Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.     

Deep Desulfurization of Diesel Oil and Crude Oils by a Newly Isolated Rhodococcus erythropolis Strain

The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as α-hydroxy-β-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30°C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30°C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.     

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.     

Desulfurization of light gas oil by a novel recombinant strain from Pseudomonas aeruginosa

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa NCIMB 9571 by using a transposon vector. Resting cells of the recombinant strain, PAR41, desulfurized 63 mg sulfur l^–1 of light gas oil (LGO) containing 360 mg S l^–1. The desulfurization activity for LGO by the resting cells of strain PAR41 grown with n-tetradecane (50% v/v) was much higher (1018-fold) than in glucose-grown cells. P. aeruginosa NCIMB 9571 is able to take up water-insoluble compounds from an oil phase which is enhanced by n-alkane.     

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.     

Microbial Desulfurization of Alkylated Dibenzothiophenes from a Hydrodesulfurized Middle Distillate by Rhodococcus erythropolis I-19

Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 μmol g dry cell weight⁻¹ min⁻¹, more than 25-fold higher than that for wild-type bacteria. According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes. Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S. XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones. I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs. Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane. Apparent saturation rate constant (K₀) and half-saturation rate constant (K₁) values were calculated to be 2.8 μmol g dry cell weight⁻¹ min⁻¹ and 130 ppm, respectively. However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior. A competitive-substrate model suggested that the apparent K₁ values for each group of Cx-DBTs increased with increasing alkylation. Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure.     

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.     

Biocatalytic desulfurization of diesel oil in an air-lift reactor with immobilized Gordonia nitida CYKS1 cells

A new type of air-lift reactor with immobilized Gordonia nitida CYKS1 cells on a fibrous support was designed and used for the biocatalytic desulfurization (BDS) of diesel oil. Its performance was evaluated at different phase ratios of the oil to the aqueous medium (or oil phase fractions) and different sucrose concentrations. When the reaction mixture contained 10% diesel oil (v/v), 61-67% of sulfur was removed as the sulfur content decreased from 202-250 to 76-90 mg L(-1) in 72 h. The sulfur content did not decrease any further because the remaining sulfur compounds were recalcitrant to BDS. During the desulfurization, the strain CYKS1 consumed hydrocarbons in the diesel oil, mainly n-alkanes with 10-26 carbons, as carbon source even though an easily available carbon source, sucrose, was supplied.     

Microbial Desulfurization of a Crude Oil Middle-Distillate Fraction: Analysis of the Extent of Sulfur Removal and the Effect of Removal on Remaining Sulfur

Rhodococcus sp. strain ECRD-1 was evaluated for its ability to desulfurize a 232 to 343°C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil. OB oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined. Gas chromatography (GC), flame ionization detection, and GC sulfur chemiluminesce detection analysis were used to qualitatively evaluate the effect of Rhodococcus sp. strain ECRD-1 treatment on the hydrocarbon and sulfur content of the oil, respectively. Total sulfur was determined by combustion of samples and measurement of released sulfur dioxide by infrared absorption. Up to 30% of the total sulfur in the middle distillate cut was removed, and compounds across the entire boiling range of the oil were affected. Sulfur K-edge X-ray absorption-edge spectroscopy was used to examine the chemical state of the sulfur remaining in the treated OB oil. Approximately equal amounts of thiophenic and sulfidic sulfur compounds were removed by ECRD-1 treatment, and over 50% of the sulfur remaining after treatment was in an oxidized form. The presence of partially oxidized sulfur compounds indicates that these compounds were en route to desulfurization. Overall, more than two-thirds of the sulfur had been removed or oxidized by the microbial treatment.     

Growth of Rhodosporidium toruloides Strain DBVPG 6662 on Dibenzothiophene Crystals and Orimulsion

Strains DBVPG 6662 and DBVPG 6739 of Rhodosporidium toruloides, a basidiomycete yeast, grew on thiosulfate as a sulfur source and glucose (2 g liter⁻¹ or 10.75 mM) as a carbon source. DBVPG 6662 has a defective sulfate transport system, whereas DBVPG 6739 barely grew on sulfate. They were compared for the ability to use dibenzothiophene (DBT) and related organic sulfur compounds as sulfur sources. In the presence of glucose as a carbon source and DBT as a sulfur source, strain DBVPG 6662 grew better than DBVPG 6739. In the presence of thiosulfate as a sulfur source, the two yeast strains did not use DBT, DBT-sulfone, benzenesulfonic acid, biphenyl, and fluorene. When the two strains were grown in the presence of glucose, strain DBVPG 6662 transformed 27% of the DBT present (10 μM) at a rate of 0.023 μmol liter⁻¹ h⁻¹ in 36 h. Traces of 2,2′-dihydroxylated biphenyl were transiently accumulated under these conditions. When the same strain was grown on glucose in the presence of a higher concentration of DBT (0.5 g liter⁻¹), mainly in an insoluble form, the whole surface of the DBT crystals was colonized by a thick mycelium. This adherent structure was imaged by confocal microscopy with fluorescent concanavalin A, a lectin that specifically binds glucose and mannose residues. When DBVPG 6662 was grown on glucose in the presence of a commercial emulsion of bitumen, i.e., orimulsion, 68% of the benzo- and dibenzothiophenes and DBTs was removed after 15 days of incubation. The fungus adhered by hyphae to orimulsion droplets. When cultivated in the presence of commercial emulsifier-free fuel oil containing alkylated benzothiophenes and DBTs and having a composition similar to that of orimulsion, strain DBVPG 6662 removed only 11% of the total organic sulfur that occurs in the medium and did not adhere to the oil droplets. These results indicate that strain DBVPG 6662 is able to utilize the organic sulfur of DBT and a large variety of thiophenic compounds that occur extensively in commercial fuel oils by physically adhering to the organic sulfur source.     

Desulfurization of model and diesel oils by resting cells of Gordona sp.

The desulfurization activity of the resting cells of Gordona sp. CYKS1 was strongly depended on harvest time and the highest value when the cells had been harvested in the early growth phase (0.12 mg sulfur g^–1 cell^–1 h^–1). For the model oil, hexadecane containing dibenzothiophene, the specific desulfurization rate decreased as the reaction proceeded. Both the specific and the volumetric desulfurization rates were not significantly affected by the aqueous-to-oil phase ratio. The diesel oils, light gas oil and a middle distillate unit feed were desulfurized at higher rates (ca. 0.34 mg sulfur g^–1 cell^–1 h^–1) than the model oil (0.12 mg sulfur g^–1 cell^–1 h^–1).     

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.     

Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis

The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l^–1 to 70 mg l^–1 in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%–41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of 9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.     

Proteomics and Metabolomics Analyses to Elucidate the Desulfurization Pathway of Chelatococcus sp.

Desulfurization of dibenzothiophene (DBT) and alkylated DBT derivatives present in transport fuel through specific cleavage of carbon-sulfur (C-S) bonds by a newly isolated bacterium Chelatococcus sp. is reported for the first time. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of DBT degradation by Chelatococcus sp. showed the transient formation of 2-hydroxybiphenyl (2-HBP) which was subsequently converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP. The relative ratio of 2-HBP and 2-MBP formed after 96 h of bacterial growth was determined at 4:1 suggesting partial conversion of 2-HBP or rapid degradation of 2-MBP. Nevertheless, the enzyme involved in this conversion process remains to be identified. This production of 2-MBP rather than 2-HBP from DBT desulfurization has a significant metabolic advantage for enhancing the growth and sulfur utilization from DBT by Chelatococcus sp. and it also reduces the environmental pollution by 2-HBP. Furthermore, desulfurization of DBT derivatives such as 4-M-DBT and 4, 6-DM-DBT by Chelatococcus sp. resulted in formation of 2-hydroxy-3-methyl-biphenyl and 2-hydroxy –3, 3^/- dimethyl-biphenyl, respectively as end product. The GC and X-ray fluorescence studies revealed that Chelatococcus sp. after 24 h of treatment at 37°C reduced the total sulfur content of diesel fuel by 12% by per gram resting cells, without compromising the quality of fuel. The LC-MS/MS analysis of tryptic digested intracellular proteins of Chelatococcus sp. when grown in DBT demonstrated the biosynthesis of 4S pathway desulfurizing enzymes viz. monoxygenases (DszC, DszA), desulfinase (DszB), and an NADH-dependent flavin reductase (DszD). Besides, several other intracellular proteins of Chelatococcus sp. having diverse biological functions were also identified by LC-MS/MS analysis. Many of these enzymes are directly involved with desulfurization process whereas the other enzymes/proteins support growth of bacteria at an expense of DBT. These combined results suggest that Chelatococcus sp. prefers sulfur-specific extended 4S pathway for deep-desulphurization which may have an advantage for its intended future application as a promising biodesulfurizing agent.     

脱硫菌Fds-1的分离鉴定及其对柴油脱硫特性的研究

从含硫土壤中分离筛选出一株专一性脱硫菌Fds-1,经生理生化指标和16S rRNA序列分析鉴定其属于枯草芽孢杆菌(Bacillus subtilis)。用Gibb’s试剂显色和气相色谱-质谱联用分析表明,该菌株通过“4S”途径脱除有机硫。实验发现Fds-1的最佳脱硫活性在30℃,在此温度下72h内能脱除约0.5mmol/L DBT中的有机硫。Fds-1菌株对有机硫化合物的利用情况和柴油脱硫前后烃组分比较都进一步证明该菌株适合于柴油生物脱硫。利用休止细胞对不同组分柴油的脱硫研究表明,脱硫菌株Fds-1对精制柴油中的DBT类化合物的降解能力强。因此,该菌株对精制低硫柴油的深度脱硫具有应用意义。     

Microbial Desulfurization of Gasoline in a Mycobacterium goodii X7B Immobilized-Cell System

Mycobacterium goodii X7B, which had been primarily isolated as a bacterial strain capable of desulfurizing dibenzothiophene to produce 2-hydroxybiphenyl via the 4S pathway, was also found to desulfurize benzothiophene. The desulfurization product was identified as o-hydroxystyrene by gas chromatography (GC)-mass spectrometry analysis. This strain appeared to have the ability to remove organic sulfur from a broad range of sulfur species in gasoline. When Dushanzi straight-run gasoline (DSRG227) containing various organic sulfur compounds was treated with immobilized cells of strain X7B for 24 h, the total sulfur content significantly decreased, from 227 to 71 ppm at 40°C. GC flame ionization detection and GC atomic emission detection analysis were used to qualitatively evaluate the effects of M. goodii X7B treatment on the contents of gasoline. In addition, when immobilized cells were incubated at 40°C with DSRG275, the sulfur content decreased from 275 to 54 ppm in two consecutive reactions. With this excellent efficiency, strain X7B is considered a good potential candidate for industrial applications for the biodesulfurization of gasoline.     

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.     

生物脱有机硫催化剂的最新研究进展

生物脱有机硫作为常规的加氢脱硫替代方法近几年受到越来越多的重视,也取得了一些重要的结果,这都推动了生物脱硫向产业化应用。本文简要综述了最近几年通过菌株改造提高催化剂活力和消除无机硫抑制以及脱硫酶系纯化方面所取得一些进展。     

Composition of Toluene-Degrading Microbial Communities from Soil at Different Concentrations of Toluene

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus Pseudomonas. An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that Pseudomonas strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by Pseudomonas strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637–645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.     

Analysis of biodesulfurization of model oil system by the bacterium,strain RIPI-22

The execution of 4S process (sulfur-specific pathway) by growing cells of the newly isolated strain RIPI-22 and its resting cells in both aqueous reaction system and in two-phase system were investigated. The time for maximum desulfurization activity of the strain in resting state and in model oil system (hexadecane containing dibenzothiophene) was 7 h. Using taguchi design procedure the effects of cell density, pH and phase ratio on the desulfurization reaction were studied. The extent of desulfurization was hardly dependent on the pH where as the volume ratio of hydrocarbon-aqueous phase significantly affected the desulfurization activity.On the basis of the determined optimum desulfurizing conditions, the biodesulfurization pattern of DBT was studied and kinetic parameters were calculated.