JP5243866B2 - Novel strain and purification method using the same - Google Patents

Description

  The present invention relates to a strain capable of decomposing a drin-based agricultural chemical compound such as dieldrin and endrin, which are persistent organic pollutants (POPs), and an environmental purification method using the same.

  Dildoline and endrin have been widely used as organochlorine pesticides since their pesticide registration in 1954. However, since then, high toxicity such as carcinogenicity has been confirmed, and manufacturing, importation and use have been prohibited since the 1970s. Nonetheless, they are persistent and remain in the soil and sediment for a long time. Bioaccumulation through the food chain still has a serious impact on human health and ecosystems. In fact, even today, more than 30 years after use, dieldrin and endrin are often detected in farmland and crops at concentrations exceeding environmental management guidelines and food standards (Hashimoto Ryoko (2003)) Proceedings of the 26th Residual Analysis Meeting: 92-97, Harumi Kondo, Eriko Amagawa, Hiroshi Sato, Kazuo Yasuda, Kenichi Onuki, Michiko Akiba, Kazuaki Kanaya (2003) Case study. Tokyo Kenanken Sen. Annual Report. 54: 132-135, Hashimoto Y (2005) Dieldrin residue in the soil and cucumber from agricultural field in Tokyo. J Pestic Sci 30: 397-402). In the 2001 POPs Convention, these are designated as persistent organic pollutants together with PCBs and dioxins, and measures are required internationally.

  As a purification method for substances such as dieldrin and endrin, which have a wide range of contamination, bioremediation is expected to be put to practical use at a lower cost than physicochemical methods.

  In recent studies on microbial degradation of dieldrin and endrin, attention has been focused on the dechlorination reaction of anaerobic microorganisms growing in sediments contaminated at high concentrations (Non-patent Documents 1 and 2). On the other hand, there is little information on aerobic microorganisms that degrade dieldrin and endrin, and little has been reported since the 1980s (Non-patent Document 3). For this reason, a method for purifying an aerobic environment such as the surface layer of farmland soil has not been established yet, although it remains at a relatively low concentration. Therefore, the response when these residues are found in farmland soil remains a passive method of cultivating crops that are difficult to absorb dieldrin and endrin.

Maule A, Plyte S, Quirk AV (1987) Dehalogenation of organochlorine insecticides by mixed anaerobic microbial populations.Pestic Biochem Physiol 27: 229-236 Baczynski TP, Grotenhuis T, Knipscheer P (2004) The dechlorination of cyclodiene pesticides by methanogenic granular sludge.Chemosphere 55: 653-659 Khan SU (1980) Pesticides in the soil environment.Elsevier scientific publishing company, Amsterdam-Oxford-New York

  Accordingly, an object of the present invention is to purify contamination in an aerobic environment such as the surface layer of farmland soil by providing an aerobic bacterial strain capable of degrading dieldrin and endrin.

  As a result of intensive studies to solve the above problems, the present inventors have found two strains having high resolution against dieldrin and endrin. The two strains are aerobic, one belonging to the genus Capriavidas and the other belonging to the genus Burkholderia. As described above, dieldrin and endrin-degrading bacteria are described in Non-Patent Documents 1 to 3. However, the microorganisms described in Non-Patent Documents 1 and 2 are anaerobic microorganisms. Non-Patent Document 3 describes microorganisms belonging to the genera Trichoderma, Fusarium, Aspergillus, Pseudomonas, and Bacillus, but Capriavidus ( Nothing is described about microorganisms belonging to the genus Cupriavidus and Burkholderia. Therefore, microorganisms belonging to the genus Capriavidas or Burkholderia and degrading dieldrin and endrin have never been known so far and have been discovered for the first time by the present inventors. The present invention has been completed based on the above findings.

That is, the present invention provides the following (1) to (8).
(1) A strain belonging to the genus Capriavidas or the genus Burkholderia and having a resolution of drin-based pesticide compounds.
(2) 16S rDNA consisting of a base sequence having 95% or more homology to the base sequence shown in SEQ ID NO: 1 or consisting of a base sequence having 95% or more homology to the base sequence shown in SEQ ID NO: 2 The strain according to (1), which has 16SrDNA.
(3) The strain according to (1), which has 16S rDNA consisting of the base sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 2.
(4) Capriavidas genus MED-5 strain deposited under the deposit number NITE P-574.
(5) Burkholderia strain MED-7 deposited under the deposit number NITE P-575.
(6) An environmental purification method comprising purifying an environment contaminated with a drin-based agricultural chemical compound with the strain according to any one of (1) to (5).
(7) The environmental purification method as described in (6), wherein the drin-based agricultural chemical compound is dieldrin or endrin.
(8) The environmental purification method according to (6) or (7), wherein the contaminated environment is an aerobic environment.

  The present invention provides a strain capable of degrading drin-based agricultural chemical compounds such as dieldrin and endrin. This strain makes it possible to efficiently purify surface soil contaminated with a drin-based agricultural chemical compound and the like.

  Hereinafter, the present invention will be described in detail.

  The strain of the present invention belongs to the genus Capriavidas or the genus Burkholderia and has a resolution of drin-based agricultural chemical compounds. Here, the “drin-based agrochemical compound” includes aldrin and the like in addition to dieldrin and endrin.

  Specific examples of the strain of the present invention include Capriavidas genus MED-5 and Burkholderia genus MED-7. These strains are strains isolated by the present inventor, and are assigned to the independent administrative corporation Product Evaluation Technology Base Organization Patent Microorganism Deposit Center (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan). Deposited as P-574 and NITE P-575 (date of deposit: May 28, 2008). MED-5 decomposes 16% of 400ppb dieldrin in 14 days using dieldrin as the sole carbon source. In addition, using endrin as the sole carbon source, it degrades 400ppb of endrin by 23% in 14 days. On the other hand, when 1,2-epoxycyclohexane is supplied as a carbon source, it degrades 38% of 400ppb dieldrin and 40% of 400ppb endrin in 14 days. MED-7 decomposes 18% of 400ppb dieldrin in 14 days using dieldrin as the sole carbon source. In addition, using endrin as the sole carbon source, it degrades 19% of 400ppb of endrin in 14 days. On the other hand, when 1,2-epoxycyclohexane is given as a carbon source, it decomposes 49% of 400ppb dieldrin and 51% of 400ppb endrin in 14 days.

  Strains having 16S rDNA showing high homology to MED-5 or MED-7 16S rDNA are also included in the strain of the present invention. Such a strain is considered to be a strain closely related to MED-5 or MED-7, and is expected to have a resolution for a drin-based agricultural chemical compound in the same manner as MED-5 and MED-7. As used herein, “high homology” refers to a homology of 95% or more, preferably 97% or more, more preferably 99% or more, most preferably 100% for MED-5. Meaning homology, MED-7 means 95% or more homology, preferably 97% or more homology, more preferably 99% or more homology, most preferably 100% homology.

  The method for culturing the strain of the present invention is not particularly limited, and the strain can be cultured by a method generally applied to microorganisms belonging to the genus Capriavidas or Burkholderia. As the medium, for example, a standard agar medium, R2A medium, LB medium or the like can be used. The culture temperature is not particularly limited, but is preferably 25 to 37 ° C, and more preferably 25 to 30 ° C.

  The microorganism of the present invention can be used for purification of an environment contaminated with a drin-based agricultural chemical compound. “Environment” as used herein mainly means soil, but also includes water, clay and the like. Since the strain of the present invention is an aerobic strain, the environment to be purified is preferably an aerobic environment. As used herein, “aerobic environment” refers to, for example, an aerobic environment forcibly created by using an oxygen supply agent or the like for surface soil, surface water, sediment (such as bottom sediment), soil or water. Say.

Purification of the environment is carried out by spraying a culture solution of microorganisms or a dry powder obtained by freeze-drying microorganisms to a contaminated environment. At this time, dry powder and inorganic salts that assist in growth may be mixed and granulated, and the powdered and granular preparations may be dispersed in a contaminated environment. The amount of microorganisms used in the treatment can be arbitrarily determined according to the environmental pollution status, etc., but is usually about 10 L for a culture solution in 100 m ² of contaminated soil and about 50 g for dry cells.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1] Isolation of microorganisms Selection of soil as an isolation source was performed as follows. Three types of uncontaminated field surface soil and one forest surface soil were used as test soils. 100 g (dry weight) of these were placed in a 500 ml glass bottle, and dieldrin and endrin were added to 1 ppm each. The soil microcosm was cultivated in a dark place at 25 ° C, and the aerobic condition was maintained by removing the lid every two weeks. Residual amounts of dieldrin and endrin in the soil were measured with a gas chromatograph mass spectrometer at the start of culture and at 3, 7, 14, and 30 weeks. As a result, high degradation was observed in the forest surface soil (Fig. 1), so this soil was determined as the separation source.

  Accumulation culture was performed using the forest surface soil as follows. 10 ml of RM2 + medium (Table 1) placed in a test tube was inoculated with 0.5 g (wet weight) of the above-mentioned forest surface soil microcosm. 1,2-epoxycyclohexane 0.1% (wt / vol) was added thereto, and cultured with shaking in the dark at 25 ° C. 1,2-Epoxycyclohexane is a structural analog of dieldrin and endrin, and can be an advantageous carbon source for degrading bacteria. This culture solution was planted once under the same conditions, and then repeatedly transplanted every 7 to 10 days in an MS medium (Table 1) supplemented with 1,2-epoxycyclohexane 0.1% (wt / vol). This culture solution was used as an accumulation culture system for degrading bacteria.

The resolution of the enrichment culture system was evaluated as follows. On the 7th day after planting, 0.5 ml of the enrichment culture system was inoculated into 9.5 ml of MS medium placed in a test tube. 1,2-epoxycyclohexane 0.1% (wt / vol) and dieldrin and endrin were added to 400 ppb, respectively, and cultured with shaking in the dark at 25 ° C. On day 14 of culture, the residual amounts of dieldrin and endrin in the culture were measured with a gas chromatograph mass spectrometer, and as a result, degradation of 32% or more was observed. (Figure 2)
Isolation of degrading bacteria from the enrichment culture system was performed as follows. The enrichment culture medium whose resolution was confirmed above was smeared on a standard agar plate medium or R2A plate medium (Table 1), and purely separated. As a result, MED-5 and MED-7 were isolated as strains having the resolution of dieldrin and endrin. In addition, 16SrDNA was extracted from the enrichment culture and subjected to denaturing gradient gel electrophoresis. As a result, bacteria that grew predominantly accumulated to several strains, which were consistent with MED-5 and MED-7. (Fig. 3).

[Example 2] Identification of microorganism
The 16S rDNA base sequences of MED-5 and MED-7 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. By comparing this nucleotide sequence with the sequence of a known microorganism, MED-5 was identified as Cupriavidus sp. And MED-7 was identified as Burkholderia sp. The closest relative to MED-5 was Cupriavidus necator with a homology rate of 99.0%, and the closest relative to MED-7 was Burkholderia terrae with a homology rate of 98.9%. The phylogenetic trees of MED-5 and MED-7 prepared by the NJ (proximity joining) method are shown in FIGS. 4 and 5, respectively. MED-5 and MED-7 had a homology rate of 97.0% or less with known pathogenic microorganisms, and were confirmed to be safe microorganisms.

  A phase contrast micrograph (1000 times) of the strain provided by the present invention is shown in FIG. MED-5 is a cocci and is consistent with the characteristics of the genus Cupriavidus. MED-7 is a gonococcus and is consistent with the characteristics of the genus Burkholderia.

[Example 3] Degradation of dieldrin and endrin in liquid medium Pre-culture was performed as follows. The R2A medium placed in the test tube was inoculated with colony 1 platinum loops from the R2A agar plate medium and cultured in the dark at 25 ° C. until the late logarithmic growth phase of each bacterium. The cells were collected from the culture solution using a centrifuge, washed and suspended in MS medium.

The resolution of the strain provided in the present invention in a liquid medium was evaluated as follows. In order to make the bacterial cell concentration uniform, the absorbance at an absorption wavelength of 600 nm was measured using a spectrophotometer. The preculture was inoculated into the MS medium placed in the test tube so that OD ₆₀₀ = 0.005. To this, dieldrin and endrin were added to 400 ppb and cultured with shaking in the dark at 25 ° C. In this liquid medium, dieldrin and endrin are the only carbon sources. On the 14th day of culture, the residual amounts of dieldrin and endrin in the culture medium were measured with a gas chromatograph mass spectrometer, and as a result, degradation of 16% or more with MED-5 and 18% or more with MED-7 was observed. (Figure 7)
[Example 4] Degradation of dieldrin and endrin in liquid medium supplemented with 1,2-epoxycyclohexane Prepared in the same manner as in Example 3, and inoculated precultured MS medium placed in a test tube. Furthermore, 0.2% (wt / vol) of 1,2-epoxycyclohexane, dieldrin and endrin were added to 400 ppb, and cultured with shaking in the dark at 25 ° C. In this liquid medium, the only carbon sources are dieldrin and endrin and 1,2-epoxycyclohexane. On day 14 of culture, the residual amounts of dieldrin and endrin in the culture medium were measured with a gas chromatograph mass spectrometer. As a result, degradation of 38% or more with MED-5 and 49% or more with MED-7 was observed. (Figure 8)

The figure which shows the time-dependent change of the dieldrin or endrin residual amount in soil. The figure which shows the time-dependent change of the dieldrin or endrin density | concentration in a culture solution. The figure which shows the result of denaturing agent concentration gradient gel electrophoresis of 16SrDNA extracted from the enrichment culture solution. The figure which shows the phylogenetic tree of MED-5 created by NJ method. The figure which shows the phylogenetic tree of MED-7 created by NJ method. Phase contrast micrographs of MED-5 and MED-7. The figure which shows the resolution with respect to dieldrin or endrin of MED-5 or MED-7. The figure which shows the resolution | decomposability with respect to dieldrin or endrin of MED-5 or MED-7 at the time of culture | cultivating in the culture medium containing 1, 2- epoxycyclohexane.

Claims (5)

  Capriavidas genus MED-5 strain deposited under accession number NITE P-574.   Burkholderia MED-7 strain deposited under accession number NITE P-575. A method for purifying an environment, comprising purifying an environment contaminated with a drin-based agrochemical compound with the strain according to claim 1 . 4. The environmental purification method according to claim 3 , wherein the drin-based agricultural chemical compound is dieldrin or endrin. The environmental purification method according to claim 3 or 4 , wherein the polluted environment is an aerobic environment.