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 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.     

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.     

Utilization of dimethyl sulfide as a sulfur source with the aid of light by Marinobacterium sp. strain DMS-S1

Strain DMS-S1 isolated from seawater was able to utilize dimethyl sulfide (DMS) as a sulfur source only in the presence of light in a sulfur-lacking medium. Phylogenetic analysis based on 16S ribosomal DNA genes indicated that the strain was closely related to Marinobacterium georgiense. The strain produced dimethyl sulfoxide (DMSO), which was a main metabolite, and small amounts of formate and formaldehyde when grown on DMS as the sole sulfur source. The cells of the strain grown with succinate as a carbon source were able to use methyl mercaptan or methanesulfonate besides DMS but not DMSO or dimethyl sulfone as a sole sulfur source. DMS was transformed to DMSO primarily at wavelengths between 380 and 480 nm by heat-stable photosensitizers released by the strain. DMS was also degraded to formaldehyde in the presence of light by unidentified heat-stable factors released by the strain, and it appeared that strain DMS-S1 used the degradation products, which should be sulfite, sulfate, or methanesulfonate, as sulfur sources.     

Utilization of Dimethyl Sulfide as a Sulfur Source with the Aid of Light by Marinobacterium sp. Strain DMS-S1

Strain DMS-S1 isolated from seawater was able to utilize dimethyl sulfide (DMS) as a sulfur source only in the presence of light in a sulfur-lacking medium. Phylogenetic analysis based on 16S ribosomal DNA genes indicated that the strain was closely related to Marinobacterium georgiense. The strain produced dimethyl sulfoxide (DMSO), which was a main metabolite, and small amounts of formate and formaldehyde when grown on DMS as the sole sulfur source. The cells of the strain grown with succinate as a carbon source were able to use methyl mercaptan or methanesulfonate besides DMS but not DMSO or dimethyl sulfone as a sole sulfur source. DMS was transformed to DMSO primarily at wavelengths between 380 and 480 nm by heat-stable photosensitizers released by the strain. DMS was also degraded to formaldehyde in the presence of light by unidentified heat-stable factors released by the strain, and it appeared that strain DMS-S1 used the degradation products, which should be sulfite, sulfate, or methanesulfonate, as sulfur sources.     

Degradation of phenanthrene,methylphenanthrenes and dibenzothiophene by a Sphingomonas strain 2mpII

Strain Sphingomonassp. (2MPII), isolated from marine sediment, was able to utilize phenanthrene (P) or 2-methylphenanthrene (2MP) as the sole carbon source. However, 9-methylphenanthrene (9MP) and dibenzothiophene (DBT) were weakly degraded. The degradation rates of 9MP and DBT increased in the presence of 2MP, whilst the degradation rate of 2MP increased in the presence of 9MP. However, the presence of DBT inhibited the degradation of 2MP. DBT sulfone, a DBT metabolite, was not assimilated by the bacteria and its presence also decreased the degradation rate of 2MP.     

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-desulfiaizing 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 supematant 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,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.     

Isolation and characterization of a moderate thermophile,Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization

An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50 degrees C M. phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1). M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.     

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.     

微生物脱除煤炭中有机硫的研究

从任丘油田分离到两株异养型细菌D-1-1和D-2-1,经鉴定分别为门多隆假单胞菌(Pseudomonas mendocoas)和争论产碱生物变型Ⅰ (Alcaligenes paradoxus biovar Ⅰ)的菌株。它们可以利用二苯噻吩(Dibenzothiophene,简称DBT)作为生长的碳源,将DBT转化成为水溶性有机硫化物。两菌于15天内可以脱除煤炭中有机硫达22.2—32.0％。     

Characterization of Gordonia sp. strain F.5.25.8 capable of dibenzothiophene desulfurization and carbazole utilization

A dibenzothiophene (DBT)-degrading bacterial strain able to utilize carbazole as the only source of nitrogen was identified as Gordonia sp. F.5.25.8 due to its 16S rRNA gene sequence and phenotypic characteristics. Gas chromatography (GC) and GC–mass spectroscopy analyses showed that strain F.5.25.8 transformed DBT into 2-hydroxybiphenyl (2-HBP). This strain was also able to grow using various organic sulfur or nitrogen compounds as the sole sulfur or nitrogen sources. Resting-cell studies indicated that desulfurization occurs either in cell-associated or in cell-free extracts of F.5.25.8. The biological responses of F.5.25.8 to a series of mutagens and environmental agents were also characterized. The results revealed that this strain is highly tolerant to DNA damage and also refractory to induced mutagenesis. Strain F.5.25.8 was also characterized genetically. Results showed that genes involved in desulfurization (dsz) are located in the chromosome, and PCR amplification was observed with primers dszA and dszB designed based on Rhodococcus genes. However, no amplification product was observed with the primer based on dszC.     

Biodesulfurization of dibenzothiophene by a newly isolated Rhodococcus erythropolis strain

A new dibenzothiophene (DBT) desulfurizing bacterium was isolated from oil-contaminated soils in Iran. HPLC analysis and PCR-based detection of the presence of the DBT desulfurization genes (dszA, dszB and dszC) indicate that this strain converts DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The strain, identified as Rhodococcus erythropolis SHT87, can utilize DBT, dibenzothiophene sulfone, thiophene, 2-methylthiophene and dimethylsulfoxide as a sole sulfur source for growth at 30 °C.The maximum specific desulfurization activity of strain SHT87 resting cells in aqueous and biphasic organic–aqueous systems at 30 °C was determined to be 0.36 and 0.47 μmol 2-HBP min⁻¹ (g dry cell)⁻¹, respectively. Three mM DBT was completely metabolized by SHT87 resting cells in the aqueous and biphasic systems within 10 h. The rate and the extent of the desulfurization reaction by strain SHT87 suggest that this strain can be used for the biodesulfurization of diesel oils.     

Degradation of dimethyl disulfide by pseudomonas fluorescens strain 76

Strain 76, which was able to utilize dimethyl disulfide (DMDS) as a sole sulfur source, was screened from our microbial collection. It was identified as Pseudomonas fluorescens by taxonomical characterization and 16S rDNA sequence analysis. It does not belong to the methylotrophs, because it did not grow on DMDS or other C1 compounds as sole carbon source, and DMDS degradation was not repressed in the presence of glucose, Na(2)SO(4), or nutrient broth. Moreover, it showed high resistance to DMDS by growing in DMDS at concentrations up to 9.04 mM. Based on these findings, strain 76 metabolizes DMDS and has dual physiological roles: sulfur assimilation and degradation. Thus it has advantages as a biological scavenger of DMDS.     

Desulfurization of Dibenzothiophene and Diesel Oils by a Newly Isolated Gordona Strain, CYKS1

A dibenzothiophene (DBT)-desulfurizing bacterial strain was isolated and identified as Gordona strain CYKS1. Strain CYKS1 was found to transform DBT to 2-hydroxybiphenyl via the 4S pathway and to be able to also use organic sulfur compounds other than DBT as a sole sulfur source. Its desulfurization activity was susceptible to sulfate repression. Active resting cells for desulfurization could be prepared only in the early growth phase. When two types of diesel oils, middle distillate unit feed (MDUF) and light gas oil (LGO) containing various organic sulfur compounds including DBT, were treated with resting cells of strain CYKS1 for 12 h, the total sulfur content significantly decreased, from 0.15% (wt/wt) to 0.06% (wt/wt) for MDUF and from 0.3% (wt/wt) to 0.25% (wt/wt) for LGO. The newly isolated strain CYKS1 is considered to have good potential for application in the biodesulfurization of fossil fuels.     

Cloning and characterization of genes encoding an enzyme which oxidizes dimethyl sulfide in Acinetobacter sp. strain 20B

Acinetobacter sp. strain 20B was isolated based on the ability to utilize dimethyl sulfide as the sole sulfur source. Since strain 20B oxidized indole as well as dimethyl sulfide, indigo production by recombinant Escherichia coli clones carrying Acinetobacter DNA was used as a selection for cloning genes encoding dimethyl sulfide oxidation genes. The gene encoding an indole-oxidizing enzyme was also found to oxidize dimethyl sulfide. The dimethyl sulfide-oxidizing enzyme genes consisted of six open reading flames designated dsoABCDEF. The deduced amino acid sequences of dsoABCDEF were homologous with those of the multicomponent phenol hydroxylases. DsoABCDEF oxidized dimethyl sulfide to dimethyl sulfoxide, and dimethyl sulfoxide to dimethyl sulfone.     

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.     

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]     

Bacterial Transformations of 1,2,3,4-Tetrahydrodibenzothiophene and Dibenzothiophene

The transformations of 1,2,3,4-tetrahydrodibenzothiophene (THDBT) were investigated with pure cultures of hydrocarbon-degrading bacteria. Metabolites were extracted from cultures with dichloromethane (DCM) and analyzed by gas chromatography (GC) with flame photometric, mass, and Fourier transform infrared detectors. Three 1-methylnaphthalene (1-MN)-utilizing Pseudomonas strains oxidized the sulfur atom of THDBT to give the sulfoxide and sulfone. They also degraded the benzene ring to yield 3-hydroxy-2-formyl-4,5,6,7-tetrahydrobenzothiophene. A cell suspension of a cyclohexane-degrading bacterium oxidized the alicyclic ring to give a hydroxy-substituted THDBT and a ketone, and it oxidized the aromatic ring to give a phenol, but no ring cleavage products were detected. GC analyses with an atomic emission detector, using the sulfur-selective mode, were used to quantify the transformation products from THDBT and dibenzothiophene (DBT). The cyclohexane degrader oxidized 19% of the THDBT to three metabolites. The cometabolism of THDBT and DBT by the three 1-MN-grown Pseudomonas strains resulted in a much greater depletion of the condensed thiophenes than could be accounted for in the metabolites detected by GC analysis, but there was no evidence of sulfate release from DBT. These 1-MN-grown strains transiently accumulated 3-hydroxy-2-formylbenzothiophene (HFBT) from DBT, but it was subsequently degraded. On the other hand, Pseudomonas strain BT1d, which was maintained on DBT as a sole carbon source, accumulated 52% of the sulfur from DBT as HFBT over 7 days, and, in total, 82% of the sulfur from DBT was accounted for by the GC method used. Lyophilization of cultures grown on 1-MN with DBT and methyl esterification of the residues gave improved recoveries of total sulfur over that obtained by DCM extraction and GC analysis. This suggested that the further degradation of HFBT by these cultures leads to the formation of organosulfur compounds that are too polar to be extracted with DCM. We believe that this is the first attempt to quantify the products of DBT degradation by the so-called Kodama pathway.     

Biodegradation of benzothiophene sulfones by a filamentous bacterium.

Previous studies showed that benzothiophene and 3- and 5-methylbenzothiophenes are oxidized by some bacteria to yield their corresponding sulfones, which were not subsequently degraded. In this study, a filamentous bacterium was isolated, which grew on each of these three sulfones as its sole carbon, sulfur, and energy source. Based on 16S rRNA gene sequencing and scanning electron microscopy, the isolate was found to belong to the genus Pseudonocardia and assigned the strain designation DB1. Benzothiophene sulfone and 3-methylbenzothiophene sulfone were more readily biodegraded than 5-methylbenzothiophene sulfone, and growth on these three compounds resulted in the release of 57, 62, and 28% of the substrate carbon as CO2, respectively. The thiophene ring was also cleaved, and between 44 and 88% of the sulfur from the consumed substrate was found as sulfate and (or) sulfite. Strain DB1 grew on benzoate, dibenzothiophene sulfone, and hexadecanoic acid, but it could not grow on benzofuran, dibenzothiophene, dibenzothiophene sulfoxide, hexadecane, indole, naphthalene, phenol, 2-sulfobenzoic acid, sulfolane, benzothiophene, or 3- or 5-methylbenzothiophenes. In addition, it did not oxidize the latter three compounds to their sulfones.