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.     

Biodesulfurization of refractory organic sulfur compounds in fossil fuels

The stringent new regulations to lower sulfur content in fossil fuels require new economic and efficient methods for desulfurization of recalcitrant organic sulfur. Hydrodesulfurization of such compounds is very costly and requires high operating temperature and pressure. Biodesulfurization is a non-invasive approach that can specifically remove sulfur from refractory hydrocarbons under mild conditions and it can be potentially used in industrial desulfurization. Intensive research has been conducted in microbiology and molecular biology of the competent strains to increase their desulfurization activity; however, even the highest activity obtained is still insufficient to fulfill the industrial requirements. To improve the biodesulfurization efficiency, more work is needed in areas such as increasing specific desulfurization activity, hydrocarbon phase tolerance, sulfur removal at higher temperature, and isolating new strains for desulfurizing a broader range of sulfur compounds. This article comprehensively reviews and discusses key issues, advances and challenges for a competitive biodesulfurization process.     

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。     

Biodesulfurization.

Microbial sulfur-specific transformations have been identified that selectively desulfurize organic sulfur compounds in fossil fuels. Recent discoveries related to biodesulfurization mechanisms may lead to commercial applications of biodesulfurization through engineering recombinant strains for over-expression of biodesulfurization genes, removal of end product repression, and/or by combining relevant industrial and environmental traits with improvements in bioprocess design.     

Thermophilic biodesulfurization and its application in oil desulfurization

To reduce the harm caused to the environment by fuel combustion and meet the increasingly stringent emission standards, the sulfur content of fuels should be reduced. Dibenzothiophene, benzothiophene, and their derivatives are sulfur-containing components of fuels that are difficult to desulfurize and can therefore cause great environmental damage. Biodesulfurization is a desulfurization method that has the advantage of being able to remove dibenzothiophene and its derivatives removed easily under conditions that are relatively mild when compared with hydrodesulfurization. This paper introduces the advantages of thermophilic biodesulfurization compared with mesophilic biodesulfurization; analyzes the desulfurization mechanism, including the desulfurization pathways and enzymic systems of desulfurization bacteria; and discusses the application of biodesulfurization in oil desulfurization. The main problems existing in biodesulfurization and possible solutions are also analyzed in this paper. Biological desulfurization is a promising method for desulfurization; accordingly, more studies investigating biodesulfurization of actual oil are needed to enable the industrialized application of biodesulfurization.

     

Bacterial biodegradation of aliphatic sulfides under aerobic carbon- or sulfur-limited growth conditions

AIMS: To isolate bacteria capable of cleaving aliphatic carbon-sulfur bonds as potential biological upgrading catalysts for the reduction of molecular weight and viscosity in heavy crude oil. METHODS AND RESULTS: Thirty-one bacterial strains isolated from enrichment cultures were able to biotransform model compounds representing the aliphatic sulfide bridges found in asphaltenes. Using gas chromatography and mass spectrometry, three types of attack were identified: alkyl chain degradation, allowing use as a carbon source; nonspecific sulfur oxidation; and sulfur-specific oxidation and carbon-sulfur bond cleavage, allowing use as a sulfur source. Di-n-octyl sulfide degradation produced octylthio- and octylsulfonyl-alkanoic acids, consistent with terminal oxidation followed by beta-oxidation reactions. Utilization of dibenzyl sulfide or 1,4-dithiane as a sulfur source was regulated by sulfate, indicating a sulfur-specific activity rather than nonspecific oxidation. Finally, several isolates were also able to use dibenzothiophene as a sulfur source, and this was the preferred organic sulfur substrate for one isolate. CONCLUSIONS: The use of commercially available alkyl sulfides in enrichment cultures gave isolates that followed a range of metabolic pathways, not just sulfur-specific attack. SIGNIFICANCE AND IMPACT OF THE STUDY: These results give new insight into biodegradation of organosulfur compounds from petroleum and for biotreatment of such compounds in chemical munitions.     

Medium composition overturns the widely accepted sulfate-dependent repression of desulfurization phenotype in Rhodococcus qingshengii IGTS8

Microbial desulfurization has been extensively studied as a promising alternative to the widely applied chemical desulfurization process. Sulfur removal from petroleum and its products becomes essential, as the environmental regulations become increasingly stringent. Rhodococcus qingshengii IGTS8 has gained ground as a naturally occurring model biocatalyst, due to its superior specific activity for desulfurization of dibenzothiophene (DBT). Recalcitrant organic sulfur compounds—DBT included—are preferentially removed by selective carbon-sulfur bond cleavage to avoid a reduction in the calorific value of the fuel. The process, however, still has not reached economically sustainable levels, as certain limitations have been identified. One of those bottlenecks is the repression of catalytic activity caused by ubiquitous sulfur sources such as inorganic sulfate, methionine, or cysteine. Herein, we report an optimized culture medium for wild-type stain IGTS8 that completely alleviates the sulfate-mediated repression of biodesulfurization activity without modification of the natural biocatalyst. Medium C not only promotes growth in the presence of several sulfur sources, including DBT, but also enhances biodesulfurization of resting cells grown in the presence of up to 5 mM sulfate. Based on the above, the present work can be considered as a step towards the development of a more viable commercial biodesulfurization process.     

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.     

Mycobiota of Oil-Contaminated Soil Samples and Their Abilities for Dibenzothiophene Desulfurization

High sulfur content of crude oil leads to poor quality of oil products and many other negative consequences such as corrosion, catalyst poisoning and environmental pollution. Saudi Arabia is seeking to reduce sulfur content in diesel and gasoline to 10 ppm and to lower benzene content in gasoline to 1%. Biotechnological processes such as biodesulfurization can be considered an alternative or complement to conventional oil refining technologies. So, the objective of the present project is to isolate and identify endogenous fungal isolates capable of oil biodesulfurization. From 60 oil-contaminated soil samples collected from Saudi Arabia, 15 species belonged to 9 fungal genera were collected and identified morphologically and with ITS sequencing. Members of Aspergillus, Penicillium and Fusarium were the most prevalent in the investigated samples. Among the collected fungal species, only Stachybotrys bisbyiisolates were able to utilize dibenzothiophene (DBT) as the sole sulfur source. Stachybotrys bisbyi TUSb1 could desulfurize 99% of the DBT (0.3 mM) as the sulfur source by a co-metabolism reaction with other carbon sources through the same pathway as 4S (involves sequential oxidation of the sulfur part and cleaving of the C–S bonds), and produced 2-hydroxy biphenyl (2-HBP) during 7 days of incubation at 30°C and 180 rpm. Stachybotrys bisbyi TUSb1 showed broad specificity for removing sulfur in different sulfur-containing compounds.     

Designing recombinant Pseudomonas strains to enhance biodesulfurization.

The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains. The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host. Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as production of biosurfactants, with the enhanced biodesulfurization phenotype.     

Microbial desulfurization of gasoline by free whole-cells of Rhodococcus erythropolis XP

Rhodococcus erythropolis XP could grow well with condensed thiophenes, mono-thiophenic compounds and mercaptans present in gasoline. Rhodococcus erythropolis XP was also capable of efficiently degrading the condensed thiophenes in resting cell as well as biphasic reactions in which n-octane served as a model oil phase. Free whole cells of R. erythropolis XP were adopted to desulfurize fluid catalytic cracking (FCC) and straight-run (SR) gasoline oils. About 30% of the sulfur content of FCC gasoline and 85% of sulfur in SR gasoline were reduced, respectively. Gas chromatography analysis with atomic emission detection also showed depletion of sulfur compounds in SR gasoline. Rhodococcus erythropolis XP could partly resist the toxicity of gasoline and had an application potential to biodesulfurization of gasoline.     

Biodesulfurization: a model system for microbial physiology research

Biological desulfurization (biodesulfurization) of dibenzothiophene (DBT) by the 4S pathway is a model system for an enviromentally benign way to lower the sulfur content of petroleum. Despite a large amount of effort the efficiency of the 4S pathway is still too low for a commercial oil biodesulfurization process, but the 4S pathway could potentially be used now for commercial processes to produce surfactants, antibiotics, polythioesters and other chemicals and for the detoxification of some chemical warfare agents. Proteins containing disulfide bonds are resistant to temperature, pH, and solvents, but the production of disulfide-rich proteins in microbial hosts is challenging. The study of the 4S pathway can provide insights as to how to maximize the production of disulfide-rich proteins. Engineering of the operon encoding the 4S pathway to contain a greater content of methionine and cysteine may be able to link use of DBT as a sole sulfur source to increasing 4S pathway activity by increasing the nutritional demand for sulfur. This strategy could result in the development of biocatalysts suitable for use in an oil biodesulfurization process, but the study of the 4S pathway can also lead to a better understanding of microbial physiology to optimize activity of a mult-step co-factor-requiring pathway, as well as the production of highly stable industrially relevant enzymes for numerous applications.     

硫氧化细菌源单质硫的生成、转运和回收

单质硫(硫粒)是硫化物生物氧化的中间产物.按化学计量式精准调控O/S比(溶解氧与硫化物的摩尔比),单质硫可成为硫氧化细菌(Sulfur-oxidizing bacteria,SOB)的主要代谢产物.根据单质硫的分布,单质硫可分为胞内硫粒和胞外硫粒.单质硫由胞内向胞外的跨膜转运过程是泌硫型SOB的重要生理特征.从生物脱硫...     

Dehalogenation, denitration, dehydroxylation, and angular attack on substituted biphenyls and related compounds by a biphenyl dioxygenase

The attack by the bph-encoded biphenyl dioxygenase of Burkholderia sp. strain LB400 on a number of symmetrical ortho-substituted biphenyls or quasi ortho-substituted biphenyl analogues has been investigated. 2,2'-Difluoro-, 2,2'-dibromo-, 2,2'-dinitro-, and 2,2'-dihydroxybiphenyl were accepted as substrates. Dioxygenation of all of these compounds showed a strong preference for the semisubstituted pair of vicinal ortho and meta carbons, leading to the formation of 2'-substituted 2,3-dihydroxybiphenyls by subsequent elimination of HX (X = F, Br, NO(2), or OH). All of these products were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenases of Burkholderia sp. strain LB400 or of Rhodococcus globerulus P6. Dibenzofuran and dibenzodioxin, which may be regarded as analogues of doubly ortho-substituted biphenyls or diphenylethers, respectively, were attacked at the "quasi ortho" carbon (the angular position 4a) and its neighbor. This shows that an aromatic ring-hydroxylating dioxygenase of class IIB is able to attack angular carbons. The catechols formed, 2,3,2'-trihydroxybiphenyl and 2,3,2'-trihydroxydiphenylether, were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenase. While angular attack by the biphenyl dioxygenase was the main route of dibenzodioxin oxidation, lateral dioxygenation leading to dihydrodiols was the major reaction with dibenzofuran. These results indicate that this enzyme is capable of hydroxylating ortho or angular carbons carrying a variety of substituents which exert electron-withdrawing inductive effects. They also support the view that the conversions of phenols into catechols by ring-hydroxylating dioxygenases, such as the transformation of 2,2'-dihydroxybiphenyl into 2,3,2'-trihydroxybiphenyl, are the results of di- rather than of monooxygenations. Lateral dioxygenation of dibenzofuran and subsequent dehydrogenation and extradiol dioxygenation by a number of biphenyl-degrading strains yielded intensely colored dead-end products. Thus, dibenzofuran can be a useful chromogenic indicator for the activity of the first three enzymes of biphenyl catabolic pathways.     

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 degrees 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 degrees 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.     

石油生物催化脱硫的研究进展

石油生物催化脱硫技术是新兴的极具潜力的石油非加氢脱硫技术,在降低轻质油品生产成本、提高油品质量和环境保护等方面显示出潜在的优势,被誉为21世纪的石油脱硫技术。本文主要对石油生物催化脱硫技术特点、各种降解路线和研究现状进行了综述,指出了石油生物催化脱硫技术存在的问题,并提出了进一步研究发展的方向。     

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.     

Biodesulfurization of water-soluble coal-derived material by Rhodococcus rhodochrous IGTS8

Rhodococcus rhodochrous IGTS8 was previously isolated because of its ability to use coal as its sole source of sulfur for growth. Subsequent growth studies have revealed that IGTS8 is capable of using a variety of organosulfur compounds as sources of sulfur but not carbon. In this article, the ability of IGTS8 to selectively remove organic sulfur from water-soluble coal-derived material is investigated. The microbial removal of organic sulfur from coal requires microorganisms capable of cleaving carbon-sulfur bonds and the accessibility of these bonds to microorganisms. The use of water-soluble coal-derived material effectively overcomes the problem of accessibility and allows the ability of microorganisms to cleave carbon-sulfur bonds present in coal-derived material to be assessed directly. Three coals, two coal solubilization procedures, and two methods of biodesulfurization were examined. The results of these experiments reveal that the microbial removal of significant amounts of organic sulfur from water-soluble coal-derived material with treatment times as brief as 24 h is possible. Moreover, the carbon content and calorific value of biotreated products are largely unaffected. Biotreatment does result, however, in an increased hydrogen and nitrogen content and a decreased oxygen content of the coal-derived material. The aqueous supernatant obtained from biodesulfurization experiments does not contain sulfate, sulfite, or other forms of soluble sulfur at increased concentrations in comparison with control samples. Sulfur removed from water-soluble coal-derived material appears to be incorporated into biomass. (c) 1992 John Wiley & Sons, Inc.     

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

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