Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp. strain WU-K2R

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.     

Thermophilic Biodesulfurization of Various Heterocyclic Sulfur Compounds and Crude Straight-Run Light Gas Oil Fraction by a Newly Isolated Strain Mycobacterium phlei WU-0103

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50°C. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45°C. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography–mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45°C with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.     

Thermophilic biodesulfurization of naphthothiophene and 2-ethylnaphthothiophene by a dibenzothiophene-desulfurizing bacterium, Mycobacterium phlei WU-F1

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degrees C), could also grow at 50 degrees C in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 degrees C, the resting cells of WU-Fl degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-Fl can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.     

Desulfurization of Benzothiophene by the Gram-Negative Bacterium, Sinorhizobium sp. KT55

Sinorhizobium sp. KT55 was the first Gram-negative isolate to be capable of utilizing benzothiophene as the sole source of sulfur. By GC-MS analysis of metabolites of benzothiophene by this strain, benzothiophene sulfone, benzo[e][1,2]oxathiin S-oxide and o-hydroxystyrene were detected, suggesting that the benzothiophene desulfurization pathway of this strain is benzothiophene → benzothiophene sulfoxide → benzothiophene sulfone → benzo[e][1,2]oxathiin S-oxide →o-hydroxystyrene. Desulfurization activity of this strain was significantly repressed by methionine, cysteine, sulfate, dimethyl sulfoxide, and Casamino acids. Received: 5 January 2001/Accepted: 6 February 2001     

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1.

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.     

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1.

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.     

Biodesulfurization of organic-sulfur compounds

A screening assay in which dibenzothiophene (DBT) or DBT-sulfone served as the only bioavailable source of sulfur was used to obtain two new bacterial isolates, strains UM9 and UM3, that desulfurized either substrate. Strain UM9 produced the desulfurized product, 2-hydroxybiphenyl (HBP); no other identifiable desulfurized products or released sulfate or sulfite were detected. Biodesulfurization activity occurred only for growing cultures and was depressed by free sulfate. Neither isolate grew on DBT, DBT-sulfone, or HBP as sole carbon sources. Under optimized conditions of pH and temperature, strain UM9 exhibited up to 35% greater biodesulfurization of DBT-sulfone than did UM3, and both isolates also desulfurized several other organic-sulfur compounds. The kinetics and characteristics of biodesulfurization by either UM3 or UM9, tentatively identified as species ofRhodococcus, indicated mechanisms different from those reported in the literature for other bacteria.     

Desulfurization of various organic sulfur compounds and the mixture of DBT + 4,6-DMDBT by Mycobacterium sp. ZD-19

A new isolated dibenzothiophene (DBT) desulfurizing bacterium, identified as Mycobacterium sp. ZD-19 can utilize a wide range of organic sulfur compounds as a sole sulfur source. Thiophene (TH) or benzothiophene (BTH) was completely degraded by strain ZD-19 within 10h or 42 h, and 100% DBT or 4,6-dimethyldibenzothiophene (4,6-DMDBT) was removed within 50h or 56 h, respectively. Diphenylsulfide (DPS) possessed the lowest desulfurization efficiencies with 60% being transformed within 50h and 80% at 90 h. The desulfurization activities of five substrates by resting cells are in order of TH>BTH>DPS>DBT>4,6-DMDBT. In addition, when DBT and 4,6-DMDBT were mixed, they could be simultaneously desulfurized by strain ZD-19. However, DBT appeared to be attacked prior to 4,6-DMDBT. The desulfurization rate of DBT or 4,6-DMDBT in mixture is lower than they are desulfurized separately, indicating that the substrate competitive inhibition is existent when DBT and 4,6-DMDBT are mixed.     

Selective Desulfurization of Dibenzothiophene by Rhodococcus erythropolis D-1

A dibenzothiophene (DBT)-degrading bacterium, Rhodococcus erythropolis D-1, which utilized DBT as a sole source of sulfur, was isolated from soil. DBT was metabolized to 2-hydroxybiphenyl (2-HBP) by the strain, and 2-HBP was almost stoichiometrically accumulated as the dead-end metabolite of DBT degradation. DBT degradation by this strain was shown to proceed as DBT → DBT sulfone → 2-HBP. DBT at an initial concentration of 0.125 mM was completely degraded within 2 days of cultivation. DBT at up to 2.2 mM was rapidly degraded by resting cells within only 150 min. It was thought this strain had a higher DBT-desulfurizing ability than other microorganisms reported previously.     

Microbial desulfurization of dibenzothiophene: A sulfur-specific pathway

Abstract Rhodococcus rhodochrous strain IGTS8 metabolizes dibenzothiophene, a model compound for organic sulfur in fossil fuels, in a sulfur-specific manner. Two routes of desulfurization have been identified. Under growth conditions, the intermediates are dibenzothiophene sulfoxide, dibenzothiophene sulfone, 2'-hydroxybiphenyl-2-sulfonate, and 2,2'-dihydroxybiphenyl. Stationary phase cells produce 2-hydroxybiphenyl as the desulfurized product and use the 2'-hydroxybiphenyl-2-sulfonate, rather than the sulfonate, as key intermediate.     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.     

Use of a Novel Fluorinated Organosulfur Compound To Isolate Bacteria Capable of Carbon-Sulfur Bond Cleavage

The vacuum residue fraction of heavy crudes contributes to the viscosity of these oils. Specific microbial cleavage of C—S bonds in alkylsulfide bridges that form linkages in this fraction may result in dramatic viscosity reduction. To date, no bacterial strains have been shown conclusively to cleave C—S bonds within alkyl chains. Screening for microbes that can perform this activity was greatly facilitated by the use of a newly synthesized compound, bis-(3-pentafluorophenylpropyl)-sulfide (PFPS), as a novel sulfur source. The terminal pentafluorinated aromatic rings of PFPS preclude growth of aromatic ring-degrading bacteria but allow for selective enrichment of strains capable of cleaving C—S bonds. A unique bacterial strain, Rhodococcus sp. strain JVH1, that used PFPS as a sole sulfur source was isolated from an oil-contaminated environment. Gas chromatography-mass spectrometry analysis revealed that JVH1 oxidized PFPS to a sulfoxide and then a sulfone prior to cleaving the C—S bond to form an alcohol and, presumably, a sulfinate from which sulfur could be extracted for growth. Four known dibenzothiophene-desulfurizing strains, including Rhodococcus sp. strain IGTS8, were all unable to cleave the C—S bond in PFPS but could oxidize PFPS to the sulfone via the sulfoxide. Conversely, JVH1 was unable to oxidize dibenzothiophene but was able to use a variety of alkyl sulfides, in addition to PFPS, as sole sulfur sources. Overall, PFPS is an excellent tool for isolating bacteria capable of cleaving subterminal C—S bonds within alkyl chains. The type of desulfurization displayed by JVH1 differs significantly from previously described reaction results.     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC) catalysis, the first and also the key step in the microbial DBT desulfurization, is the conversion of DBT to DBT sulfone (DBTO₂). In this study, dszC of a DBT-desulfurizing bacterium Rhodococcus sp. DS-3 was cloned by PCR. The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank. The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E. coli BL21 strain. The expression amount of DszC was about 20% of total supernatant at low temperature. The soluble DszC in the supernatant was purified by Ni²⁺ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity. Only one band was detected by Western-blotting, which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3, paC5) and Rhodococcus sp. DS-3. The activity of purified DszC was 0.36 U. DszC can utilize the organic compound such as DBT and methyl-DBT, but not DBT derivates such as DBF, which has no sulfur or inorganic sulfur. __________ Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2005, 38(6): 1–6 [译自: 南开大学学报 (自然科学版), 2005, 38(6): 1–6]     

Sulfur from benzothiophene and alkylbenzothiophenes supports growth of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. strain JVH1

Rhodococcus sp. strain JVH1 was previously reported to use a number of compounds with aliphatic sulfide bridges as sulfur sources for growth. We have shown that although JVH1 does not use the three-ring thiophenic sulfur compound dibenzothiophene, this strain can use the two-ring compound benzothiophene as its sole sulfur source, resulting in growth of the culture and loss of benzothiophene. Addition of inorganic sulfate to the medium reduced the conversion of benzothiophene, indicating that benzothiophene metabolism is repressed by sulfate and that benzothiophene is therefore used specifically as a sulfur source. JVH1 also used all six isomers of methylbenzothiophene and two dimethylbenzothiophene isomers as sulfur sources for growth. Metabolites identified from benzothiophene and some methylbenzothiophenes were consistent with published pathways for benzothiophene biodesulfurization. Products retaining the sulfur atom were sulfones and sultines, the sultines being formed from phenolic sulfinates under acidic extraction conditions. With 2-methylbenzothiophene, the final desulfurized product was 2-methylbenzofuran, formed by dehydration of 3-(o-hydroxyphenyl) propanone under acidic extraction conditions and indicating an oxygenative desulfination reaction. With 3-methylbenzothiophene, the final desulfurized product was 2-isopropenylphenol, indicating a hydrolytic desulfination reaction. JVH1 is the first microorganism reported to use all six isomers of methylbenzothiophene, as well as some dimethylbenzothiophene isomers, as sole sulfur sources. JVH1 therefore possesses broader sulfur extraction abilities than previously reported, including not only sulfidic compounds but also some thiophenic species.     

In vivo evolution of the Rhodococcus sp. strain DS7: selection of recombinants able to desulfurize both dibenzothiophene and benzothiophene

Rhodococcus sp. DS7, isolated from a polluted soil, has shown good desulfurizing activity towards dibenzothiophene (DBT) and its derivatives, but is not able to desulfurize benzothiophene (BT), the other thiophenic molecule recalcitrant to the chemical hydrodesulfurization (HDS) process, and most abundant in gasoline. To select a Rhodococcus DS7 derivative strain able to desulfurize both DBT and BT, we took advantage of the verified capacity of this strain to integrate exogenous DNA randomly, with a good efficiency. Heterologous chromosomal DNA, digested with restriction enzymes, from two BT but not DBT desulfurizing strains, Rhodococcus sp. ATCC 27778 and Gordonia sp. ATCC 19067, was electroporated into Rhodococcus DS7. Selection on minimal medium with BT as sole sulfur source allowed us to isolate several DS7 derivatives with the capacity to desulfurize both thiophenic molecules. Two strains, one derived from the integration and recombination of DNA from ATCC 27778, and the other from ATCC 19067, have been partially characterized. These recombinant microorganisms are an interesting starting point to develop new biodesulfurization processes.     

Conversion of dibenzothiophene by the mushrooms <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and <Emphasis Type="Italic">Coprinellus radians</Emphasis> and their extracellular peroxygenases

The conversion of the heterocycle dibenzothiophene (DBT) by the agaric basidiomycetes Agrocybe aegerita and Coprinellus radians was studied in vivo and in vitro with whole cells and with purified extracellular peroxygenases, respectively. A. aegerita oxidized DBT (110 μM) by 100% within 16 days into eight different metabolites. Among the latter were mainly S-oxidation products (DBT sulfoxide, DBT sulfone) and in lower amounts, ring-hydroxylation compounds (e.g., 2-hydroxy-DBT). C. radians converted about 60% of DBT into DBT sulfoxide and DBT sulfone as the sole metabolites. In vitro tests with purified peroxygenases were performed to compare the product pattern with the metabolites formed in vivo. Using ascorbic acid as radical scavenger, a total of 19 and seven oxygenation products were detected after DBT conversion by the peroxygenases of A. aegerita (AaP) and C. radians (CrP), respectively. Whereas ring hydroxylation was favored over S-oxidation by AaP (again 2-hydroxy-DBT was identified), CrP formed DBT sulfoxide as major product. This finding suggests that fungal peroxygenases can considerably differ in their catalytic properties. Using H₂ ¹⁸O₂, the origin of oxygen was proved to be the peroxide. Based on these results, we propose that extracellular peroxygenases may be involved in the oxidation of heterocycles by fungi also under natural conditions.     

Mutagenic activity of methyl-substituted tri-and tetracyclic aromatic sulfur heterocycles

A number of polycyclic aromatic sulfur heterocycles have been identified in coal-derived products and in shale oils. The mutagenic activity of some of these compounds, including dibenzothiophene, benzo[b]naphtho[1,2-d]thiophene, benzo[b]naphtho[2,1-d]thiophene and benzo[b]naphtho[2,3-d]thiophene have been determined using the Salmonella/microsome mutagenicity test. These compounds demonstrated either very weak or no mutagenic activity. The methyl derivatives of each of these four compounds were assayed for mutagenic activity. Salmonella typhimurium TA98 was used as the tester strain. All assays required a rat-liver homogenate metabolic activator. Five of the methylated derivatives, 1-methylbenzo[b]naphtho[1,2-d]thiophene, 3-methylbenzo[b]naphtho[1,2,-d]thiophene, 1-methylbenzo[b]-naphtho[2,1-d]thiophene, 6-methylbenzo[b]naphtho[2,1-d]thiophene and 4-methylbenzo[b]naphtho[2,3-[d]thiophene demonstrated mutagenic activity. However, activity was observed only at high concentrations of the metabolic activator.     

Synthesis of carbon-11-labeled 4-(phenylamino)-pyrrolo[2,1-f][1,2,4]triazine derivatives as new potential PET tracers for imaging of p38α mitogen-activated protein kinase

The reference standards methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10b) and corresponding precursors 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11b) were synthesized from methyl crotonate and 3-amino-4-methylbenzoic acid in multiple steps with moderate to excellent yields. The target tracer [¹¹C]methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10a) and [¹¹C]methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10b) were prepared from their corresponding precursors with [¹¹C]CH₃OTf under basic condition through O-[¹¹C]methylation and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields at end of bombardment (EOB) with 185–555 GBq/μmol specific activity at end of synthesis (EOS).     

Multi-criteria comparison of resting cell activities of bacterial strains selected for biodesulfurization of petroleum compounds

Five isolates able to utilize dibenzothiophene (DBT) as a sole sulfur source and to convert it to 2-hydroxybiphenyl (HBP) with high rates were selected to investigate their potentialities as biocatalysts of a diesel oil biodesulfurization process. Conventional and chemotaxonomic analyses and 16S ribosomal DNA (rDNA) sequencing showed that these strains belonged to the Rhodococcus/Gordonia cluster. The desulfurizing activities of resting cells were compared under various conditions to evaluate their stability in both aqueous and organic media, their sensitivity to the presence of hexadecane and their sulfur substrate selectivity. In spite of their taxonomic similarity, the five strains exhibited different properties. This diversity was not confirmed by the analysis of the desulfurizing genes by amplification and sequencing of large fragments of dszA, dszB, dszC, and dszD genes which revealed that four of the five selected strains had a dsz genotype identical to those of the reference strain, Rhodococcus erythropolis IGTS8.