JPH06319530A - Yeast capable of decomposing petroleum - Google Patents

Abstract

PURPOSE:To provide the yeast having petroleum degradative activity, belonging to Candida, capable of efficiently decomposing and removing petroleum, excellent in resistance to environmental pollution, thus useful in petroleum spill accidents, etc. CONSTITUTION:The objective yeast, Candida sp. M23-2 (FERM P-13540), having petroleum degradative activity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism belonging to the genus Candida, which has a remarkable decomposing activity for petroleum, particularly crude oil, and has an excellent decomposition rate.

[0002]

2. Description of the Related Art In recent years, pollution of the ocean by various petroleums has become a very serious environmental problem. Such pollution is caused by a marine accident of a large tanker, an oil field accident, an accident of various industrial plants, or the like, and in any case, a huge amount of oil is leaked, which causes a large-scale environmental damage.

Conventionally, many attempts of bioremediation have been made to decompose and remove such petroleum pollution by microorganisms, but there are many problems to be solved for practical use. In the case of petroleum decomposition by microorganisms, it is particularly problematic because of its low decomposition rate,
In order for the effects of bioremediation to become apparent,
I need at least one month. If it is possible to increase the rate of petroleum decomposition, the effects such as reduction of costs required for bioremediation and reduction of environmental damage will be great.

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present inventor diligently searched for marine samples from around the world for microorganisms excellent in growth rate in a crude oil-containing medium. We succeeded in isolating a novel yeast, Candida sp. M23-2, which has a remarkable degradation activity and an excellent degradation rate, and completed the present invention.

An object of the present invention is to provide a novel yeast, Candida sp. M 23-2, which has a remarkable decomposition activity on petroleum, particularly crude oil, and has an excellent decomposition rate.

[0006]

The present invention resides in Candida sp. M 23-2 having a petroleum-degrading activity. And, as the petroleum, crude oil and the like are mentioned here. INDUSTRIAL APPLICABILITY The novel Candida sp. M 23-2 of the present invention is useful for decomposing and removing petroleum that has flowed out to the ocean and the like.

The present invention will be described in detail below. The present inventors searched for microorganisms having a petroleum-degrading activity as follows. That is, 5 g of coastal mud from Mizushima, Okayama Prefecture, 1 g of ammonium nitrate, 2 mg of potassium phosphate, 20 mg of iron citrate,
Add 50 g of yeast extract, 800 ml of seawater, and 200 ml of pure water to a basic medium containing 1 g of weathering crude oil (the remainder of the crude oil having a boiling point of 270 ° C or less to distill off) and add it to 40 ml of an accumulation medium at 3 ° C at 20 ° C Accumulation culture was carried out by shaking for a day. 0.1 ml of the end product of this enrichment culture is added to 5 ml of new enrichment medium
The cells were transplanted to and cultured at 20 ° C. for 24 hours to carry out a second enrichment culture. The third enrichment culture was performed in the same manner as the second enrichment culture using 0.1 ml of the end product of the second enrichment culture. This operation was repeated, and the product obtained after the sixth accumulation culture was spread on a plate medium in which 20 mg / ml of agar was added to the accumulation medium and cultured at 20 ° C. for 7 days to form colonies. These colonies were isolated by blunting with a bacterium. For the isolates, 1-10 at 20 ° C. in medium supplemented with 2 mg / ml weathering crude oil.
After culturing for one day, the degradation activity was compared. Decomposition activity, residual oil in the end product of the main culture, chloroform extraction, analyzed by thin layer chromatography-flame ionization detection method, and calculate the decomposition rate and decomposition rate of each strain from this quantitative value. Compared. The M23-2 strain with the highest degradation rate was selected by comparing the degradation rate and rate of weathering crude oil.

This strain M23-2 has the following mycological properties. (1) Growth condition on medium 1) Microscopic observation by corn meal agar slide culture Vegetative cell size Length: 4.2 (± 0.8) μm Width: 2.6 (± 0.6) μm Vegetative cell shape Oval Proliferation method Multipolar budding No hypha formation No pseudohyphae formation 2) Growth on YM liquid medium Surface growth Membrane formation Medium cloudiness Strongly cloudy precipitate formation Powder 3) Growth on YM agar medium Good growth Peripheral edge of colony Raised state of colony Cone shape of surface of colony Scratch-like appearance of colony Matte of colony Wetness and stickiness, no color tone White (2) Ascospore formation YM Agar No formation McClary et al. No medium formed Gorodkowa medium not formed V8 agar medium not formed (3) Injection spore formation YM Agar not formed (4) Physiological properties Growth temperature 6-37 ℃ Optimal growth temperature 20-31 ℃ Growth pH 2.0 ~ 9.0 Optimal growth pH 3.0 ~ 8.0 No assimilation of nitrate No decomposition of fat No decomposition of urea No liquefaction of gelatin Maximum concentration of viable glucose 50% Optimal concentration of growth 2-10% Maximum concentration of viable concentration of sodium chloride 10% Optimal concentration of growth 0-5% No formation of carotenoids Remarkable organic No acid formation No starch-like substance formation No vitamin requirement No DNase productivity No DBB colony color reaction No GC content 37.8 mol% Ubiquinone type Q9 (5) Sugar fermentability Glucose-Galactose-α-Methyl Glucoside-saccharose-maltose-cellobiose-trehalose-lactose-melibiose-raffinose-meliditose-inulin-soluble starch- (6) sugar assimilation D-glucose + D-galactose + saccharose + maltose + cellobiose + lactohalose. Sucrose + melibiose-raffinose-meriditol + inulin ± soluble starch + D-xylose + L-arabinose-ethanol + α-Me-D-glucoside + succinate + inosit-arbutin + a book having the above-mentioned mycological properties As a result of examining the taxonomic position of the strain with reference to Yeasts (Yeasts JA Barnett et al., 1990 edition), this strain belongs to a strain of Candida that does not form pseudohyphae, but the strain is strictly known. Since no description to identify was found, it was determined to be a new strain, and this was named Candida sp. M23-2 strain.

This Candida sp. M23-2 was transferred to the Institute of Biotechnology, Institute of Industrial Science, on March 23, 1993 by FERM P-13.
Deposited as 540. For culturing the strain, various media containing a carbon source, a nitrogen source, and an inorganic substance, which are commonly used for culturing microorganisms, can be used. As a carbon source,
For example, various sugars such as glucose, fructose and starch, crude oil such as weathering crude oil, various hydrocarbons such as n-alkanes, etc., and nitrogen sources include organic nitrogens such as yeast extract, meat extract, peptone and various amino acids. Also, various inorganic nitrogens such as ammonium sulfate. The carbon source and the nitrogen source may be used alone or in combination. Other inorganic substances such as phosphorus, sodium, potassium, calcium, and trace metals are added as needed. The culturable pH is in a wide range of 2.0 to 9.0, and the sodium chloride concentration can be cultivated at 0 to 10%, and there is no particular limitation within these ranges.

The morphology of the bacterial cells in using this strain is
It may be a live cell, a frozen product or a lyophilized product and is not particularly limited.

[0011]

EXAMPLES The present invention will be specifically described below with reference to examples. However, these examples do not limit the technical scope of the present invention. Example 1 Candida sp. M23-2 was added to YM broth in 5 ml of the above basic medium supplemented with 5 mg / ml of the above weathering crude oil.
0.1 ml of a culture-finished product obtained by culturing at 20 ° C. for 3 days was inoculated and shake-cultured at 20 ° C. for 3 days. Next, the residual oil content in the culture-finished product was analyzed as described above, and the crude oil decomposition rate of the bacterium was calculated from this quantitative value.

The results are shown in Table 1. Candida sp.
The decomposition rate of weathering crude oil at 20 ° C according to 23-2 is 0.
It was 55 mg / ml / day. Comparative Example 1 Instead of Candida sp. M23-2, a known petroleum degrading yeast,
Candida petroleum ATCC 20226, Yarrowia
Lipolitica ATCC34922, Yarrowia Lipolitica IFO 0
746, Saccharomyces cerevisiae ATCC 20252 or Torulaspora hansenii IFO 0083 were used, but the same procedure as in Example 1 was performed. The results are shown in Table 1, and the decomposition rate of crude oil was 0.42 mg / ml / day with the highest Yarrowia lipolytica ATCC 34922.

[0013]

[Table 1]

Example 2 The procedure of Example 1 was repeated, except that the culture was carried out at 30 ° C. The results are shown in Table 2, and the decomposition rate of weathering crude oil by Candida sp. M23-2 at 30 ° C was 0.69 mg /
ml / day. Comparative Example 2 Instead of Candida sp. M23-2, a known petroleum degrading yeast,
Candida petroleum ATCC 20226, Yarrowia
Lipolitica ATCC 34922, Yarrowia Lipolitica IFO
0746, Saccharomyces cerevisiae ATCC 20252 or Torulaspora hansenii IFO 0083 was used, but the same procedure as in Example 2 was performed.

The results are shown in Table 2, and the decomposition rate of crude oil was 0 even with the highest Yarrowia lipolytica IFO 0746.
It was 42 mg / ml / day.

[0016]

[Table 2]

Example 3 Candida sp. M23-2 was cultured for 3 days in YM broth at 30 ° C. for 3 days in 5 ml of a medium prepared by adding 5 mg / ml of the above-mentioned weathering crude oil to the above-mentioned basic medium, 0.1 was inoculated and shake-cultured at 30 ° C. for 10 days. Then, the residual oil content in the culture-finished product was analyzed as described above, and the crude oil decomposition rate of the bacterium was calculated from this quantitative value.

The results are shown in Table 3. Candida sp.
The decomposition rate of weathering crude oil according to 23-2 was 55.5%. Comparative Example 3 Instead of Candida sp. M23-2, a known petroleum-degrading yeast,
Candida petroleum ATCC 20226, Yarrowia
Lipolitica ATCC 34922, Yarrowia Lipolitica IFO
0746, Saccharomyces cerevisiae ATCC 20252 or Traspora hansenii IFO 0083, except that the same procedure as in Example 3 was performed. The results are shown in Table 3, and the decomposition rate of crude oil is highest in Yarrowia lipolytica.
Even IFO 0746 was 45.1%.

[0019]

[Table 3]

[0020]

INDUSTRIAL APPLICABILITY The present invention provides a novel yeast. And, this novel yeast has a remarkable decomposition activity on petroleum, particularly crude oil, and has an excellent decomposition rate, so that various yeasts can be efficiently decomposed and removed using this yeast, Environmental pollution due to petroleum can be prevented. Therefore, the present invention is an industrially very useful invention.

Claims (1)

[Claims] 1. A Candida sp. M having a petroleum degrading activity.
23-2