Evidence of aerobic utilization of di-ortho-substituted trichlorobiphenyls as growth substrates by Pseudomonas sp. SA-6 and Ralstonia sp. SA-4

Robust and effective bioremediation strategies have not yet been developed for polychlorinated biphenyl (PCB)-contaminated soils. This is in part a result of the fact that ortho - or ortho - and para -substituted congeners, frequent dead-end products of reductive dechlorination of PCB mixtures, have greatly reduced aerobic biodegradability. In this study, we report substantial evidence of utilization of diortho -substituted trichlorobiphenyls (triCBs) as growth substrates by Ralstonia sp. SA-4 and Pseudomonas sp. SA-6 in which ortho -substitution resulted in no obvious patterns of recalcitrance. These stains exhibited unusual preferences for growth on congeners chlorinated on both rings. Substrate uptake studies with benzoate-grown cells revealed that the isolates attacked the 2-chlorophenyl rings of 2,2',4- and 2,2',5-triCB. Between 71% and 93% of the initial 0.23–0.34 mM dose of congeners were transformed in less than 261 h concomitant with non-stoichiometric production of respective dichlorobenzoates and chloride ion. In enzyme assays, activity of 2,3-dihydroxybiphenyl-1,2-dioxygenase was constitutive. Additionally, these strains harboured no detectable plasmids which, coupled with exponential growth on the two triCB congeners, suggested chromosomal location of PCB degradative genes. In addition to the fact that there is a paucity of information on degradation of PCBs by tropical isolates, growth on triCBs as a sole carbon and energy source has never been demonstrated for any natural or engineered microorganisms. Such isolates may help prevent accumulation of ortho -substituted congeners in natural systems and offer the hope for development of effective bioaugmentation or sequential anaerobic–aerobic bioremediation strategies.     

Analysis of changes in congener selectivity during PCB degradation by Burkholderia sp. strain TSN101 with increasing concentrations of PCB and characterization of the bphBCD genes and gene products

We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 μg Kaneclor 300/ml, a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls in medium containing 25 μg Kaneclor 300/ml. Using PCB concentrations of 50–150 μg of Kaneclor 300/ml, the congener selectivity pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 μg Kaneclor 300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2′- and 4′-positions. A 5-kb DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated 99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2,3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the cloned bph gene in biphenyl metabolism. Received: 18 February 1997 / Accepted: 19 August 1997     

Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.     

Growth on dichlorobiphenyls with chlorine substitution on each ring by bacteria isolated from contaminated African soils

Until recently, it was generally believed that the presence of more than one chlorine substituent prevented chlorinated biphenyls from serving as a sole source of carbon and energy for aerobic bacteria. In this study, we report the isolation of three aerobic strains, identified as Enterobacter sp. SA-2, Ralstonia sp. SA-4, and Pseudomonas sp. SA-6 from Nigerian polluted soils, that were able to grow on a wide range of dichlorobiphenyls (diCBs). In addition to growing on all monochlorobiphenyls (monoCBs), the strains were all able to utilize 2,2′-, 2,4′-, and 2,3-diCB as a sole source of carbon and energy. With the exception of strain SA-2, growth was also sustainable on 3,3′-, and 3,5-diCB. Washed benzoate-grown cells were typically able to degrade 68 to 100% of the diCB (100 ppm) within 188 h, concomitant with a cell number increase of up to three orders-of-magnitude and elimination of varying amounts of chloride. In many cases, stoichiometric production of a chlorobenzoate (CBA) as a product was observed. During growth on 2,2′-, and 2,4′-diCB, organisms exclusively attacked an o-chlorinated ring resulting in the production of 2-CBA and 4-CBA, respectively. A gradual decline in the concentration of the latter was observed, which suggested that the product was being degraded further. In the case of 2,3-diCB, the unsubstituted ring was preferentially metabolized. Initial diCB degradation rates were greatest for 2,4′-diCB (11.2 ± 0.91 to 30.3 ± 7.8 nmol/min per 10⁹ cells) and lowest for 2,2′-diCB (0.37 ± 0.12 to 2.7 ± 1.2 nmol/min per 10⁹ cells).     

Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes

The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4^′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction. Received: 15 April 1998 / Received revision: 12 June 1998 / Accepted: 19 June 1998     

Influence of chroline substitution pattern on the degradation of polychlorinated biphenyls by eight bacterial strains

We compared the metabolism of eight di- and trichlorobiphenyls by eight bacterial strains chosen to represent a broad range of degradative activity against polychlorinated biphenyls (PCBs). The PCB congeners used were 2,3-, 2,3′-, 2,4′-, 3,3′-, 2,3,3′-, 2,4,4′-, 2,5,3′-, and 3,4,2′-chlorobiphenyl. The bacterial strains used wereCorynebacterium sp. MB1,Alcaligenes strainsA. eutrophus H850 andA. faecalis Pi434, andPseudomonas strains LB400 and H1130,P. testosteroni H430 and H336, andP. cepacia H201. The results indicated that both the relative rates of primary degradation of PCBs and the choice of the ring attacked were dependent on the bacterial strain used. The bacterial strains exhibited considerable differences in their relative reactivity preferences for attack on mono- and dichlorophenyl groups and in the degree to which the attack was affected by the chlorine substitution pattern on the nonreacting ring. For MB1 the reactivity pattern was 3-≥4-≫2-chlorophenyl with no attack on 2,4- or 2,5-chlorophenyl groups. This strain was relatively insensitive to the chlorine substitution pattern on the nonreacting ring. Strains H1130, H430, H201, and Pi434 exhibited the same reactivity preferences as MB1, but for these strains (and for all others tested) the chlorination pattern on the nonreacting ring had a strong effect. For strain H336 the reactivity preference was 4-≥2->2,4-≥3-chlorophenyl, with no evidence of attack on 2,5-chlorophenyl rings. For strains H850 and LB400 the relative reactivity was 2->2,5->3-≫2,4->4-chlorophenyl. On this basis we propose that the eight bacterial strains represent four distinct classes of biphenyl/PCB-dioxygenase activity. The types of products formed were largely strain-independent and were determined primarily by the chlorine substitution pattern on the reacting ring. When the reacting ring was an unsubstituted phenyl or a 2-chlorophenyl group, the products were chlorobenzoic acids in high yields; for a 3-chlorophenyl ring, both chlorobenzoic acids and chloroacetophenones in moderate yields; and for a 4- or 2,4-chlorophenyl group, chlorobenzoic acids in low yields with an apparent accumulation ofmeta ring-fission product. Strains H850 and LB400 were able to degrade the 3-chlorobenzoic acid that they produced from the degradation of 2,3′-chlorobiphenyl. We conclude that despite differences among strains in the specificity of the initial dioxygenase, the specificities of the enzymes responsible for the subsequent degradation to chlorobenzoic acid and/or chloroacetophenone are quite similar for all strains.     

Isolation and characterization of a novel polychlorinated biphenyl-degrading bacterium, Paenibacillus sp. KBC101

The biphenyl-utilizing bacterial strain KBC101 has been newly isolated from soil. Biphenyl-grown cells of KBC101 efficiently degraded di- to nonachlorobiphenyls. The isolate was identified as Paenibacillus sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various biological and physiological characteristics. In the case of highly chlorinated biphenyl (polychlorinated biphenyl; PCB) congeners, the degradation activities of this strain were superior to those of the previously reported strong PCB degrader, Rhodococcus sp. RHA1. Recalcitrant coplanar PCBs, such as 3,4,3,4-CB, were also efficiently degraded by strain KBC101 cells. This is the first report of a representative of the genus Paenibacillus capable of degrading PCBs. In addition to growth on biphenyl, strain KBC101 could grow on dibenzofuran, xanthene, benzophenone, anthrone, phenanthrene, naphthalene, fluorene, fluoranthene, and chrysene as sole sources of carbon and energy. Paenibacillus sp. strain KBC101 presented heterogeneous degradation profiles toward various aromatic compounds.     

Influence of chlorine substituents on rates of oxidation of chlorinated biphenyls by the biphenyl dioxygenase of Burkholderia sp. strain LB400

Biphenyl dioxygenase from Burkholderia (Pseudomonas) sp. strain LB400 catalyzes the first reaction of a pathway for the degradation of biphenyl and a broad range of chlorinated biphenyls (CBs). The effect of chlorine substituents on catalysis was determined by measuring the specific activity of the enzyme with biphenyl and 18 congeners. The catalytic oxygenase component was purified and incubated with individual CBs in the presence of electron transport proteins and cofactors that were required for enzyme activity. The rate of depletion of biphenyl from the assay mixture and the rate of formation of cis-biphenyl 2,3-dihydrodiol, the oxidation product, were almost equal, indicating that the assay accurately measured enzyme-specific activity. Four classes of CBs were defined based on their oxidation rates. Class I contained 3-CB and 2,5-CB, which gave rates that were approximately twice that of biphenyl. Class II contained 2,5,3',4'-CB, 2,3,2',5'-CB, 2,3,4,5-CB, 2,3,2',3'-CB, 2,4, 5,2',5'-CB, 2,5,3'-CB, 2,5,4'-CB, 2-CB, and 3,4,5-CB, which gave rates that ranged from 97 to 35% of the biphenyl rate. Class III contained only 2,3,4,2',5'-CB, which gave a rate that was 4% of the biphenyl rate. Class IV contained 2,4,4'-CB, 2,4,2',4'-CB, 3,4,5, 2'-CB, 3,4,5,3'-CB, 3,5,3',5'-CB, and 3,4,5,2',5'-CB, which showed no detectable depletion. Rates were not significantly correlated with the aqueous solubilities of the CBs or the number of chlorine substituents on the rings. Oxidation products were detected for all class I, II, and III congeners and were identified as chlorinated cis-dihydrodiols for classes I and II. The specificity of biphenyl dioxygenase for the CBs examined in this study was determined by the relative positions of the chlorine substituents on the aromatic rings rather than the number of chlorine substituents on the rings.     

Rhodococcus sp. strain TM1 plays a synergistic role in the degradation of piperidine by Mycobacterium sp. strain THO100

Mycobacterium sp. strain THO100 and Rhodococcus sp. strain TM1 were isolated from a morpholine-containing enrichment culture of activated sewage sludge. Strain THO100, but not strain TM1, was able to degrade alicyclic amines such as morpholine, piperidine, and pyrrolidine. The mixed strains THO100 and TM1 showed a better growth on piperidine as the substrate than the pure strain THO100 because strain TM1 was able to reduce the level of glutaraldehyde (GA) produced during piperidine degradation. GA was toxic to strain THO100 (IC₅₀ = 28.3 μM) but less toxic to strain TM1 (IC₅₀ = 215 μM). Strain THO100 possessed constitutive semialdehyde dehydrogenases, namely Sad1 and Sad2, whose activities toward succinic semialdehyde (SSA) were strongly inhibited by GA. The two isozymes were identified as catalase–peroxidase (KatG = Sad1) and semialdehyde dehydrogenase (Sad2) based on mass spectrometric analyses of tryptic peptides and database searches of the partial DNA sequences of their genes. In contrast, strain TM1 containing another constitutive enzyme Gad1 could oxidize both SSA and GA. This study suggested that strain TM1 possessing Gad1 played a synergistic role in reducing the toxic and inhibitory effects of GA produced in the degradation of piperidine by strain THO100.     

Derivatization of bioactive carbazoles by the biphenyl-degrading bacterium <Emphasis Type="Italic">Ralstonia</Emphasis> sp. strain SBUG 290

Different 9H-carbazole derivatives have been investigated within the last decades due to their broad range of pharmacological applications. While the metabolism of 9H-carbazole has previously been reported, nothing was known about the bacterial transformation of 2,3,4,9-tetrahydro-1H-carbazole and 9-methyl-9H-carbazole. Thus, for the first time, the bacterial biotransformation of 2,3,4,9-tetrahydro-1H-carbazole and 9-methyl-9H-carbazole was analyzed using biphenyl-grown cells of Ralstonia sp. strain SBUG 290 expressing biphenyl 2,3-dioxygenase. This strain accumulated 3-hydroxy-1,2,3,5,6,7,8,9-octahydrocarbazol-4-one and 6′-iminobicyclohexylidene-2′,4′-dien-2-one as major products during the incubation with 2,3,4,9-tetrahydro-1H-carbazole. Carbazol-9-yl-methanol was verified as the primary oxidation product of 9-methyl-9H-carbazole. In addition, 9H-carbazol-1-ol, 9H-carbazol-3-ol, and 3-hydroxy-1,2,3,9-tetrahydrocarbazol-4-one where detected in lower concentrations during the transformation of carbazol-9-yl-methanol and 9-methyl-9H-carbazole. Products were identified by high-performance liquid chromatography, gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, as well as ¹H and ¹³C nuclear magnetic resonance analyses.     

Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.     

Biodegradation of seven polychlorinated biphenyls by a newly isolated aerobic bacterium (<Emphasis Type="Italic">Rhodococcus</Emphasis> sp. R04)

An aerobic bacterial strain, designated R04, belonging to the genus Rhodococcus has been isolated and characerized by 16S rDNA analysis. The capability of this strain to degrade seven different polychlorinated biphenyls (CBs), 500 ppm 3-CB, 3,4-CB, 4,4-CB, 2,4,6-CB, 2,4,5-CB, 2,3,4,5-CB and 3,4,3,4-CB in liquid medium, was evaluated. After 5 days of incubation, the concentration of chloride increased to 0.35 mM in cultures containing 3-CB and R04, whereas in cultures with 3,4-CB, 2,3,4,5-CB or 3,4,3,4-CB plus R04 the chloride content increased to 0.1 mM. However, non-stoichiometric amounts of chloride were produced in cultures with R04 and 4,4-CB, 2,4,6-CB and 2,4,5-CB. The spectrum of supernatants from R04 grown on seven PCBs had a UV-visible (UV-VIS) absorption at 200–500 nm, characteristic of biphenyl-derived cleavage products. Gas-chromatographic (GC) analysis showed that R04 was able to transform 100% of 3-CB and 3,4-CB after 1 day of incubation, and 95% of 4,4-CB, 2,4,6-CB, 2,4,5-CB, 2,3,4,5-CB and 3,4,3,4-CB after 5 days of incubation. The position of the chlorine substituents on the rings strongly influenced the degradation of polychlorinated biphenyls (PCBs) and their intermediate metabolites by Rhodococcus sp. R04. The degradation of PCBs was further evaluated by monitoring intermediate metabolites of PCBs.     

Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90

Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 degrading short ethoxy (EO) chain-nonylphenol (NP) [NPEO_av2.0 containing NP mono- ∼ tetraethoxylates (NP1EO ∼ NP4EO); average 2.0 EO units] were isolated by enrichment cultures. Both strains grew on NP but not on octyl- and nonylphenol polyethoxylates (NPEOs) (average 10 EO units). Growth and degradation of NPEO_av2.0 was increased with increased concentrations of yeast extract (0.02–0.5%) in a culture medium. Culture supernatants of both strains grown on NPEO_av2.0 were analyzed by high-performance liquid chromatography, showing degradation of NP4EO–NP1EO. The metabolites from nonylphenol diethoxylate (NP2EO) by resting cells of both strains were identified by gas chromatography–mass spectrometry as nonylphenoxyethoxyacetic acid, NP1EO, nonylphenoxyacetic acid (NP1EC), and NP, while those from NP1EO were identified as NP1EC and NP. Cell-free extracts from strain AS08 grown on NPEO_av2.0 dehydrogenated NPEOs, NPEO_av2.0, NP2EO, NP1EO, and PEG 400, but the extracts were inactive toward di- ∼ tetraethylene glycol. Aldehydes were formed in the reaction mixture of each substrate with cell-free extracts. From these results, the aerobic metabolic pathway for short EO chain-NP is proposed: A terminal alcohol group of the EO chain is oxidized to a carboxylic acid via an aldehyde, and then one EO unit is removed. This process is repeated until NP is produced.English edition: The paper was edited by a native speaker through KN international ()     

Production and characteristics of the exocellular polysaccharide of a mutantMycobacterium strain

Mutant strains ofMycobacterium sp. V-649 producing highly mucous colonies on a solid cultivation medium were prepared after treatment with N-methyl-N′-nitro-N-nitrosoguanidine and production of the exocellular polysaccharide was tested. The strains were cultivated in media with suitable sugar sources under submerged conditions. It was found thatMycobacterium sp. V649/15 produces a maximum of 15–19% polymer after a 5–6-d cultivation. Gas chromatography indicated that the exocellular polysaccharide produced by this strain is of glucan type.     

Degradation of Mono-chlorinated Dibenzo-p-Dioxins by Janibacter sp. Strain YA Isolated from River Sediment

Strain YA was newly isolated from an enrichment culture of river sediment and was identified as Janibacter sp. It was able to utilize dibenzofuran as the sole source of carbon and energy. Strain YA degraded > 90% of 1-chloro-dibenzo-p-dioxin (1-CDD) and > 80% of 2-chloro-dibenzo-p-dioxin in 18 hours with each initial concentration at 40 mg/L. A novel metabolite, 2-chloro-2′,6-dihydroxydiphenylether, was observed in 1-CDD degradation. From the metabolites detected by gas chromatography–mass spectrometry, strain YA was supposed to have at least two types of oxidation pathways in 1-CDD degradation.     

Influence of chlorobenzoic acids on the growth and degradation potentials of PCB-degrading microorganisms

The biodegradation of polychlorinated biphenyls (PCBs) by diverse bacteria including those utilized in this study is often incomplete, a concomitant accumulation of chlorobenzoic acids (CBAs) are released as dead-end products. The build-up of these metabolites in the growth medium may result in feed-back inhibition and impede PCB biotransformation. In this investigation using GC-ECD and HPLC analyses, we confirmed that CBAs inhibit growth and PCB biodegradation potentials of five tropical bacteria namely, Pseudomonas aeruginosa SA-1, Enterobacter sp. SA-2, Ralstonia sp. SA-3, Ralstonia sp. SA-5 and Pseudomonas sp. SA-6. Among the four CBAs (2-CBA, 3-CBA, 4-CBA acids and 2,3-diCBA), 3-CBA was the strongest inhibitor followed by 4-CBA. Furthermore, we found that 3-CBA heavily inhibited growth of SA-3 and SA-6 on monochlorobiphenyls by 82–90% while elimination rate was inhibited by 71–88%. In the case of 2,3-diCBA, inhibition was generally less than 60%. However, effects of both acids were stronger in SA-3 than SA-6. We also found that 3-CBA and 2,3-diCBA completely inhibited carbon-chloride cleavage of 2-CB and 3-CB since cultivation in the absence of the acids resulted in recovery of 23–50% chloride in the culture fluids of organisms. These findings may therefore, have practical and ecological significance and are useful for improving the efficiency and the stability of some biological treatment processes.     

Microbial Reductive Dechlorination of Weathered and Exogenous Co-planar Polychlorinated Biphenyls (PCBs) in an Anaerobic Sediment of Venice Lagoon

The occurrence of reductive dechlorination processes towards pre-existing PCBs and five exogenous coplanar PCBs were investigated in a contaminated sediment of Porto Marghera (Venice Lagoon, Italy) suspended, under strictly anaerobic conditions, in water collected from the same site. PCB dechlorination started after five months of incubation, when sulfate initially occurring in the microcosms was completely depleted and methanogenesis was in progress. It was ascribed to sulfate-reducing bacteria. Several pre-existing hexa-, penta- and tetra-chlorinated biphenyls were slowly bioconverted into tri- and di-, ortho-substituted PCBs from the 5th to the 16th month of experiment. Spiked coplanar PCBs, i.e., 3,3′,4,4′-tetrachlorobiphenyl, 3,3′,4,4′,5- and 2,3′,4,4′,5-pentachlorobiphenyls, 3,3′,4,4′,5,5′- and 2,3,3′,4,4′,5-hexachlorobiphenyls, were extensively transformed (by about 90%) into lower chlorinated congeners, such as 3,3′,5,5′-/2,3′,4,4′-tetrachlorobiphenyl, 3,3′,5-, 2,4,4′-, 2,3′,4- and 2,3′,5-trichlorobiphenyl, 3,4-/3,4′- and 3,3′-dichlorobiphenyl and 2-chlorobiphenyl. The reductive dechlorination of spiked PCBs did not influence significantly the biotransformation rate and extent of pre-existing PCBs.     

Molecular genetics and evolutionary relationship of PCB-degrading bacteria

Biphenyl-utilizing soil bacteria are ubiquitously distributed in the natural environment. They cometabolize a variety of polychlorinated biphenyl (PCB) congeners to chlorobenzoic acids through a 2,3-dioxygenase pathway, or alternatively through a 3,4-dioxygenase system. Thebph genes coding for the metabolism of biphenyl have been cloned from several pseudomonads. The biochemistry and molecular genetics of PCB degradation are reviewed and discussed from the viewpoint of an evolutionary relationship.Abbreviations BP biphenyl - bph BP/PCB-degradative gene - 23DHBP 2,3-dihydroxybiphenyl - HPDA 2-hydroxy-6-oxo-6-phenylhexa 2,4-dienoic acid - KF707 P. pseudoalcaligenes strain KF707 - LB400 Pseudomonas sp. strain LB400 - PCB polychlorinated biphenyls - Q1 P. paucimobilis strain Q1tod; toluene catabolic gene     

Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.     

Mineralization of PCBs by the genetically modified strain Cupriavidus necator JMS34 and its application for bioremediation of PCBs in soil

Polychlorobiphenyls (PCBs) are classified as “high-priority pollutants.” Diverse microorganisms are able to degrade PCBs. However, bacterial degradation of PCBs is generally incomplete, leading to the accumulation of chlorobenzoates (CBAs) as dead-end metabolites. To obtain a microorganism able to mineralize PCB congeners, the bph locus of Burkholderia xenovorans LB400, which encodes one of the most effective PCB degradation pathways, was incorporated into the genome of the CBA-degrading bacterium Cupriavidus necator JMP134-X3. The bph genes were transferred into strain JMP134-X3, using the mini-Tn5 transposon system and biparental mating. The genetically modified derivative, C. necator strain JMS34, had only one chromosomal insertion of bph locus, which was stable under nonselective conditions. This modified bacterium was able to grow on biphenyl, 3-CBA and 4-CBA, and degraded 3,5-CBA in the presence of m-toluate. The strain JMS34 mineralized 3-CB, 4-CB, 2,4′-CB, and 3,5-CB, without accumulation of CBAs. Bioaugmentation of PCB-polluted soils with C. necator strain JMS34 and with the native B. xenovorans LB400 was monitored. It is noteworthy that strain JMS34 degraded, in 1 week, 99% of 3-CB and 4-CB and approximately 80% of 2,4′-CB in nonsterile soil, as well as in sterile soil. Additionally, the bacterial count of strain JMS34 increased by almost two orders of magnitude in PCB-polluted nonsterile soil. In contrast, the presence of native microflora reduced the degradation of these PCBs by strain LB400 from 73% (sterile soil) to approximately 50% (nonsterile soil). This study contributes to the development of improved biocatalysts for remediation of PCB-contaminated environments.