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。     

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.     

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。     

Microbial desulfurization of motor fuel

Deep desulfurization of oil and its fractions is currently performed by hydration at high temperature and hydrogen pressure, which makes the process rather expensive. Searches for alternative modes for desulfurization, among which is biodesulfurization, are intensely in progress. In this review, the following subjects are discussed: microorganisms capable of desulfurizing petroleum products, mechanisms of their activity, achievements in the field of process development, and disadvantages of the method. The existing level of knowledge is insufficient for immediate implementation of an industrial biotechnological process for sulfur elimination from oil and motor fuel and it can only be regarded as a medium-term (10–15 years) prospect.     

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.     

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 alpha-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.     

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.

     

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.     

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.     

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.     

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.     

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.     

Flux-based analysis of sulfur metabolism in desulfurizing strains of Rhodococcus erythropolis

Rhodococcus erythropolis has been studied widely for potential applications in biodesulfurization. Previous works have been largely experimental with an emphasis on the characterization and genetic engineering of desulfurizing strains for improved biocatalysis. A systems modeling approach that can complement these experimental efforts by providing useful insights into the complex interactions of desulfurization reactions with various other metabolic activities is absent in the literature. In this work, we report the first attempt at reconstructing a flux-based model to analyze sulfur utilization by R. erythropolis. The model includes the 4S pathway for dibenzothiophene (DBT) desulfurization. It predicts closely the growth rates reported by two independent experimental studies, and gives a clear and comprehensive picture of the pathways that assimilate the sulfur from DBT into biomass. In addition, it successfully elucidates that sulfate promotes higher cell growth than DBT and its presence in the medium reduces DBT desulfurization rates. A study using eight carbon sources suggests that ethanol and lactate yield higher cell growth and desulfurization rates than citrate, fructose, glucose, gluconate, glutamate, and glycerol.     

Relation between bacterial strain resistance to solvents and biodesulfurization activity in organic medium

Microorganisms used in biodesulfurization of petroleum products have to withstand high concentrations of hydrocarbons. The capacities of seven desulfurizing strains of Rhodococcus to be active in the presence of solvents were evaluated. Octanol and toluene (log P=2.9) were selected as toxic solvents. The effect of the solvents was determined by measuring either inhibition of growth or the decrease in respiratory activity of the cells. Differences among strains in their resistance to solvent responses were observed, but these variations were dependent on the test used. Resistance to solvents was then compared to the capacity of the different strains to retain biodesulfurization activity in the presence of hexadecane. Inhibition of desulfurization by high concentrations of hexadecane was found to be well correlated to the sensitivity of the strains to respiration inhibition by toluene, but not to growth inhibition. This result also showed that the respirometric test was a rapid and reliable test to select solvent-resistant strains for use as resting cells in biocatalysis processes, such as biodesulfurization, in organic media.     

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.     

Roles of sulfite oxidoreductase and sulfite reductase in improving desulfurization by Rhodococcus erythropolis

Rhodococcus erythropolis has been widely studied for desulfurization. However, activity levels required for commercial application have not been achieved. A major limitation of the current work in biodesulfurization is inadequate information regarding sulfur metabolism generally, and in particular the metabolism of the sulfur obtained from dibenzothiophene (DBT) metabolism via the 4S pathway. In this work, we have investigated the possible routes taken by the sulfur from DBT to convert into biomass or other metabolites. We propose two alternate hypotheses. In the first, we hypothesize that the cell can convert via sulfite reductase (SR) the sulfite from the metabolism of DBT into sulfide that can be assimilated into biomass. However, in the process, it may convert any excess sulfite into extracellular sulfate via sulfite oxidoreductase (SOR) to avoid the toxic effects of sulfite. In the second, we speculate that the cell cannot assimilate the sulfite directly into biomass via SR. It must first use SOR to produce extracellular sulfate, and then recapture that sulfate into biomass via SR. Thus, either way, we propose that SOR and SR activities, in addition to dsz genes and cofactors, may be critical in increasing desulfurization levels significantly. In particular, we suggest that the simultaneous increase in SOR activity and decrease in SR activity can enable increased desulfurization activity.     

血红蛋白基因在脱硫菌R-8中的表达及其在柴油脱硫中的应用

摘要：【目的】旨在构建一株优良的工程菌株,对血红蛋白基因在柴油的生物脱硫领域的应用做初步的探索。【方法】以德氏假单胞菌（Pseudomonas delafieldii） R-8为出发菌株,通过基因工程的手段,构建透明颤菌（Vitreoscilla）血红蛋白基因表达质粒并电击导入原始菌株,得到重组菌P. delafieldii R-8-2。【结果】R-8-2菌株的CO差光谱在419 nm处有特征峰出现,表明血红蛋白在脱硫菌中得到了有效表达。R-8-2菌株和R-8菌株相比,生长得到改善,相同培养条件下菌体密度比R-8提高了20%,最大脱硫活性能够达到R-8的2.4倍。在实际柴油脱硫实验中,R-8-2菌株能将柴油的硫含量降至96.6 mg/L,脱硫率达到69.9%,而R-8仅为57.2%。【结论】R-8-2是在较低溶氧条件下仍能保持较高的菌体密度和脱硫活性的基因工程菌株,具有良好的应用前景,该研究为血红蛋白基因在生物脱硫工业的应用提供参考。     

Cloning of a rhodococcal promoter using a transposon for dibenzothiophene biodesulfurization

The expression of biodesulfurization genes (dsz) in Rhodococcus erythropolis strain KA2-5-1 is repressed by sulfate which is the product of biodesulfurization. The application of a sulfate non-repressible promoter could be effective in enhancing biodesulfurization. A promoter-probe transposon was constructed using the promoterless, red-shifted green fluorescence protein gene (rsgfp). A 340 bp putative promoter element, designated kap1, was isolated from a strain KA2-5-1 recombinant that had shown high fluorescence intensity. The activity of kap1 was not affected by 1 mM sulfate. It gave about a 2-fold greater activity than the 16S ribosomal RNA promoter in R. erythropolis strain KA2-5-1 and is therefore useful for expressing desulfurization genes in rhodococcal strains.     

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...     

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.