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.     

Biphenyl uptake by psychrotolerant Pseudomonas sp. strain Cam-1 and mesophilic Burkholderia sp. strain LB400

We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400.     

Novel maltotriose esters enhance biodegradation of Aroclor 1242 by Burkholderia cepacia LB400

The objective of this research was to evaluate the effect of enzymatically synthesized maltotriose fatty acid monoesters (Ferrer, M., et al. 2000 Tetrahedron 56, 4053–4061) on Aroclor 1242 solubilization and biodegradation. Three forms of the surfactant, laurate, palmitate and stearate monoester, were tested. Potential enhancement of solubilization of hydrophobic substances mediated by these non-ionic surfactants was exploited in this study. A polychlorinated biphenyl (PCB) degrading organism, Burkholderia cepacia LB400, was also selected. It was found that all surfactants were effective in solubilizing Aroclor 1242 but the rate of Aroclor 1242 biodegradation proceeded rapidly only in the presence of 6-O-palmitoylmaltotriose. For example, the addition of 48 mg 6-O-palmitoylmaltotriose/l increased the apparent solubility from 140 to 305 g/l. As a result, only 8% of the Aroclor remained at the end of 24 h incubation. In contrast, 49.2% of the Aroclor 1242 remained in the absence of surfactant. It appears that maltotriose fatty acid monoesters can significantly increase the bioavailability, and thereby accelerate the biodegradation of highly chlorinated PCBs, particularly Aroclor 1242, by Burkholderia cepacia LB400. The possibility of obtaining these biodegradable surfactants with high yield, easy recovery and high purity by using a new enzymatic methodology, makes maltotriose esters available for bioremediation purposes.     

Phenotypic and Phylogenetic Characterization of Burkholderia (Pseudomonas) sp. Strain LB400

The relevant phenotypic traits and phylogenetic relationships between Burkholderia (Pseudomonas) sp. strain LB400 and B. cepacia ATCC 25416^T were compared to determine the degree to which these two strains might be related. Strain LB400 degrades chlorinated biphenyls and has been a model system for potential use in the bioremediation of polychlorinated biphenyls, while some strains of B. cepacia are plant and human pathogens. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that strain LB400 was a member of the genus Burkholderia. The 16S rRNA gene sequence showed that this strain was not as closely related to B. cepacia as previously suspected or to other known pathogens of this genus, but is closely related to B. phenazinium, B. caribensis, B. graminis, and three unnamed Burkholderia spp. not known to be pathogenic. Received: 16 August 2000 / Accepted: 27 September 2000     

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.     

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.     

Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

Oxidation of biphenyl and nine chlorinated biphenyls (CBs) by the biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400 was examined. The purified terminal oxygenase required the addition of partially purified electron transport components, NAD(P)H, and ferrous iron to oxidize biphenyl and CBs. cis-Biphenyl 2,3-dihydrodiol was produced with biphenyl as the substrate. Dihydrodiols were produced from all CBs, and more than one compound was produced with most substrates. Catechols were produced when the dioxygenase-catalyzed reaction occurred at the 2,3 position of a 2-chlorophenyl ring, resulting in dechlorination of the substrate. Oxidation at the 3,4 position of a 2,5-dichlorophenyl ring produced a 3,4-dihydrodiol. Compounds resulting from both types of reaction were produced during oxidation of 2,5,2'-trichlorobiphenyl. The broad substrate specificity and the ability to oxidize at different ring positions suggest that the biphenyl 2,3-dioxygenase is responsible for the wide range of CBs oxidized by Pseudomonas sp. strain LB400.     

Catabolic potential of multiple PCB transformation systems in Rhodococcus sp. strain RHA1

Summary Polychlorinated biphenyl (PCB) transformation activity of a strong PCB degrader, Rhodococcus sp. strain RHA1, was examined in different concentrations of PCBs. A extremely strong PCB transformation activity was observed on 30 g PCB/ml. At 50 and 100 g/ml, transformation activities were diminished. In the case of bphA insertion mutant, RDA1, transformation activity in the presence of ethylbezene was poor even at 30 g/ml. This indicated that the bphA dependent system would play a major role in PCB transformation by RHA1. Greater transformation activity of RHA1 was observed in the presence of ethylbenzene than in the presence of biphenyl.     

Growth inhibition of Rhodococcus sp. strain RHA1 in the course of PCB transformation

Summary Growth of a PCB degrader Rhodococcus sp. RHA1 on biphenyl and ethylbenzene was inhibited by 100 g/ml PCB 48. A PCB tolerant derivative of RHA1 designated RCD1 was deficient in growth on biphenyl. Southern hybridization experiments suggested that RCD1 has the bphDE gene deletion in a 390-kb linear plasmid of RHA1. The bphD gene complementation restored growth deficiency on biphenyl and growth inhibition on ethylbenzene by PCB 48, indicating that PCB metabolites are the cause of growth inhibition.     

Catabolism of benzoate and phthalate in Rhodococcus sp. strain RHA1: redundancies and convergence

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the beta-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the beta-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent beta-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.     

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.     

Anaerobic dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated microorganisms from sediments.

A polychlorobiphenyl (PCB)-dechlorinating inoculum eluted from upper Hudson River sediments was treated with either heat or ethanol or both. The treated cultures retained the ability to dechlorinate PCBs (Aroclor 1242) under strictly anaerobic conditions. The dechlorination activity was maintained in serial cultures inoculated with transfers of 1% inoculum when the transferred inoculum was treated each time in the same manner. No methane production was detected in any treated culture, although dechlorination of PCBs in the untreated cultures was always accompanied by methane production. All treated cultures preferentially removed meta chlorines, yielding a dechlorination pattern characterized by accumulation of certain ortho- and para-subsituted congeners such as 2-4-chlorobiphenyl (2-4-CB), 2,4-2-CB, and 2,4-4-CB. In contrast, the untreated cultures showed more extensive dechlorination activities, which almost completely removed both meta and para chlorines from Aroclor 1242. These results suggest that microorganisms responsible for the dechlorination of PCBs in the upper Hudson River sediments can be grouped into two populations according to their responses to the heat and ethanol treatments. Microorganisms surviving the heat and ethanol treatments preferentially remove meta chlorines, while microorganisms lost from the enrichment mainly contribute to the para dechlorination activity. These results indicate that anaerobic sporeformers are at least one of the physiological groups responsible for the reductive dechlorination of PCBs. The selection of a dechlorinating population by such treatments may be an important step in isolation of PCB-dechlorinating microorganisms.