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.     

Desulfurization of 2,4,6,8-tetraethyl dibenzothiophene by recombinant Mycobacterium sp. strain MR65

Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (C_x-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C₈-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C₆-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for Cx-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated C_x-DBTs is the transfer process from the oil phase into the cell.     

红球菌Rhodococcus sp.DS-3 dszABC基因和dszD基因在大肠杆菌中的共表达

二苯并噻吩(DBT)及其衍生物微生物脱硫的4S途径需要4个酶(DszA,DszB,DszC and DszD)参与催化。其中DBT单加氧酶(DszC or DBT-MO)和DBT-砜单加氧酶(DszA or DBTO2-MO)都是黄素依赖型氧化酶,它们的催化反应需要菌体中还原型的黄素单核苷酸(FMNH2),FMNH2由辅酶黄素还原酶(DszD)再生。因此,共表达DszA,DszB,DszC和DszD可以提高整个脱硫途径的速率。构建了两个不相容性表达载体pBADD和paN2并在大肠杆菌中实现了4个脱硫酶基因的共表达。DszA,DszB,DszC和DszD的可溶性蛋白表达量分别占菌体总蛋白质的7.6%,3.5%,3.1%和18%。共表达时的脱硫活性是单独用paN2表达时的5.4倍,并对工程菌休止细胞脱除模拟柴油中DBT的活性进行了研究。     

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.     

Deep desulfurization of extensively hydrodesulfurized middle distillate oil by Rhodococcus sp. strain ECRD-1

Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by Rhodococcus sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.     

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.     

Deep Desulfurization of Extensively Hydrodesulfurized Middle Distillate Oil by Rhodococcus sp. Strain ECRD-1

Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by Rhodococcus sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.     

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.     

Kinetic analysis of biodesulfurization of model oil containing multiple alkyl dibenzothiophenes

Biodesulfurization is regarded as a promising alternative technology for desulfurization from diesel oil due to its mild operating conditions and its ability to remove sulfur from alky dibenzothiophenes (C_x-DBTs). The diesel oil contains complex mixtures of C_x-DBTs in which individual microbial biodesulfurization may be altered. In this work, interactions among three typical C_x-DBTs such as dibenzothiophenes (DBT), 4-methyldibenzothiophene (4-MDBT), and 4,6-dimethyldibenzothiophene (4,6-DMDBT) were investigated using Mycobacterium sp. ZD-19 in an airlift reactor. The experimental results indicated that the desulfurization rates would decrease in the multiple C_x-DBTs system compared to the single C_x-DBT system. The extent of inhibition depended upon the substrate numbers, concentrations, and affinities of the co-existing substrates. For example, compared to individual desulfurization rate (100 %), DBT desulfurization rate decreased to 75.2 % (DBT + 4,6-DMDBT), 64.8 % (DBT + 4-MDBT), and 54.7 % (DBT + 4,6-DMDBT + 4-MDBT), respectively. This phenomenon was caused by an apparent competitive inhibition of substrates, which was well predicted by a Michaelis–Menten competitive inhibition model.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Batch and Fed-Batch Cultures

The DbtS(sup+) phenotype (which confers the ability to oxidize selectively the sulfur atom of dibenzothiophene [DBT] or dibenzothiophene sulfone [DBTO(inf2)]) of Rhodococcus erythropolis N1-36 was quantitatively characterized in batch and fed-batch cultures. In flask cultures, production of the desulfurization product, monohydroxybiphenyl (OH-BP), was maximal at pH 6.0, while specific productivity (OH-BP cell(sup-1)) was maximal at pH 5.5. Quantitative measurements in fermentors (in both batch and fed-batch modes) demonstrated that DBTO(inf2) as the sole sulfur source yielded a greater amount of product than did DBT. Specifically, 100 (mu)M DBT maximally yielded (apprx=)40 (mu)M OH-BP, while 100 (mu)M DBTO(inf2) yielded (apprx=)60 (mu)M OH-BP. Neither maintaining the pH at 6.0 nor adding an additional carbon source increased the yield of OH-BP. The presence of SO(inf4)(sup2-) in growth media repressed expression of desulfurization activity, but SO(inf4)(sup2-) added to suspensions of cells grown in DBT or DBTO(inf2) did not inhibit desulfurization activity.     

Biodesulfurization of DBT in tetradecane and crude oil by a facultative thermophilic bacterium Mycobacterium goodii X7B

Mycobacterium goodii X7B, a facultative thermophilic bacterium, cleaving the C-S bond of dibenzothiophene via a sulfur-specific pathway, was investigated for DBT in tetradecane and crude oil desulfurization. The extent of growth was improved by fed-batch culture controlled at a constant pH. The total sulfur level of dibenzothiophene in tetradecane, was reduced by 99%, from 200 to 2 ppm within 24h at 40 degrees C. After 72 h treatment, 59% of the total sulfur content in Liaoning crude oil was removed, from 3600 to 1478 ppm.     

脱硫工程菌的构建及其脱硫性能分析

以专一性脱硫菌德氏假单胞菌Pseudomonas delafieldii R-8为出发菌株, 利用pPR9TT穿梭质粒构建脱硫操纵子表达载体, 转化原始菌培养得到1株多拷贝脱硫基因的脱硫工程菌R-8-1, 并对其脱硫性能进行了研究。结果表明, 在同样的生物催化脱硫反应条件下, 工程菌的脱硫活性达到6.25 mmol DBT/g dry cell/h, 是原始菌的2倍; 柴油的脱硫试验表明, 在12 h内工程菌静息细胞能将柴油硫含量从310.8 mg/L降至100.1 mg/ L, 脱硫率达到68%, 而原始菌为53%。进一步比较了重组质粒pPR-dsz在工程菌株中传代的稳定性, 试验表明pPR-dsz在工程菌株R-8-1中具有良好的遗传稳定性。此研究为生物脱硫提供了1株优良的工程菌株, 并为该技术的应用提供了参考。     

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.     

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.     

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。     

Enhanced biodesulfurization by expression of dibenzothiophene uptake genes in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The transfer of dibenzothiophene (DBT) and its derivatives into cells is a critical step for biodesulfurization. The desulfurization reactions of resting cells and cell lysate were studied, which showed that the desulfurization rate of DBT, especially 4, 6-dimethyldibenzothiophene (4, 6-DMDBT) in Rhodococcus erythropolis LSSE8-1 was seriously affected by the transfer into cells. The inhibited effect of NaN₃ on desulfurization reactions was studied, which confirmed that the transfer of DBT into cells was an active transport in R. erythropolis LSSE8-1. The uptake-genes of DBT and its derivatives (HcuABC) of Pseudomonas delafieldii R-8 were introduced into the specific desulfurization bacterium, R. erythropolis LSSE8-1. Compared with the wild type, the strains bearing HcuABC genes showed a higher desulfurization activity. The desulfurization ratio of DBT showed a 19% increase, and 13% increase of 4, 6-DMDBT.     

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.     

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 micro M) at a rate of 0.023 micro mol 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.     

Deep desulfurization of hydrodesulfurization-treated diesel oil by a facultative thermophilic bacterium Mycobacterium sp. X7B

The dibenzothiophene (DBT) desulfurization pathway of a facultative thermophilic bacterium Mycobacterium sp. X7B was investigated. Metabolites were identified by gas chromatography-mass spectrometry, and the results showed that 2-hydroxybiphenyl, the end product of the previously reported sulfur-specific pathway (also called 4S pathway), was further converted to 2-methoxybiphenyl. This is the first strain to possess this ability and therefore, an extended 4S pathway was determined. In addition, the DBT-desulfurizing bacterium Mycobacterium sp. X7B was able to grow on DBT derivatives such as 4-methylDBT and 4,6-dimethylDBT. Resting cells could desulfurize diesel oil (total sulfur, 535 ppm) after hydrodesulfurization. GC flame ionization detection and GC atomic emission detection analyses were used to qualitatively evaluate the effect of Mycobacterium sp. X7B treatment on the content of the diesel oil. The total sulfur content of the diesel oil was reduced 86% using resting cell biocatalysts for 24 h at 45 degrees C.     

Microbial desulfurization of alkylated dibenzothiophene and alkylated benzothiophene by recombinant Rhodococcus sp. strain T09

The dibenzothiophene (DBT) desulfurizing operon, dsz, was introduced into various benzothiophene (BT)-desulfurizing bacteria using a Rhodococcus-E. coli shuttle vector. Of the tested recombinant bacteria, only those from Rhodococcus sp. strain T09 grew with both DBT and BT as the sole sulfur source. These recombinant cells desulfurized not only alkylated BTs, but also various alkylated DBTs, producing alkylated hydroxybiphenyls as the desulfurized products. Recombinant strain T09 also desulfurized alkylated DBT in an oil-water, two-phase resting-cell reaction. The dsz operon had the same desulfurizing activity when inserted into the vector in either orientation, indicating that the promoter region of the operon was functional in strain T09.