Degradation and dechlorination of low-chlorinated biphenyls by a three-membered bacterial co-culture

A Pseudomonas sp. strain, designated CPE1, was found to be capable of completely mineralizing 4-chlorobiphenyl via 4-chlorobenzoate and of partially dechlorinating 3,4-dichlorobiphenyl in the presence of biphenyl. A three-membered bacterial consortium, designated ECO3, prepared by combining CPE1 with two chlorobenzoate (CBA)-degrading strains, was capable of extensively degrading and dechlorinating all the monochlorinated biphenyls and several dichlorinated biphenyls in the presence of bipheny. Both CPE1 and ECO3 were capable of co-metabolizing several low-chlorinated biphenyl congeners of Fenclor 42 in the presence of biphenyl; however, only in ECO3 cultures were high degradation rates and chloride release observed. The higher rate of degradation and mineralization of some polychlorinated biphenyls (PCBs) of Fenclor 42 due to the concerted action of ECO3 members both on PCBs and CBAs suggested that the removal of CBAs from the culture medium may favour PCB degradation, and, therefore, that CBAs may be ivollved in the regulation of the degradation process of several chlorinated biphenyl congeners.Correspoeence to: F. Fava     

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 M) and 3,4-CBA (50 M). Although 2,5-CBA and 2,6-CBA were not utilised as carbon sources by strain JHR22, they did not inhibit 2-CBA utilisation at the concentrations studied, whereas 2,4-CBA was co-metabolised with 2-CBA. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.Abbreviations PCB Polychlorinated biphenyl - CBA Chlorobenzoate     

Pathways for 3-chloro- and 4-chlorobenzoate degradationin Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l^-1) and 4-chlorobenzoate (4-CBA 12 g l^-1) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoatewas readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.     

Influence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids

The effect of yeast extract and its less complex substituents on the rate of aerobic dechlorination of 2-chlorobenzoic acid (2-ClBzOH) and 2,5-dichlorobenzoic acid (2,5-Cl₂BzOH) by Pseudomonas sp. CPE2 strain, and of 3-chlorobenzoic acid (3-ClBzOH), 4-chlorobenzoic acid (4-ClBzOH) and 3,4-dichlorobenzoic acid (3,4-Cl₂BzOH) by Alcaligenes sp. CPE3 strain were investigated. Yeast extract at 50 mg/l increased the average dechlorination rate of 200 mg/l of 4-ClBzOH, 2,5-Cl₂BzOH, 3,4-Cl₂BzOH, 3-ClBzOH and 2-ClBzOH by about 75%, 70%, 55%, 7%, and 1%, respectively. However, in the presence of yeast extract the specific dechlorination activity of CPE2 and CPE3 cells (per unit biomass) was always lower than without yeast extract, although it increased significantly during the exponential growth phase. When a mixed vitamin solution or a mixed trace element solution was used instead of yeast extract the rate of 4-ClBzOH dechlorination increased by 30%–35%, whereas the rate of 2,5-Cl₂BzOH and 3,4-Cl₂BzOH dechlorination increased by only 2%–10%. The presence of vitamins or trace elements also resulted in a specific dechlorination activity that was generally higher than that observed for the same cells grown solely on chlorobenzoic acid. The results of this work indicate that yeast extract, a complex mixture of readily oxidizable carbon sources, vitamins, and trace elements, enhances the growth and the dechlorination activity of CPE2 and CPE3 cells, thus resulting in an overall increase in the rate of chlorobenzoic acid utilization and dechlorination.     

Simultaneous degradation of 3-chlorobenzoate and phenolic compounds by a defined mixed culture ofPseudomonas spp.

A mixed culture of a chlorobenzoate-(3-CBA)-degradingPseudomonas aeruginosa, strain 3mT, and a phenol/cresols-degradingPseudomonas sp., strain CP4, simultaneously and efficiently degraded mixtures of 3-CBA and phenol/cresols. However, strains 3mT and CP4 usedortho- andmeta-ring cleavage pathways, respectively. Degradation of 3-CBA was complete when the 3-CBA was equal in amount to or less than that of phenol. CP4/3mT inoculum ratios (w/w) of 1:1 or 1:2 gave the most effective degradation of both the substrates in the mixture. The mixed culture degraded equimolar mixtures of 3-CBA/phenol up to 10mm. Equimolar mixtures of 3-CBA ando-, m- orp-cresol were also degraded by the mixed culture.The authors are with the Microbiology and Bioengineering Department, Central Food Technological Research Institute, Mysore-570013, India;     

Cloning, Expression, and Nucleotide Sequence of the Pseudomonas aeruginosa 142 ohb Genes Coding for Oxygenolytic ortho Dehalogenation of Halobenzoates

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISP_OHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISP_OHB utilized electron transfer components provided by the heterologous hosts. ISP_OHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.     

Biodegradation Kinetics of Four Substituted Chlorobenzoic Acids by Enterobacter aerogenes

Enterobacter aerogenes is generally found in soil, sewage plants, and human gastrointestinal tract. Thus, this study was conducted to evaluate the ability of Enterobacter aerogenes to degrade four chlorobenzoic acid compounds (2-chlorobenzoic acid (2-CBA), 3-chlorobenzoic acid (3-CBA), 4-chlorobenzoic acid (4-CBA), and 3,4-dichlorobenzoic acid (3,4-dCBA)) in minimal salt medium. Enterobacter aerogenes was partially able to degrade and dechlorinate these CBAs at concentration of 3.5 mM within 72 h of incubation. According to Haldane single-substrate model, the values of maximum predicted growth rate (μ_max), half saturation constant (K _s), and inhibition constant (K _i) fell in the range of 0.2–0.8 h^?1, 8–41 mM, and 5–53 mM, respectively. Based on the estimated values of both α, a growth-associated constant, and β, a non–growth-associated constant, the production of chloride was predominantly growth associated, since negligible values of the β were determined. Haldane model gave a good prediction of the CBA substrate utilization and degradation, and was in a very good agreement with the experimental data. Because of the capability of Enterobacter aerogenes to utilize these aromatic compounds as carbon and energy sources, this microorganism can be a valuable and promising candidate for use in the biotreatment of wastewater and soil samples contaminated with mixtures of chlorobenzoates.     

Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5alphaF'(pOD22) and DH5alphaF'(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (beta-ISP) and 48,243 Da (alpha-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.     

Degradation of polychlorinated phenols by Streptomyces rochei 303

The strain Streptomyces rochei 303 (VKM Ac-1284D) is capable of utilizing 2-chloro-,2,4-,2,6-dichloro- and 2,4,6-trichlorophenols as the sole source of carbon. Its resting cells completely dechlorinated and degraded 2-, 3-chloro-; 2,4-, 2,6-, 2,3-, 2,5-, 3,4-, 3,5-dichloro-; 2,4-, 2,6-dibromo-; 2,4,6-, 2,4,5-, 2,3,4-, 2,3,5-, 2,3,6-trichlorophenols; 2,3,5,6-tetrachloro- and pentachlorophenol. During chlorophenol degradation, a stoichiometric amount of chloride ions was released and chlorohydroquinols were formed as intermediates. In cell-free extracts of S. rochei, the activity of hydroxyquinol 1,2-dioxygenase was found. The enzyme was induced with chlorophenols. Of all so far described strains degrading polychlorophenols, S. rochei 303 utilized a wider range of chlorinated phenols as the sole sourse of carbon and energy.Abbreviations CP chlorophenol - DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - DBrP dibromophenol - CHQ chlorohydroquinol - DCHQ dichlorohydroquinol - HHQ hydroxyhydroquinol - CHHQ chlorohydroxyhydroquinol - CC chlorocatechol - TLC thin layer chromatography - GC/MC chromato-mass-spectrometry - HPLC high-performance liquid chromatography     

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls – Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2–4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose–response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

Rhodopseudomonas palustris strain RCB100 degrades 3-chlorobenzoate (3-CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3-CBA and determined its N-terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate-CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3-CBA as a sole carbon source. The aliA gene from the 3-CBA degrading strain differed by a single nucleotide from the aliA gene from strain CGA009, causing the substitution of a serine for a threonine at position 208. Both AliA enzymes, purified as His-tagged fusion proteins, had comparable activities with cyclohexanecarboxylate. However, AliA from the 3-CBA degrading strain was 10-fold more active with 3-CBA (kcat/Km of 4.3 x 10(4) M(-1) s(-1)) than the enzyme from the sequenced strain (kcat/Km 0.32 x 10(4) M(-1) s(-1)). The CGA009 enzyme was not sufficiently active with 3-CBA to complement an RCB100 aliA mutant for growth on this compound. Here, whole genome sequence information enabled us to identify a single nucleotide among 5.4 million nucleotides that contributes to the substrate preference of a coenzyme A ligase.     

Effect of yeast extract on growth kinetics during aerobic biodegradation of chlorobenzoic acids

The Monod or Andrews kinetic parameters describing the growth of Pseudomonas sp. CPE2 strain on 2,5-dich!orobenzoic acid and 2-chlorobenzoic acid, and Al-caligenes sp. CPE3 strain on 3,4-dichlorobenzoic acid, 4-chlorobenzoic acid, and 3-chlorobenzoic acid were determined from batch and continuous growth experiments conducted in the presence or absence of yeast extract (50 mg/L). Strain CPE2 displayed inhibitory growth kinetics in the absence of yeast extract and a noninhibitory kinetics in the presence of yeast extract. Similar results were obtained for CPE3. The presence of yeast extract also resulted in a significant increase in the affinity of the strains for the chlorobenzoic acids they degraded. (c) 1995 John Wiley & Sons, Inc.     

Biological degradation of 4-chlorobenzoic acid by a PCB-metabolizing bacterium through a pathway not involving (chloro)catechol

Cupriavidus sp. strain SK-3, previously isolated on polychlorinated biphenyl mixtures, was found to aerobically utilize a wide spectrum of substituted aromatic compounds including 4-fluoro-, 4-chloro- and 4-bromobenzoic acids as a sole carbon and energy source. Other chlorobenzoic acid (CBA) congeners such as 2-, 3-, 2,3-, 2,5-, 3,4- and 3,5-CBA were all rapidly transformed to respective chlorocatechols (CCs). Under aerobic conditions, strain SK-3 grew readily on 4-CBA to a maximum concentration of 5 mM above which growth became impaired and yielded no biomass. Growth lagged significantly at concentrations above 3 mM, however chloride elimination was stoichiometric and generally mirrored growth and substrate consumption in all incubations. Experiments with resting cells, cell-free extracts and analysis of metabolite pools suggest that 4-CBA was metabolized in a reaction exclusively involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoic acid, which was then hydroxylated to protocatechuic acid (PCA) and subsequently metabolized via the β-ketoadipate pathway. When strain SK-3 was grown on 4-CBA, there was gratuitous induction of the catechol-1,2-dioxygenase and gentisate-1,2-dioxygenase pathways, even if both were not involved in the metabolism of the acid. While activities of the modified ortho- and meta-cleavage pathways were not detectable in all extracts, activity of PCA-3,4-dioxygenase was over ten-times higher than those of catechol-1,2- and gentisate-1,2-dioxygenases. Therefore, the only reason other congeners were not utilized for growth was the accumulation of CCs, suggesting a narrow spectrum of the activity of enzymes downstream of benzoate-1,2-dioxygenase, which exhibited affinity for a number of substituted analogs, and that the metabolic bottlenecks are either CCs or catabolites of the modified ortho-cleavage metabolic route.     

Degradation of mono- and dichlorobenzoic acid isomers by two natural isolates of Alcaligenes denitrificans

Two strains of Alcaligenes denitrificans, designated BRI 3010 and BRI 6011, were isolated from polychlorinated biphenyl (PCB)-contaminated soil using 2,5-dichlorobenzoic acid (2,5-DCBA) and 2,4-DCBA, respectively, as sole carbon and energy sources. Both strains degraded 2-chlorobenzoic acid (2-CBA), 2,3-DCBA, and 2,5-DCBA, and were unable to degrade 2,6-DCBA. BRI 6011 alone degraded 2,4-DCBA. Growth of BRI 6011 in yeast extract and 2,6-DCBA induced pyrocatechase activity, but 2,6-DCBA was not degraded, suggesting the importance of an unsubstituted carbon six of the aromatic ring. Metabolism of the chlorinated substrates resulted in the stoichiometric release of chloride, and degradation proceeded by intradiol cleavage of the aromatic ring. Growth of both strains on 2,5-DCBA induced pyrocatechase activities with catechol and chlorocatechols as substrates. In contrast to dichlorobenzoic acids, growth on 2-CBA, benzoic acid, mono- and dihydroxybenzoic acids induced a pyrocatechase activity against catechol only. Although 2,4-DCBA was a more potent inducer of both pyrocatechase activities, its utilization by BRI 6011 was inhibited by 2,5-DCBA. Specific uptake rates using resting cells were highest with 2-CBA, except when the resting cells had been previously grown on 2,5-DCBA, in which case 2,5-DCBA was the preferred substrate. The higher rates of 2,5-DCBA uptake obtained by growth on that substrate, suggested the existence of a separately induced uptake system for 2,5-DCBA.     

Hydrolytic dehalogenation of 4-chlorobenzoic acid by an Acinetobacter sp

Acinetobacter sp. strain ST-1, isolated from garden soil, can mineralize 4-chlorobenzoic acid (4-CBA). The bacterium degrades 4-CBA, starting with dehalogenation to yield 4-hydroxybenzoic acid (4-HBA) under both aerobic and anaerobic conditions, suggesting that the dehalogenating enzyme in the strain is not an oxygenase; the enzyme may catalyze halide hydrolysis. To identify the oxygen source of the C(4)-hydroxy groups in the dehalogenation step, we used H(2)(18)O as the solvent under anaerobic conditions. When resting cells were incubated in the presence of 4-CBA and H(2)(18)O under a nitrogen gas stream, the hydroxy group on the aromatic nucleus of the 4-HBA produced was derived from water, not from molecular oxygen. This dehalogenation was hydrolytic, because analysis of the mass spectrum of the trimethylsilyl derivative of one of the metabolites, (18)O-labeled 4-HBA, showed that 80% of the C4-hydroxy groups were labeled with (18)O. Hydrolytic dehalogenation of 4-CBA in intact cells has not been reported earlier. To identify substrate specificity, we next examined the ability of the strain to dehalogenate 4-CBA analogues and dichlorobenzoic acids. The results of metabolite analysis by high-pressure liquid chromatography showed that the strain dehalogenated 4-bromobenzoic acid and 4-iodobenzoic acid, yielding 4-HBA, suggesting that these compounds could be further degraded and mineralized by the strain via the beta-ketoadipate pathway, as occurs with 4-CBA. This strain, however, did not dehalogenate 4-fluorobenzoic acid, 2- and 3-chlorobenzoic acids, or 2,4-, 3,4-, and 3,5-dichlorobenzoic acids during 4 days of incubation, implying that the dehalogenating enzyme of the strain has high substrate specificity.     

Biosynthesis and cytoplasmic accumulation of a chlorinated catechol pigment during 3-chlorobenzoate aerobic co-metabolism in Pseudomonas fluorescens

A strain of Pseudomonas fluorescens was capable of co-metabolizing 3-chlorobenzoic acid with the production of a chlorinated catechol black pigment. A peroxidase and another enzymatic activity referred to as a polyphenol oxidase were found to be involved in the oxidation of 4-chlorocatechol to 4-chloro-1,2-benzoquinone, i.e. in the production of highly reactive substrates for pigment formation. Therefore, P. fluorescens cells were seen to take an active part not only in 3-chlorobenzoate mineralization but also in overall pigment production. pH was found to be a key parameter in the regulation of the activity of P. fluorescens oxidoreductive enzymes. Ultrastructural investigations showed that electron dense granules of pigment were distributed throughout the cytoplasm of Pseudomonas fluorescens cells grown in presence of 3-chlorobenzoate, as confirmed also by Thiéry cytochemical investigations.In these cells, an extensive contraction of the cytoplasm as well as a significant damage to the cell wall after two days of incubation, suggested that pigment production caused a premature death of the cells accompanied by the leakage of the cell content. Pigment production seemed to occur mostly in the cytoplasmic context where the electron dense material accumulates until it is released in the medium after the cell lysis.Abbreviations 3-CBA 3-chlorobenzoic acid - BA benzoic acid - 4-CC 4-chlorocatechol - 3-CC 3-chlorocatechol - MBTH 3-methyl-2-benzothiazolinone hydrazone - l-DOPA l-3,4-dihydroxyphenyl-alanine - SPB sodium phosphate buffer     

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.     

Degradation of 4-Chlorobenzoate by Facultatively Alkalophilic Arthrobacter sp. Strain SB8

A facultative alkalophile capable of utilizing 4-chlorobenzoate (4-CBA), strain SB8, was isolated from soil with an alkaline medium (pH 10.0) containing the haloaromatic compound as the carbon source. The strain, identified as an Arthrobacter sp., showed rather extensive 4-CBA-degrading ability. 4-CBA utilization by the strain was possible in the alkaline medium containing up to 10 g of the compound per liter. The 4-CBA-dechlorinating activity of resting cells was almost completely uninhibited by substrate concentrations up to 150 mM. The bacterium dehalogenated 4-CBA in the initial stage of the degradation and metabolized the compound via 4-hydroxybenzoate and protocatechuate. O₂ was needed for 4-CBA dechlorination by resting cells but not by cell extracts. O₂ was inhibitory to the 4-CBA dechlorination activity of cell extracts. These facts suggest dechlorination of 4-CBA by halide hydrolysis and an energy requirement for the transport of 4-CBA into cells.     

Biodegradation of 3-chlorobenzoate and formation of black color in the presence and absence of benzoate

Summary Four isolates of Pseudomonas from soil and sewage utilized 3-chlorobenzoate (3-CBA) adaptively as sole source of carbon and energy. Two of these were studied in detail. Their doubling times in batch culture were about twice as long on chlorobenzoate as on benzoate or glucose. Both isolates showed oxygen uptake on catechol, without lag, when grown on either benzoate or 3-CBA. One strain, designated Pseudomonas H1, could oxidize a key intermediate, 4-chlorocatechol, only when grown on 3-CBA. Pseudomonas H2 could oxidize the chlorocatechol not only when grown on 3-CBA but also when grown on benzoate. Benzoate-adapted P. H1 therefore accumulated chlorocatechols when incubated with a mixture of 3-CBA and benzoate, whereas P. H2 under the same conditions did not. The accumulated chlorocatechols inhibited further oxygen uptake, and in alkaline media they polymerized to a black, melanin-like pigment. Intense black pigment, similar to that formed by P. I, was formed if raw sewage was incubated with a mixture of 3-CBA and benzoate. The pigment was not formed if the sewage was first adapted by incubation with 3-CBA.     

Metabolism of meta- or/and para-chlorinated benzoic acids by the crude extract of Alcaligenes sp. strain CPE3 grown on 3,4-dichlorobenzoic acid

Summary A crude extract of Alcaligenes sp. CPE3 strain grown on 3,4-dichlorobenzoic acid metabolised 3- and 4-chlorobenzoic acid by reactions requiring O₂ and NADH, and 3,4-dichlorobenzoic acid by a reaction requiring O₂, NADH, FAD and FMN. The specific activity of the extract vs. 3-chlorobenzoic acid was described by the Michaelis-Menten kinetics, that vs. 4-chlorobenzoic acid was described by a substrate inhibitory kinetics and that vs. 3,4-dichlorobenzoic acid exhibited a two-peaked profile.