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.     

Sulfur-Specific Microbial Desulfurization of Sterically Hindered Analogs of Dibenzothiophene

Dibenzothiophenes (DBTs) bearing alkyl substitutions adjacent to the sulfur atom, such as 4,6-diethyldibenzothiophene (4,6-DEDBT), are referred to as sterically hindered with regard to access to the sulfur moiety. By using enrichment cultures with 4,6-DEDBT as the sole sulfur source, bacterial isolates which selectively remove sulfur from sterically hindered DBTs were obtained. The isolates were tentatively identified as Arthrobacter species. 4,6-DEDBT sulfone was shown to be an intermediate in the 4,6-DEDBT desulfurization pathway, and 2-hydroxy-3,3(prm1)-diethylbiphenyl (HDEBP) was identified as the sulfur-free end product.     

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。     

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.     

Biotechnology of desulfurization of diesel: prospects and challenges

To meet stringent emission standards stipulated by regulatory agencies, the oil industry is required to make a huge investment to bring down the sulfur content in diesel to the desired level, using conventional hydrodesulfurization (HDS) technology, by which sulfur is catalytically converted to hydrogen sulfide in the presence of hydrogen. These reactions proceed rapidly only at high temperature and pressure and therefore the capital cost as well as the operating cost associated with HDS very high. Biological desulfurization has the potential of being developed as a viable technology downstream of classical HDS. Various attempts have been made to develop biotechnological processes based on microbiological desulfurization employing aerobic and anaerobic bacteria. However, there are several bottlenecks limiting commercialization of the process. This review discusses various aspects of microbial desulfurization and the progress made towards its commercialization.     

Metabolic response against sulfur-containing heterocyclic compounds by the lignin-degrading basidiomycete Coriolus versicolor

The fungal conversions of sulfur-containing heterocyclic compounds were investigated using the lignin-degrading basidiomycete Coriolus versicolor. The fungus metabolized a series of sulfur compounds--25 structurally related thiophene derivatives--via several different pathways. Under primary metabolic conditions, C. versicolor utilized thiophenes, such as 2-hydroxymethyl-, 2-formyl-, and 2-carboxyl-thiophenes, as a nutrient sulfur source for growth; thus, the fungus degraded these compounds more effectively in a non-sulfur-containing medium than in conventional medium. The product analysis revealed that several redox reactions, decarboxylation reactions, and C-S cleavage reactions were involved in the fungal conversion of non-aromatic thiophenes. On the other hand, benzothiophene (BT) and dibenzothiophene (DBT) skeletons were converted to water-soluble products. All the products and metabolic intermediates were more hydrophilic than the starting substrates. These metabolic actions seemed to be a chemical stress response against exogenously added xenobiotics. These metabolic reactions were optimized under ligninolytic conditions, also suggesting the occurrence of a fungal xenobiotic response. Furthermore, the fungus converted a series of BTs and DBTs via several different pathways, which seemed to be controlled by the chemical structure of the substrates. DBT, 4-methylDBT, 4, 6-dimethylDBT, 2-methylBT, and 7-methylBT were immediately oxidized to their S-oxides. BTs and DBTs with the hydroxymethyl substituent were converted to their xylosides without S-oxidation. Those with carboxyl and formyl substituents were reduced to form a hydroxymethyl group, then xylosidated. These observations strongly suggested the involvement of a fungal substrate-recognition and metabolic response mechanism in the metabolism of sulfur-containing heterocyclic compounds by C. versicolor.     

Thermophilic Carbon-Sulfur-Bond-Targeted Biodesulfurization

Petroleum contains many heterocyclic organosulfur compounds refractory to conventional hydrodesulfurization carried out with chemical catalysts. Among these, dibenzothiophene (DBT) and DBTs bearing alkyl substitutions are representative compounds. Two bacterial strains, which have been identified as Paenibacillus strains and which are capable of efficiently cleaving carbon-sulfur (C--S) bonds in DBT at high temperatures, have been isolated for the first time. Upon attacking DBT and its various methylated derivatives at temperatures up to 60(deg)C, both growing and resting cells of these bacteria can release sulfur atoms as sulfate ions and leave the monohydroxylated hydrocarbon moieties intact. Moreover, when either of these paenibacilli was incubated at 50(deg)C with light gas oil previously processed through hydrodesulfurization, the total sulfur content in the oil phase clearly decreased.     

Kinetic analysis of microbial desulfurization of model and light gas oils containing multiple alkyl dibenzothiophenes

The reaction mechanism of biodesulfurization was investigated using whole cells of Rhodococcus erythropolis KA2-5-1, which have the ability to convert dibenzothiophene (DBT) into 2-hydroxybiphenyl. The desulfurization patterns of alkyl DBTs were represented by the Michaeis-Menten equation. The values of rate constants, the limiting maximal velocity (Vmax) and Michaelis constant (Km), for desulfurization of alkyl DBTs were calculated. The relative desulfurization activities of various alkyl DBTs were reduced in proportion to the total carbon numbers of alkyl substituent groups. Alkyl DBTs that had a total of six carbons of alkyl substituent groups were not desulfurized. The type or position of alkyl substituent groups had little effect on desulfurization activity. The desulfurization activity of each alkyl DBT, when mixed together, was reduced. This phenomenon was caused by apparent competitive inhibition of substrates. Using the apparent competitive inhibition model, the desulfurization pattern of a multiple components system containing alkyl DBTs was elucidated. This model was also applicable for biodesulfurization of light gas oil.     

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.     

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.     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Microwave effects on NiMoS and CoMoS single-sheet catalysts

Single-sheet nanoclusters of MoS₂, NiMoS or CoMoS are widely used in hydrodesulfurization (HDS) catalysis in the petroleum industry. In HDS reactions under microwave irradiation, experiments indirectly pointed out that for pristine MoS₂ reaction rates are accelerated because hot spots are generated on the catalyst bed. In this work, we investigated NiMoS and CoMoS isolated single-sheet substituted catalysts before and after thiophene adsorption focusing on quantifying the effect of microwave irradiation. For that purpose, density functional theory (DFT) molecular charge densities of each system were decomposed according to the distributed multipole analysis (DMA) of Stone. Site dipole values of each system were directly associated with a larger or smaller interaction with the microwave field according to a proposed general approach. We showed that microwave enhancement of HDS reaction rates can occur more efficiently in the CoMoS and NiMoS promoted clusters compared to pristine MoS₂ in the following order: CoMoS > NiMoS > MoS₂. The atomic origin of the catalyst hot spots induced by microwaves was clearly established in the promoted clusters.     

Comparison of the substrate specificity of the two bacterial desulfurization systems

Using cell-free extracts of a desulfurizing mesophile, Rhodococcus erythropolis KA2-5-1 (the Dsz system) and Escherichia coli JM109, which possesses the desulfurizing genes of a thermophile Paenibacillus sp. A11-2 (the Tds system), the reactivity of desulfurizing enzymes toward 4,6-dialkyl dibenzothiophenes (4,6-dialkyl DBTs) and 7-alkyl benzothiophenes (7-alkyl BTs) was investigated. Both systems desulfurized all the 4,6-dialkyl DBTs, except 4,6-dibutyl DBT. Although some alkylated BTs were degraded by the Dsz system, no desulfurized compounds were detected. The reactivity of the Tds system toward alkylated BTs was higher than that of DBT. In contrast to the Dsz system, the Tds system yielded desulfurized compounds from all of the alkylated BTs examined.     

Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11-2

Paenibacillus A11-2 can efficiently cleave two carbon&bond;sulfur bonds in dibenzothiophene (DBT) and alkyl DBTs, which are refractory by conventional petroleum hydrodesulfurization, to remove sulfur atom at high temperatures. An 8.7-kb DNA fragment containing the genes for the DBT desulfurizing enzymes of A11-2 was cloned in Escherichia coli and characterized. Heterologous expression analysis of the deletion mutants identified three open reading frames that were required for the desulfurization of DBT to 2-hydroxybiphenyl (2-HBP). The three genes were designated tdsA, tdsB, and tdsC (for thermophilic desulfurization). Both the nucleotide sequences and the deduced amino acid sequences show significant homology to dszABC genes of Rhodococcus sp. IGTS8, but there are several local differences between them. Subclone analysis revealed that the product of tdsC oxidizes DBT to DBT-5,5'-dioxide via DBT-5-oxide, the product of tdsA converts DBT-5,5'-dioxide to 2-(2-hydroxyphenyl) benzene sulfinate, and the product of tdsB converts 2-(2-hydroxyphenyl)benzene sulfinate to 2-HBP. Cell-free extracts of a recombinant E. coli harboring all the three desulfurization genes converted DBT to 2-HBP at both 37 and 50 degrees C. In vivo and in vitro exhibition of desulfurization activity of the recombinant genes derived from a Paenibacillus indicates that an E. coli oxidoreductase can be functionally coupled with the monooxygenases of a gram-positive thermophile.     

Contact versus energy transfer fluorescence quenching in the sulfur substituted form of the enzyme rhodanese: a study using cesium ion resolved emission spectra.

The intrinsic fluorescence of the enzyme rhodanese (EC 2.8.1.1) can be resolved into separate contributions from solvent accessible and solvent inaccessible tryptophan residues by comparing spectra run in 2 $\text{M}$ NaCl with those run in the quenching solution, 2 $\text{M}$ CsCl. Both these classes of tryptophan residues are quenched when sulfur is transferred to rhodanese forming a sulfur substituted enzyme which is an intermediate in the catalytic cycle. This observation is consistent with a non-radiative energy transfer mechanism for quenching as opposed to a mechanism requiring direct contact between the bound sulfur and an active site tryptophan. Therefore, the data supports the hypothesis that the primary stabilizing influence in forming the substituted enzyme intermediate is a persulfide bond between an active site sulfhydryl group and the transferred sulfur.     

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。     

Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp. strain A11-2

A benzothiophene (BT) and dibenzothiophene (DBT) monooxygenase (TdsC), which catalyzes the oxidation of the sulfur atoms in BT and DBT molecules, was purified from Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme and its subunit were determined to be 200 kDa and 43 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating a tetrameric structure. The N-terminal amino acid sequence of the purified TdsC completely matched the amino acid sequence deduced from the nucleotide sequence of the tdsC gene reported previously [Ishii et al. (2000) Biophys Biochem Res Commun 270:81-88]. The optimal temperature and pH for the TdsC reaction were 65 degrees C and pH 9, respectively. TdsC required NADH, FMN and TdsD, a NADH-dependent FMN oxidoreductase, for its activity, as was observed for TdsA. FAD, lumiflavin and/or NADPH had some effect on the maintenance of TdsC activity. A comparison of the substrate specificity of TdsC and DszC, the homologous monooxygenase purified from Rhodococcus erythropolis strain KA2-5-1, demonstrated a contrasting pattern towards alkylated DBTs and BTs.     

Substrate Preferences in Biodesulfurization of Diesel Range Fuels by Rhodococcus sp. Strain ECRD-1

The range of sulfur compounds in fuel oil and the substrate range and preference of the biocatalytic system determine the maximum extent to which sulfur can be removed by biodesulfurization. We show that the biodesulfurization apparatus in Rhodococcus sp. strain ECRD-1 is able to attack all isomers of dibenzothiophene including those with at least four pendant carbons, with a slight preference for those substituted in the α-position. With somewhat less avidity, this apparatus is also able to attack substituted benzothiophenes with between two and seven pendant carbons. Some compounds containing sulfidic sulfur are also susceptible to desulfurization, although we have not yet been able to determine their molecular identities.     

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snailHelix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mountin situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion.In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snailLymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of theHelix family and the light yellow cells ofLymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.     

Partitioning of gonococcal LPS into phenol and water: Different LPS composition of in vivo and in vitro selected variants

Abstract Depending on the culture conditions, Pyrodictium occultum cells revealed two different types of fibers with significant differences in their width in the electron microscope. During growth on elemental sulfur preferentially fibres with a diameter of about 23 nm (type I) were produced. When elemental sulfur was substituted by thiosulfate fibers with a diameter of around 15 nm (type II), were the main appendages. Both types form hollow cylinders consisting of helically arranged sub-units with a wall thickness of 2–3 nm. A triple- layered unit membrane could not be found.