JPH06125767A - Method for separating organic solvent-resistant microorganisms - Google Patents

Abstract

(57) [Summary] [Structure] After mixing the sample with water and an organic solvent and culturing with shaking, the culture mixture is allowed to stand and separated into an aqueous phase and an organic solvent phase, and an appropriate amount of the organic solvent phase is added to the medium. A method for separating an organic solvent-resistant microorganism, which comprises culturing in addition to the above and isolating a growing microorganism. [Effect] Microbes with excellent organic solvent resistance can be
Can be efficiently separated.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for separating organic solvent-resistant microorganisms.

[0002]

2. Description of the Related Art In recent years, marine pollution by oil has frequently occurred and has become a problem. As a means for solving such a problem, a microorganism capable of efficiently and easily decomposing all hydrocarbons contained in polluted petroleum flowing out to the sea is desired, but a microorganism sufficiently equipped with such ability is It has not been discovered yet. Utilizes a single hydrocarbon contained in petroleum,
Until now, Bacillus (Bacillus)
s) genus, Acinetobacter genus, Pseudomonas genus, Moraxella (Moraxella) genus,
Several microorganisms belonging to the genus Arcadia or the genus Candida have been isolated from soil and seawater.These are the salt tolerance and organic solvent tolerance required for the decomposition of polluted petroleum at sea. It does not have the tolerance of and is not satisfactory.

On the other hand, in order to decompose polluted petroleum and the like that have flowed out to the sea, it is required that petroleum products such as kerosene and heavy oil can be efficiently decomposed, and it is a kind of organic solvent contained in these products. Hexane, benzene,
Resistance to toxicity such as toluene and xylene, resistance to salt contained in seawater at a high concentration, and resistance to pressure are also required. Conventionally, in order to separate microorganisms having such resistance, particularly organic solvent resistance, a method of adding a sample to a medium containing an organic solvent and culturing, and isolating microorganisms that grow in the medium have been adopted. . However, this method has a problem that not only time is required, but also the separation efficiency is extremely low.

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for separating microorganisms from a sample in a short time, efficiently and excellent in organic solvent resistance.

[0005]

Means for Solving the Problems The above object of the present invention is to mix a sample with water and an organic solvent, perform shaking culture, and then leave the culture mixture stationary to separate into an aqueous phase and an organic solvent phase, This has been achieved by a method for separating an organic solvent-resistant microorganism, which comprises adding an appropriate amount of an organic solvent phase to a medium and culturing the same to isolate a growing microorganism. In the present invention, the shaking culture is 4 to 3
It is preferable to carry out the treatment at 0 ° C. for 1 to 7 days. The water used in the present invention is not particularly limited. For example, deionized water, distilled water, seawater or artificial seawater may be used. The pH of the water used is preferably 4-9, more preferably 5-5.
8, most preferably around neutral. The volume ratio of water and organic solvent to be mixed with the sample is preferably 1: 9 to 9: 1, and the sample is 1 to 10 parts by volume (weight part) with respect to the total amount of water and organic solvent (100 parts by volume). Part) is preferably added. Examples of the medium for culturing the appropriate amount of the separated organic solvent phase include agar medium. The culture of the organic solvent phase is carried out under the condition that the organic solvent-resistant microorganism grows, for example, 20 to 40.
C., preferably 30.degree. C., for 1 to 10 days. The organic solvent used in the present invention is for separating microorganisms having resistance to the organic solvent, and is typically a hydrocarbon that is liquid at room temperature, particularly an aromatic hydrocarbon,
Examples thereof include aliphatic hydrocarbons and alicyclic hydrocarbons. Aromatic hydrocarbons having 6 to 8 carbon atoms, such as benzene, toluene, xylene, etc., and aliphatic hydrocarbons having 5 to 16 carbon atoms, such as pentane, hexane, heptane, octane. Etc., the alicyclic hydrocarbon has 5 to 8 carbon atoms, for example, cyclopentane,
Examples thereof include cyclohexane, cycloheptane, cyclooctane and the like. Further, a mixture of these organic solvents such as thinner and petroleum benzine can be used.
The organic solvent-resistant microorganism thus isolated can be determined, for example, as follows. Pre-incubation: Add 5 ml of liquid medium to a test tube, and at 2
Sterilize by autoclaving for 0 minutes. A platinum loop is taken from the isolated microorganism, inoculated into a sterilized medium, and shake culture is carried out at a temperature of 35 ° C. for 1 day to prepare a precultured microorganism suspension. Determination method: Add 5 ml of liquid medium to a test tube and sterilize under the above conditions. 100 μ of the above microbial suspension in a sterilized medium
1 of the above, 50 μl of an organic solvent is added, a silicone rubber stopper is attached, and shaking culture is carried out at a temperature of 30 ° C. for 3 days. After shaking, the wavelength of medium 6 compared to the control without inoculation of microorganisms
Those having an increase in the absorbance at 60 nm are judged as organic solvent microorganisms.

[0006]

According to the present invention, microorganisms having excellent resistance to organic solvents can be efficiently separated from a sample in a short time.

[0007]

EXAMPLES The present invention will be described in more detail below with reference to examples. In addition,% is weight% unless otherwise specified. Example 1 Separation of Organic Solvent-Resistant Microorganisms : 1 g of a sample was placed in a test tube, 5 ml of distilled water or artificial seawater (both pH 6) was added, and the mixture was sufficiently stirred to disperse the sample. To this, an organic solvent was added so that the total amount was 10 ml, a silicone rubber stopper was attached, and vigorous shaking culture was carried out at a temperature of 10 ° C. for 7 days. During this period, microorganisms that can survive or grow in the organic solvent are transferred to the organic solvent phase. After completion of the culture, the culture was placed in a separating funnel and allowed to stand for 1 hour to separate it into an aqueous phase and an organic solvent phase. 100 μl of the organic solvent phase was taken, smeared and deposited on an agar medium, and cultured at a temperature of 30 ° C. for 7 days to isolate the grown microorganism. Composition of artificial seawater : Sodium chloride 3%, calcium chloride 0.1%, magnesium chloride 0.1% and potassium chloride 0.1%.
Including 1%. Composition of agar medium : Proteose peptone 0.5%, phytonpeptone 0.25%, calcium chloride 0.1%, magnesium chloride 0.01%, sodium chloride 5.8%, sodium sulfite 50ppm and agar 2% thing. Or yeast extract 0.5%, tryptone 1%, sodium chloride 0.5%
And agar 2%, calcium chloride 0.1
% And 0.01% magnesium chloride. Composition of liquid medium : Agar removed from the above composition Judgment of organic solvent resistant microorganisms : 5 ml of liquid medium was added to a test tube and autoclaved at 120 ° C. for 20 minutes. A platinum loop was taken from the isolated microorganism, inoculated into the medium, and shake culture was carried out at a temperature of 35 ° C. for 1 day to prepare a precultured microorganism suspension. 5 ml of the liquid medium was added to the test tube and sterilized under the above conditions. 100 μl of the above microbial suspension was inoculated into a sterilized medium, 50 μl of an organic solvent was further added, a silicone rubber stopper was attached, and shaking culture was carried out at a temperature of 30 ° C. for 3 days. After shaking culture, an organic solvent microorganism was judged to be one in which an increase in the absorbance of the medium at a wavelength of 660 nm was observed as compared with a control in which no microorganism was inoculated. The results are shown in Table 1. As a result of various examinations on the separation source, it is found that the organic solvent-resistant microorganism can be efficiently separated by the method of the present invention with any separation source.

[0008]

[Table 1] Table 1 Number of organic solvent resistant microorganisms separated from 1 g of each sample ──────────────────────────────── ──── Soil sample Deep sea sample ────────────────────────────── A B C D E F G F H I J ─ ────────────────────────────────── Number of shares 10 7 11 16 19 37 48 52 9 4 ────── ────────────────────────────── A, B, C, and D are randomly sampled at each point of RIKEN. Soil sample. E, F, and G are mud samples collected from the seabed of Suruga Bay. H, I, and J are mud samples collected from the sea floor of Sagami Bay. The water phase is distilled water from A to D, and artificial seawater from E to J.

In this example, the microorganism isolated from Sample G, that is, the microorganism isolated from the seabed soil at a depth of 1945 meters in Suruga Bay, is a novel microorganism, and is a Flavobacterium genus DS-711 strain. Was named. This DS-711 strain was deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science on August 27, 1991, and the microorganism deposit number is Micromachine Research Institute No. 4010 (FERM).
BP-4010).

(Example 2) Using the sample G in accordance with Example 1, changing the ratio of the organic solvent phase and the aqueous phase (artificial seawater) as shown in Table 2 below, the isolated organic solvent-resistant microorganism I checked the number. The results are shown in Table 2. Efficient separation is
Although it can be obtained at a ratio of 5: 5, it can be seen that organic solvent-resistant microorganisms can be obtained at other ratios.

[0011]

[Table 2] Table 2 Relationship between the ratio of organic solvent / water and the number of separated organic solvent-resistant microorganisms ──────────────────────────── ──────── Organic solvent used Organic solvent / water Separated organic solvent resistance (V / V) Microorganism count / Sample 1g ──────────────────── ──────────────── Benzene 1: 9 17 3: 7 7 29 5: 5 48 7: 3 32 9: 1 41 Toluene 5: 5 79 Xylene 5: 5 98 ─── ────────────────────────────────

Example 3 Benzene was used as the organic solvent, benzene: water (artificial seawater) was set to 5: 5, and the culturing time was changed as shown in Table 3 below. The number of isolated organic solvent-resistant microorganisms was investigated. The results are shown in Table 3. It is desirable that the number of culture days is 7 days, but it can be seen that a large number of target organic solvent-resistant microorganisms can be collected even with one day of culture.

[0013]

[Table 3] Table 3 Relationship between culture time and number of separated organic solvent-resistant microorganisms ──────────────────────────────── ──── Number of organic solvent-resistant microorganisms separated per 1g of sample Culture time (days) ─────────────────────────── Sample F Sample G ─────────────────────────────────── 1 21 30 2 26 26 32 5 27 39 39 7 37 48 48 ── ──────────────────────────────────

Example 4 Benzene was used as an organic solvent, benzene: water (artificial seawater) was set to 5: 5, and the culture temperature was changed as shown in Table 4 below. The number of isolated organic solvent-resistant microorganisms was investigated. The results are shown in Table 4. In the case of any of the samples, most organic solvent-resistant microorganisms can be obtained by culturing at a temperature of 10 ° C, but in the temperature range of 4 ° C to 30 ° C, organic solvent-resistant microorganisms can be efficiently obtained in any case. To be

[0015]

[Table 4] Table 4 Relationship between culture temperature and the number of separated organic solvent-resistant microorganisms ──────────────────────────────── ──── Number of isolated organic solvent-resistant microorganisms per 1g of sample Culture temperature (℃) ─────────────────────────── Sample F Sample G ─────────────────────────────────── 4 31 31 34 10 37 48 48 20 28 29 29 30 26 26 28 ── ──────────────────────────────────

(Example 5) Benzene was used as an organic solvent, and benzene: water (artificial seawater) was set to 5: 5, and sample G was used.
Was used in the same manner as in Example 1 except that the pH of the aqueous phase was changed as shown in Table 5, and the number of separated organic solvent-resistant microorganisms was examined. The pH was adjusted with sodium hydroxide and sulfuric acid. The results are shown in Table 5. Sample suspension aqueous phase is pH 5 to pH 8
In the range up to, organic solvent-resistant microorganisms were efficiently obtained. On the other hand, when the aqueous phase was under acidic conditions of pH 3 and alkaline conditions of pH 10, the number of organic solvent-resistant microorganisms obtained was drastically reduced. Therefore, it can be said that the pH of the treatment aqueous phase is preferably around neutral.

[0017]

[Table 5] Table 5 Relationship between pH of aqueous phase and number of separated organic solvent-resistant microorganisms ─────────────────────────────── ───── Number of solvent-resistant microorganisms separated per 1g of sample Water phase pH ─────────────────────────── Sample F Sample G ─ ────────────────────────────────── (when using distilled water) 3 2 7 5 14 19 6 15 19 7 12 20 8 10 18 10 10 2 (When artificial seawater is used) 3 4 8 5 29 29 41 6 37 37 48 7 33 33 39 8 30 38 38 10 3 9 9 ─────────────── ─────────────────────

Example 6 Preincubation of kerosene-degrading cells with DS-711 strain: A large test tube was charged with 10 ml of M-medium containing 1 mol of sodium chloride, and a silicon stopper was attached to the tube to sterilize it. After sterilization,
One platinum loop of the DS-711 strain was scraped from the stock slant, inoculated into the medium, and vigorously shake-cultured at 37 ° C for one day. Kerosene decomposition experiment : A 500 ml pleated Erlenmeyer flask was charged with 100 ml of M-medium containing 1 mol of sodium chloride, sterilized, and inoculated with 1 ml of the above preculture suspension. After the inoculation, 10 ml of kerosene was overlaid and shake-cultured at 35 ° C. for 1 week. After culturing, centrifugation was performed to remove the bacterial cells, and the supernatant was transferred to a separatory funnel to separate the remaining kerosene phase and the aqueous phase of the medium,
50 ml of benzene containing 0.1 g of octacosan was added to each to extract hydrocarbons. After extraction, anhydrous sodium sulfate was added to benzene, dehydrated and concentrated, and individual linear hydrocarbons having 6 to 16 carbon atoms were analyzed by gas chromatography. The results are shown in Table 6 below. Gas chromatography conditions: Gas chromatography:
Shimadzu GC-14A, Detector: Single-flame ionizatio
n detector, column: glass column (2.1m × 3.2m / m),
Carrier: silicon 10% OV-101, 60/80 mesh Chromosorb W AW-DMCS, carrier gas: N ₂ (40-50
ml / min), temperature: 35-300 ° C (program speed 10 ° C /
Minutes)

[0019]

[Table 6] Table 6 Decomposition of hydrocarbons contained in kerosene ─────────────────────────────────── Carbonization Hydrogen content Control area Hydrocarbons Experimental area Hydrocarbons (without inoculation of cells) (mg) Kerosene phase Water phase Kerosene phase Water phase ─────────────────────── ──────────── n-hexane 0.8 not detected not detected not detected not detected n-heptane 56 34 not detected not detected 0.02 n-octane 72 39 not detected 7 not detected n-nonane 236 190 not detected Detected 34 Not detected n-decane 312 245 Not detected 70 Not detected n-undecane 320 270 0.04 85 1.4 n-dodecane 704 652 0.08 150 1.7 n-tridecane 596 501 0.09 134 2.4 n-tetradecane 364 332 0.09 78 1.7 n-pentadecane 212 188 0.07 12 1.3 n-hexadecane 48 39 0.02 7 0.6 ──────────────────────── ─────────── total concentration of hydrocarbons from C6 included in the test kerosene to 16 corresponds to 29.2% by volume per kerosene.

(Example 7) Pre-incubation of cells degrading hydrocarbons contained in 5% n-hexane-added kerosene by DS-711 strain: The same method as in Example 6 was carried out. Kerosene degradation test : Put 100 ml of M-medium containing 1 mol of salt into a 500 ml Erlenmeyer flask with folds, sterilize,
1 ml of the above culture suspension of strain DS-711 was inoculated. After inoculation, 10 ml of kerosene prepared so that the hexane concentration was 5% by weight was overlaid, and the experiment was conducted under the same conditions as in Example 6 below. The results are shown in Table 7.

[0021]

[Table 7] Table 7 Decomposition of hydrocarbons contained in kerosene ─────────────────────────────────── Carbonization Hydrogen content Control area Hydrocarbons Experimental area Hydrocarbons (without inoculation of cells) (mg) Kerosene phase Water phase Kerosene phase Water phase ─────────────────────── ──────────── n-hexane 500.8 224 not detected 210 0.25 n-heptane 56 34 not detected not detected 0.02 n-octane 72 39 not detected 11 not detected n-nonane 236 190 not detected 55 not Detected n-decane 312 245 Not detected 85 Not detected n-undecane 320 270 0.04 96 0.05 n-dodecane 704 652 0.08 167 0.09 n-Tridecane 596 501 0.09 100 1.7 n-Tetradecane 364 332 0.09 45 1.5 n-Pentadecane 212 188 0.07 16 2.3 n-hexadecane 48 39 0.02 9 0.9 ──────────────────────────── ───────

(Example 8) Pre- decomposition culture of heavy oil by strain DS-711: The same method as in Example 6 was carried out. Heavy oil decomposition test : 100 ml of M-medium containing 1 mol of sodium chloride was placed in a 500 ml pleated Erlenmeyer flask, sterilized, 1 ml of the culture suspension was inoculated, and 5 ml of C-heavy oil (made by Idemitsu Petroleum) was added. The cells were overlaid and gently agitated and cultured at a temperature of 28 ° C. for 2 weeks. After culturing, centrifuge to remove bacterial cells,
The supernatant was transferred to a separatory funnel, 100 ml of hexane was added to extract hydrocarbons, and the extract was dehydrated with magnesium sulfate and then subjected to silicic acid column chromatography (silicic acid / celite = 9 /
Hydrocarbons were eluted with 1, 20 g, column 25 mm x 100 mm, elution solvent n-hexane 150 ml). Concentrate the eluate under reduced pressure with a rotary evaporator until it reaches a certain volume,
Individual straight chain hydrocarbons having 9 to 16 carbon atoms were analyzed by gas chromatography. The results are shown in Table 8.

[0023]

[Table 8] Table 8 Decomposition of hydrocarbons contained in heavy oil ─────────────────────────────────── Carbonization Hydrogen amount Control field Hydrocarbon field Experimental field Hydrocarbon (mg) (without inoculation of bacterial cells) ──────────────────────────────── ──── n-nonane 15 11 0.7 n-decane 21 16 0.8 n-undecane 25 22 1.1 n-dodecane 45 39 2.6 n-tridecane 49 41 2.4 n-tetradecane 40 36 2.2 n-pentadecane 35 29 1.9 n-hexadecane 29 22 1.2 ─────────────────────────────────── Hexane, heptane, and octane were not measured.

Claims (8)

[Claims] 1. A sample is mixed with water and an organic solvent, shake-cultured, and then the culture mixture is allowed to stand, separated into an aqueous phase and an organic solvent phase, and an appropriate amount of the organic solvent phase is added to a medium to culture. And a method for separating an organic solvent-resistant microorganism, which comprises isolating a growing microorganism. 2. The method according to claim 1, wherein the shaking culture is performed at 4 to 30 ° C. for 1 to 7 days. 3. The method according to claim 1 or 2, wherein the medium is an agar medium. 4. The volume ratio of water to the organic solvent is 1: 9 to 9: 1.
The method according to any one of claims 1 to 3, wherein 5. Incubation of the organic solvent phase at 20-40 ° C.
The method according to claim 1, wherein the method is performed for 10 days. 6. The method according to claim 1, wherein the water is deionized water, distilled water, seawater or artificial seawater. 7. The method according to claim 1, wherein the organic solvent is a hydrocarbon. 8. The method according to claim 1, wherein the hydrocarbon is an aromatic hydrocarbon.