JP4931451B2 - New microorganism - Google Patents

Description

  The present invention relates to a microorganism that efficiently decomposes hydrocarbons derived from petroleum products, in particular, aromatic hydrocarbons such as benzene, toluene, and xylene.

As a technique for purifying groundwater in soil containing hydrocarbons derived from petroleum products, purification techniques using microorganisms are being studied.
However, the degradation rate of hydrocarbons derived from petroleum products by microorganisms is generally relatively slow, and it takes several months to several years to purify the groundwater in the soil containing hydrocarbons derived from petroleum products. Therefore, there is a demand for microorganisms that can be degraded in a short period of time.

It is well known that soil and groundwater contain microorganisms that decompose hydrocarbons derived from petroleum products (see Non-Patent Document 1).
So far, several species of microorganisms that decompose hydrocarbons derived from petroleum products have been reported, and microorganisms that decompose aromatic hydrocarbons such as benzene, toluene, or xylene have been obtained (patent documents). 1, refer to Non-Patent Document 2). However, the degradability of the microorganisms for each component such as benzene, toluene, xylene, etc. has been confirmed. However, if the aromatic hydrocarbons are derived from petroleum products, usually a plurality of aromatic hydrocarbons are mixed. In many cases, it has not yet been confirmed to what extent each of the three components of benzene, toluene, and xylene coexists.
In addition, regarding another microorganism, it has been reported that, in a situation where benzene, toluene and xylene coexist, only toluene and xylene are decomposed, and benzene is not decomposed at all (see Non-Patent Document 3).
JP-A-11-266858 Bioremediation Engineer -Design and Application-, pp. 104-114, published on January 23, 1997 Lee, SK, Appl Microbiol Biotechnol, 56, 270-275 (2001) Prenafeta-Boldu., Applied and Environmental Microbiology., 2660-2665 (2002)

Soil and groundwater usually have multiple petroleum hydrocarbons in common, but as mentioned above, all these components are efficiently used in the situation where the three components of benzene, toluene and xylene coexist. No microorganism is known that has the ability to degrade well.
Accordingly, an object of the present invention is to provide a novel microorganism capable of efficiently decomposing aromatic hydrocarbons, particularly benzene, toluene and xylene, even in the situation where a plurality of petroleum hydrocarbons coexist.

  In view of such a situation, the present inventors screened known microorganisms and unknown microorganisms from soils in various places in Japan. As a result, microorganisms belonging to the genus Capriavidus are petroleum hydrocarbons, particularly benzene, toluene. The present invention was completed by finding that benzene, toluene, and xylene can be efficiently decomposed even in soil and groundwater in which a plurality of petroleum hydrocarbons coexist, having a high ability to decompose aromatic hydrocarbons such as xylene. It came to be.

That is, the present invention provides a C-102 strain (FERM P-20819) or a C-112 strain (FERM P-20820) belonging to the genus Capriavidus, which has the ability to decompose hydrocarbons derived from petroleum products. Is.

  The microorganism of the present invention can efficiently decompose hydrocarbons derived from petroleum products such as gasoline, kerosene, light oil, heavy oil and lubricating oil, particularly aromatic hydrocarbons such as benzene, toluene and xylene. In addition, since the microorganism of the present invention can decompose all of these components even in the presence of three components of benzene, toluene and xylene, it contains hydrocarbons derived from petroleum products by using the microorganism. Soil and groundwater can be efficiently purified.

  In the present invention, petroleum products refer to gasoline, kerosene, light oil, heavy oil, lubricating oil, etc., and hydrocarbons derived from petroleum products include aliphatic hydrocarbons such as alkanes and alkenes; benzene, toluene, xylene, etc. Monocyclic aromatic hydrocarbons, or polycyclic aromatic hydrocarbons such as naphthalene and anthracene.

The microorganism of the present invention belongs to the genus Cupriavidus and efficiently decomposes hydrocarbons derived from petroleum products. Such a microorganism belonging to the genus Capriavidas is not particularly limited as long as it is a microorganism having the ability to decompose hydrocarbons derived from petroleum products, but a microorganism having the ability to decompose aromatic hydrocarbons is preferred, and in particular, benzene, Microorganisms having the ability to decompose two or three mixtures of toluene and xylene are preferred, and the total decomposition rate for the three in the presence of benzene, toluene and xylene is 50 to 100%, particularly 90 to What is 100% is preferable. Specifically, the C-102 strain and the C-112 strain isolated by the present inventors from soil in Saitama Prefecture, among which the C-112 strain is preferred.
The C-102 and C-112 strains have the following mycological properties.
(1) Mycological properties of C-112 strain

(2) Mycological properties of C-102 strain

  From these mycological properties, the base sequence of ribosomal 16S rDNA and its homology, (BRENNER, (DJ), KRIEG, (NR), STALEY, (JT) and GARRITY, (GM), Bergey's manual of Systematic Bacteriology Vol. Two, The Proteobacteria, Part C The Alpha-, Beta-, Delta-, and Epsilonproteobacteria. 2005, Springer). As a result of classification and identification, the C-112 strain belongs to the genus Cupriavidus. Identified. However, at the seed level, nitrate is reduced, no indole is produced, no activity such as urease and protease is shown, toluene is assimilated, glucose is not assimilated, growth at 41 ° C., starch is not hydrolyzed Since the point is different from the known species contained in the genus Capriavidas, it was identified as a novel microorganism and deposited as the Capriavidas genus C-112 strain at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary (FERM P-20820).

  The C-102 strain was also identified as belonging to the genus Cupriavidus from its bacteriological properties, the base sequence of 16S rDNA of ribosome and its homology. However, at the seed level, Capriavidas genus includes the point that it does not reduce nitrate, produce indole, does not show activity such as urease and protease, assimilates toluene, does not assimilate glucose, and does not hydrolyze starch. Unlike the known species, the cell size and the point that it does not show growth at 41 ° C are also different from the C-112 strain. Therefore, it was identified as a novel microorganism and the Capriavidas genus C-102 strain was established as an independent administrative agency. Deposited at the Institute for Biological Deposits (FERM P-20819).

  C-102 strain and C-112 strain belong to the genus Capriavidas, reduce nitrate, do not produce indole, do not show activity of urease and protease, etc., assimilate toluene, do not assimilate glucose, It is considered to be a new species having the property of not hydrolyzing. Examples of the related strains of the C-102 strain and the C-112 strain include those having these properties.

  The medium for cultivating the novel microorganism of the present invention is not particularly limited, and a medium having any composition may be used as long as it is a medium for culturing microorganisms and does not prevent the decomposition of hydrocarbons derived from petroleum products. be able to. Examples of the carbon source include aromatic hydrocarbons such as benzene, toluene and xylene; petroleum products such as gasoline containing these; organic acids such as acetic acid and pyruvic acid; and the like. Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, and urea; organic nitrogen sources such as peptone, meat extract, yeast extract, and casein. Furthermore, inorganic salts and the like can be added as appropriate.

  The culture conditions are not particularly limited and may be any culture conditions that generally do not kill microorganisms. For example, it is preferable to aerobically culture for 1 to 60 days under conditions of pH 6 to 8 and temperature of 20 to 35 ° C. .

  As a method for purifying soil containing petroleum products-derived hydrocarbons or groundwater in the soil using the novel microorganism of the present invention, microorganisms may be added to soil or groundwater containing petroleum products-derived hydrocarbons. That's fine. As the microorganism to be added, a culture solution obtained by culturing may be used as it is, or a microorganism collected by centrifugation or the like may be used. Moreover, you may use what was freeze-dried, and what was carried on the well-known support | carrier.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 Acquisition of Bacteria:
10 mL of the medium 1 shown in Table 7 was put in a test tube having an inner diameter of 21 mm, and sterilized at 121 ° C. for 15 minutes to prepare a liquid medium. 1 g each of 150 samples of soil collected in Japan including Saitama Prefecture was added thereto, and aerobic culture was performed for 1 year under conditions of 30 ° C. and 250 rpm with a test tube shaker. As a carbon source, 10 μL of commercial gasoline was added every 3 days to 1 week. Every 1 to 2 months, 1 mL of the culture solution was newly planted into the medium 1. 100 μL of the culture solution in which growth was observed was applied to a nutrient plate medium and cultured at 30 ° C. for 2 days. The appearing colonies were respectively picked up by platinum ears, inoculated into the above-mentioned medium 1, and cultured for 2 weeks under the condition where commercial gasoline was added. About the sample by which growth was recognized, it apply | coated to the nutrient plate culture medium again, and the single colony was formed. In this way, C-102 and C-112 strains were obtained as microorganisms having a high ability to decompose petroleum hydrocarbons.

Example 2 Decomposition test of benzene, toluene and xylene:
In a 50 ml glass centrifuge tube, 10 mL of the medium 1, 1 mL of the culture solution of the C-112 strain and the C-102 strain was centrifuged, the supernatant was removed, and the microorganism suspension prepared to 1 mL again with sterilized water. After inoculating the solution, 0.04 mg of benzene, toluene, and xylene was added and cultured at 30 ° C. and 100 rpm for 15 hours. Thereafter, the decomposition ability was measured by measuring the remaining benzene, toluene, and xylene contained in the culture solution.

  The remaining amount of the substrate was determined by introducing 10 mL of the culture solution after culturing into a 20 mL vial containing 3 g of sodium chloride, sealing it, heating the vial at 80 ° C. for 30 minutes, and gas phase after heating by gas chromatography. Quantitatively determined. The remaining amount of the substrate was compared with the added amount, and the decomposition rate was determined from the decrease rate. Further, as a control test, microorganisms contained in soil obtained at the initial stage of the process obtained by the method in Example 1 were used to confirm the ability to decompose benzene, toluene and xylene.

  As a result of measuring the residual amounts of benzene, toluene and xylene after 15 hours, as shown in Table 8, the system to which C-112 strain and C-102 strain were added had particularly high decomposition ability for benzene. Indicated. It was also shown that these three components can be decomposed simultaneously in an environment where benzene, toluene and xylene coexist. The numerical values in the table indicate the percentage reduction with respect to the added substrate.

Claims (1)

C-102 strain (FERM P-20819) or C-112 strain (FERM P-20820) belonging to the genus Cupriavidus having the ability to decompose hydrocarbons derived from petroleum products.