Degradation of mixtures of chloroaromatic compounds in soil slurry by mixed cultures of specialized organisms

The degradation of a mixture of 13 chloroaromatics, 2-chloro-, 3-chloro-, 4-chloro- and 3,4-dichloroaniline,2-chloro-, 3-chloro-,4-chloro-, 3,4-dichloro-and 3,5-dichlorobenzoate, and chloro-,1,2-dichloro-, 1,4-dichloro- and 1,2,4-trichlorobenzene in soil slurries by a mixed culture of Pseudomonas acidovorans strain BN 3.1, Pseudomonas ruhlandii strain FRB2, Pseudomonas cepacia strain JH230 and Pseudomonas aeruginosa strain RHO1 was studied. About 70% of the organic bound chlorine was eliminated after 25 days from soil with a carbon content of 8% (soil 1) when 2–3 × 10⁵ cells/g soil of each of the strains were added to the slurries. The effect of the clean-up was demonstrated by a biological test using cress and wheat. Both plants showed good germination and growth on both non-contaminated soils and the contaminated soil 1 after the biotreatment with the strains. No growth was observed when the plants were incubated with the contaminated soil 1 and with the contaminated and biotreated soil 2 (carbon content 2.6%). This indicates that the remaining 30% of organic chlorine in soil 1 after biotreatment does not influence the germination and growth of the two plants tested. *** DIRECT SUPPORT *** AG903062 00010     

Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51.

Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.     

Streptomyces sp. is a powerful biotechnological tool for the biodegradation of HCH isomers: biochemical and molecular basis

Actinobacteria are well-known degraders of toxic materials that have the ability to tolerate and remove organochloride pesticides; thus, they are used for bioremediation. The biodegradation of organochlorines by actinobacteria has been demonstrated in pure and mixed cultures with the concomitant production of metabolic intermediates including γ-pentachlorocyclohexene (γ-PCCH); 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN); 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB), or 1,4-dichlorobenzene (1,4-DCB); 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,4-trichlorobenzene (1,2,4-TCB), or 1,3,5-trichlorobenzene (1,3,5-TCB); 1,3-DCB; and 1,2-DCB. Chromatography coupled to mass spectrometric detection, especially GC–MS, is typically used to determine HCH-isomer metabolites. The important enzymes involved in HCH isomer degradation metabolic pathways include hexachlorocyclohexane dehydrochlorinase (LinA), haloalkane dehalogenase (LinB), and alcohol dehydrogenase (LinC). The metabolic versatility of these enzymes is known. Advances have been made in the identification of actinobacterial haloalkane dehydrogenase, which is encoded by linB. This knowledge will permit future improvements in biodegradation processes using Actinobacteria. The enzymatic and genetic characterizations of the molecular mechanisms involved in these processes have not been fully elucidated, necessitating further studies. New advances in this area suggest promising results. The scope of this paper encompasses the following: (i) the aerobic degradation pathways of hexachlorocyclohexane (HCH) isomers; (ii) the important genes and enzymes involved in the metabolic pathways of HCH isomer degradation; and (iii) the identification and quantification of intermediate metabolites through gas chromatography coupled to mass spectrometry (GC–MS).     

ATP synthesis associated with the conversion of hexachlorocyclohexane related compounds

Clostridium rectum strain S-17 converts -1,2,3,4,5,6-hexachlorocyclohexane (HCH) related compounds to chlorobenzenes. The metabolites from -1,2,3,4,5,6-hexachlorocyclohexene and -1,3,4,5,6-pentachlorocyclohexene are identified as 1,2,4-trichlorobenzene and 1,4-dichlorobenzene, respectively. ATP synthesis, converting these chlorinated compounds, is observed in the cell suspension of C. rectum as indicated by luciferase-luciferin reaction and phosphorylation of ³²P-labeled phosphate. These observation lead to the conclusion that HCH and related compounds serve as artificial electron acceptors of the Stickland reaction, and therefore, the reductive dechlorination is associated with ATP synthesis.Abbreviations HCH -1,2,3,4,5,6-hexachlorocyclohexane - HCCH -1,2,3,4,5,6-hexachlorocyclohexene - PCCH -1,3,4,5,6-pentachlorocyclohexene - TCCH -3,4,5,6-tetrachlorocyclohexene - 1,2,4-TCB 1,2,4-trichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene - DTT 1,4-dithiothreitol - IAA monoiodoacetic acid     

Degradation of 1,2,4-Trichloro- and 1,2,4,5-Tetrachlorobenzene by Pseudomonas Strains

Two Pseudomonas sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon and energy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grew on monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). Strain PS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds both strains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and 1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. The intermediary cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCB was rearomatized by an NAD⁺-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from the initially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent ortho cleavage was catalyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which was channeled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present case included 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitro group was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for the oxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on these haloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.     

Biodegradation of lindane using a novel yeast strain, Rhodotorula sp. VITJzN03 isolated from agricultural soil

Lindane is a notorious organochlorine pesticide due to its high toxicity, persistence in the environment and its tendency to bioaccumulate. A yeast strain isolated from sorghum cultivation field was able to use lindane as carbon and energy source under aerobic conditions. With molecular techniques, it was identified and named as Rhodotorula strain VITJzN03. The effects of nutritional and environmental factors on yeast growth and the biodegradation of lindane was investigated. The maximum production of yeast biomass along with 100 % lindane mineralization was noted at an initial lindane concentration of 600 mg l^?1 within a period of 10 days. Lindane concentration above 600 mg l^?1 inhibited the growth of yeast in liquid medium. A positive relationship was noted between the release of chloride ions and the increase of yeast biomass as well as degradation of lindane. The calculated degradation rate and half life of lindane were found to be 0.416 day^?1 and 1.66 days, respectively. The analysis of the metabolites using GC–MS identified the formation of seven intermediates including γ-pentachlorocyclohexane(γ-PCCH), 1,3,4,6-tetrachloro-1,4-cyclohexadiene(1,4-TCCHdiene), 1,2,4-trichlorobenzene (1,2,4 TCB), 1,4-dichlorobenzene (1,4 DCB), chloro-cis-1,2-dihydroxycyclohexadiene (CDCHdiene), 3-chlorocatechol (3-CC) and maleylacetate (MA) derivatives indicating that lindane degradation follows successive dechlorination and oxido-reduction. Based on the results of the present study, the possible pathway for lindane degradation by Rhodotorula sp. VITJzN03 has been proposed. To the best of our knowledge, this is the first report on lindane degradation by yeast which can serve as a potential agent for in situ bioremediation of medium to high level lindane-contaminated sites.     

Dehalorespiration with hexachlorobenzene and pentachlorobenzene by<Emphasis Type="Italic"> Dehalococcoides</Emphasis> sp. strain CBDB1

The chlororespiring anaerobe Dehalococcoides sp. strain CBDB1 used hexachlorobenzene and pentachlorobenzene as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with hexachlorobenzene or pentachlorobenzene as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with hexachlorobenzene or pentachlorobenzene when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield (Y) of 2.1±0.24 g protein/mol Cl^– released with hexachlorobenzene as electron acceptor; with pentachlorobenzene, the growth yield was 2.9±0.15 g/mol Cl^–. Hexachlorobenzene was reductively dechlorinated to pentachlorobenzene, which was converted to a mixture of 1,2,3,5- and 1,2,4,5-tetrachlorobenzene. Formation of 1,2,3,4-tetrachlorobenzene was not detected. The final end-products of hexachlorobenzene and pentachlorobenzene dechlorination were 1,3,5-trichlorobenzene, 1,3- and 1,4-dichlorobenzene, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene exclusively to 1,2,4-trichlorobenzene. The organism therefore catalyzes two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-trichlorobenzene, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-dichlorobenzene via 1,2,4-trichlorobenzene singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive dehalogenases might be induced by the different electron acceptors. To our knowledge, this is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorobenzene via dehalorespiration by a pure bacterial culture.     

Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1.

Xanthobacter flavus 14p1 was isolated from sludge of the river Mulde by selective enrichment with 1,4-dichlorobenzene as the sole source of carbon and energy. The bacterium did not use other aromatic or chloroaromatic compounds as growth substrates. During growth on 1,4-dichlorobenzene, stoichiometric amounts of chloride ions were released. Degradation products of 1,4-dichlorobenzene were identified by gas chromatography-mass spectrometry analysis. 3,6-Dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene and 3,6-dichlorocatechol were isolated from culture fluid. 2,5-Dichloromuconic acid and 2-chloromaleylacetic acid as well as the decarboxylation product 2-chloroacetoacrylic acid were identified after enzymatic conversion of 3,6-dichlorocatechol by cell extract. 1,4-Dichlorobenzene dioxygenase, dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase activity were induced in cells grown on 1,4-dichlorobenzene. The results demonstrate that 1,4-dichlorobenzene degradation is initiated by dioxygenation and that ring opening proceeds via ortho cleavage.     

Reductive dehalogenation of chlorinated benzenes and toluenes under methanogenic conditions.

The anaerobic metabolism of chlorinated benzenes and toluenes was evaluated in soil slurry microcosms under methanogenic conditions. A mixture of hexachlorobenzene, pentachlorobenzene, and 1,2,4-trichlorobenzene (TCB) in soil slurries was biotransformed through sequential reductive dechlorination to chlorobenzene (CB). The metabolic pathway for hexachlorobenzene and pentachlorobenzene decay proceeded via 1,2,3,4-tetrachlorobenzene (TTCB)-->1,2,3-TCB + 1,2,4-TCB-->1,2-dichlorobenzene (DCB) + 1,4-DCB-->CB. In a mineral salts medium, the CB-adapted soil microorganisms dehalogenated individual 1,2,4,5-TTCB, 1,2,3,4-TTCB, 1,2,3-TCB, and 1,2,4-TCB but not 1,2,3,5-TTCB or 1,3,5-TCB. Similarly, a mixture of 2,3,6-trichlorotoluene (TCT), 2,5-dichlorotoluene (DCT), and 3,4-DCT was reductively dechlorinated in soil slurries to predominantly toluene and small amounts of 2-, 3-, and 4-chlorotoluene (CT). Toluene was further degraded. When tested individually in a mineral salts medium, the CT-adapted soil microorganisms dechlorinated several TCT and DCT isomers. Key metabolic routes for TCTs followed: 2,3,6-TCT-->2,5-DCT-->2-CT-->toluene; 2,4,5-TCT-->2,5-DCT + 3,4-DCT-->3-CT + 4-CT-->toluene. Among DCTs tested, 2,4-DCT and 3,4-DCT were dechlorinated via the removal of o- and m-chlorine, respectively, to 4-CT and subsequently to toluene via p-chlorine removal. Likewise, 2,5-DCT was dechlorinated via 2-CT to toluene. Evidently, microorganisms capable of removing o-, m-, and p-chlorines are present in the soil system, as reflected by the dechlorination of different isomers of CBs and CTs to CB and toluene, respectively. These findings help clarify the metabolic fate of chlorinated benzenes and toluenes in anaerobic environments.     

Enrichment and Properties of a 1,2,4-Trichlorobenzene-Dechlorinating Methanogenic Microbial Consortium

A methanogenic microbial consortium capable of reductively dechlorinating 1,2,4-trichlorobenzene (1,2,4-TCB) was enriched from a mixture of polluted sediments. 1,2,4-TCB was dechlorinated via 1,4-dichlorobenzene (1,4-DCB) to chlorobenzene (CB). Lactate, which was used as an electron donor during the enrichment, was converted via propionate and acetate to methane. Glucose, ethanol, methanol, propionate, acetate, and hydrogen were also suitable electron donors for dechlorination, whereas formate was not. The addition of 5% (wt/vol) sterile Rhine River sand was necessary to maintain the dechlorinating activity of the consortium. The addition of 2-bromoethanesulfonic acid (BrES) inhibited methanogenesis completely but had no effect on the dechlorination of 1,2,4-TCB. The consortium was also able to dechlorinate other chlorinated benzenes via various simultaneous pathways to 1,3,5-TCB, 1,2-DCB, 1,3-DCB, or CB as an end product. The addition of BrES inhibited several of the simultaneously occurring dechlorination pathways of 1,2,3,4- and 1,2,3,5-tetrachlorobenzene and of pentachlorobenzene, which resulted in the formation of CB as the only final product. Hexachlorobenzene and polychlorinated biphenyls (PCBs) were dechlorinated after a lag phase of ca. 15 days, showing a dechlorination pattern that is different from those observed for lower chlorinated benzenes: only chlorines with two adjacent chlorines were removed. The results show that the consortium possesses at least three distinct dechlorination activities toward chlorinated benzenes and PCBs.     

Reductive dechlorination of all trichloro- and dichlorobenzene isomers

Abstract All three isomers of trichlorobenzene were reductively dechlorinated to monochlorobenzene via dichlorobenzenes in anaerobic sediment columns. The dechlorination was specific: 1,2,3- and 1,3,5-trichlorobenzene were solely transformed to 1,3-dichlorobenzene, while 1,4-dichlorobenzene was the only product of 1,2,4-trichlorobenzene transformation. Microorganisms were responsible for the observed transformations. Since monochlorobenzene and dichlorobenzene are mineralized by bacteria in the presence of oxygen, the process of reductive dechlorination may be an important initial step to obtain complete mineralization of otherwise recalcitrant trichlorobenzenes. This is especially true for the 1,3,5-isomer, which seems to resist biodegradation in oxic environments.     

Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175

An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source. During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released. Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene. It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.     

Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175.

An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source. During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released. Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene. It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.     

Degradation of chlorobenzenes at nanomolar concentrations by Burkholderia sp. strain PS14 in liquid cultures and in soil.

The utilization of 1,2,4,5-tetrachloro-, 1,2,4-trichloro-, the three isomeric dichlorobenzenes and fructose as the sole carbon and energy sources at nanomolar concentrations was studied in batch experiments with Burkholderia sp. strain PS14. In liquid culture, all chlorobenzenes were metabolized within 1 h from their initial concentration of 500 nM to below their detection limits of 0.5 nM for 1,2,4,5-tetrachloro- and 1,2,4-trichlorobenzene and 7.5 nM for the three dichlorobenzene isomers, with 63% mineralization of the tetra- and trichloroisomers. Fructose at the same initial concentration was, in contrast, metabolized over a 4-h incubation period down to a residual concentration of approximately 125 nM with 38% mineralization during this time. In soil microcosms, Burkholderia sp. strain PS14 metabolized tetrachlorobenzene present at 64.8 ppb and trichlorobenzene present at 54.4 ppb over a 72-h incubation period to below the detection limits of 0.108 and 0.09 ppb, respectively, with approximately 80% mineralization. A high sorptive capacity of Burkholderia sp. strain PS14 for 1,2,4, 5-tetrachlorobenzene was found at very low cell density. The results demonstrate that Burkholderia sp. strain PS14 exhibits a very high affinity for chlorobenzenes at nanomolar concentrations.     

The lin genes for γ-hexachlorocyclohexane degradation in Sphingomonas sp. MM-1 proved to be dispersed across multiple plasmids

A γ-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of γ-HCH led to the detection of five γ-HCH metabolites, γ-pentachlorocyclohexene, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol, 2,5-dichlorohydroquinone, 1,2,4-trichlorobenzene, and 2,5-dichlorophenol, strongly suggesting that MM-1 has the lin genes for γ-HCH degradation originally identified in the well-studied γ-HCH-degrading strain Sphingobium japonicum UT26. Southern blot, PCR amplification, and sequencing analyses indicated that MM-1 has seven lin genes for the conversion of γ-HCH to β-ketoadipate (six structural genes, linA to linF, and one regulatory gene, linR). MM-1 carried four plasmids, of 200, 50, 40, and 30 kb. Southern blot analysis revealed that all seven lin genes were dispersed across three of the four plasmids, and that IS6100, often found close to the lin genes, was present on all four plasmids.     

Metabolism of dichloromethylcatechols as central intermediates in the degradation of dichlorotoluenes by Ralstonia sp. strain PS12

Ralstonia sp. strain PS12 is able to use 2,4-, 2,5-, and 3,4-dichlorotoluene as growth substrates. Dichloromethylcatechols are central intermediates that are formed by TecA tetrachlorobenzene dioxygenase-mediated activation at two adjacent unsubstituted carbon atoms followed by TecB chlorobenzene dihydrodiol dehydrogenase-catalyzed rearomatization and then are channeled into a chlorocatechol ortho cleavage pathway involving a chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase. However, completely different metabolic routes were observed for the three dichloromethylcatechols analyzed. Whereas 3,4-dichloro-6-methylcatechol is quantitatively transformed into one dienelactone (5-chloro-2-methyldienelactone) and thus is degraded via a linear pathway, 3,5-dichloro-2-methylmuconate formed from 4,6-dichloro-3-methylcatechol is subject to both 1,4- and 3,6-cycloisomerization and thus is degraded via a branched metabolic route. 3,6-Dichloro-4-methylcatechol, on the first view, is transformed predominantly into one (2-chloro-3-methyl-trans-) dienelactone. In situ (1)H nuclear magnetic resonance analysis revealed the intermediate formation of 2,5-dichloro-4-methylmuconolactone, showing that both 1,4- and 3,6-cycloisomerization occur with this muconate and indicating a degradation of the muconolactone via a reversible cycloisomerization reaction and the dienelactone-forming branch of the pathway. Diastereomeric mixtures of two dichloromethylmuconolactones were prepared chemically to proof such a hypothesis. Chloromuconate cycloisomerase transformed 3,5-dichloro-2-methylmuconolactone into a mixture of 2-chloro-5-methyl-cis- and 3-chloro-2-methyldienelactone, affording evidence for a metabolic route of 3,5-dichloro-2-methylmuconolactone via 3,5-dichloro-2-methylmuconate into 2-chloro-5-methyl-cis-dienelactone. 2,5-Dichloro-3-methylmuconolactone was transformed nearly exclusively into 2-chloro-3-methyl-trans-dienelactone.     

Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria

Summary Three strains, RHO1, R3 and B1, tentatively identified as a Pseudomonas sp., an Alcaligenes sp. and a Pseudomonas sp. which were able to use 1,4-dichlorobenzene as the sole carbon and energy source were isolated from water of the Rhine river and from the sewage plant at Leverkusen-Bürrig. A hybrid strain, WR1313, which uses chlorobenzene as the growth substrate, was obtained by mating the benzene-growing Pseudomonas putida strain F1 with strain B13, a Pseudomonas sp. degrading chlorocatechols. Further selection of this strain for growth on 1,4-dichlorobenzene allowed the isolation of strain WR1323. During growth on 1,4-dichlorobenzene the strains released stoichiometric amounts of chloride. The affinity of the organisms to 1,4-dichlorobenzene was measured with strain R3 showing a K_s value of 1.2 mg/l. Respiration data and enzyme activities in cell extracts as well as the isolation of 3,6-dichlorocatechol from the culture fluid are consistent with the degradation of 1,4-dichlorobenzene via 3,6-dichlorocatechol, 2,5-dichloro-cis,cis-muconate, 2-chloro-4-carboxymethylenebut-2-en-4-olide.     

Degradation of Chlorobenzenes at Nanomolar Concentrations by Burkholderia sp. Strain PS14 in Liquid Cultures and in Soil

The utilization of 1,2,4,5-tetrachloro-, 1,2,4-trichloro-, the three isomeric dichlorobenzenes and fructose as the sole carbon and energy sources at nanomolar concentrations was studied in batch experiments with Burkholderia sp. strain PS14. In liquid culture, all chlorobenzenes were metabolized within 1 h from their initial concentration of 500 nM to below their detection limits of 0.5 nM for 1,2,4,5-tetrachloro- and 1,2,4-trichlorobenzene and 7.5 nM for the three dichlorobenzene isomers, with 63% mineralization of the tetra- and trichloroisomers. Fructose at the same initial concentration was, in contrast, metabolized over a 4-h incubation period down to a residual concentration of approximately 125 nM with 38% mineralization during this time. In soil microcosms, Burkholderia sp. strain PS14 metabolized tetrachlorobenzene present at 64.8 ppb and trichlorobenzene present at 54.4 ppb over a 72-h incubation period to below the detection limits of 0.108 and 0.09 ppb, respectively, with approximately 80% mineralization. A high sorptive capacity of Burkholderia sp. strain PS14 for 1,2,4,5-tetrachlorobenzene was found at very low cell density. The results demonstrate that Burkholderia sp. strain PS14 exhibits a very high affinity for chlorobenzenes at nanomolar concentrations.     

好氧氯苯降解菌的分离鉴定

【目的】分离好氧氯苯降解菌,并通过研究降解特性为应用提供理论依据。【方法】利用富集培养技术分离菌株,通过形态、生理生化反应特征及16S rRNA基因序列分析鉴定菌株,测定培养液中氯苯、其它氯苯类化合物和氯离子的浓度以及菌体细胞的密度和菌体细胞粗提液中邻苯二酚双加氧酶的活性,研究菌株的降解特性。【结果】16S rRNA基因序列相似性比较表明,分离出的菌株与乙酸钙不动杆菌(Acinetobacter calcoaceticus)的相似性高达98.5%。以初始浓度为50mg/L的氯苯为唯一碳源和能源时,120h内菌株对氯苯的降解率高达98.2%,氯离子净释放量和氯苯降解量的摩尔比范围为1:1.85-1:1.39,菌体细胞粗提液中邻苯二酚1,2-双加氧酶的平均活性为0.538U/mg蛋白质。加入葡萄糖后,菌体细胞数量和氯离子浓度明显增加,但单位细胞的氯苯降解能力明显下降。在二氯苯和三氯苯共存时,菌株对氯苯的降解能力受到明显的抑制作用,但对二氯苯有一定的降解作用,降解能力大小顺序为:1,3-二氯苯1,2-二氯苯1,4-二氯苯。【结论】分离出的好氧氯苯降解菌属于Acinetobacter属菌株,该菌株对氯苯和二氯苯均具有降解作用,可能通过邻位裂环途径降解氯苯,氯苯对菌株的降解能力和邻苯二酚1,2-双加氧酶的活性具有明显的增强作用。     

Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1

Xanthobacter flavus 14p1 used 1,4-dichlorobenzene as the sole source of carbon and energy but did not grow on other (chloro)aromatic compounds. 1,4-Dichlorobenzene was attacked by a chlorobenzene dioxygenase, and the intermediate chlorocatechol was metabolized by the modified ortho pathway. All enzymes necessary to convert 1,4-dichlorobenzene to 3-oxoadipate showed a low substrate specificity and also accepted the respective intermediates of chlorobenzene or 1,3-dichlorobenzene degradation. Of the three compounds chlorobenzene, 1,4-dichlorobenzene, and 1,3-dichlorobenzene, the latter was the most toxic for X. flavus 14p1. Furthermore, 1,3-dichlorobenzene did not induce chlorocatechol 1,2-dioxygenase activity of the organism. Chlorobenzene, however, induced chlorocatechol 1,2-dioxygenase, dienelactone hydrolase, and maleylacetate reductase activities. As demonstrated by chloride release, also chlorobenzene dioxygenase, chlorobenzene cis-dihydrodiol dehydrogenase, and chloromuconate cycloisomerase activities were present in chlorobenzene-induced cells, but chlorobenzene failed to support growth. Presumably a toxic compound was formed from one of the intermediates. Received: 10 June 1996 / Revision received: 23 December 1996 / Accepted: 18 January 1997