JPH11181446A - Production of desulfurization-active microogranism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the desulfurization process in which benzothiophene, dibenzothiophene and their derivatives contained in fossil fuel such as petroleum are decomposed by a microorganism. SOLUTION: A microorganism having excellent desulfurization activity is prepared by culturing a microorganism that is capable of decomposing dibenzothiophene, benzothiophene and their alkyl-substituted derivatives in a culture medium containing water-soluble sulfonic acid compounds as only one sulfur source. The microorganism or its treated products are used to desulfurize petroleum or petroleum products. An easily handleable water-soluble sulfur compound is given as a substrate for the desulfurization microbial strain a to allow this desulfurization strain to stabilize and express high desulfurization activity. In addition, in comparison with the case that and insoluble organic sulfur compound is used as a sulfur source, the cell bodies having the desulfurization activity can be cultured in an increased concentration in a shortened time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for decomposing thiophene-based compounds using microorganisms, that is, benzothiophene, dibenzothiophene and substituted derivatives thereof. In particular, it relates to a method for decomposing and desulfurizing benzothiophene and dibenzothiophene and their substituted derivatives contained in fossil fuels such as petroleum by a microorganism.

[0002]

2. Description of the Related Art Microorganisms that decompose not only crude oil and coal but also model heterocyclic compounds containing sulfur and selectively remove sulfur, which is a heteroatom, to produce sulfates and hydroxylated compounds have been reported. Have been. This type of reaction is considered to be a reaction that specifically cleaves the C—S bond in the sulfur compound and releases sulfur in the form of sulfate, in view of the structure of the metabolite. The decomposition reaction of the organic sulfur compound of the type that specifically cuts the C—S bond but leaves the C—C bond uncut without cutting does not destroy the carbon skeleton and can be desulfurized without losing its value as energy. Is considered to be a desirable method for desulfurizing petroleum. Dibenzothiophene is a typical model compound used as a sulfur source in the cultivation of microorganisms that decompose hardly decomposable heterocyclic sulfur compounds contained in fossil fuels into CS bonds specifically and perform desulfurization. However, it is known that many kinds of thiophene compounds such as dibenzothiophene compounds and benzothiophene compounds are present in oil as heterocyclic sulfur compounds. In particular, even in light oil that has undergone normal hydrodesulfurization and microbial desulfurization,
Certain alkylated dibenzothiophenes and alkylated benzothiophenes remain. That is, alkylated dibenzothiophenes and alkylated benzothiophenes are present as highly difficult-to-remove sulfur compounds. Both types of alkylated heterocyclic sulfur compounds can be specifically bonded to a C--S bond. If a fungus capable of decomposing and desulfurizing is searched for and used for desulfurization, its efficiency is expected to increase. Further, it is not necessary to use a plurality of types of desulfurizing bacteria, and it is expected that the microbial desulfurization process will be simplified. In conclusion, biodesulfurization is the use of microorganisms that simultaneously have the activity of cleaving C—S bonds in dibenzothiophene and alkyl-substituted benzothiophene molecules at room temperature and produce desulfurization products in the form of water-soluble substances. Most desirable as a process.

[0003] As described above, microorganisms that perform dibenzothiophene degradation reactions of the CS bond cleavage type are known among bacteria of several genera. For example, IGTS of Rhodococcus sp.
Eight strains (ATCC 53968) are the most studied dibenzothiophene-degrading strains, adding an oxygen atom to the sulfur atom of dibenzothiophene, producing dibenzothiophene sulfone from dibenzothiophene sulfoxide, and then 2'-
Via hydroxybiphenyl-2-sulfinate, 2-
Perform a reaction to produce hydroxybiphenyl. But,
It uses benzothiophene as its sole sulfur source and cannot grow (Kayser, KJ, Bielaga-J
ones, BA, Jackowski, K., Odusan, O., and Kilbane,
JJJ Gen. Microbiol., 139, 3123-3129 (1993)).
This fact means that Rhodococcus sp. Strain IGTS8 cannot degrade benzothiophene.
Moreover, we do not know that such a description exists up to the present regarding the degradability of this strain to alkylated benzothiophenes. To our knowledge, Rh
In addition to odococcus sp. IGTS8 strain, there is a report of a sulfur attack type biodesulfurization reaction system as shown in Table 1.

[0004]

[Table 1]

[0005] However, although the activity of decomposing dibenzothiophene is known for all of these CS bond-cleaving desulfurizing bacteria, the activity of decomposing benzothiophene compounds is not described. On the other hand, as a result of extensive screening of microorganisms having CS bond-cleaving activity, Rhodoc was identified as a strain capable of degrading and desulfurizing alkylated dibenzothiophene and alkylated benzothiophene.
Acquired occus erythropolis strain KA2-5-1 (Japanese Patent Application No. 9-164351).

As described above, a hardly decomposable heterocyclic sulfur compound contained in a fossil fuel is specifically decomposed by a CS bond,
A typical compound used as a sulfur source in the cultivation of a desulfurizing microorganism is dibenzothiophene. Dibenzothiophene is actually a heterocyclic sulfur compound contained in fossil fuels, and its alkylated derivative has been reported to be resistant to the hydrodesulfurization process. It is used as an optimal model compound when investigating reactivity. Also,
When screening for a CS bond-cleaved microorganism, it is often added to the medium as the sole sulfur source. However, dibenzothiophene is insoluble in water, and thus is suspended in an aqueous medium as an insoluble substance. For this reason, the uptake of dibenzothiophene by bacteria is limited, and when culturing cells having desulfurization activity, it takes a long time to grow, and therefore, there is a problem when culturing cells having high desulfurization activity in large quantities. . In addition, adsorption or adhesion to the culture vessel wall is likely to occur, and it has been difficult to strictly and quantitatively control the amount of dibenzothiophene remaining in the culture medium and used for desulfurizing bacteria. For this reason, when dibenzothiophene is used as a medium component, cultivation is carried out for a long time while strictly controlling the desulfurization activity of the bacterium, and a large amount of desulfurized cells with high resulfurization activity with high reproducibility are obtained. Was difficult to obtain.
If a water-soluble sulfur compound is used in place of dibenzothiophene, it can be more easily used by microorganisms as a sulfur source than dibenzothiophene, so that it is possible to achieve high growth and obtain a large amount of cells. It will be easier.

On the other hand, typical water-soluble sulfur compounds that can be used as a medium component include sodium sulfate and sulfur-containing amino acids such as methionine and cysteine. However, it is known that all of these sulfur compounds suppress the expression of the desulfurization activity of the microorganism when added to the medium as the sole sulfur source. Another problem is that sulfur-containing amino acids are expensive as medium components for mass culture. When bacteria are cultured in a medium containing dimethyl sulfoxide, a type of sulfoxide, as the sole sulfur source,
It has been reported for the Rhodococcus sp. IGTS8 strain that sulfate and sulfur-containing amino acids cysteine and methionine do not inhibit desulfurization activity at concentrations similar to those required to show inhibition (Li, MZ, Squires , CH, Mon
ticello, DJ and Childs, JDJBacteriol., 178, 6
409-6418 (1996)).

[0008] For strains that specifically decompose dibenzothiophene by CS bond, the degradability of these sulfur compounds to a wide range is determined by using the growth of bacteria in a medium containing various sulfur compounds as the sole sulfur source as an index. The report we examined included:
Kayser et al. (Kayser, KJ, Bielaga-Jones, BA, Jacko
wski, K., Odusan, O. and Kilbane, JJJGen. Micro
biol. 139, 3123-3129 (1993)) and Izumi et al. (Izum
i, Y., Ohshiro, T., Ogiino, H., Hine, Y. and Shima
o, M. Appl.Environ.Microbiol., 60, 223-226 (199
4)) is reported. Among them, Izumi et al. Also examined some sulfonic acids, which are water-soluble sulfur compounds,
Rhodococcus erythropolis, a dibenzothiophene-degrading strain
Strain D-1 showed that benzenesulfonic acid was not available as the sole sulfur source, but methanesulfonic acid was available. However, no report has been made on the dibenzothiophene decomposition activity and desulfurization activity of R. erythropolis strain D-1 cultured in a medium containing methanesulfonic acid as the sole sulfur source.

The Pseudomonas aeruginosa PAO1 strain is known to grow using various alkanesulfonic acids as sole sulfur sources (Kertresz, AM, FEMS Microbiol.
Lett., 137, 221-225, 1996). The supply of sulfur by the desulfonation of the alkanesulfonic acid is suppressed by sulfates and thiocyanates, and is suppressed under conditions free of these substances. However, it is not known at all whether there is any relationship between the expression of the decomposing activity for benzothiophene, dibenzothiophene and substituted derivatives thereof and the desulfonation of alkanesulfonic acid.

[0010] We do not know any report on the case of culturing the aforementioned Rhodococcus sp. IGTS8 strain in a medium containing a sulfur compound of sulfonic acids as the sole sulfur source. Also,
We also do not know about the degrading activity of dibenzothiophene, benzothiophene and their substituted derivatives by such cultured cells.

[0011]

DISCLOSURE OF THE INVENTION The present invention has improved the activity of decomposing dibenzothiophene and the like by culturing a microorganism having the ability to degrade alkylated benzothiophene or alkylated dibenzodithiophene in a medium containing a specific compound. Another object of the present invention is to provide a method for producing a microorganism which is stably retained.

[0012]

The present invention provides a method for producing a microorganism having excellent desulfurization activity, which comprises culturing a microorganism having the ability to degrade dibenzothiophene in a medium containing a water-soluble sulfonic acid compound as a sole sulfur source. It is. Examples of the compound include an aliphatic sulfonic acid compound or an aromatic sulfonic acid compound, and examples of the aliphatic sulfonic acid compound include 2-aminoethanesulfonic acid, cysteic acid, and methanesulfonic acid. p-aminobenzenesulfonic acid.

The above microorganisms include microorganisms belonging to the genus Rhodococcus, and specific examples thereof include Rhodococcus.
sp.IGTS8 or Rhodococcus erythropolis KA2-5-
1 Further, the present invention is a method for desulfurizing petroleum or petroleum products, which comprises desulfurizing petroleum or petroleum products using the microorganisms or processed products thereof. Here, the petroleum or petroleum products include heavy oil, heavy oil, light oil and the like.

[0014]

DETAILED DESCRIPTION OF THE INVENTION The microorganism used in the present invention is Rhodococcus sp. IGTS8 (ATCC 53968).
Or Rhodococcus erythropolis (Rhodococcus erythropolis) KA2-5-1 is mentioned. Among them, Rhodococcus erythropolis KA2-5-1 has been isolated from nature by the present inventors, and its bacteriological properties are as follows.

Cell morphology Bacillus and cocci color beige gram staining + sporulation-motility-catalase test + oxidase-growth at 37 #C + growth at 41 #C-growth at 45 #C-portion of 16S rDNA Sequence Shows 99.8% homology with the partial sequence (region 95-730) of 16S rDNA of Rhodococcus erythropolis.

As a diamino acid in peptidoglycan, meso-diaminopimelic acid was detected. The chain length of mycolic acid, a fatty acid unique to bacteria belonging to the genus Mycobacterium , was determined by high-temperature gas chromatography, and the separation pattern was used to determine the pattern of mycolic acid of Rhodococcus using a database. Showed high similarity to that of Rhodococcus erythropolis. As a result of analysis of other fatty acids, tubercrostearic acid was detected in addition to unbranched saturated and unsaturated fatty acids. This fatty acid pattern, all of the members and Mycobacterium (M ycobacterium) of the genus Rhodococcus, Nocardia (Nocardi
a), Jietchia (Dietzia), Tsukamurella (Tsukamurella)
Bacteria of the genus Genus and certain corynebacteria (Corynebacteri)
a) Found in species of bacteria. Among these bacteria, the Rhodococcus erythropolis strain KA2-5-1 showed the most similar fatty acid pattern to that of Rhodococcus erythropolis strain KA2-5-1.
It was erythropolis.

[0017]

Further, when it was examined whether or not 35 kinds of carbon sources were used in Rhodococcus erythropolis strain KA2-5-1, the pattern completely coincided with the use of the carbon source pattern of Rhodococcus erythropolis. These results are KA2-5-1 strain was identified as Rhodococcus erythropolis (Rhodococcus erythropolis) strain. This Rhodococcus erythropolis KA2-5-1 strain was established in June 1997
FERM P-162 joined the Institute of Biotechnology and Industrial Technology on the 17th
Deposited as 77. The treated bacterial cells used in the present invention include a bacterial cell culture solution, a ground bacterial cell, a crude enzyme obtained from the bacterial cells, and the like.

As the water-soluble sulfonic acid compound used in the present invention, an aliphatic sulfonic acid compound or an aromatic sulfonic acid compound is used. Examples of the aliphatic sulfonic acid compound include 2-aminoethanesulfonic acid and cysteine. Examples of the acid and methanesulfonic acid include aromatic sulfonic acid compounds such as p-aminobenzenesulfonic acid. The amount used is not particularly limited, and an effective amount is added to the medium.

The cultivation of the microorganism is carried out according to a usual culturing method of the 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, and for example, glucose, sucrose, lactose, fructose, ethanol 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. In addition, phosphates, carbonates, magnesium,
Calcium, potassium, sodium, iron, manganese, zinc, molybdenum, tungsten, copper, vitamins, and the like are used as needed. Culture is performed at pH 6-8, temperature 30
It is carried out aerobically for 1 to 2 days under shaking or aeration conditions at a temperature around 0 ° C.

Rhodococcus erythropolis strain KA2-5-1 comprises 1-methyldibenzothiophene, 2-methyldibenzothiophene, 2-ethyldibenzothiophene, 3-methyldibenzothiophene, 3-ethyldibenzothiophene, 4-methyldibenzothiophene, 4,6-dimethyldibenzothiophene, 2,8-dimethyldibenzothiophene, 3,4,6-trimethyldibenzothiophene,
It has the property of decomposing heterocyclic sulfur compounds such as 3,4,6,7-tetramethyldibenzothiophene and 3-methylbenzothiophene. For example, as a result of degradation of alkylated dibenzothiophene by the microorganism Rhodococcus erythropolis KA2-5-1, the main compound produced as a desulfurization product was a hydroxy compound. In order to analyze the structure of the product in the present invention, for example, the microorganism strain is cultured at, for example, 30 ° C. in a medium containing dibenzothiophene as a sole sulfur source to obtain a culture solution. The obtained culture solution was centrifuged, and the pH of the supernatant was adjusted to about 2 and then extracted with ethyl acetate. The extract was analyzed mainly by gas chromatography, gas chromatography / mass spectrum analysis. As necessary, ¹ H-nuclear magnetic resonance spectrum analysis and ultraviolet absorption spectrum analysis were performed. as a result,
It has been confirmed that dibenzothiophene is desulfurized and converted into 2-hydroxybiphenyl, and other alkylated dibenzothiophenes are desulfurized and converted into hydroxy forms corresponding to the respective substrate compounds.

A room-temperature desulfurizing bacterium, for example, the Rhodococcus erythropolis KA2-5-1 strain described in the present invention is obtained by culturing the cells and then bringing the cultured cells into contact with an organic sulfur compound intended for decomposition. Thereby, the organic sulfur compound can be decomposed. Such a quiescent cell reaction can be performed, for example, as follows. Cell preparation
This can be achieved 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 30 ° 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. A normal temperature desulfurization bacterium medium is suitable as the medium, 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, and for example, glucose, sucrose, lactose, fructose, ethanol 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. In addition, phosphates, carbonates, magnesium, calcium, potassium, sodium, iron, manganese, zinc, molybdenum, tungsten, copper, vitamins, and the like are used as needed. Normal cultivation is performed aerobically for 1 to 2 days at about 30 ° C. under shaking or aeration conditions. However, the culture temperature is preferably 30 ° C, but may be any temperature in the temperature range of 25 ° C to 37 ° C.

It is preferable 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 resting cell reaction is performed by adding petroleum or a petroleum product containing a substrate to a cell suspension prepared by suspending cells in a suitable buffer. Various buffers can be used as the buffer. The pH of the buffer is preferably pH 6-7, but other pH
But it doesn't matter. In addition, water, a medium, or the like may be used instead of the buffer solution. The concentration of the cell suspension is preferably between OD ₆₆₀ of 1 to 50, but can be increased or decreased as necessary.

Examples of the substrate for the quiescent cell reaction include, for example,
It is possible to use petroleum or petroleum products containing dibenzothiophene, dibenzothiophene derivatives, benzothiophene or benzothiophene derivatives. The concentration is
50 ppm to 5000 ppm is preferred, but can be increased or decreased as needed. Further, the reaction solution may be preheated to the same temperature as the reaction temperature before adding the substrate. The resting cell reaction is preferably performed at 30 ° C., but may be performed at any temperature between 25 ° C. and 37 ° C.
The reaction time is preferably 1-2 hours, but can be increased or decreased as necessary.

The resting cell reaction may be carried out in an oil-water two-phase system to which an organic solvent such as tetradecane has been added. In this case, the organic solvent used is C8-C
20 n-paraffin, kerosene, light oil, heavy oil, etc. may be used. If necessary, the gas phase above the reaction solution may be replaced and sealed with oxygen. Further, instead of the resting cells in the above reaction, an enzyme involved in the selective cleavage of the CS bond of the alkylated benzothiophene and / or the alkylated dibenzothiophene compound isolated and purified from the cells may be used. Good.

The extraction of the reaction product can be performed 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 the separation is not limited to these methods, and any method may be used as long as the reaction product can be separated. The reaction product is analyzed using gas chromatography, gas chromatography / mass spectrum analysis, gas chromatography / atomic emission detection analysis, gas chromatography / Fourier transform infrared spectroscopy, nuclear magnetic resonance, and the like. 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. Hereinafter, the present invention will be described specifically with reference to examples.

[0028]

The present invention will be described below in more detail with reference to examples. However, the present invention is not limited to these examples. [Example 1] A desulfurized bacterium R.erythropolis KA2-5-1 containing a desulfurized bacterium basic medium (Table 2) containing 2-aminoethanesulfonic acid (taurine) as a sole sulfur source, which is a water-soluble sulfur compound.
Try to culture the strain or Rhodococcus sp. IGTS-8 strain,
A dibenzothiophene degradation test was performed by a quiescent cell method. To the test tube, 5 ml of a desulfurized bacterium basic medium containing 0.14 mM of 2-aminoethanesulfonic acid was added, and a 1% pre-cultured desulfurized bacterium was inoculated and cultured at 30 ° C. for 2 nights.

[0029]

[Table 2]

The turbidity (measurement wavelength: 660 nm) of the culture solution was measured with a spectrophotometer.
7. Rhodococcus sp. IGTS-8 strain was 4.8, indicating that the strain grew well. After washing the culture with phosphate buffer,
The suspension was suspended in the same buffer solution to prepare a reaction cell fluid. The final concentration is 15
Dibenzothiophene was added so that the concentration became 0 ppm, and the mixture was shaken at 30 ° C. for 2 hours to conduct a dibenzothiophene degradation test by a resting cell method. The reaction solution was adjusted to acidic conditions by adding hydrochloric acid, extracted with ethyl acetate to obtain a measurement sample, analyzed by gas chromatography, and analyzed by R. erythropolis strain KA2-5-1 and Rh.
The amount of 2-hydroxybiphenyl, a dibenzothiophene desulfurization product of odococcus sp. strain IGTS-8, was quantified.
Gas chromatogram showing that dibenzothiophene as a substrate was decomposed by the desulfurizing bacterium R. erythropolis KA2-5-1 strain cultured in a medium containing 2-aminoethanesulfonic acid as the sole sulfur source to produce 2-hydroxybiphenyl 1 is shown in FIG. Bacterial desulfurization activity is expressed as the amount of 2-hydroxybiphenyl (μmole unit) produced per hour per 1 g of dried cells.

[0031]

[Table 3]

From the results shown in Table 3, the dibenzothiophene degrading activity of the desulfurized bacteria cultured using 2-aminoethanesulfonic acid as the sole sulfur source is observed. It is clear that 2-aminoethanesulfonic acid can be used as a sulfur source for satisfactorily growing desulfurized bacterial strains and expressing desulfurization activity. Moreover, as compared with the case where the R. erythropolis KA2-5-1 strain was cultured using a medium containing dibenzothiophene as the sole sulfur source, the proliferation was higher. In addition, compared to the case of culturing R.erythropolis KA2-5-1 strain using a medium containing dibenzothiophene as the sole sulfur source, it was confirmed that the degree of pH decrease of the culture solution was small,
This is considered to be one of the causes of high proliferation.

Example 2 Induction of Desulfurization Activity by Methanesulfonic Acid A basic medium for desulfurization bacteria containing a water-soluble sulfur compound, sodium methanesulfonate, as the sole sulfur source.
The culture of ythropolis KA2-5-1 was attempted, and a dibenzothiophene degradation test was performed by the resting cell method. 5 ml of desulfurized bacteria basic medium containing 0.14 mM sodium methanesulfonate in a test tube
, And 1% of a desulfurized bacterium pre-cultured was inoculated and cultured at 30 ° C. for 2 nights. Turbidity of culture solution (measurement wavelength 660n
m) was 5.0, indicating that the strain grew well. Furthermore, as a result of performing a resting bacterial cell reaction according to the method described in Example 1,
The proliferating cells showed a desulfurization activity of 8.0 μmole g ⁻¹ hr ⁻¹ . From these results, when sodium methanesulfonate was cultured as the sole sulfur source, R.erythropolis KA2
It is clear that the -5-1 strain exhibits dibenzothiophene degrading activity. That is, these results indicate that methanesulfonic acid can be used instead of dibenzothiophene as a sulfur source for satisfactorily growing desulfurized bacterial strains and expressing desulfurization activity.

[Example 3] Induction of desulfurization activity by cysteic acid [0034] A desulfurization bacterium R. erythropolis KA2 containing a water-soluble sulfur compound, cysteic acid, as a sole sulfur source.
-5-1 strain was cultivated, and a dibenzothiophene degradation test was performed by a resting cell method. To a test tube, 5 ml of a desulfurized bacterium basic medium containing 0.14 mM cysteic acid was added, and 1% of a desulfurized bacterium which had been precultured in advance was inoculated and cultured at 30 ° C for 2 nights. The turbidity (measurement wavelength 660 nm) of the culture solution was 5.7, and the strain grew well. Furthermore, as a result of performing a resting bacterial cell reaction according to the method described in Example 1, the proliferating bacteria showed 15.4 μmole g ⁻¹ hr.
^It showed a desulfurization activity of ^-1 . From these results, it was found that R.erythropol could be cultured when cysteic acid was used as the sole sulfur source.
It is clear that the strain KA2-5-1 shows high dibenzothiophene degrading activity. That is, these results indicate that cysteic acid can be used instead of dibenzothiophene as a sulfur source for satisfactorily growing desulfurized bacterial strains and expressing desulfurization activity.

Example 4 Induction of Desulfurization Activity by p-Aminobenzenesulfonic Acid A desulfurization bacteria basic medium containing p-aminobenzenesulfonic acid, which is a water-soluble sulfur compound, as the sole sulfur source.
Culture of erythropolis KA2-5-1 strain was attempted, and a dibenzothiophene degradation test was performed by a resting cell method. P-
5% of a desulfurized bacterium basic medium containing 0.14 mM of aminobenzenesulfonic acid was added thereto, and 1% of a desulfurized bacterium which had been precultured in advance was inoculated and cultured at 30 ° C. for 2 nights. Reached 5.1 and showed good growth. Furthermore,
As a result of performing a resting bacterial cell reaction according to the method described in Example 1, the growing bacterial cells exhibited a desulfurization activity of 16.3 μmole g ⁻¹ hr ⁻¹ . From these results, R.erythrop was also found when p-aminobenzenesulfonic acid was used as the sole sulfur source.
It is clear that olis KA2-5-1 strain shows high dibenzothiophene degrading activity. In other words, these results indicate that p-type was used instead of dibenzothiophene as a sulfur source for satisfactorily growing desulfurized bacterial strains and expressing desulfurization activity.
It means that aminobenzenesulfonic acid can be used.

Example 5 Stability of Desulfurization Activity Expressed by Sulfonic Acids The desulfurized bacterial strain R. erythropolis KA2-5-1 grown and subcultured in a medium containing sulfonic acids as the sole sulfur source is dibenzo. Shows thiophene degradation activity. The dibenzothiophene-degrading activity observed in these cases is similar to that of desulfurized bacteria grown in a medium containing dibenzothiophene as the sole sulfur source, and the group of desulfurization enzymes (Dsz Protein group). On the other hand, the dibenzothiophene degradation activity by the dsz gene group is strongly affected by sulfates and sulfur-containing amino acids, and if these sulfur compounds are present at high concentrations in the medium, the degradation activity may not be detected. (Piddington, CS, Kovacevich, BR, and Rambos
ek, J. Appl. Env. Microbiol., 61: 468-475, (1995);
Wang, P., and Krawiec, S. Appl. Env. Microbiol., 6
2: 1670-1675 (1996); Monticello., DJ, Ji, W., Child
s, JD, McDonalld, CA, Johnson, SW, and Squir
es, CH 1995.p.471-481.In Srivastava, V., Marku
szewski, R, Smith, J., and Hayes, TD (ed.), Gas, o
il, and environmental biotechnology, vol.VII.Ins
titute of Gas Technology, Des Plaines, III.). It has been suggested that this loss of desulfurization activity is due to suppression of the expression of the dsz gene group (Li, M, Z, Squires, CH,
Monticello, DJ, and Childs, JDJBacteriol, 17
8: 6409-6418 (1996)). Therefore, the cells were actually cultured in a medium containing 2-aminoethanesulfonic acid as a sole sulfur source.
In the R.erythropolis KA2-5-1 cell line, the desulfurization gene constituting the dsz operon is expressed efficiently, and DszA, DszB, and DszC
In order to confirm that the three desulfurization gene products were produced, proteins were extracted from the cells, and the reactivity of each Dsz protein with antisera was examined. First, a medium containing 2-aminoethanesulfonic acid as the sole sulfur source is OD660 7.
The R. erythropolis KA2-5-1 strain grown to 0 was washed with a phosphate buffer, suspended in the same solution to 1 g / 10 ml, and sonicated to prepare a cell-free extract. In a similar manner, dibenzothiophene is used as the sole sulfur
A cell-free extract was also prepared from the strain grown to 6605.0. 1.5 μg of these cell-free extracts were electrophoresed on an 8-25% polyacrylamide gradient gel in the presence of SDS, and the protein fraction was applied to a polyvinylidene difluoride (PVDF) membrane by Western blotting. Transcribed. The proteins separated on the PVDF membrane were reacted with antisera reacting with desulfurizing enzymes DszA, DszB and DszC, respectively, and the reaction was detected by immunoblotting. The antiserum used here is known R.erythropolis
This peptide was prepared by synthesizing peptides thought to correspond to the epitope based on the amino acid sequence of the desulfurase of the IGTS-8 strain, and using these as antigens. as a result,
In the R. erythropolis KA2-5-1 strain cultured in a medium containing 2-aminoethanesulfonic acid as a sole sulfur source, three desulfurization-related genes dszA, dszB, Dsz protein encoded by dszC, DszA, DszB, It was confirmed that all of DszC was efficiently produced. FIG. 2 shows the results of the obtained Western blot analysis. In conclusion, even in the absence of dibenzothiophene, in the presence of sulfonic acids such as 2-aminoethanesulfonic acid, bacteria continue to express the desulfurization gene group dsz operon that degrades dibenzothiophene, and the desulfurization enzyme group Dsz protein It is clear that by continuing to synthesize the group, it expresses dibenzothiophene degrading activity and further retains this ability.

In order to examine the stability of expression and retention of desulfurization activity by sulfonic acids, R. erythropolis KA2-5 was used in a medium containing 2-aminoethanesulfonic acid as the sole sulfur source.
One subculture was carried out for several generations. As a result of measuring the dibenzothiophene decomposing activity for each subculture, it was confirmed that the desulfurization activity expression ability was stably maintained at least up to 18 times in the subculture.

It is considered that sulfonic acids such as 2-aminoethanesulfonic acid are more easily used as a sulfur source than when dibenzothiophene is used as the sole sulfur source, and higher proliferation was observed. . This means that using sulfonic acids as a sulfur source makes it easier to obtain a larger amount of bacterial cells than using dibenzothiophene. In addition, R.eryt is a medium containing dibenzothiophene as the sole sulfur source.
Compared with the culture of hropolis KA2-5-1 strain, it was also confirmed that the decrease in pH due to the growth of cells was smaller when cultured in a medium containing sulfonic acids as the sole sulfur source. Was. The small decrease in pH is also considered to be one of the causes of the desulfurized bacterial cells having high proliferation in a medium containing sulfonic acids.

Example 6 Induction of Degradation Activity of Alkyl-Substituted Dibenzothiophenes R. was prepared by the same method as described in Example 1 using a medium containing 2-aminoethanesulfonic acid as the sole sulfur source. Using erythropolis strain KA2-5-1 cells, the desulfurization ability of methyl-substituted benzothiophene and dibenzothiophene was examined. As a result, the examined 3-methylbenzothiophene, 4-methyldibenzothiophene,
For all of 4,6-dimethyldibenzothiophene, formation of a desulfurization product was confirmed. This result indicates that the desulfurization activity induced by 2-aminoethanesulfonic acid acts not only on dibenzothiophene but also on methyl-substituted dibenzothiophene and methyl-substituted benzothiophene.

[0040]

According to the present invention, a stable and high desulfurization activity can be exhibited and maintained in a desulfurized bacterial strain by giving a water-soluble sulfur compound which is easy to handle as a substrate to the desulfurized bacterial strain. Further, according to the present invention, compared with the case where an organic sulfur compound insoluble in water is used as a sulfur source,
Cells having desulfurization activity can be cultured at a high concentration in a short time.

[Brief description of the drawings]

FIG. 1 shows that dibenzothiophene is converted by resting cells of R. erythropolis strain KA2-5-1 cultured in a medium containing 2-aminoethanesulfonic acid as a sole sulfur source to produce 2-hydroxybiphenyl. Gas chromatogram shown.

FIG. 2. R.erythropolis strain KA2-5-1 cultured in a medium containing 2-aminoethanesulfonic acid as sole sulfur source,
Western blot analysis showing that Dsz protein is efficiently produced.

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Claims (7)

[Claims] 1. A method for producing a microorganism having excellent desulfurization activity, comprising culturing a microorganism having the ability to degrade dibenzothiophene as a sole sulfur source in a medium containing a compound of a water-soluble sulfonic acid. 2. The method for producing a microorganism according to claim 1, wherein the compound is an aliphatic sulfonic acid compound or an aromatic sulfonic acid compound. 3. The method for producing a microorganism according to claim 2, wherein the aliphatic sulfonic acid compound is 2-aminoethanesulfonic acid, cysteic acid, or methanesulfonic acid. 4. The method for producing a microorganism according to claim 2, wherein the aromatic sulfonic acid compound is p-aminobenzenesulfonic acid. 5. The method for producing a microorganism according to claim 1, wherein the microorganism is a microorganism belonging to the genus Rhodococcus. 6. The microorganism belonging to the genus Rhodococcus sp. IGTS8 or Rhodococcus erythropolis.
The method for producing a microorganism according to claim 5, which is KA2-5-1. 7. A desulfurization method for petroleum or petroleum products, comprising desulfurizing petroleum or petroleum products using the microorganism or a processed product thereof according to any one of claims 1 to 6.