JP2001231546A - Method for desulfurizing with microorganism at high temperature - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable desulfurization with microorganisms under a wide temperature range condition including a high temperature range. SOLUTION: The method for degrading an organic sulfur compound, characterized by degrading the organic sulfur compound with Mycobacterium phlei WU-F1 strain, a variant of the strain, or a transformant containing the stain as a host.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for decomposing organic sulfur compounds such as benzothiophene and dibenzothiophene using microorganisms. Since the method of the present invention can be used to decompose organic sulfur compounds contained in fossil fuels such as petroleum, it is easy to remove sulfur, which is diffused into the air by combustion of fossil fuels and becomes a source of environmental pollution, from fossil fuels. To

[0002]

2. Description of the Related Art (1) Conventional hydrodesulfurization method A desulfurization method for removing sulfur from a hydrocarbon fuel such as petroleum includes:
Although methods such as alkali cleaning and solvent desulfurization are also known,
At present, hydrodesulfurization is the mainstream. Hydrodesulfurization is a method in which a sulfur compound in a petroleum fraction is reacted with hydrogen in the presence of a catalyst, and removed as hydrogen sulfide to reduce the sulfur content of the product. As the catalyst, a metal catalyst such as cobalt, molybdenum, nickel, tungsten or the like using alumina as a carrier is used. In the case of a molybdenum-supported alumina catalyst, cobalt or nickel is usually added as a co-catalyst in order to improve the catalytic performance. There is no doubt that hydrodesulfurization using metal catalysts is an extremely complete process that is currently widely used worldwide. However, there are several issues in terms of the process for making petroleum products in compliance with stricter environmental regulations. The following is a brief description of an example.

[0003] Metal catalysts generally have low substrate specificity and are therefore suitable for the purpose of decomposing various kinds of sulfur compounds and reducing the sulfur content of the entire fossil fuel,
It is believed that the desulfurization effect may be insufficient for certain groups of sulfur compounds. For example, various heterocyclic organic sulfur compounds still remain in the gas oil after desulfurization. One of the causes of the insufficient desulfurization effect of the metal catalyst is considered to be steric hindrance due to substituents around sulfur atoms in these organic sulfur compounds. Of these substituents, the effect of the presence of the methyl substituent on the reactivity of the metal catalyst in hydrodesulfurization is
Studies are being made on thiophene, benzothiophene, dibenzothiophene, and the like. According to these results, it is clear that the desulfurization reactivity generally decreases as the number of substituents increases, but the effect of the position of the substituent on the reactivity is extremely large. A report comparing the desulfurization reactivity of methyldibenzothiophenes and showing that the steric hindrance by the substituent has a very large effect on the reactivity of the metal catalyst has been reported, for example, by Houalla, M., Broderick, DH, Sapre,
AV, Nag, NK, de Beer, VHJ, Gates, BC, Kwa
rt, HJ Catalt., 61.523-527 (1980). Indeed, it is known that various alkylated derivatives of these dibenzothiophenes are present in significant amounts in gas oils (eg, Kabe, T., Ishihara, A. and Tajima, H .;
Ind. Eng. Chem. Res., 31, 1577-1580 (1992)).

[0004] As described above, desulfurization of an organic sulfur compound having resistance to hydrodesulfurization requires a higher reaction temperature and pressure than currently used, and the amount of hydrogen to be added is also reduced. It is expected to increase significantly. Improvements in such hydrodesulfurization processes are expected to require significant capital and operating costs. As the main sulfur compound species which contains such an organic sulfur compound having resistance to hydrodesulfurization, for example, there is gas oil, and when performing a more advanced desulfurization of gas oil (ultra deep desulfurization), as described above. A significant improvement in the hydrodesulfurization process is required.

On the other hand, the enzymatic reaction performed by living organisms proceeds under relatively mild conditions, and the rate of the enzymatic reaction itself is not inferior to the rate of a reaction using a chemical catalyst. Furthermore, it is necessary to properly cope with a wide variety of biological reactions that occur in vivo, so that there are numerous types of enzymes, and these enzymes generally show very high substrate specificity. Have been. Such a feature is expected to be utilized in a so-called biodesulfurization reaction that removes sulfur in sulfur compounds contained in fossil fuels using microorganisms (Monticello, DJ, Hydrocarbo).
n Processing 39-45 (1994)).

Many methods for removing sulfur from petroleum using bacteria have been reported in the past, but most of them utilize a microbial metabolic reaction that proceeds under a temperature condition around 30 ° C. On the other hand, it is known that the rate of a chemical reaction generally increases depending on temperature. In the desulfurization step in the petroleum refining process, fractional distillation and desulfurization reaction are performed under high temperature and high pressure conditions. Therefore, if the bio-desulfurization step is incorporated into the petroleum refining process, it would be desirable to be able to perform the bio-desulfurization reaction at a higher temperature during the cooling without cooling the petroleum fraction to near normal temperature. Reports on high temperature biodesulfurization include:

Most attempts to perform desulfurization reactions at elevated temperatures using microorganisms can be found in coal desulfurization. Coal contains various sulfur compounds. Although the main inorganic sulfur compound is pyrite, a wide variety of organic sulfur compounds are mixed and many are known to contain thiol, sulfide, disulfide, and thiophene groups. The microorganisms used were bacteria of the genus Sulfolobus, all of which are thermophilic bacteria. Leaching of metals from mineral sulfides (Brierley CL & Murr, LE, Science 179, 448-490
(1973)) and the use of various different sulfolobus strains for sulfur removal of pyrite from coal has been reported (K
argi, F. & Robinson, JM, Biotechnol. Bioeng. 24,
2115-2121 (1982); Kargi, F. & Robinson, JM, App
l. Environ. Microbiol., 44, 878-883 (1982); Kargi,
F. & Gervoni. TD, Biotechnol. Letters 5, 33-38
(1983); Kargi, F. and Robonson, JM, Biotechnol.
Bioeng., 26, 687-690 (1984); Kargi, F. & Robinson,
JM, Biotechnol. Bioeng. 27, 41-49 (1985); Karg
i, F., Biotechnol. Lett., 9, 478-482 (1987)). Kar
gi and Robinson (Kargi.F. and Robinson, JM, Appl.
According to nviron. Microbiol., 44, 878-883 (1982)),
Sulfolobus acidocardarius (Sulfolobus a) isolated from an acidic hot spring in Yellowstone National Park, USA
cidocaldarius) grows at 45-70 ° C,
Oxidizes elemental sulfur at optimal pH 2. Pyrite oxidation by two other Sulfolobus acidocardarius strains has also been reported (Tobita, M., Yokozeki, M., Nishika
wa, N. & Kawakami. Y., Biosci. Biotech. Biochem. 5
8, 771-772 (1994)).

[0008] Among the organic sulfur compounds contained in fossil fuels, dibenzothiophene and its derivatives are known to be less susceptible to hydrodesulfurization in ordinary petroleum refining processes. The dibenzothiophene sulfolobus
High-temperature decomposition by Acidocardarius has also been reported (K
argi, K. & Robinson, JM, Biotechnol. Bioeng. 26,
687-690 (1984); Kargi, F., Biotechnol. Letters
9, 478-482 (1987)). According to these reports, when model aromatic heterocyclic sulfur compounds such as thianthrene, thioxanthene and dibenzothiophene were reacted with this microorganism at high temperature, these sulfur compounds were oxidized and decomposed. Oxidation of these aromatic heterocyclic sulfur compounds by Sulfolobus acidocardarius has been observed at 70 ° C., producing sulfate ions as reaction products. However, this reaction is a reaction in a medium that does not contain a carbon source other than the sulfur compound, and uses the sulfur compound as a carbon source. In view of the above, it is apparent that this reaction is not a CS bond cleavage type reaction but a CC bond cleavage type reaction. The C-C bond-cleaving type reaction has a problem that it decomposes aromatic hydrocarbons and the like in fossil fuels and causes a reduction in energy content. Furthermore, this Sulfolobus acidocardarius can only grow in acidic media, and the oxidative degradation of dibenzothiophene requires progression under harsh acidic conditions (pH 2.5). It is considered that such severe conditions cause deterioration of petroleum products, and at the same time, require an acid-resistant material in a step relating to desulfurization, which is considered to be undesirable in the process. When grown under autotrophic conditions, Sulfolobus acidocardarius gains the required energy from reduced iron-sulfur compounds and uses carbon dioxide as a carbon source. Also, Sulfolobus
Acidocardarius, when grown under heterotrophic conditions, can utilize various organic compounds as carbon and energy sources. That is, it is considered that the presence of fossil fuel is utilized as a carbon source.

Finnerty et al. Reported that strains belonging to Pseudomonas stutzeri, Pseudomonas alcaligenes, and Pseudomonas putida belong to dibenzothiophene, benzothiophene, thioxanthen,
Reports that thianthrene is degraded and converted to water-soluble substances (Finnerty, WR, Shockiey, K., Atta
way, H. in MicrobialEnhanced Oil Recovery, Zajic,
JE et al. (Eds.) Penwell.Tulsa, Okla., 83-91 (198
3)) In this case, the oxidation reaction proceeds even at 55 ° C. However, the degradation product of dibenzothiophene by these Pseudomonas strains was 3-hydroxy-2-formylbenzothiophene, the product of a CC bond cleavage reaction (Monticello, DJ, Bakker, D., Finn
erty, WR Appl.Environ.Microbiol., 49, 756-760
(1985)). Further, the oxidation activity of dibenzothiophene by these Pseudomonas strains is induced by naphthalene and salicylic acid, which are aromatic hydrocarbons containing no sulfur, and is blocked by chloramphenicol. From these facts, it is clear that the decomposition reaction of dibenzothiophene by these Pseudomonas strains is based on the decomposition by breaking the CC bond in the aromatic ring. In addition, valuable aromatic hydrocarbons contained in the petroleum fraction other than the sulfur compound may be decomposed at the same time, which lowers the value of the fuel and the quality of the petroleum fraction.

As described above, the bacteria discovered so far that can decompose dibenzothiophene at a high temperature cleave a CC bond in a dibenzothiophene molecule and catalyze a reaction utilizing the same as a carbon source. Instead of a CC bond,
If microorganisms that specifically cleave CS bonds can be used,
Desulfurization can be performed without reducing the energy content of the fossil fuel. Also, in the CS bond cleavage type reaction, dibenzothiophene is finally decomposed to sulfate, but in the CC bond cleavage type reaction, a small amount of dibenzothiophene is decomposed to sulfate. , Mostly 3
It is only decomposed to -hydroxy-2-formylbenzothiophene. Therefore, even if the decomposition products are exposed, C
A microorganism that cleaves a CS bond is superior to a microorganism that cleaves a -C bond.

Microorganisms that perform CS bond cleavage type dibenzothiophene degradation reactions are known among bacteria belonging to several genera. However, very few examples have described that all of these bacteria exhibited an activity of decomposing dibenzothiophene under a high temperature condition of at least 42 ° C. For example, Rhodococcus sp.
ATCC 53968 is a well-studied dibenzothiophene-degrading strain that adds an oxygen atom to the sulfur atom of dibenzothiophene to form dibenzothiophene sulfone from dibenzothiophene sulfoxide, followed by 2'-hydroxybiphenyl-2-sulfinate. Perform a reaction to produce 2-hydroxybiphenyl. However, it has been reported that even at 37 ° C. and 43 ° C., which is slightly higher than the normal culturing temperature of 30 ° C., the cultivation of the bacterium is very slow or stops growing after 48 hours (Japanese Unexamined Patent Publication No. No. 54695). From this, in order to carry out the high-temperature desulfurization reaction, organic sulfur compounds, particularly heterocyclic sulfur compounds including dibenzothiophene and its substituted derivative compounds, can be grown at a high temperature in a CS bond-specific manner. It is considered optimal to use a microorganism that can be cleaved.

Suzuki et al. Reported that Paenibacil
lus) sp. A11-2 strain can grow under high temperature conditions and
It has been reported that the CS bond of an organic sulfur compound can be specifically cleaved (Suzuki, M., Konishi, J., Ishii,
Y., Okumura, K., Appl.Environ.Microbiol., 63, 31
64-3169 (1997)). According to this report, this strain is 30-
It can grow under a temperature condition of 60 ° C. and has a C value of dibenzothiophene or dibenzothiophene derivatives such as 4-methyldibenzothiophene and 4,6-dimethyldibenzothiophene.
-S bond can be specifically cleaved. Therefore, this strain is suitable for performing a high-temperature desulfurization reaction. However, this strain has a high desulfurization activity at 45 ° C. or higher, which requires heating depending on the environment. However, the desulfurization activity at 45 ° C. or lower rapidly decreases in an environment where heating is not required, and the medium temperature range (30 ° C.) (In the vicinity) has a disadvantage that the activity is extremely low. Moreover, the desulfurization activity of this strain at a high temperature range is significantly lower than that of Rhodococcus erythropolis, which is known to have a desulfurization activity at a medium temperature range.

In the biodesulfurization process, a high temperature range (hereinafter, “high temperature range” refers to a temperature around 50 to 60 ° C.)
Maintaining high desulfurization activity under a wide range of temperature conditions (30 to 50 ° C.) is effective in simplifying the temperature control of petroleum fractions. Furuya et al. Reported that Bacillus subtilis WU-S2B strain maintained relatively high desulfurization activity under a wide range of temperature conditions including a high temperature range, and specifically inhibited the CS bond of organic sulfur compounds. (Furuya, Nishii, Nakagawa, Kirimura, Usami,
Abstracts of Annual Meeting of the Japanese Society of Agricultural Chemistry, 384 (1999)). According to this report, this strain maintains a relatively high desulfurization activity under a wide range of temperature conditions from 30 to 50 ° C, and dibenzothiophene or dibenzothiophene such as 2,8-dimethyldibenzothiophene and 4,6-dimethyldibenzothiophene. The CS bond of the thiophene derivative can be specifically cleaved. However, this strain has a remarkably low desulfurization activity as compared with ATCC53968 of Rhodococcus sp. Which shows a very high desulfurization activity around 30 ° C. Therefore, in order to carry out the high-temperature desulfurization reaction, an extremely high desulfurization activity is maintained under a wide range of temperature conditions including a middle temperature range and a high temperature range, and an organic sulfur compound, especially dibenzothiophene, is maintained at a middle temperature range and a high temperature range. It is considered optimal to use a microorganism capable of specifically cleaving the C—S bond of heterocyclic sulfur compounds including the compound thereof and its substituted derivative compound.

[0014]

As described above, as microorganisms used in the desulfurization step of petroleum, extremely high desulfurization activity is maintained under a wide range of temperature conditions from a medium temperature range to a high temperature range, and organic microorganisms are used. Desirably, it is one that can specifically cleave the CS bond of a sulfur compound, but as described above, such a microorganism has not yet been found. The present invention
Under such a technical background, the purpose is to maintain a very high desulfurization activity under a wide range of temperature conditions including a middle temperature range and a high temperature range, and to produce an organic sulfur compound. An object of the present invention is to provide a means for desulfurizing petroleum or the like using a microorganism capable of specifically cleaving a CS bond, separating the microorganism from nature.

[0015]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, it has been found that strains belonging to the genus Mycobacterium and Frei can be grown in a wide temperature range including a medium temperature range and a high temperature range. Under the conditions, it is possible to maintain a very high desulfurization activity and at the same temperature as Rhodococcus erythropolis, whose desulfurization activity in the medium temperature range is widely known, and to be able to specifically cleave the CS bond of the organic sulfur compound. We have found and completed the present invention.

That is, the present invention is a method for decomposing an organic sulfur compound, which comprises decomposing an organic sulfur compound with the following microorganisms (1) to (3). (1) Mycobacterium ph
lei) WU-F1 strain (2) Mutant strain of Mycobacterium frei WU-F1 strain (3) Transformant using Mycobacterium frei WU-F1 strain as a host Mycobacterium frei strain WU-F1 having the ability to degrade compounds.

Further, the present invention provides a Mycobacterium
It is a transformant using Frey WU-F1 as a host.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
First, the Mycobacterium frei strain WU-F1 (hereinafter, simply referred to as "WU-F1 strain") of the present invention will be described. W
The U-F1 strain was discovered by the present inventors through screening using various types of soils collected from various parts of Japan as isolation sources. The WU-F1 strain has the following mycological properties.

Cell morphology: rod-like (width 1 μm, length 2
μm) Gram stain test: + Spore formation:-Oxidase test:-Catalase test: + Diamino acid of peptidoglycan: Mesodiaminopimelic acid Menaquinone: MK-9 (H2) Mycolic acid pattern: Mycobacterium frei group Fatty acid pattern with homology to mycolic acid pattern of the bacterium belonging to: Partial sequence of 16Sr DNA having homology to fatty acid pattern of bacterium belonging to Mycobacterium group: 16SrDNA of bacterium belonging to Mycobacterium frei group Has 100% homology with partial sequence

The cultivation of the WU-F1 strain is carried out according to a usual culture method of a microorganism. The form of culture is preferably liquid culture. Commonly used nutrients for the medium are widely used. Any available carbon compound may be used as the carbon source. For example, glucose or the like is used. Any available nitrogen compound may be used as the nitrogen source. For example, organic nutrients such as peptone, polypeptone, meat extract, yeast extract, soybean powder, and casein hydrolyzate can be used. If it is desired to culture in a medium that does not contain sulfur compounds that can affect the desulfurization reaction, inorganic nitrogen compounds such as ammonium chloride can also be used.
In addition, phosphates, carbonates, magnesium, calcium, potassium, sodium, iron, manganese, zinc, molybdenum, tungsten, copper, vitamins, and the like are used as needed. The cultivation is carried out aerobically for 2 to 3 days at pH 6 to 8 and at a temperature of 30 to 50 ° C under shaking or aeration conditions.
The WU-F1 strain has been deposited with the National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-17717 (Deposit date: February 8, 2000).

Next, the method for decomposing an organic sulfur compound of the present invention will be described. The method for decomposing an organic sulfur compound of the present invention comprises the steps of: preparing an organic sulfur compound from a WU-F1 strain, a mutant strain of WU-F1 strain, or
It is characterized by being degraded by a transformant using the WU-F1 strain as a host. Here, the mutant strain of WU-F1 strain is WU-F1
A strain originating from a strain, having the same properties as the WU-F1 strain in terms of the ability to decompose organic sulfur compounds, but different from WU-F1 in at least one other property. The transformant using the WU-F1 strain as a host means a strain in which a part of the characteristics of the WU-F1 strain is changed by introducing a foreign gene into the WU-F1 strain. Examples of the foreign gene to be introduced here include a desulfurase gene, a high-temperature desulfurase gene, and an oxidoreductase gene.
As desulfurase genes, for example, Rhodococcus (Rhod
ococcus) sp. ATCC53968-derived desulfurase gene (Piddi
ngton, CS, Kovacevich, BR and Rambosek, J. Appl.
Environ. Microbiol. 61 (2), p468-475 (1995)) and a desulfurizing enzyme gene derived from Mycobacterium sp. G3 (Nobuhiko Nomura, Mariko Nakahara, Toshiaki Nakajima (Kobe), Tadatoshi Nakahara, Petroleum Institute Conference Abstracts p24
3 (1998).). Examples of high temperature desulfurization enzyme genes include, for example, Paenibacil
lus) sp. A11-2 strain-derived high temperature desulfurase gene
-341987). Examples of oxidoreductase genes include, for example, Rhodococcus
s) The oxidoreductase gene from ATCC53968 of sp. (Childs,
JD, Li, MZ, Mitchell, K., Emanule, J., Gray, KA
and Squires, CH Genbank AccNo. AF048979) and oxidoreductase genes derived from Vibrio harveyi (Izumoto Y., Mori T., Ymamoto K., Biochem Bio)
phys Acta 1185, p243-246 (1994)). There is no need to use a special method to introduce a foreign gene into the WU-F1 strain, and a general method can be employed.

The organic sulfur compound in the present invention mainly means a heterocyclic organic sulfur compound, but is not limited thereto. As the heterocyclic organic sulfur compound,
For example, dibenzothiophene and its derivatives can be mentioned. The dibenzothiophene derivative is not particularly limited, and examples thereof include alkyldibenzothiophene,
More specifically, 2,8-dimethyldibenzothiophene, 4,8
Examples thereof include 6-dimethyldibenzothiophene and 3,4-benzodibenzothiophene.

As a specific decomposition method, a method of culturing microorganisms in a liquid containing an organic sulfur compound (culturing method), a method of contacting microbial cells with a liquid containing an organic sulfur compound (resting cell method), and the like However, the present invention is not limited to these. The culture method can be performed, for example, as follows.

The decomposition of the organic sulfur compound is performed by adding an appropriate amount to a fresh medium to which a liquid containing an appropriate organic sulfur compound has been added.
For example, it can be obtained by inoculating a 1 to 2% inoculum and performing reciprocating or rotary shaking culture at 50 ° C. At this time, the inoculum is preferably in the late logarithmic phase, but may be in any state from the early logarithmic phase to the stationary phase. In addition, the volume of the inoculation may be increased or decreased as necessary. As the medium, the high-temperature desulfurizing bacteria medium used in Examples is suitable, but other mediums may be used. Commonly used nutrients for the medium are widely used. Any available carbon compound may be used as the carbon source. For example, glucose and the like are used. Any available nitrogen compound may be used as the nitrogen source. For example, organic nutrients such as peptone, polypeptone, meat extract, yeast extract, soybean powder, and casein hydrolyzate can be used. If it is desired to culture in a medium that does not contain a sulfur compound that may affect the desulfurization reaction, an inorganic nitrogen compound such as ammonium chloride can be used instead of the above organic nutrient. In addition, phosphates, carbonates, magnesium, calcium, sodium, iron, manganese, zinc, molybdenum, tungsten, copper, vitamins and the like are used as necessary. Ordinary culture is carried out aerobically for 2 to 3 days at about 50 ° C. under shaking or aeration culture conditions. However,
The culture temperature is preferably 50 ° C., but may be any temperature between 30 ° C. and 50 ° C. Further, the culture time may be increased or decreased as necessary.

As the liquid containing an organic sulfur compound, for example, fractions of crude oil, heavy oil, light oil, kerosene, gasoline, etc. can be used, but any liquid containing an organic sulfur compound may be used. The concentration of the organic sulfur compound in the liquid is preferably from 50 to 500 ppm, but can be increased or decreased as necessary. Further, before adding the liquid containing the organic sulfur compound, the culture solution may be pre-heated to the same temperature as the reaction temperature. The decomposition of the organic sulfur compound by the cell culture method using the present thermophilic desulfurizing bacterium may be performed in an oil-water two-phase system to which an organic solvent such as tridecane is added. In this case, the organic solvent used may be hydrocarbon, kerosene, light oil, heavy oil, or the like which is in a liquid state at the temperature at which the decomposition reaction is performed, in addition to tridecane.
If necessary, the gas phase above the culture solution may be replaced and sealed with oxygen. Further, air or oxygen may be introduced into the culture solution.

The resting cell method can be performed, for example, as follows. The cells can be prepared by inoculating a fresh medium with an appropriate amount of, for example, 1 to 2% by volume of inoculum, and performing reciprocating or rotary shaking culture at 45 ° C. At this time, the inoculum is preferably in the late logarithmic phase, but may be in any state from the early logarithmic phase to the stationary phase. Also, the amount of inoculation can be increased or decreased as needed. As the medium, a high-temperature desulfurization bacterium medium is suitable, but another medium may be used. Commonly used nutrients for the medium are widely used. Any available carbon compound may be used as the carbon source. For example, glucose or the like is used. Any available nitrogen compound may be used as the nitrogen source, for example, peptone, polypeptone, meat extract,
Organic nutrients such as yeast extract, soy flour, casein hydrolyzate, etc. can also be used. If it is desired to culture in a medium that does not contain sulfur compounds that can affect the desulfurization reaction, inorganic nitrogen compounds such as ammonium chloride can also be used. In addition, phosphates, carbonates, magnesium, calcium, potassium, sodium, iron, manganese, zinc, molybdenum, tungsten, copper, vitamins, and the like are used as needed. Normal culture is about
Perform aerobically for 2 to 3 days at 45 ° C under shaking or aerated conditions. However, the culture temperature is preferably 45 ° C.
Any temperature between 50 ° C can be used.

It is desirable that the cells obtained by culturing are separated and collected by means such as centrifugation, and the cells are washed, collected again, and used for a resting cell reaction. At this time, the cells are preferably collected in the middle to late logarithmic growth phases, but may be in the early to stationary logarithmic growth phases. As a means for separating and collecting bacteria, any method such as filtration or sedimentation may be used in addition to centrifugation. For washing the cells,
Any buffer such as physiological saline, phosphate buffer and Tris buffer can be used, and the cells may be washed using water.

The quiescent cell reaction is carried out by adding a liquid containing an organic sulfur compound to a cell suspension prepared by suspending cells in an appropriate buffer. Various buffers can be used as the buffer. The pH of the buffer is preferably pH 7 to 8, but may be other pH. In addition, water, a medium, or the like may be used instead of the buffer solution. The concentration of the cell suspension, OD ₆₆₀ is 1-5
A value between 0 is preferred, but can be increased or decreased as needed.

As the liquid containing an organic sulfur compound, for example, a petroleum fraction such as crude oil, heavy oil, light oil, kerosene, and gasoline can be used, but any liquid containing an organic sulfur compound may be used. The concentration of the organic sulfur compound in the liquid is preferably from 50 ppm to 5000 ppm, but can be increased or decreased as necessary. Before adding the liquid containing the organic sulfur compound, the reaction solution may be preheated to the same temperature as the reaction temperature. The resting cell reaction is preferably performed at 50 ° C.
Any temperature of 20 ° C to 52 ° C may be used, and the reaction time is 1 to
2 hours is preferred, but can be increased or decreased as needed. The resting cell reaction may be performed in an oil-water two-phase system to which an organic solvent such as tridecane is added. In this case, the organic solvent used may be hydrocarbon, kerosene, light oil, heavy oil, or the like that is in a liquid state at the temperature at which the decomposition reaction is performed, in addition to tridecane. If necessary, the gas phase above the reaction solution may be replaced and sealed with oxygen.

As described above, the decomposition method of the present invention can be carried out. The extraction and analysis of the reaction product generated by the decomposition can be performed, for example, as follows. After adjusting the pH of the reaction solution to about 2 using 6N hydrochloric acid, the mixture is extracted with stirring using ethyl acetate. However, the solvent used for extraction is not limited to ethyl acetate, and any solvent may be used as long as the desired reaction product can be extracted. The amount of ethyl acetate is preferably equal to the amount of the reaction solution, but can be increased or decreased as needed. The reaction product can be separated using a reverse-phase C18 column or a normal-phase silica column, but another column may be used if necessary.

The method used for separation is not limited to these methods, and any method may be used as long as the reaction product can be separated. The analysis of the reaction product is performed using gas chromatography, gas chromatography / mass spectrum analysis, gas chromatography / atomic emission detection analysis, gas chromatography / Fourier transform infrared spectroscopy, nuclear magnetic resonance, etc. be able to. Further, other analysis methods may be used as needed. Furthermore, the method used for analysis is not limited to these methods, and any method may be used as long as the reaction product can be analyzed.

[0032]

The present invention will be described below in more detail with reference to examples. [Example 1] Isolation of strain WU-F1 Isolation of strain WU-F1 was performed as follows. For the accumulation and isolation of this strain, a medium shown in Table 1 containing dibenzothiophene, which is a typical organic sulfur compound contained in a petroleum fraction, as a sole sulfur source (hereinafter referred to as a "thermophilic desulfurization medium") )It was used.

First, a test tube with silicon (capacity 27 ml, diameter
18 ml x 180 mm length), 5 ml of high-temperature desulfurization bacteria medium and 1 spatelle (approximately 0.5 g) such as soil collected from various parts of Japan were added, and cultured at 50 ° C for 3-5 days with shaking (shaking speed 240 rpm). Was done. For samples in which turbidity was observed in the medium, 0.1 ml of the culture was added to fresh same medium, and the enrichment culture was repeated three to four times. A culture solution was prepared using a high-temperature desulfurized bacterium medium for a sample in which the growth of cells was recognized by these enrichment cultures. 5 ml of these cultures are collected in the above-mentioned test tube with a silicon stopper, and 6N hydrochloric acid is added dropwise.
After adjusting the pH to 2.5 or less, an equal amount of ethyl acetate was added, and the product was extracted by stirring. The extracted product was analyzed by high performance liquid chromatography and gas chromatography. As a result, for the sample in which the production of 2-hydroxybiphenyl was observed, the enriched culture solution was used and the separation operation described below was performed. .

The culture solution in which the production of 2-hydroxybiphenyl was observed was diluted using a high-temperature desulfurizing bacteria medium. The obtained diluted solution was added to an LB medium (tryptone: 10 g / l, yeast extract: 5 g / l, sodium chloride: 10 g / l, pH 7) at a final concentration of 1.5.
% On agar plates prepared by adding agar and then culturing at 30 ° C. to form colonies.
Next, a part of the formed colony was inoculated in a high-temperature desulfurization bacterium medium, and liquid culture was performed by the method used for the above-described enrichment culture. Using the culture solution thus obtained, the production of 2-hydroxybiphenyl was examined by the method described above. A colony in which the production of 2-hydroxybiphenyl was observed was selected, and the above series of operations was repeated twice or three times to isolate a target strain, that is, the WU-F1 strain.

[0035]

[Table 1]

[Example 2] High-temperature decomposition of dibenzothiophene by WU-F1 strain Using a high-temperature desulfurizing bacterium medium containing dibenzothiophene, which is an organic sulfur compound, as the sole sulfur source, WU-WU was used at high and medium temperatures.
-F1 strain was cultivated, and the degradation test of dibenzothiophene was performed using the growth of cells and the production of 2-hydroxybiphenyl as indices.

5 ml of a high-temperature desulfurized bacterium medium was added to a test tube with a silicon stopper, and after preheating at a predetermined temperature for 1 hour, a 1% inoculation of the preculture solution prepared in the same manner as described in Example 1 was performed. Then, a decomposition test was performed under the culture conditions described in Example 1. The turbidity of each culture at a measurement wavelength of 660 nm was measured by a spectrophotometer, and the turbidity was used to evaluate the growth of the microorganism. The analysis of the decomposition products was performed by high performance liquid chromatography and gas chromatography in the same manner as in Example 1 to quantify the amount of 2-hydroxybiphenyl produced. Table 2 shows the growth degree of the WU-F1 strain and the amount of 2-hydroxybiphenyl produced.

[0038]

[Table 2]

From the results shown in Table 2, it can be seen that significant growth of desulfurized bacteria was observed at 50 ° C., with which dibenzothiophene was decomposed and 2-hydroxybiphenyl was produced. Bacillus subtilis WU-S2B, which exhibits desulfurization activity under a wide range of temperature conditions from 30 to 50 ° C, takes 5 days to degrade 0.54 mM DBT at 50 ° C, whereas WU-F1 strain can be degraded in 3 days It is. Although not shown in the table, even at 30 ° C, WU-F
Significant growth of one strain was observed, and it was confirmed that dibenzothiophene was decomposed. That is, the WU-F1 strain can perform more efficient desulfurization under a wide range of temperature conditions including a medium temperature range to a high temperature range.

Example 3 Decomposition of Dibenzothiophene Derivatives at High Temperature by WU-F1 Strain WU-F1 was cultured in a thermophilic desulfurizing bacterium medium containing an alkyl derivative of dibenzothiophene as the sole sulfur source. Degradability of the derivative was investigated. As alkyl derivatives of dibenzothiophene, 2,
8-dimethyldibenzothiophene, 4,6-dimethyldibenzothiophene, and 3,4-benzodibenzothiophene were used.
In order to confirm that the WU-F1 strain decomposes the alkyl derivative of dibenzothiophene under the conditions where the hydrocarbon solvent actually contained in petroleum coexists, the above dibenzothiophene was adjusted to have a sulfur concentration of 100 ppm. Each derivative
The solution dissolved in n-tridecane was added to a thermophilic desulfurization bacterium medium and cultured.

5 ml of a medium and 1 ml of n-tridecane containing an organic sulfur compound were added to a test tube with a silicon stopper, and about 1% of the precultured bacterial solution prepared by the method described in Example 1 was inoculated at a temperature of 50 ° C. The cells were shaken at a rotation speed of 240 rpm and cultured. Measurement wavelength
The turbidity of each culture at 660 nm was measured using a spectrophotometer, and the turbidity was used to evaluate the growth of the microorganism.

[0042]

[Table 3]

As shown in Table 3, since the desulfurizing bacteria are growing, various dibenzothiophene derivatives dissolved in the hydrocarbon-based solvent contained in petroleum are decomposed under the coexistence condition of the solvent. You can see that there is. This example shows that the dibenzothiophene derivative is efficiently degraded with the growth of high temperature desulfurized bacteria. In addition, the extracted product was analyzed by gas chromatography and gas chromatography / mass spectrometry, and as a result, it was estimated that compounds in which sulfur atoms were specifically removed from various dibenzothiophene derivatives were produced. Was.

Example 4 Decomposition of Dibenzothiophene at High Temperature by Resting Cells The details of the resting cell reaction using the WU-F1 strain are shown below.

To a test tube with a silicon stopper, 5 ml of a high-temperature desulfurized bacterium medium was added, and glycerol stock for storage (10% glycerol was added to a suspension of cells grown on the high-temperature desulfurized bacterium medium, (Frozen at -80 ° C.) 0.1 ml was inoculated and shake-cultured at 50 ° C. for 2 days, and the resulting culture was used as a seed culture. Cells for use in the quiescent cell reaction were prepared as follows. In a 500 ml Erlenmeyer flask with a baffle, 50 mg of dibenzothiophene
200 ml of a high-temperature desulfurization bacterium culture medium containing a concentration of / l (50 ppm) was added. After inoculating 5 ml of the inoculum culture solution, reciprocal shaking culture was performed at 45 ° C. until the late logarithmic growth phase was reached (about 45 hours). Next, 16,000 × g of the obtained culture solution, 10
The cells were collected by centrifugation for minutes. After removing the supernatant, the obtained cells were washed with 200 ml of a 0.1 M phosphate buffer (pH 7.6) and centrifuged again under the same conditions to collect the cells. After repeating this washing operation twice, the obtained cells were resuspended using the same phosphate buffer so that the OD ₆₆₀ became about 50. This resuspension was used as a cell suspension for a quiescent cell reaction.

The resting cell reaction was carried out as follows. 30
To an L-shaped test tube with a silicon stopper having a capacity of ml, add 0.6 ml of the bacterial suspension prepared by the method described above, and add 15 ml at each reaction temperature.
The temperature of the cell suspension was adjusted so as to be the same as the temperature at which the quiescent cell reaction was actually performed. To this cell suspension, 9 μl of a 10,000 ppm dibenzothiophene solution (dissolved in tridecane) was added (final concentration: 150 ppm), and reciprocal stirring was performed at each temperature for 2 hours to perform a resting cell reaction.

After the reaction, 6N hydrochloric acid was added to the cell suspension.
After adjusting the pH to around 2, 3 ml of ethyl acetate was added,
Dibenzothiophene and resting cell reaction products were extracted. High performance liquid chromatography analysis of the resulting extract confirmed that 2-hydroxybiphenyl, a decomposition product of dibenzothiophene, was formed.

[0048]

[Table 4]

The results shown in Table 4 demonstrate that the desulfurization activity is maintained under a wide range of temperature conditions from 20 ° C. to 52 ° C., and that the desulfurization activity is particularly maintained at 30 ° C. to 50 ° C. Things. That is, the WU-F1 strain exhibits overwhelmingly higher activity than the B. subtilis WU-S2B strain, which exhibits desulfurization activity under a wide range of temperature conditions of 30 to 50 ° C (Furuya, Nishii, Nakagawa, Kirimura, Usami) , Proceedings of the Japanese Society of Agricultural Chemistry, 384 (199
9)). Also, the maximum desulfurization activity at 50 ° C is 0.08mg / min / mg-dryc
ell showing higher activity than the strain of Paenibacillus sp. A11-2, and the maximum desulfurization activity at 30 ° C. 0.27 mg / min / mg-drycell
Showing activity comparable to ATCC 53968 of Rhodococcus sp. (Suzuki, M., Konishi, J., Ishii, Y., Okumora,
K., Appl.Environ.Microbiol., 63, 3164-3169 (199
7)).

Example 5 Decomposition of Dibenzothiophene Derivative by Resting Cells at High Temperature The resting cells reaction was carried out by the method described in Example 4. To an L-shaped test tube with a silicon stopper with a capacity of 30 ml, add 0.6 ml of the bacterial suspension prepared by the method described above, perform preheating for 15 minutes at each reaction temperature, and adjust the temperature of the bacterial cell suspension to The temperature was adjusted to be the same as the temperature at which the resting cell reaction was performed. To this cell suspension was added 9 μl of a 10,000 ppm dibenzothiophene derivative (2,8-dimethyldibenzothiophene, 4,6-dimethyldibenzothiophene, 3,4-benzodibenzothiophene) solution (dissolved in tridecane) (final solution). (Concentration 150 ppm)
A resting cell reaction was carried out by reciprocating stirring at 50 ° C. for 8 hours.

After the reaction, 6N hydrochloric acid was added to the cell suspension.
After adjusting the pH to around 2, 3 ml of ethyl acetate was added,
The dibenzothiophene derivative and the quiescent cell reaction product were extracted. The extracted product was analyzed by gas chromatography and gas chromatography / mass spectrometry, and as a result, it was presumed that various dibenzothiophene derivatives were formed with a compound in which a sulfur atom was specifically removed.

[0052]

[Table 5]

The results shown in Table 5 demonstrate that the dibenzothiophene derivative is efficiently decomposed in response to the quiescent cell reaction of the high-temperature desulfurizing bacteria.

[0054]

According to the present invention, a heterocyclic organic sulfur compound can be specifically decomposed in a C--S bond under a wide range of temperature conditions including a high temperature range, much more efficiently than heretofore possible. . This enables efficient desulfurization under a wide range of temperature conditions.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12R 1:32) C12R 1:32) (C12N 1/21 (C12N 1/21 C12R 1:32) C12R 1:32) (72) Inventor Yoshitaka Ishii 23-73 Yada, Shizuoka City, Shizuoka Prefecture Art Hill II 305 F-term (reference) 4B065 AA36X AC20 BA16 BA23 BB11 BB13 CA56

Claims (6)

[Claims] 1. The method according to claim 1, wherein the organic sulfur compound is selected from the group consisting of:
A method for decomposing an organic sulfur compound, wherein the method is decomposed by a microorganism. (1) Mycobacterium frei WU-F1 strain (2) Mutant strain of Mycobacterium frei WU-F1 strain (3) Transformant using Mycobacterium frei WU-F1 strain as host 2. The method for decomposing an organic sulfur compound according to claim 1, wherein the organic sulfur compound is a heterocyclic organic sulfur compound. 3. The method for decomposing an organic sulfur compound according to claim 2, wherein the heterocyclic organic sulfur compound is dibenzothiophene or a derivative thereof. 4. The method for decomposing an organic sulfur compound according to claim 3, wherein the dibenzothiophene derivative is an alkyl dibenzothiophene. 5. A Mycobacterium frei WU-F1 strain capable of decomposing an organic sulfur compound at a high temperature. 6. A transformant using Mycobacterium frei WU-F1 as a host.