Degradation Of Polychlorinated Biphenyls Research Articles

Photochemical of Polychlorinated biphenyl by the photolysis and solvent

Polychlorinated biphenyls (PCBs) are one group of persistent organic pollutants (POPs) that are of international concern because of global distribution, persistence, and toxicity. Removal of these compounds from the environment remains a very difficult challenge because the compounds are highly hydrophobic and have very low solubility in water. The photochemical reactor was of annular geometry with a cylindrical low-pressure mercury lamp. The whole Lamp was immersed in a reactor thermostat controlling the temperature at 32 ± 2 °C. The Polychlorinated biphenyls (PCBs) were analyzed by GC/ECD. The degradation of PCBs in terms of one, two and three lamp was 91.9%, 92.7% and 93% respectively. The degradation of PCBs in terms of use of 10% and 20% of total volume of solution of H 2 O 2 were 88.8% and 93% respectively. The degradation of PCBs in terms of ratio to ethanol with oil transformer in 1:1, 2:1 and 3:1 was 83.4%, 92.5% and 93% respectively. The experiments show that UVC-photolysis of H 2 O 2 leads to a degradation efficiency of PCBs in the presence of ethanol. @ JASEM J. Appl. Sci. Environ. Manage . December, 2010, Vol. 14 (4) 107 - 112

Expression inEscherichia coliof Biphenyl 2,3-Dioxygenase Genes from a Gram-Positive Polychlorinated Biphenyl Degrader,Rhodococcus jostiiRHA1

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.

Dechlorination of polychlorinated biphenyls using magnesium and acidified alcohols

Polychlorinated biphenyls (PCBs) were widely used in industry until their regulation in the 1970s. However, due to their inherent stability, they are still a widespread environmental contaminant. A novel method of degradation of PCBs (via hydrodehalogenation) has been observed using magnesium powder, a carboxylic acid, and alcohol solvents and is described in this paper. The rates of degradation were determined while varying the type of acid (formic, acetic, propionic, butyric, valeric, benzoic, ascorbic, and phosphoric), the amount of magnesium from 0.05 to 0.25g, the amount of acetic acid from 0.5 to 50μL and the concentration of PCB-151 from 0.1 to 50μg/mL, as well as the alcohol solvent (methanol, ethanol, propanol, butanol, octanol, and decanol). The results of these studies indicate that the most rapid PCB dechlorination is achieved using a matrix consisting of at least 0.02gMg/mL ethanol, and 10μL acetic acid/mL ethanol in which case 50ng/μL of PCB-151 is dechlorinated in approximately 40min.

The effect of hydrogen peroxide and solvent on photolysis of PCBs to reduce occupational exposure

Background: Polychlorinated biphenyls (PCBs) are toxic bio-accumulate components and may increase risk of adverse effects on human health and the environment. For different social, technical and economic reasons, significant quantities of PCBs contaminated transformer oil are still in use or storied. The study aimed to determine the effect of hydrogen peroxide and solvent on photolysis of PCBs to reduce occupational exposure. Methods: The photochemical annular geometry (500 ml volume) reactor was designed with a cylindrical low-pressure mercury lamp (UV-C Cathodeon TUV 6WE) with emission at 254 nm. The radiant power emitted by the lamp was 6w. The whole lamp was immersed in a reactor thermostat controlling of temperature at 32 ± 2 oC. The PCBs was determined using GC/ECD and data was analyzed by SPSS. Results: Degradation of total PCBs in terms of %10 and %20 of volume of H2O2 were 41%, 75% and 94%, respectively. The degradation of total PCBs in terms of ratio to solvent with oil transformer in 1:1, 2:1 and 3:1 was 61%, 75% and 94%, respectively. Conclusion: Our study show that UV-C photolysis of H2O2 leads to a degradation efficiency of PCBs only in the presence of isooctane, therefore indicating that the intermediates formation after ethanol oxidation are able to initiate PCBs degradation.

Expression, purification and functional characterization of a recombinant 2,3-dihydroxybiphenyl-1,2-dioxygenase from Rhodococcus rhodochrous

A 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene from a Rhodococcus sp. strain, named RrbphCI and involved in the degradation of polychlorinated biphenyls (PCBs), was synthesized. RrbphCI was expressed in Escherichia coli and its encoded enzyme was purified. SDS-PAGE analysis indicated that the size of the protein encoded by RrbphCI was about 32 kDa. The activity of the 2,3-DHBP dioxygenase was 82.8 U/mg when the substrate was 2,3-DHBP, with optimum pH 8.0 at 30°C, and optimum temperature was 40°C at pH 8.0. The RrbphCI gene was transformed into Pseudomonas putida strain EG11, to determine the ability of the enzyme to degrade 2,3-DHBP. The wild type EG11 degraded 61.86% of supplied 2,3-DHBP and the transformed EG11 (hosting the RrbphCI gene) utilized 52.68% after 2 min of treatment at 30°C. The overexpressed and purified enzyme was able to degrade 2,3-DHBP. The 2,3-DHBP dioxygenase is a key enzyme in the PCB degradation pathway. RrbphCI and its encoded 2,3-DHBP dioxygenase may have transgenic applications in bioremediation of PCBs.

폴리염화비페닐류의 생분해 우수 백색부후균 선발 및 분해율 분석

본 연구에서는 다양한 목재부후균의 polychlorinated biphenyls (PCBs) 생분해 가능성을 구명하고자 7종의 백색부후균을 이용하여 PCB 혼합물 중 가장 분해가 어렵다고 알려진 Aroclor 1254와 1260을 대상으로 분해력이 우수한 목재부후균을 선발하였다. 그리고 선발된 목재부후균을 이용하여 GC 분석을 통해 Aroclor 1254, 1260의 분해율을 조사하였다. 고체배지 저항성 테스트를 통해 우수분해 균주로 선발된 Cystidodontia isubellina 는 다른 균주에 비해 균사생장량의 억제가 가장 적었으며 상대적으로 생장속도도 빠른 특징을 나타냈다. 선발된 C. isubellina에 의한 Aroclor 1254와 1260의 분해율 분석 결과, Aroclor 1254배양 13일째 57.57%로 가장 높은 분해율을 나타냈다. Aroclor 1260 역시 배양일수가 증가함에 따라 분해율 역시 증가하는 경향을 보였으며, 배양 13일째 49.43%로, 두 혼합물 모두 기존의 다른 연구에서보다 높은 분해율을 나타냈다. In this study, the possibility of biodegradation of polychlorinated biphenyls (PCBs) by various white rot fungi was evaluated, and outstanding white rot fungi for the degradation of PCBs were selected. Seven white rot fungi were used to degrade Aroclor 1254 and 1260, which are widely considered to be toxic and difficult to degrade. And the degradation rates of Aroclors by selected white rot fungi were performed by GC analysis. Through the resistance test of white rot fungi on different concentrations of PCBs, the inhibition of mycelial growth of Cystidodontia isubellina was much less than that of others, and this fungus grew faster than others, relatively. Based on this result, it was considered that C. isubellina was selected as degrading fungus for Aroclors. As a result of biodegradation rate of Aroclors by Cystidodontia isubellina, the degradation rate of Arolor 1254 was reached to 57.57% in 13 days, which showed very high degradation rate. Also the degradation rate of Aroclor 1260 by C. isubellina had a tendency of increasing along with increasing incubation day. Maximal degradation rate of Aroclor 1260 was 49.43% at 13 days. Based on this results, it indicated that in comparison with a previous study, high degradation rate was obtained by C. isubellina.

Congener Selectivity During Polychlorinated Biphenyls Degradation by Enterobacter sp. LY402

The relationship between the selectivity of a particular polychlorinated biphenyls (PCBs) congener and its biodegradability under the same concentration, especially by Enterobacter sp. LY402, is less well studied. To measure congener selectivity of Enterobacter sp. LY402, several influencing factors were studied. The results showed LY402 effectively degraded coplanar 3,4,3',4'-chlorobiphenyl (CB) at a concentration of 0.05 μM, but not 0.5 μM. The degradation rates of 2,4,5,2',3'-CB and 2,4,5,2',4',5'-CB were increased significantly when the sample constituents were changed from 12 to 5 congeners or to one congener. This indicated that bioremediation of individual congener was affected by other congeners present in the mixture. Moreover, for PCBs containing one chlorine on each phenyl ring, the reactivity preference of LY402 was 2,2'-CB ≥ 3,3'-CB ≫ 4,4'-CB. For two ortho chlorines congeners of PCBs, 2,2'-CB was degraded faster than 2,6-CB. Although 2,6-CB and 4,4'-CB were poorly degraded, the addition of one (i.e., 2,4,4'-CB and 2,6,3'-CB) or two more chlorines (i.e., 2,4,2',4'-CB) on the phenyl ring significantly increased their biodegradability. In addition, comparing the two congeners of ortho-meta-chlorinated biphenyl, 2,3,2',3'-CB with neighbor meta chlorines was degraded slower than 2,5,2',5'-CB with interval meta chlorines. All these indicated that the transformation rates of PCBs were not consistent with the number of chlorines, and PCBs containing the same numbers of chlorines but at different positions also resulted in different conversions. In principle, the extents of effect caused by the position of chlorine substituents on the degradation of PCBs by LY402 were ortho- > meta- > para-CB. In conclusion, the congener selectivity of LY402 was determined by many factors, including the composition of the congeners, their concentrations in the mixture and location and number of chlorine substituents on the phenyl rings.

Pilot-Plant Study of the PCB Degradation at Ambient Temperature and Pressure

A continuous pilot plant for the degradation of polychlorinated biphenyls (PCBs) by the palladium on carbon (Pd/C)-catalyzed hydrogenation in the presence of triethylamine was designed and constructed. Both undiluted PCBs obtained from a capacitor and diluted PCBs with desulfurized trans oil were smoothly decomposed at ambient temperature and pressure. Desulfurization of the trans oil was found to be essential for the efficient degradation due to the possible deactivation of the Pd/C by catalysis poisoning due to the sulfur-containing materials in the oil. The combined use of the present degradation method and the catalytic desulfurization technology for the purification of gasoline and kerosene could be used in practical applications.

Dual Two-Component Regulatory Systems Are Involved in Aromatic Compound Degradation in a Polychlorinated-Biphenyl Degrader, Rhodococcus jostii RHA1

A Gram-positive polychlorinated-biphenyl (PCB) degrader, Rhodococcus jostii RHA1, degrades PCBs by cometabolism with biphenyl. A two-component BphS1T1 system encoded by bphS1 and bphT1 (formerly bphS and bphT) is responsible for the transcription induction of the five gene clusters, bphAaAbAcAdC1B1, etbAa1Ab1CbphD1, etbAa2Ab2AcD2, etbAdbphB2, and etbD1, which constitute multiple enzyme systems for biphenyl/PCB degradation. The bphS2 and bphT2 genes, which encode BphS2 and BphT2, virtually identical to BphS1 (92%) and BphT1 (97%), respectively, were characterized. BphS2T2 induced the activation of the bphAa promoter in a host, Rhodococcus erythropolis IAM1399, in the presence of a variety of aromatics, including benzene, toluene, ethylbenzene, xylenes, isopropylbenzene, and chlorinated benzenes, as effectively as BphS1T1. The substrate spectrum of BphS2T2 was the same as that of BphS1T1, except for biphenyl, which is a substrate only for BphS1T1. BphS2T2 activated transcription from the five promoters of biphenyl/PCB degradation enzyme gene clusters as effectively as BphS1T1. The targeted disruptions of the bphS1, bphS2, bphT1, and bphT2 genes indicated that all these genes are involved in the growth of RHA1 on aromatic compounds. The hybrid system with bphS1 and bphT2 and that with bphS2 and bphT1 were constructed, and both systems conducted induced activation of the bphAa promoter, indicating cross-communication. These results indicated that RHA1 employs not only multiple enzyme systems, but also dual regulatory systems for biphenyl/PCB degradation. Comparison of the sequences, including bphS2T2, with the bphS1T1-containing sequences and the corresponding sequences in other rhodococcal degraders suggests that bphS2T2 might have originated from bphS1T1.

Effect of Cocontaminants on the Remediation of PCB-Impacted Soils by Hydrogen Peroxide

The effects of cocontaminants, such as extractable organics and transformer oil (TO) on the remediation of polychlorinated biphenyl (PCB) impacted soils by hydrogen peroxide ( H2 O2 ) , have been investigated. Presence of transformer oil is found to adversely affect the PCB degradation. About 20 and 60% of Aroclor 1254 were remediated by 15 and 30% H2 O2 , respectively, in soils which did not contain TO. When TO was introduced, no Aroclor 1254 degradation was observed with 15% H2 O2 and a limited amount of about 20% of Aroclor 1254 degraded with 30% H2 O2 treatment. The impact of cocontaminants on major Aroclor 1254 congeners 101, 87, 110, 118, 105, and 138 were also found to be limited in the presence of TO.

Genetically modified Pseudomonas biosensing biodegraders to detect PCB and chlorobenzoate bioavailability and biodegradation in contaminated soils

Whole cell microbial biosensors offer excellent possibilities for assaying the complex nature of the bioavailable and bioaccessible fraction of pollutants in contaminated soils, which currently cannot be easily addressed. This paper describes the application and evaluation of three microbial biosensor strains designed to detect the bioavailability and biodegradation of PCBs (and end-products) in contaminated soils and sediments. Polychlorinated biphenyls (PCBs) are considered to be one of the most wide spread, hazardous and persistent pollutants. Herein we describe that there was a positive correlation between the PCB levels within the samples and the percentage of biosensor cells that were expressing their reporter gene; gfp. Immobilisation of the biosensors in calcium alginate beads allowed easy and accurate detection of the biosensor strains in contaminated soil and sludge samples. The biosensors also showed that PCB degradation activity was occurring at a much greater level in Pea inoculated planted soil compared to inoculated unplanted soil indicating rhizoremediation (the removal of pollutants by plant root associated microbes) shows considerable promise as a solution for removing organic xenobiotics from the environment.

β-cyclodextrin enhanced phytoremediation of aged PCBs-contaminated soil from e-waste recycling area

The objective in the first phase of this study was to screen four plant species (alfalfa, ryegrass, tall fescue and rice) for phytoremediation of aged polychlorinated biphenyl (PCB)-contaminated soil from an electronic and electric waste (e-waste) recycling site. Glucose, biphenyl and three surfactants (TritonX-100, randomly methylated-beta-cyclodextrins and beta-cyclodextrin) were used to enhance the phytoremediation process. During the second phase, the focus was rhizosphere characteristics and plant uptake to investigate the mechanism of PCB removal from soil. In the first phase, all the tested plant species showed a significantly greater PCB removal percentage compared to the unplanted controls, while different amendments showed no significant difference. The most effective plant (ryegrass) combined with beta-cyclodextrin was selected for further studies. During the rhizosphere characteristics and plant uptake study, the highest PCB removal percentage (38.1%) was observed in the ryegrass planted soil when beta-cyclodextrin was amended at 1.0% (w/w). The presence of plants significantly increased the biological activity (microbial counts and enzyme activity) of both beta-cyclodextrin amended and non-amended soils. Higher levels of PCB removal were closely related to greater microbial counts and soil enzyme activities by correlation analysis. After 120 days of plant growth, ryegrass accumulated 708.7-820.1 ng PCBs/g in the root and 71.7-110.8 ng PCBs/g in the shoot, resulting in about 0.08% PCBs removal from soil. It was concluded that high PCB degradation was due to the increased PCB bioavailability as well as biostimulation of microbial communities after plantation and beta-cyclodextrin addition. Furthermore, results suggested that PCB removal was mainly contributed by microbial degradation rather than plant uptake or abiotic dissipation.

Enhanced removal of polychlorinated biphenyls from alfalfa rhizosphere soil in a field study: The impact of a rhizobial inoculum

Polychlorinated biphenyls (PCB) are persistent pollutants in soil environments where they continue to present considerable human health risks. Successful strategies to remediate contaminated soils are needed that are effective and of low cost. Bioremediation approaches that include the use of plants and microbial communities to promote degradation of PCB have significant potential but need further assessment under field conditions. The effects of growth of alfalfa ( Medicago sativa L.) and inoculation with a symbiotic nitrogen fixing bacterium ( Rhizobium meliloti) on the removal of polychlorinated biphenyls (PCB) from rhizosphere soil were evaluated in a field experiment. The initial PCB content of the soil ranged from 414 to 498 µg kg − 1. PCB removal for the rhizosphere soil was enhanced in the planted treatments, an average of 36% decrease in PCB levels compared to a 5.4% decrease in the unplanted soil, and further enhanced when plants were inoculated with the symbiotic Rhizobium (an average of 43% decrease) when evaluated at 90 days after planting. Plant biomass production was higher in the inoculated treatment. The total PCB content was increased from 3.30 µg kg − 1 to 26.72 µg kg − 1 in plant shoots, and from 115.07 µg kg − 1 to 142.23 µg kg − 1 in roots in the inoculated treatment compared to the planted treatment. Increased colony forming units (cfu) of total heterotrophic bacteria, biphenyl-degrading bacteria and fungi were observed in the rhizosphere of inoculated plants. PCB removal from the rhizosphere soil was not significantly correlated with the direct PCB uptake by the plants in any of the treatments but was significantly correlated with the stimulation of rhizosphere microflora. Changes in the soil microbial community structure in the planted and inoculated treatment were observed by profiling of bacterial ribosomal sequences. Some bacteria, such as Flavobacterium sp., may have contributed to the effective degradation of PCB and deserve further investigation.

Phytoremediation of polychlorinated biphenyls: new trends and promises.

Transgenic plants and associated bacteria constitute a new generation of genetically modified organisms for efficient and environment-friendly treatment of soil and water contaminated with polychlorinated biphenyls (PCBs). This review focuses on recent advances in phytoremediation for the treatment of PCBs, including the development of transgenic plants and associated bacteria. Phytoremediation, or the use of higher plants for rehabilitation of soil and groundwater, is a promising strategy for cost-effective treatment of sites contaminated by toxic compounds, including PCBs. Plants can help mitigate environmental pollution by PCBs through a range of mechanisms: besides uptake from soil (phytoextraction), plants are capable of enzymatic transformation of PCBs (phytotransformation); by releasing a variety of secondary metabolites, plants also enhance the microbial activity in the root zone, improving biodegradation of PCBs (rhizoremediation). However, because of their hydrophobicity and chemical stability, PCBs are only slowly taken up and degraded by plants and associated bacteria, resulting in incomplete treatment and potential release of toxic metabolites into the environment. Moreover, naturally occurring plant-associated bacteria may not possess the enzymatic machinery necessary for PCB degradation. To overcome these limitations, bacterial genes involved in the metabolism of PCBs, such as biphenyl dioxygenases, have been introduced into higher plants, following a strategy similar to the development of transgenic crops. Similarly, bacteria have been genetically modified that exhibit improved biodegradation capabilities and are able to maintain stable relationships with plants. Transgenic plants and associated bacteria bring hope for a broader and more efficient application of phytoremediation for the treatment of PCBs.

The effects of individual PCB congeners on the soil bacterial community structure and the abundance of biphenyl dioxygenase genes

Polychlorinated biphenyls (PCBs) are toxic environmental contaminants that represent a class of 209 congeners characterized by different degrees of chlorination and substitution patterns. Most of experimental studies about microbial degradation of PCBs have been conducted on PCB mixtures, even though evidence accumulated in bacteria and other organisms shows that exposure to different congeners may have different biological effects. Microcosm experiments were conducted using aerobic agitated soil slurries individually exposed to PCB congeners with different degrees of chlorination: PCB-3, 15, 28, and 77, and the commercial mixture Aroclor 1242. After four weeks of incubation, PCBs were analyzed by gas chromatography/mass spectrometry (GC/MS) showing different transformation extents: With the exception of PCB-15 that was not significantly transformed (7%), biodegradation rates decreased with the degree of chlorination, from 75% for PCB-3 to 22% for PCB-77 and Aroclor 1242. The bacterial abundance, as measured by colony counting and 16S rDNA quantification by real-time PCR, was lower (of about 40%) in soil microcosms exposed to the higher-chlorinated congeners, PCB-28, PCB-77, and Aroclor 1242, as compared to non-exposed soils and soils exposed to the lower-chlorinated congeners, PCB-3 and PCB-15. The relative abundance of different taxonomic groups, as determined by real-time PCR, revealed an increase of β-Proteobacteria and Actinobacteria in all microcosms exposed to PCBs, as compared with non-exposed soil. In addition, exposure to PCB-77 and Aroclor 1242 resulted in a higher abundance of α-Proteobacteria and Acidobacteria. Globally, these results suggest that exposure to PCBs (and especially to higher-chlorinated congeners and Aroclor 1242) selected bacterial groups involving most known PCB degraders, i.e., β-Proteobacteria and Acidobacteria. The quantification of biphenyl dioxygenase (BPH) genes — involved in the aerobic degradation of PCBs — using real-time PCR showed that exposure to all PCB congeners and Aroclor 1242 resulted in a marked increase of two out of the four BPH genes tested, similarly suggesting the selection of PCB-degrading bacteria. This paper showed that exposure to different PCB congeners leads to different structures of the soil bacterial community and BPH genes expression patterns.

Environmentally relevant parameters affecting PCB degradation: carbon source- and growth phase-mitigated effects of the expression of the biphenyl pathway and associated genes in Burkholderia xenovorans LB400

The principal means for microbial degradation of polychlorinated biphenyls (PCBs) is through the biphenyl pathway. Although molecular aspects of the regulation of the biphenyl pathway have been studied, information on environmental facets such as the effect of alternative carbon sources on (polychlorinated) biphenyl degradation is limited. Here we explore the effect of environmental conditions (e.g., carbon source and growth phase) on the variation in PCB degradation profiles of Burkholderia xenovorans LB400. Genome-wide expression patterns reveal 25 genes commonly up-regulated during PCB degradation and growth on biphenyl to be upregulated in the transition to stationary phase (relative to growth on succinate) including two putative detoxification pathways. Quantitative reverse transcription PCR (Q-RT-PCR) analysis of the upper biphenyl pathway (bphA, bphD, and bphR1), and detoxification genes in response to environmental conditions suggest associated regulation of the biphenyl pathway and chloroacetaldehyde dehydrogenase. The response of genes in the upper biphenyl pathway to carbon source competition and growth phase reveals inhibition of the biphenyl pathway by PCBs. Although PCBs are not degraded during growth on succinate with PCBs, expression data indicate that the biphenyl pathway is induced, suggesting that post-transcriptional regulation or active transport of biphenyl maybe limiting PCB degradation. Identification of the involvement of peripheral pathways in degradation of PCBs is crucial to understanding PCB degradation in an environmental context as bacteria capable of biodegradation experience a range of carbon sources and growth phases.

Characterization of an Aldolase−Dehydrogenase Complex That Exhibits Substrate Channeling in the Polychlorinated Biphenyls Degradation Pathway

An aldolase and dehydrogenase complex from the polychlorinated biphenyl degradation pathway of the bacterium Burkholderia xenovorans LB400 was purified. The aldolase, BphI, had the highest activity with Mn(2+) as the cofactor and was able to transform 4-hydroxy-2-oxopentanoate and 4-hydroxy-2-oxohexanoate to pyruvate and acetaldehyde or propionaldehyde with similar specificity constants. Aldolase activity was competitively inhibited by the pyruvate enolate analogue, oxalate, with a K(ic) of 0.93 microM. The pH-rate profiles suggested the involvement of a pK(a) 7.7 catalytic base in the reaction mechanism. BphI activity was activated 15-fold when substrate turnover was occurring in the dehydrogenase, BphJ, which can be attributed partially to nicotinamide coenzyme binding to BphJ. BphJ had similar specificity constants for acetaldehyde or propionaldehyde and was able to utilize aliphatic aldehydes from two to five carbons in length as substrates, although K(m) values for these aldehyes were >20 mM. When 4-hydroxy-2-oxopentanoate was provided as a substrate to the BphI-BphJ complex in a coupled enzyme assay, no lag in the progress curve of BphJ was observed. When 1 mM propionaldehyde was added exogenously to a reaction mixture containing 0.1 mM 4-hydroxy-2-oxopentanoate, 95% of the CoA esters produced was acetyl CoA. Conversely, 99% of the CoA esters produced was propionyl CoA when a 10-fold molar excess of exogenous acetaldehyde was added in a reaction mixture containing 4-hydroxy-2-oxohexanoate. These results demonstrate that acetaldehyde and propionaldehyde, products of the BphI reaction, are not released in the bulk solvent but are channeled directly to the dehydrogenase.

Mechanism of the degradation of individual PCB congeners using mechanically alloyed Mg/Pd in methanol

Polychlorinated biphenyls (PCBs) are a continuing concern in the environment, although legislation restricting the production and use of PCBs was introduced more than 30 years ago. The combination of zero-valent metals and hydrogenation catalysts has been proven effective in the remediation of PCBs, although the exact mechanism of degradation is not known as of yet. The use of mechanically alloyed zero-valent magnesium and palladium (on graphite) has shown great success in the dechlorination of PCBs. Knowing the mechanism for this dechlorination would be helpful in optimizing the bimetallic Mg/Pd for use in the field. A variety of experiments have been performed on a single PCB congener (PCB-151, 2,2′,3,5,5′,6-polychlorobiphenyl) in an attempt to determine the mechanism by which the degradation occurs. The studies are carried out in methanol to mimic the solvent system which will be used in field applications. Results of these studies have suggested three possible mechanisms, all of which include the removal of the chlorine atom by a hydrogen as the rate-limiting step, varying only in the exact nature of the hydrogen species (radical, hydride, or “hydride-like” radical). Brief: A series of studies has suggested three possible mechanistic pathways for the degradation of PCBs in methanol by Mg/Pd.

Bioinformatic analysis and in vitro site-directed mutagenesis of conserved amino acids in BphK LB400, a specific bacterial glutathione transferase

Glutathione transferases [GSTs: EC 2.5.1.18)] are ubiquitous multifunctional prokaryotic and eukaryotic enzymes involved in the cellular detoxification and excretion of a large variety of compounds. However, our understanding of the role of bacterial GSTs in metabolism is still in its infancy. The association of bacterial GST DNA with other genes involved in degradation of toxic pollutants, including polychlorinated biphenyls (PCBs), indirectly suggests a role for bacterial GSTs in biodegradation. Previously, in this laboratory, a specific bacterial GST, BphK LB400 isolated from Burkholderia xenovorans LB400, was shown to be capable of dehalogenating chlorinated organic substrates rendering them less toxic. However, little is known about the specific amino acids in BphK LB400 involved in catalysis in vitro. In this study, bioinformatic analysis of BphK LB400 and other bacterial GSTs, including PCB degraders, identified a number of amino acids that were identical in all bacterial GST sequences analysed. Two amino acids, Cys10 and His106, were selected for in vitro site-directed mutagenesis studies. In vitro GST activity assay results suggest that these two amino acids play a role in determining the catalytic activity of BphK LB400. Studies of bacterial cell extracts expressing BphK LB400 (wildtype and mutant) identified a specific mutant, Cys10Phe, with increased GST activity towards 1-chloro-2,4-dinitrobenzene (the model substrate for GSTs). BphK LB400 (mutant) with increased activity towards toxic chlorinated organic compounds could have potential for bioremediation of contaminated soil in the environment.

The effect of bacterial glutathione S‐transferase on morpholine degradation

Glutathione S-transferases (GSTs) constitute a large family of enzymes that catalyze the addition of glutathione to endogenous, or xenobiotic, often toxic electrophilic compounds. The effect of this enzyme in facilitating polychlorinated biphenyls degradation has been studied previously. Here the effects of induced cell-free extracts of Acinetobacter calcoaceticus and Pseudomonas aeruginosa (grown on hexadecane), and E. coli BL21 (induced with pGEX-2T plasmid on isothiopropylgalactoside) were recruited to facilitate morpholine degradation by Mycobacterium and were compared with non-induced strains. The results showed that all induced strains had significantly more GST activity compared to non-induced ones, and the strain with most GST activity, A. calcoaceticus BS, removed morpholine faster. Eukaryotic GST gene expressed in E. coli BL21 also could facilitate morpholine degradation by Mycobacterium, The same experiments performed with cell-free extracts of non-induced cells did not show any significant effects on morpholine removal. These results showed that there is a correlation between GST activity and acceleration of morpholine degradation.