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.     

The nature of the cellulose-binding domain effects the activities of a bacterial endoglucanase on different forms of cellulose

Abstract The hybrid Pseudomonas cepacia strain JHR22 was tested for its ability to degrade Aroclor 1221 in soil. The influence of supplements—mineral salts and trace elements—on the degradation was investigated. Disapperance of Aroclor 1221 congeners, occurence of metabolites, and release of chloride were measured under different conditions. After 45 days the hybrid organism, strain JHR22, was still present at high numbers in soil, independently of whether the soil had been sterilized prior to inoculation or not. There was only a minor difference in degradation efficiency between sterilized and untreated soil with about 70% release of chloride when 10⁷ cells/g soil were inoculated. The whole hybrid pathway, originating from three different strains, was found to be stable under the conditions tested. Mineral salts did not significantly affect the degradation rate or survival of the hybrid strain.     

Catabolic characteristics of biphenyl-utilizing isolates which cometabolize PCBs

As there are at least three types of bacteria involved in the aerobic mineralization of polychlorinated biphenyls (PCBs), this study was undertaken to determine what catabolic features are lacking in biphenyl-degraders and to determine if chlorobenzoate- and chloroacetate-utilizing bacteria are as indigenous to soil as biphenyl-degraders. Bacteria were tested for their ability to utilize chlorinated acids and to cometabolize Aroclor 1254 and dibenzo-p-dioxane (dioxin). The broad and variable substrate specificity of the biphenyl dioxygenase among strains was noted by the range of <1 to 53% cometabolism of total PCB congeners and by the oxidation of dioxin, which was not a growth substrate. Growth on chloroalkanoic acids was more frequent with 2-chloropropionate (87% of all strains), 3-chloropropionate (72%), 4-chlorobutyrate (66%), and less frequent (28%) withtrans-3-chlorocrotonate. However, only one strain,Pseudomonas fluorescens K3, could utilize chloroacetate. No biphenyl-utilizers grew on 2- or 4-chlorobenzoate, and only five strains grew on 3-chlorobenzoate. Acetate and benzoate-utilizers were found in all three soils tested at levels near 10⁶/g, whereas chloroacetate- or chlorobenzoate-utilizers were not detected. The inability of biphenyl-degraders to dehalogenate the products of PCB cometabolism is clearly unrelated to metabolism of saturated chloroaliphatic acids, with the notable exception of chloroacetate, since most strains grew on them. Thus, the inability to utilize chloroacetate, a central intermediate in the meta fission pathway, may be relevant to the incomplete catabolism of PCBs by biphenyl-utilizers.     

The degradation of biphenyl and chlorobiphenyls by mixed bacterial cultures

Abstract Pseudomonas sp. HV3 grows on naphthalene but not on biphenyl, as the sole source of carbon. When the cells of Pseudomonas sp. HV3 grown on naphthalene were shaken with biphenyl as the carbon source in a mineral salt solution, a yellow metabolite identified as the meta -cleavage product of biphenyl was excreted. The degradation of biphenyl stopped here, but was completed if either 2-methyl-4-chlorophenoxy acetic acid (MCPA)-degrading mixed culture or a Nocardia strain was added to the growth solution. Neither of these uses naphthalene or biphenyl as growth substrate. The mixed culture of Pseudomonas sp. HV3 and Nocardia sp. also degrades the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221. A yellow metabolite was likewise produced in the degradation, and sometimes two different peaks of the yellow metabolite were observed. The gas chromatography-mass spectrometry (GC-MS) analyses showed that 40–87% of Aroclor 1221 was degraded during an incubation time of 6–21 days. Chlorobenzoic acids were found as metabolites.     

Microorganisms Degrading Polychlorinated Biphenyls

Four strains belonging to the genus Bacilluscapable of degrading polychlorinated biphenyls (PCBs) were isolated by screening collection strains of soil bacteria degrading an organochlorine pesticide, hexachlorocyclohexane (HCCH). A method for production of tritium-labeled PCBs was developed. Consumption and degradation of PCBs by the soil bacterial strains selected were studied using tritium-labeled PCBs and GLC. It was demonstrated that PCBs are degradable both in culture media and in model soil samples.     

Cyclodextrins enhance the aerobic degradation and dechlorination of low-chlorinated biphenyls

Summary The aerobic dechlorination of 4-chlorobiphenyl and of Aroclor 1221 by Pseudomonas sp. CPE1 strain was enhanced by the presence of hydroxypropyl--cyclodextrin in the medium. The best dechlorination results were obtained when 4-chlorobiphenyl or Aroclor 1221 and the cyclodextrin were used at the molar ratios 1:1 and 1:1.5. This agent can be adopted to enhance the efficiency of PCB degradation tests.     

Isolation and Characterization of a Mixed Culture That Degrades Polychlorinated Biphenyls

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

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     

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.     

Eco-physiological responses of Polytrichum commune to soil contamination by polychlorinated biphenyls

 通过微体繁殖技术在多氯联苯(PCBs)污染土壤基质上进行大金发藓(Polytrichum commune)的室内培养, 研究了不同浓度(5、10和20 mg·kg^–1)低氯PCBs (Aroclor 1242)和高氯PCBs (Aroclor 1254)对大金发藓生理生态指标的影响。经6个月的培养, 大金发藓的密度和盖度分别达93%和50株·cm^–2以上, PCBs处理组与对照组相比无显著差异, 表明PCBs对大金发藓茎叶碎片再生成新植株体的能力没有产生不利影响。大金发藓鲜质量和株高随低氯PCBs (Aroclor 1242)浓度增加而增加、随高氯PCBs (Aroclor 1254)浓度增加而减小, 但均高于对照, 表明PCBs处理对大金发藓的生长具有一定的促进作用。PCBs处理组大金发藓叶绿素a、b以及叶绿素a + b含量较对照组有所增加, 叶绿素a/b值与对照组相比基本没有变化。PCBs处理组大金发藓膜脂过氧化产物丙二醛含量和超氧化物歧化酶活性与对照组相比无显著差异, 谷胱甘肽含量较对照组显著增加, 表明谷胱甘肽在大金发藓体内活性氧清除过程中起重要作用。总体来看, 大金发藓能在所设浓度的PCBs范围内正常生长, 对PCBs有较强的耐性。     

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)。     

Bioremediation of Aroclor 1242 by a consortium culture in marine sediment microcosm

Plant terpenes have proven to be effective in stimulation of polychlorinated biphenyls (PCBs) biodegradation in soil systems. However, data on the application of plant terpenes in marine sediments contaminated with PCBs remains limited. The aim of this study was to ascertain the roles of a PCB degrading consortium and plant terpenes in stimulation of PCB biodegradation in marine sediments. The consortium culture 1-2Mix (strains 1-2M and 1-2T in commensalism), a utilizer of biphenyl and a natural substrate was enriched and isolated from marine sediments from the Busan coast, South Korea. PCB degradation by this culture was shown to be more effectively induced by tangerine peel extract than other known substrates (limonene, pinene, and cymene). Coastal sediment microcosms inoculated with 1-2Mix were set up to elucidate the effect of the consortium and plant terpenes on degradation of Aroclor 1242. After four weeks, the highest removal rates of PCBs, compared with the control (autoclaved sediment and no inoculation of 1-2Mix), were observed in order of the inducers tested; biphenyl (71.1%), tangerine peel extract (69.5%), surfactant (66.0%), and limonene (63.0%). Bioaugmentation effect was doubled in the presence of natural substrates such as tangerine peel extract and limonene, indicating effectiveness of these substrates in biostimulation. It was concluded that the tangerine peel extract could replace biphenyl as a feasible induction substrate for effective remediation of PCBs in the marine sediment.     

水稻土泥浆体系中多氯联苯的微生物厌氧脱氯

考察了厌氧水稻土泥浆体系中高氯代多氯联苯混合物Aroclor1260的脱氯过程,并对体系中的微生物群落结构变化进行分析.结果表明： Aroclor1260可在厌氧水稻土泥浆体系中发生脱氯,经过128 d,总消减率达到55.5%,在泥浆体系中引入驯化的脱氯富集培养体反而使脱氯效果下降,消减率为46.9%.Aroclor1260的主要脱氯过程发生在五、六、七氯联苯,其中七氯联苯脱氯过程最显著,五氯联苯作为脱氯产物有一定累积.有机物厌氧发酵产生的H₂会被脱氯过程所消耗,从而将体系中的氢分压维持在较低水平,抑制产甲烷过程而保证脱氯过程的持续进行.不同条件和培养方式驯化得到的微生物群落结构差异较大,富集培养体引入可能导致其与原体系中脱氯相关菌群竞争,从而改变体系原有菌群结构,这可能是导致其脱氯效率下降的原因.     

Degradation of aroclor 1242 dechlorination products in sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. strain RHA1(fcb)

Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (10(4) or 10(6) cells g(-1) sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase.     

Degradation of 3-chlorobenzoate by thermophilic micro-organisms

Thermophilic (75°C), anaerobic biodegradation of chlorobenzoates was investigated using different inocula from geothermal and non-geothermal environments. Microbial dehalogenation of 3-chlorobenzoate (0·5 mmol l⁻¹) was achieved by two mixed cultures growing anaerobically at 75°C. One culture consisted of a facultative anaerobe and two obligate anaerobes, one of which was a methanogen, isolated from terrestrial sediments from hot springs in New Zealand. The other culture, derived from a non-geothermal environment, consisted of a Clostridium spp. and a non-spore-forming obligate anaerobe. No degradation of either 2-chlorobenzoate or 4-chlorobenzoate was achieved by these thermophilic cultures over the same time period. This is the first reported biotransformation of this chlorinated aromatic at a temperature of 75°C.     

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.     

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

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

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

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

Degradation of Aroclor 1242 Dechlorination Products in Sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. Strain RHA1(fcb)

Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (10⁴ or 10⁶ cells g⁻¹ sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase.     

Functional Establishment of Introduced Chlorobenzoate Degraders Following Bioaugmentation with Newly Activated Soil

Introduced degraders often do not survive when applied to polluted sites; however, the potential for successful bioaugmentation may be increased if newly activated soil (containing indigenous degrader populations recently exposed to the contaminant) or potentially active soil (containing indigenous degrader populations not previously exposed to the contaminant) is used as the inoculant. To investigate this concept, Madera and Oversite soils were amended with 0 or 500 micrograms of 2-, 3-, or 4-chlorobenzoate per gram soil. The Madera degraded 2-chlorobenzoate while the Oversite degraded 3- and 4-chlorobenzoate. After 22 days of incubation, non-active soils that had not degraded chlorobenzoate were bioaugmented with the appropriate activated soil that had been exposed to and degraded chlorobenzoate. Thus, Oversite soil that had not degraded 2-chlorobenzoate was bioaugmented with Madera soil that had degraded 2-chlorobenzoate. Likewise, Madera soil that had not degraded 3- or 4-chlorobenzoate was bioaugmented with the Oversite soil that had degraded 3- or 4-chlorobenzoate. Additionally, the non-active soils were bioaugmented with the corresponding potentially active soils. The Oversite soil amended with activated Madera soil degraded the 2-chlorobenzoate within 3 days of bioaugmentation. The Madera soil amended with activated Oversite soils degraded the 3- and 4-chlorobenzoate within 20 and 6 days, respectively. Large degrader populations developed in microcosms bioaugmented with activated soil, and shifts in the 3- and 4-CB degrader community structures occurred following bioaugmentation. In contrast, bioaugmentation with potentially active soil did not impact degradation. The results indicate the potential for bioaugmentation with newly activated soil to enhance contaminant degradation.