locus from Burkholderia cepacia LB400 and the bphA genes from different sources was compared. The analysis of PCB congeners for which hydroxylation was observed but no formation of the corresponding yellow meta-cleavage product demonstrated that only lightly chlorinated congeners including one tetrachlorobiphenyl (2,2',4,4'-CB) were transformed into their corresponding yellow meta-cleavage products. Although many other tetrachlorobiphenyls (2, 2',5,5'-CB, 2,2',3,5'-CB, 2,4,4',5-CB, 2,3',4',5-CB, 2,3',4,4'-CB) and one pentachlorobiphenyl (2,2',4,5,5'-CB) tested were depleted from resting cell suspensions, no yellow meta-cleavage products were observed. For most of these congeners, dihydrodiol compounds accumulated as the endproducts, indicating that the bphB-encoded biphenyl-2,3-dihydrodiol-2,3-dehydrogenase is a key limiting step for further degradation of highly chlorinated congeners. These results suggest that engineering the biphenyl dioxygenase alone is insufficient for an improved removal of PCB. Rather, improved degradation of PCBs is more likely to be achieved with recombinant strains containing metabolic pathways not only specifically engineered for expanding the initial dioxygenation but also for the mineralization of PCBs.     

Cometabolic Degradation of Polychlorinated Biphenyls at Low Temperature by Psychrotolerant Bacterium Hydrogenophaga sp. IA3-A

A biphenyl-utilizing bacterium isolated from polychlorinated biphenyls (PCBs)-contaminated soils grew on tryptic soy at temperatures between 4 and 40°C. The Gram-negative rod bacterium formed yellow colonies on nutrient agar and it denitrified nitrate to nitrogen. Analysis of cellular fatty acids showed that it was most closely related to Hydrogenophaga taeniospiralis. At 5°C, biphenyl-grown cells cometabolically degraded di- and trichlorinated isomers of PCBs in 10 ppm of Aroclor 1248. At 30°C, PCBs that were removed included a congener with four chlorine substituents. At 5°C, cells transformed 2,4′-dichlorobiphenyl (2,4′-DCB) and accumulated ortho-chlorinated meta-cleavage product as a stable metabolite. Analysis of extracts of culture supernatant by gas chromatography–mass spectrometry indicated that products of transformation of 2,4′-DCB included 2- and 4-chlorobenzoic acid (2- and 4-CBA), suggesting that (chloro)biphenyl-degrading upper-pathway enzymes of the bacterium are active at low temperature. The bacterium Hydrogenophaga sp. IA3-A is a PCB-degrading psychrotolerant strain.     

Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms.

We examined the degradation of biphenyl and the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221 by indigenous Arctic soil microorganisms to assess both the response of the soil microflora to PCB pollution and the potential of the microflora for bioremediation. In soil slurries, Arctic soil microflora and temperate-soil microflora had similar potentials to mineralize [14C]biphenyl. Mineralization began sooner and was more extensive in slurries of PCB-contaminated Arctic soils than in slurries of uncontaminated Arctic soils. The maximum mineralization rates at 30 and 7 degrees C were typically 1.2 to 1.4 and 0.52 to 1.0 mg of biphenyl g of dry soil-1 day-1, respectively. Slurries of PCB-contaminated Arctic soils degraded Aroclor 1221 more extensively at 30 degrees C (71 to 76% removal) than at 7 degrees C (14 to 40% removal). We isolated from Arctic soils organisms that were capable of psychrotolerant (growing at 7 to 30 degrees C) or psychrophilic (growing at 7 to 15 degrees C) growth on biphenyl. Two psychrotolerant isolates extensively degraded Aroclor 1221 at 7 degrees C (54 to 60% removal). The soil microflora and psychrotolerant isolates degraded all mono-, most di-, and some trichlorobiphenyl congeners. The results suggest that PCB pollution selected for biphenyl-mineralizing microorganisms in Arctic soils. While low temperatures severely limited Aroclor 1221 removal in slurries of Arctic soils, results with pure cultures suggest that more effective PCB biodegradation is possible under appropriate conditions.     

A new aerobic gram-positive bacterium with a unique ability to degrade ortho- and para-chlorinated biphenyls

Strain B51 capable of degrading polychlorinated biphenyls (PCB) was isolated from soil contaminated with wastes from the chemical industry. Based on its morphological and chemotaxonomic characteristics, the strain was identified as a Microbacterium sp. Experiments with washed cells showed that strain B51 is able to degrade ortho- and para-substituted mono-, di-, and trichlorinated biphenyls (MCB, DCB, and TCB, respectively). Unlike the known PCB degraders, Microbacterium sp. B51 is able to oxidize the ortho-chlorinated ring of 2,2'-DCB and 2,4'-DCB and the para-chlorinated ring of 4.4'-DCB. The degradation of 2,4'-DCB and 4,4'-DCB was associated with the accumulation of 4-chlorobenzoic acid (4-CBA) in the medium in amounts comprising 80-90% of the theoretical yield. The strain was able to utilize 2-MCB, 2,2'-DCB, and their intermediate 2-CBA and to oxidize the mono(ortho)-chlorinated ring of 2,4,2'-TCB and the di(ortho-para)-chlorinated ring of 2,4,4'-TCB. A mixed culture of Microbacterium sp. B51 and the 4-CBA-degrading bacterium Arthrobacter sp. H15 was found to grow well on 1 g/l 2,4'-DCB as the sole source of carbon and energy.     

In vitro effects of polychlorinated biphenyls (PCBs) on the contractility of bovine myometrium from the periovulatory stage of the estrous cycle

Strips of longitudinal myometrium from cows were obtained on days 19-21 and 1-5 of the estrous cycle and incubated (aerated atmosphere; 4 degrees C; 24, 48 or 72 h) with a mixture of PCBs Aroclor (Ar) 1248 or with one of three PCBs (77, 126 or 153), all at doses of 10 or 100 ng/ml. The force and frequency of spontaneous and oxytocin (OT; 10(-7)M)-stimulated contractions of each strip was registered by means of HSE Schuler Organbath. Contractions of myometrial strips in the presence and absence of PCBs were observed after 24, 48 and 72 h of incubation. All PCBs significantly affected myometrial contractions. A mixture of PCBs increased the spontaneous force of contractions after 24 h but decreased after 48 h. Individual congeners of PCB also amplified the force of contractions and in most cases this effect was dose-dependent. Response of myometrium to PCB-126 and PCB-153 or PCB-77 appeared after 24 h or 48 h of incubation. Incubation of myometrial strips with PCB congeners markedly amplified OT-stimulated contractions. This effect was less evident when tissue was pre-treated with a higher dose of PCBs. Pre-treatment with estrogen-like PCB-153 increased the spontaneous and OT-evoked frequency of myometrial contractions from days 19-21. The spontaneous force of myometrial strips' contractions as well as the effects evoked by PCBs and OT was higher before than after ovulation. In summary, PCBs affected both the force and frequency of uterine contractions. Thus, it can be concluded that PCBs may impair both ovum fertilization and blastocyst implantation in cows.     

Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.     

Effects of Polychlorinated Biphenyls on Macromolecular Synthesis by a Heterotrophic Marine Bacterium

Growth rates and final cell yields of a polychlorinated biphenyl (PCB)-sensitive pseudomonad isolated from the open ocean were reduced in a dose-dependent manner by 10 to 100 μg of Aroclor 1254 per liter, a commercial mixture of PCB isomers added to its culture medium. Effects on growth rates were detected within 1 h (approximately one doubling time) of treatment. By 4 h posttreatment, the amounts of deoxyribonucleic acid and ribonucleic acid per cell in exponentially growing populations treated with sublethal doses of Aroclor were detectably lower than in appropriate controls. Corresponding cell protein values were slightly higher than in controls. Selective degradation of cell proteins or nucleic acids was not detected in cells whose growth was totally suppressed for 4 h by PCBs. Cells whose growth rate was inhibited 20 to 50% by Aroclor synthesized protein at normal rates for periods in excess of 5 h from the time the chlorinated hydrocarbons were added. In contrast, rates per cell of adenine uptake and adenine incorporation into deoxyribonucleic acid and total nucleic acids by the cells treated with PCBs were significantly lower than in control cells. Intracellular adenine pools of cells whose growth was inhibited to 20% of the control rate by PCBs were 30% smaller and appeared to require a longer interval to equilibrate than those of untreated cells. This may indicate impaired transport and/or efflux of this nucleic acid precursor through the membrane of affected cells. Inhibition of nucleic acid synthesis in this sensitive bacterium by PCBs could explain the observed inhibitory effects of the chlorinated hydrocarbons on its growth.     

Degradation of nonionic surfactants and polychlorinated biphenyls by recombinant field application vectors

Degradation of polychlorinated biphenyls (PCBs) in the environment is limited by their aqueous solubility and the degradative competence of indigenous populations. Field application vectors (FAVs) have been developed in which surfactants are used to both increase the solubility of the PCBs and support the growth of surfactant-degrading strains engineered for PCB degradation. Surfactant and PCB degradation by two recombinant strains were investigated. Pseudomonas putida IPL5 utilizes both alkylethoxylate [polyoxyethylene 10 lauryl ether (POL)] and alkylphenolethoxylate [Igepal CO-720 (IGP)] surfactants as growth substrates, but only degrades the ethoxylate moiety. The resulting degradation products from the alkyl- and alkylphenolethoxylate surfactants were 2-(dodecyloxy)ethanol and nonylphenoldiethoxylates, respectively. Ralstonia eutropha B30P4 grows on alkylethoxylate surfactants without the appearance of solvent-extractable degradation products. It also degrades the 2-(dodecyloxy)ethanol produced by strain IPL5 from the alkylethoxylate surfactants. The extent of degradation of the alkylethoxylate surfactant (POL) was greater for strain IPL5 (90%) than for B30P4 (60%) as determined by the cobaltothiocyanate active substances method (CTAS). The recombinant strain B30P4::TnPCB grew on biphenyl. In contrast, the recombinant strain IPL5::TnPCB could not grow on biphenyl, and PCB degradation was inhibited in the presence of biphenyl. The most extensive surfactant and PCB degradation was achieved by the use of both recombinant strains together in the absence of biphenyl. PCB (Aroclor 1242) and surfactant (POL) concentrations were reduced from 25 ppm and 2000 ppm, respectively, to 6.5 ppm and 225 ppm, without the accumulation of surfactant degradation products. Given the inherent complexity of commercial surfactant preparations, the use of recombinant consortia to achieve extensive surfactant and PCB degradation appears to be an environmentally acceptable and effective PCB remediation option. Received 04 October 1996/ Accepted in revised form 04 August 1997     

Effect of novel biosurfactants on biodegradation of polychlorinated biphenyls by pure and mixed bacterial cultures.

A study was conducted to determine the potential positive effect of novel biosurfactants on the enhancement of Aroclor 1248 metabolization in both in vitro and in situ experiments. Among two lipopeptides tested the highest activity was found in experiments with a hydrolytically opened form of lichenysin A. Lichenysin A itself did not enhance the degradation activity of chosen microorganism-degraders and in most cases inhibited their PCB mineralization rates. Glucolipid surfactant from marine bacterium Alcanivorax borkumensis showed in several tests a strong enhancing effect on microbial metabolization of Aroclor 1248 congeners. Biosurfactants appeared to act very specifically, i.e. depending on strain and concentration used. Experiments set up with soil samples did not give a clear answer whether bioemulsifiers applied at low concentration could sufficiently increase the rates of biodegradation in situ. Only A. borkumiensis glucose lipid caused the most marked enhancement of Aroclor 1248 metabolization in soil microcosm. We suggest that taking into account the specificity of surface- and biological activities of various biosurfactants they may promote the mineralization of sorbed PCBs in polluted soils, when the optimized biosurfactant-degrader combination is used.     

Biodegradation of PCBs in two-phase partitioning bioreactors following solid extraction from soil

This article demonstrates the feasibility of a novel process concept for the remediation of PCB contaminated soil. The proposed process consists of PCB extraction from soil using solid polymer beads, followed by biodegradation of the extracted PCBs in a solid-liquid two-phase partitioning bioreactor (TPPB), where PCBs are delivered from the polymer beads to the degrading organisms. The commercially available thermoplastic polymer Hytrel was used to extract Aroclor 1242 from contaminated artificial soil in bench scale experiments. Initial PCB contamination levels of 100 and 1,000 mg kg(-1) could be reduced to 32% +/- 1 to 41% +/- 7 of the initial value after 48 h mixing in the presence of a mobilizing agent at polymer-to-soil ratios of 1% (w/w) and 10% (w/w). The decrease of detectable PCBs in the soil was consistent with an increase of PCBs in the polymer beads. It was further shown that Aroclor 1242 could be delivered to the PCB degrading organism Burkholderia xenovorans LB400 in a solid-liquid TPPB via Hytrel beads. A total of 70 mg Aroclor 1242 could be degraded in a 1 L solid-liquid TPPB within 80 h of operation.     

Microbial reductive dechlorination of PCBs

Reductive dechlorination is an advantageous process to microorganisms under anaerobic conditions because it is an electron sink, thereby allowing reoxidation of metabolic intermediates. In some organisms this has been demonstrated to support growth. Many chlorinated compounds have now been shown to be reductively dechlorinated under anaerobic conditions, including many of the congeners in commercial PCB mixtures. Anaerobic microbial communities in sediments dechlorinate Aroclor at rates of 3 µg Cl/g sediment × week. PCB dechlorination occurs at 12° C, a temperature relevant for remediation at temperate sites, and at concentrations of 100 to 1000 ppm. The positions dechlorinated are usually meta > para > ortho. The biphenyl rings, and the mono-ortho- and diorthochlorobiphenyls were not degraded after a one year incubation. Hence subsequent aerobic treatment may be necessary to meet regulatory standards. Reductive dechlorination of Arochlors does reduce their dioxin-like toxicity as measured by bioassay and by analysis of the co-planar congeners. The most important limitation to using PCB dechlorination as a remediation technology is the slower than desired dechlorination rates and no means yet discovered to substantially enhance these rates. Long term enrichments using PCBs as the only electron acceptor resulted in an initial enhancement in dechlorination rate. This rate was sustained but did not increase in serial transfers. Bioremediation of soil contaminated with Aroclor 1254 from a transformer spill was dechlorinated by greater than 50% following mixing of the soil with dechlorinating organisms and river sediment. It is now reasonable to field test reductive dechlorination of PCBs in cases where the PCB concentration is in the range where regulatory standards may be directly achieved by dechlorination, where a subsequent aerobic treatment is feasible, where any co-contaminants do not pose an inhibitory problem, and where anaerobic conditions can be established.This paper was presented at the Pacific Basin Conference on Hazardous Waste, April, 1992, Bangkok, Thailand. Published by permission of the Pacific Basin Consortium for Hazardous Waste Research, East-West Center, Honolulu, HI