Degradation of Aroclor 1221 and survival of strains in soil microcosms

Three bacterial strains able to use different aromatic compounds as the sole carbon and energy source were tested for their potential to degrade Aroclor 1221 in soil microcosms when present in mixed culture. Disappearance of polychlorinated biphenyls (PCBs), occurrence of metabolites, release of chloride, and survival of the laboratory-selected strains were investigated under different conditions. In principle, complete mineralization of various congeners of Aroclor 1221, a technical mixture of PCBs, by the mixed culture was possible. The autochthonous microflora negatively affected the degradation due to formation of a toxic compound from 4-chlorobenzoate. 4-Chlorobenzoate was produced by one of the added strains, Pseudomonas sp. JHK, during degradation of 4-chlorobiphenyl. The unknown metabolite of 4-chlorobenzoate led to a rapid decrease in viable counts of the laboratory-selected strains in the soil microcosm.Correspondence to: J. Havel     

Degradation of 3-chlorobiphenyl by in vivo constructed hybrid pseudomonads

Abstract 3-Chlorobiphenyl-degrading bacteria were obtained from the mating between Pseudomonas putida strain BN10 and Pseudomonas sp. strain B13. Strains such as BN210 resulted from the transfer of the genes coding the enzyme sequence for the degradation of chlorocatechols from B13 into BN10, whereas B13 derivatives such as B131 have acquired the biphenyl degradation sequence from BN10. During growth of the hybrid strains on 3-chlorobiphenyl 90% chloride was released. Activities of phenylcatechol 2,3-dioxygenase, benzoate dioxygenase, catechol 1,2-dioxygenase, chloromuconate cyloisomerase and 4-carboxymethyl-enebut-2-en-4-olide hydrolase were found in 3-chlorobiphenyl-grown cells. The hybrid strains were found to convert some congeners of the Aroclor 1221 mixture such as mono- and dichloro-substituted biphenyls.     

一株联苯降解菌的特性及鉴定*

从华北油田污染土壤中筛选出一株能够以联苯为唯一碳源和能源生长的菌株。该菌生长的最适联苯浓度为0．2％～0．4％,在联苯浓度为0．1％的培养基中培养36h后降解率达99．8%。该菌还可以降解苯甲酸钠、邻苯二酚、间苯二酚、对苯二酚和多氯联苯Aroclorl221、Aroclorl242等芳香族化合物。通过16S rDNA基因序列分析鉴定该菌为嗜吡啶红球菌(Rhodococcus pyridinovorans)。     

Bacterial metabolism of hydroxylated biphenyls.

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

Bacterial metabolism of hydroxylated biphenyls

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

Synergistic growth of two members from a mixed microbial consortium growing on biphenyl

Abstract A stable nine-membered aerobic bacterial consortium (BSEN-2) growing on biphenyl as the sole carbon and energy source was isolated from a polychlorinated biphenyl (PCB) contaminated soil. Characterisation of the members, strains BPSI-1 to 9, revealed three principal genera, Pseudomonas, Sphingomonas and Alcaligenes . Phenotypic analysis based on standard microbiological tests and Biolog identification, showed close relationship between community members with the exception of Sphingomonas paucimobilis strain BPSI-3. Some clusters revealed relationships unrelated to genus groupings. Strain BPSI-3 produced a bright yellow water soluble compound from biphenyl having absorption maxima at 412 and 337 nm at neutral pH. This is similar, but not identical, to those results reported for muconic semialdehydes, cleavage products of biphenyl and other aromatic compounds. Only four of the nine isolates, BPSI-2, 3, 4 and 7, were capable of growth on biphenyl as sole carbon and energy source. Two isolates, Alcaligenes faecalis type II strain BPSI-2 and S. paucimobilis strain BPSI-3, were isolated together and were difficult to separate into pure cultures. Growth studies in liquid culture showed that a co-culture of these two achieved a specific growth rate (μ) approximately twice as high as strain BPSI-2 and four times that of BPSI-3. Both strains grew equally well on benzoate with no significant difference in their specific growth rates. When compared to the original mixed culture, BSEN-2, the co-culture achieved 39% greater biomass and a specific growth rate twice as high. In the co-culture, the yellow colour seen with pure cultures of BPSI-3 was not observed. BPSI-2 was found to be able to utilise the yellow metabolites more effectively than BPSI-3. A model for the interaction of these two strains, based on the utilisation of biphenyl catabolites and degradation at the genetic level, has been proposed.     

Aerobic mineralization of 4,4′-dichlorobiphenyl and 4-chlorobenzoic acid by a novel natural bacterial strain that grows poorly on benzoate and biphenyl

Achromobacter xylosoxidans strain IR08 was isolated from soil contaminated with electrical transformer fluid by enrichment culture containing Aroclor 1221 as the sole carbon source. This strain was found to grow on all monochlorobiphenyls, 4,4′-dichlorobiphenyl (4,4′-diCB) and a wide range of other xenobiotic compounds. During growth on 4,4′-diCB, a near-stoichiometric amount of chloride was excreted into the culture fluid in less than 5 days and growth yield was more than twice that achieved on biphenyl. The production of 4-CBA or chlorocatechol as a metabolite was not observed. Quite unusually, coincubation of strain IR08 with 4,4′-diCB and biphenyl at relatively equal concentrations showed preferential utilization of the chlorobiphenyl: 4,4′-diCB was mineralized in less than 5 days concomitant with stoichiometric release of chloride, while biphenyl was poorly degraded. Growth on 2.5 mM CBA also resulted in complete disappearance of the substrate, however, inorganic chloride recovered from the culture broth was less than 65%. The isolation of a dichlorobiphenyl-mineralizing rather than transformation strain such as IR08 is an important advance in an effort to develop effective bioremediation strategy for polychlorinated biphenyl-contaminated soil.     

Motility and chemotaxis of Pseudomonas sp. B4 towards polychlorobiphenyls and chlorobenzoates

The polychlorinated biphenyl (PCB)-degrading Pseudomonas sp. B4 was tested for its motility and ability to sense and respond to biphenyl, its chloroderivatives and chlorobenzoates in chemotaxis assays. Pseudomonas sp. B4 was attracted to biphenyl, PCBs and benzoate in swarm plate and capillary assays. Chemotaxis towards these compounds correlated with their use as carbon and energy sources. No chemotactic effect was observed in the presence of 2- and 3-chlorobenzoates. Furthermore, a toxic effect was observed when the microorganism was exposed to 3-chlorobenzoate. A nonmotile Pseudomonas sp. B4 transformant and Burkholderia xenovorans LB400, the laboratory model strain for PCB degradation, were both capable of growing in biphenyl as the sole carbon source, but showed a clear disadvantage to access the pollutants to be degraded, compared with the highly motile Pseudomonas sp. B4, stressing the importance of motility and chemotaxis in this environmental biodegradation.     

Isolation and characterization of a thermophilic Bacillus sp. JF8 capable of degrading polychlorinated biphenyls and naphthalene

Bacillus sp. strain JF8, which was isolated from compost, utilizes naphthalene and biphenyl as carbon sources at 60 degrees C. Biphenyl grown cells of strain JF8 barely degraded naphthalene while naphthalene grown cells did not degrade p-chlorobiphenyl, suggesting the existince of two independent degradation pathways. Isolation of JF8N, a mutant strain which can not utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, supports this hypothesis. Biphenyl grown cells of strain JF8 can degrade several polychlorinated biphenyl congeners including tetra- and pentachlorobiphenyl. bph and nah probes from mesophilic organisms failed to hybridize to strain JF8 DNA.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

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.     

Enhancement of Pyrroloquinoline Quinone Production and Polyvinyl Alcohol Degradation in Mixed Continuous Cultures of Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C with Mixed Carbon Sources

In a mixed continuous culture of Pseudomonas putida VM15A and Pseudomonas sp. strain VM15C with polyvinyl alcohol (PVA) as the sole source of carbon, growth of the PVA-degrading bacterium VM15C and, hence, PVA degradation were limited by the growth factor, pyrroloquinoline quinone, produced by VM15A. Feeding of a carbon source for VM15A, ethanol, with PVA enhanced pyrroloquinoline quinone production and caused increases in the VM15C population and PVA degradation in a mixed continuous culture. There was an optimum range for PVA degradation of the ethanol concentration, although pyrroloquinoline quinone concentrations in continuous mixed cultures increased with increasing ethanol concentration.     

Yields of Bacterial Cells from Hydrocarbons

A strain of Nocardia and one of Pseudomonas, both isolated on pristane (2,6,10,14-tetramethylpentadecane), gave cell yields of approximately 100% on n-octadecane and pristane. Both organisms grew more rapidly on the n-octadecane than on the pristane. A mixed culture, isolated on 3-methylheptane, whose two components were identified as species of Pseudomonas and of Nocardia, gave approximately 100% cell yields and grew with generation times of about 5 hr on n-heptane, n-octane, and 2-methylheptane. The generation time on 3-methylheptane was 8.6 hr and the cell yield was only 79%. A strain of Pseudomonas isolated from naphthalene enrichments and one from phenanthrene enrichments both gave a cell yield of 50% on naphthalene. The phenanthrene isolate gave a cell yield of 40% on phenanthrene. A Nocardia species isolated on benzene gave a 79% cell yield on benzene. The generation times of the bacteria isolated on aromatic hydrocarbons were related to the solubility of the aromatic hydrocarbons on which they were grown; the more insoluble hydrocarbons gave slower growth.     

Bacteria--degraders of polycyclic aromatic hydrocarbons, isolated from soil and bottom sediments in salt-mining areas

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical and salt producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1-1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp.

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Construction of a Novel Polychlorinated Biphenyl-Degrading Bacterium: Utilization of 3,4'-Dichlorobiphenyl by Pseudomonas acidovorans M3GY

Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4'-dichlorobiphenyl (3,4'-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Kr?ckel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CC1 and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4'-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 mug ml and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction of P. acidovorans M3GY, with the novel capability to utilize 3,4'-DCBP, thus involves the complete use of meta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.     

Characterization of multiple novel aerobic polychlorinated biphenyl (PCB)-utilizing bacterial strains indigenous to contaminated tropical African soils

Contaminated sites in Lagos, Nigeria were screened for the presence of chlorobiphenyl-degrading bacteria. The technique of continual enrichment on Askarel fluid yielded bacterial isolates able to utilize dichlorobiphenyls (diCBs) as growth substrates and six were selected for further studies. Phenotypic typing and 16S rDNA analysis classified these organisms as species of Enterobacter, Ralstonia and Pseudomonas. All the strains readily utilized a broad spectrum of xenobiotics as sole sources of carbon and energy. Growth was observed on all monochlorobiphenyls (CBs), 2,2′-, 2,3-, 2,4′-, 3,3′- and 3,5-diCB as well as di- and trichlorobenzenes Growth was also sustainable on Askarel electrical transformer fluid and Aroclor 1221. Time-course studies using 100 ppm of 2-, 3- or 4-CB resulted in rapid exponential increases in cell numbers and CB transformation to respective chlorobenzoates (CBAs) within 70 h. Significant amounts of chloride were recovered in culture media of cells incubated with 2-CB and 3-CB, suggesting susceptibilities of both 2- and 3-chlorophenyl rings to attack, while the 4-CB was stoichiometrically transformed to 4-CBA. Extensive degradation of most of the congeners in Aroclor 1221 was observed when isolates were cultivated with the mixture as a sole carbon source. Aroclor 1221 was depleted by a minimum of 51% and maximum of 71%. Substantial amounts of chloride eliminated from the mixture ranged between 15 and 43%. These results suggest that some contaminated soils in the tropics may contain exotic micro-organisms whose abilities and potentials are previously unknown. An understanding of these novel strains therefore, may help answer questions about the microbial degradation of polychlorinated biphenyls (PCBs) in natural systems and enhance the potential use of bioremediation as an effective tool for cleanup of PCB-contaminated soils.     

Chemotaxis of Pseudomonas spp. to the polyaromatic hydrocarbon naphthalene.

Two naphthalene-degrading bacteria, Pseudomonas putida G7 and Pseudomonas sp. strain NCIB 9816-4, were chemotactically attracted to naphthalene in drop assays and modified capillary assays. Growth on naphthalene or salicylate induced the chemotactic response. P. putida G7 was also chemotactic to biphenyl; other polyaromatic hydrocarbons that were tested did not appear to be chemoattractants for either Pseudomonas strain. Strains that were cured of the naphthalene degradation plasmid were not attracted to naphthalene.