KR20220051114A - Microbial materials for degrading organochlorine pesticides - Google Patents

Abstract

The present invention provides a microbial material for decomposing organochlorine pesticides, which comprises at least one microorganism selected from a group consisting of Phanerochaete chrysosporium microorganism and Streptomyces sp. microorganism as an active ingredient.

Description

Microbial materials for degrading organochlorine pesticides

The present invention relates to a microbial preparation for decomposing organic chlorine-based pesticides, and the microbial preparation of the present invention can be used for restoration of contaminated soil.

Pesticides greatly contribute to improving agricultural productivity by protecting crops from pests and weeds, but pesticides with high persistence cause many problems in the environment and ecosystem, such as soil contamination.

Persistent organic pollutants (POPs) are toxic, bioaccumulative, persistent and long-distance substances. Adopted in May and entered into force in May 2004, Korea also prepared it in 2007 and is managing POPs in accordance with the 'Residual Organic Pollutants Control Act'.

Among POPs, residual organochlorine pesticides (ROCPs) that contain chlorine and are used as pesticides are aldrin, endrin, dieldrin, toxaphene, chlordane, heptachlor, mirex, hexachlorobenzene (HCB), dichloro diphenyl trichloro ethane ( DDT), α-hexachlorocyclohexane (α-HCH), β-HCH, pentachlorobenzene (PeCB), and endosulfan 13 species. Among them, 11 species excluding chlordane and mirex have been registered and used as insecticides in Korea.

The Ministry of Food and Drug Safety establishes and manages residual tolerance standards as the sum of single components or metabolites of residual organic chlorine pesticides among agricultural products that are toxic. For example, the residual tolerance standards for aldrin and dieldrin are the sum of these. 0.01-0.1, endirn (sum of endrin and δ-ketoendrin) is 0.01-0.05, endosulfan (sum of α-endosulfan, β-endosuflan, and endosulfan sulfate) is 0.05-0.2, heptachlor (sum of heptachlor and heptachlor epoxide) is 0.01 -0.02 mg/kg is set to manage each crop.

Organochlorine pesticides (insecticides) such as BHC, DDT, endosulfan, heptachlor, and aldrin have been extensively used to protect crops from pests ^{1 )} . Endosulfan is composed of a mixture of two isomers (α-endosulfan, β-endosulfan) as follows, and through oxidation and hydrolysis reactions, endosulfan sulfate, endosulfan diol, endosulfan hydroxyether, endosulfan lactone, endosulfan ether, dicarbonic acid, etc. An analogue of the is formed.

Endosulfan is known to have characteristics such as high toxicity, bioaccumulation, mobility, and recalcitrance. In particular, it can cause genotoxicity and neurotoxicity in fish, invertebrates, and mammals, and has a long half-life of 60-800 days. There are ^2,3) . However, it is still used in developing countries in Asia, India and China, and is detected in various environments such as soil, air, and water ^{4,5 )} . In Korea, registration of endosulfan as a pesticide has been canceled since 2011, and since 2015, it has been included in persistent organic pollutants ^{6 )} to strengthen management. However, although domestic use of endosulfan has been completely stopped, it has been detected in agricultural environments and crops such as facility cultivation areas and field soils ⁷⁾ . Although it is rare that endosulfan is detected in excess of the permissible standard, it is necessary to develop detoxification measures and environmental restoration technology in consideration of the chronic effects on the environment and humans ⁸⁾ .

Among the detoxification methods, the bioremediaton technology using microorganisms has the advantage that there is no concern of secondary contamination compared to the chemical treatment method, and it has environmental safety, economic feasibility, simplicity of operation, high efficiency and wide area application ^{9.10,11 )} .

In Korea, Shin Jae-ho et al. ^{12 )} conducted a basic study for the microbiological treatment of this endosulfan, Bacillus sp. E64-2 strain was used under laboratory conditions, and Cho et al. ^{13 )} Klebsiella on activated charcoal. After loading the oxytoca KE-8 strain, an endosulfan degradation test was conducted in the laboratory.

Overseas, Ishag et al. ^{14 )} selected candidate strains from soil contaminated with this pesticide and conducted research on endosulfan decomposition in the laboratory. Shivaramaiah et al. ^{15 )} also conducted decomposition experiments using native bacteria present in soil. .

An object of the present invention is to select microorganisms having excellent decomposition ability of organochlorine-based pesticides, and to provide a microbial composition capable of restoring soil contaminated with organochlorine-based pesticides by using them as a single microorganism or a complex microorganism.

1 The present invention Phanerochaete chrysosporium ( Phanerochaete chrysosporium ) Microorganisms for decomposing organic chlorine-based pesticides comprising one or more microorganisms selected from the group consisting of microorganisms and Streptomyces sp . microorganisms as an active ingredient provide a formulation.

In one embodiment, the Panerokite chrysosporium microorganism may be a Panerokite chrysosporium Y-2 microorganism.

In one aspect, the Streptomyces genus ( Streptomyces sp.) The microorganism may be one or more microorganisms selected from the group consisting of Streptomyces genus MJM14731, Streptomyces genus MJM14747, and Streptomyces genus MJM14850 microorganisms.

In one embodiment, the microorganism may be a complex microorganism of the Panerocite chrysosporium Y-2 microorganism and the genus Streptomyces MJM14747.

In one aspect, the microorganism is Panerokite It may be a complex microorganism of Chrysosporium Y-2 microorganism and Streptomyces genus MJM14731 .

In one aspect, the microbial preparation may further include a carrier.

In one aspect, the carrier may be sawdust or rice husk.

In one embodiment, the organochlorine pesticide is aldrin, endrin, dieldrin, toxaphene, chlordane, heptachlor, mirex, hexachlorobenzene (HCB), dichloro diphenyl trichloro ethane (DDT), α-hexachlorocyclohexane (α-HCH), β-HCH, pentachlorobenzene (PeCB) and may be at least one selected from the group consisting of endosulfan.

The microorganism composition according to the present invention has excellent decomposition ability of organochlorine-based pesticides, and can be used to effectively restore soil contaminated with organochlorine-based pesticides.

1 shows the change and degradation rate of high concentration endosulfan with time according to the microbial treatment of the present invention, (a) is a Phanerochaete chrysosporium Y-2 strain, (b) is a treatment result of a Trichosporon loubieri Y1-A strain, (c) is Decomposition rates of these microorganisms over time are shown.
Figure 2 shows the time-dependent change and decomposition rate of high concentration endosulfan according to the microbial treatment of the present invention, (a) is Streptomyces sp. MJM14731 strain, (b) Streptomyces sp. MJM14747 strain, (c) Streptomyces sp. MJM14850 strain, (d) shows the degradation rate over time of these strains.
Figure 3 shows the time-dependent change of low-concentration endosulfan according to the microbial treatment of the present invention, (a) is Phanerochaete chrysosporium Y2 strain, (b) Streptomyces sp. It is the result of treatment of strain MJM14747.
Figure 4 shows Phanerochaete chrysosporium Y2: Streptomyces sp. It shows the change and degradation rate of high concentration endosulfan with time according to the complex microbial treatment of the MJM14747 strain, (a) is a 30:70 blend, (b) is a 50:50 blend, (c) is a 70:30 blend treatment result, ( d) shows the decomposition rate over time of single microorganisms and complex microorganisms.
5 is a photograph showing the change of state with the lapse of the storage period in the conditions of sawdust or soil of each microorganism.
6 is a graph showing changes in biomass with the lapse of a storage period in the conditions of a culture solution (liquid phase) or sawdust of each microorganism.
7 shows the composition and microbial treatment process of the control group and the experimental group in the field soil test.
8 shows the soil particle size distribution ratio before starting the in situ soil test.
Figure 9 shows the time-dependent endosulfan change of the control in the field soil test.
Figure 10 shows the time-dependent endosulfan change of complex microorganisms in the field soil test.
11 shows the decomposition rate (removal rate) of endosulfan by time of the complex microorganism and the control in the field soil test.
12 shows the change in soil pH in an in situ soil test.
13 shows the soil after the test of the control group and the microbial treatment group in the field soil test.

In the present invention, 'microorganism' includes 'strain' or 'strain' unless otherwise specified.

In the present invention, 'single microorganism' or 'single microorganism' means a microorganism consisting of only one strain.

In the present invention, 'composite microorganism' refers to a microbial group consisting of two or more strains. Complex microorganisms include a mixture of a culture solution of a single microorganism or a culture of two or more strains.

In the present invention, 'fungi' may mean ' Phanerochaete chrysosporium ' unless otherwise specified, and in particular, ' Phanerochaete ' It may mean chrysosporium Y-2'.

In the present invention, 'actinomycetes' refers to ' Streptomyces ' unless otherwise specified. sp.', in particular ' Streptomyces sp. MJM14747'.

The present inventors studied various microorganisms to select microorganisms that can effectively decompose and remove organochlorine pesticides, and as a result, the fungus Panerokite Chrysosporium ( Phanerochaete chrysosporium ) microorganisms, Streptomyces sp. microorganisms, which are actinomycetes, confirmed that they have excellent decomposition ability of organic chlorine-based pesticides, and were manufactured in the form of a complex microorganism most suitable for endosulfan decomposition efficiency, and were produced in the field for 100 days. Through the empirical test, it was confirmed that the microorganism according to the present invention can be effectively used to restore endosulfan-contaminated soil.

The present invention is Panerokite Chrysosporium ( Phanerochaete ) chrysosporium ) microorganisms and Streptomyces genus ( Streptomyces ) sp.) provides a microorganism composition for decomposing organic chlorine-based pesticides, including one or more microorganisms selected from the group consisting of microorganisms.

The Panerokite Chrysosporium ( Phanerochaete ) chrysosporium ) microorganisms are known as white molds, including but not limited to Panerokite Chrysosporium Y-2 strain can be used. The Panerokite chrysosporium Y-2 strain was 2005.12.19. It has been deposited at the Korean Culture Center of Microorganisms (KCCM) with the deposit number KCCM12440P.

The Streptomyces genus sp.) microorganisms are known as actinomycetes, but are not limited to Streptomyces genus MJM14747, Streptomyces genus MJM14731, or Streptomyces genus MJM14850 strains may be used. Streptomyces genus MJM14731, Streptomyces genus MJM14747, Streptomyces genus MJM14850 strains can be purchased from Myongji University Agro-Life Biofood and Pharmaceutical Material Development Project Group, and the Streptomyces genus MJM14747 strain is available on November 13, 2018. It has been deposited with the Korean Agricultural Culture Collection (KACC) with the deposit number KACC81078BP.

The microorganisms according to the present invention may be cultured in a medium. As the culture medium, a natural medium or a synthetic medium may be used. The culture medium may include a carbon source, a nitrogen source, and inorganic salts.

The carbon source of the culture medium is not limited, but for example, glucose and sugar, fructose, maltose, lactose, dextrin, dextrose, starch ( It may be a known carbon source in the field of microbial culture, such as sugars such as starch), organic acids such as malic acid and citric acid, and fatty acids with low molecular weight.

The nitrogen source of the culture medium is not limited, but for example, peptone, meat extract, yeast extract, dried yeast, casein, whey protein, soybean, ammonium salt, nitrate and other organic or inorganic nitrogen, sulfur It may be a nitrogen source known in the field of microbial culture, such as containing compounds.

Inorganic salts of the culture medium are not limited, for example, magnesium (Mg). Manganese (Mn), Calcium (Ca). Iron (Fe), potassium (K), sodium (Na), phosphorus (P), sulfur (S), boron (B), molybdenum (Mo), copper (Cu), cobalt (Co), zinc (Zn), etc. As such, it may be an inorganic salt known in the field of microbial culture.

The culture medium may further include growth factors, if necessary, in addition to the carbon source, nitrogen source and components of inorganic salts. The growth factor may be an amino acid, a vitamin, a nucleic acid, or a compound related thereto.

The microorganisms according to the present invention are not limited, but may be cultured in a temperature range of 20°C to 40°C, preferably in a culture temperature range of 25°C to 35°C.

The microorganism according to the present invention is not limited, but may be cultured for 12 hours to 14 days, preferably 12 hours to 7 days.

The composition for soil restoration of the present invention may include single microorganisms or complex microorganisms. The complex microorganism of the present invention may be prepared by mixing a culture medium obtained by culturing each strain individually, or may be prepared by culturing two or more strains together.

The concentration of microorganisms in the radiation shielding composition of the present invention is not limited, but 0.5×10 ² CFU/ml or more, preferably 0.5×10 ³ CFU/ml or more, more preferably 0.5×10 ⁴ CFU/ml or more , more preferably 0.5×10 ⁵ CFU/ml or more.

The composition for soil restoration of the present invention may be a culture medium of microorganisms in one embodiment, a microorganism concentrated or purified from the culture medium, and may be in a dried or semi-dried form if necessary.

To the composition for soil restoration of the present invention, a carrier may be added if necessary. The carrier is not limited, but a polymer resin such as sawdust, rice husk, activated carbon, zeolite, ceramic, or polyurethane may be used.

An object of the present invention is to restore contaminated soil to maintain the original function of cultivating crops, so it is preferable that the carrier be a naturally degradable carrier. The biodegradable carrier is not limited, but sawdust and rice husk are preferred.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Phanerochaete chrysosporium Y -2 culture of strains

The fungus Phanerochaete The chrysosporium Y-2 strain was inoculated in PDB medium and cultured at 25° C./130 rpm for 24 hours. After completion of the culture, the cells were separated by centrifugation at 3000rpm/5min, the mycelia were homogenized to homogenize, and the cells were harvested by centrifugation again. The harvested cells were used for subsequent experiments with wet weight.

Example 2: Trichosporon loubieri Culture of Y1-A strain

The yeast Trichosporon loubieri Y1-A strain was inoculated in YPD medium and cultured for 24 hours at 25°C/130rpm conditions. After completion of the culture, the cells were separated by centrifugation at 3000rpm/5min, the mycelia were homogenized to homogenize, and the cells were harvested by centrifugation again. The harvested cells were used for subsequent experiments with wet weight.

Example 3: Streptomyces sp . MJM14731 culture of strains

The actinomycetes Streptomyces sp. MJM14731 strain was inoculated in BN (Barnnett's medium) medium and cultured for 7 days at 30°C/130rpm conditions. After completion of the culture, the cells were separated by centrifugation at 3000rpm/5min, the mycelia were homogenized to homogenize, and the cells were harvested by centrifugation again. The harvested cells were used for subsequent experiments with wet weight.

Example 4: Streptomyces sp . MJM14747 culture of strains

The actinomycetes Streptomyces sp. MJM14747 strain was inoculated in BN (Barnnett's medium) medium and cultured at 30° C./130 rpm for 7 days. After completion of the culture, the cells were separated by centrifugation at 3000rpm/5min, the mycelia were homogenized to homogenize, and the cells were harvested by centrifugation again. The harvested cells were used for subsequent experiments with wet weight.

Example 5: Streptomyces sp . MJM17850 culture of strains

The actinomycetes Streptomyces sp. MJM14850 strain was inoculated in BN (Barnnett's medium) medium and cultured for 7 days at 30°C/130rpm conditions. After completion of the culture, the cells were separated by centrifugation at 3000rpm/5min, the mycelia were homogenized to homogenize, and the cells were harvested by centrifugation again. The harvested cells were used for subsequent experiments with wet weight.

Example 6: High Concentration endosulfan Microbial treatment experiments in soil (single microorganisms)

(1) Soil preparation

As the experimental soil, loess for landscaping was purchased and used. After sieving through a 425μm (No. 40) mesh sieve before the experiment, the remaining microorganisms and spores in the soil were completely killed through intermittent sterilization by repeating the process of sterilization at 121℃/30min and cooling (12 hours) three times. Thereafter, it was used in the experiment after drying for 24 hours in a dryer at 60°C.

(2) Endosulfan reagent

Endosulfan was purchased from Wako Pure Chemical Industries (Osaka, Japan) and used.

(3) Culture conditions of the soil inoculated with microorganisms

As shown in the treatment conditions in Table 1 below, 172uL of endosulfan was added and homogenized based on 10g of soil so that the final concentration was 100μg/g. Each microorganism was inoculated into endosulfan-contaminated soil and cultured at 25°C, and the sample on day 0 was used as a control. Sample collection was carried out 4 times for a total of 20 days in units of 1 g of 5 days, and 5 mL of a sterile aqueous solution prepared with 0.8% commercial fertilizers F1 (actinomycetes) and F3 (yeast, mold) and 2% glucose once every 10 days during the process was added to control the supply of nutrients and humidity.

Microbial and endosulfan treatment conditions in soil soil Endosulfan (5.83mg/mL) Cell culture supernatant sterile water P. chrysosporium Y-2 10g 172uL 0.2g 1mL 5mL T. loubieri Y1-A 10g 172uL 0.2g 1mL 5mL Streptomyces sp. MJM14731 10g 172uL 0.2g 1mL 5mL Streptomyces sp. MJM14747 10g 172uL 0.2g 1mL 5mL Streptomyces sp. MJM14750 10g 172uL 0.2g 1mL 5mL

(4) Endosulfan analysis (GC analysis)

For the analysis of endosulfan in soil, Restek's QuEChERS kit (Pennsylvania, USA) was used, and samples were collected according to the AOAC 2007 method, and then gas chromatography (GC, Agilent Technologies, Santa Clara, USA) was used for the following GC-FID. It was analyzed under the instrument conditions in Table 2.

Items instrumental conditions Column DB-5MSD Agilent Tech. 30 m × 0.25 mm, 0.25 μM carrier gas He (1 mL/min) Injection vol. 1 μL Injection Mode split Inlet temp. 250 ℃ Detector temp. 300 ℃ Oven temp. Stage Rate (℃/min) Temp. (℃) Hold time (min) Initial 0 140 0 Ramp 1 8 180 One Ramp 2 4 250 16.5

(5) Experimental results

The results of the high-concentration endosulfan (100 μg/g) resolution experiment for a total of 20 days are shown in Tables 3 to 7 and FIGS. 1 and 2 below.

Figure 1 shows the change and degradation rate with time of high concentration endosulfan according to the microbial treatment of the present invention, (a) is Phanerochaete chrysosporium Y-2 strain, (b) is the treatment result of Trichosporon loubieri Y1-A strain, (c) shows the degradation rate of these microorganisms over time.

Figure 2 shows the time-dependent change and decomposition rate of high concentration endosulfan according to the microbial treatment of the present invention, (a) is Streptomyces sp. MJM14731 strain, (b) Streptomyces sp. MJM14747 strain, (c) Streptomyces sp. MJM14850 strain, (d) shows the degradation rate over time of these strains.

Phanerochaete High-concentration endosulfan degradation of chrysosporium Y-2 strain Ether Alpha Diol Beta Sulfate Total
(ng/ uL ) Endosulfan degradation rate ( % ) 0 days 1.1 135.0 0.1 35.1 0.048 171.3 - 5 days 1.0 68.1 0.3 15.2 0.120 84.8 50.5% 10 days 0.4 54.4 2.7 12.0 0.183 69.6 59.4% 15th 1.0 42.3 0.1 11.0 1.128 55.5 67.6% 20 days 0.7 33 1.8 9.0 2.800 47.3 72. 4 %

Trichosporon High-concentration endosulfan degradation of loubieri Y1-A strain Ether Alpha Diol Beta Sulfate Total
(ng/ uL ) Endosulfan degradation rate ( % ) 0 days 1.1 135.0 0.1 35.1 0.048 171.3 - 5 days 1.1 134.0 0.4 35.0 0.102 170.6 0.4% 10 days 0.3 130.6 2.8 33.0 0.118 166.9 2.6% 15th 2.3 100.8 0.1 32.0 0.091 135.4 21.0% 20 days 0.7 95.0 1.8 31.0 2.800 131.3 23.4%

Streptomyces sp. High-concentration endosulfan degradation of strain MJM14731 Ether Alpha Diol Beta Sulfate Total
(ng/ uL ) Endosulfan degradation rate ( % ) 0 days 1.1 135.0 0.1 35.1 0.048 171.3 - 5 days 0.4 78.3 0.1 27.9 0.051 106.7 37.7% 10 days 0.1 46.6 1.0 25.7 0.091 73.4 57.1% 15th 0.3 30.3 0.1 19.1 0.812 50.6 70.4% 20 days 0.2 22.7 0.7 17.2 1.220 42.0 75.5%

Streptomyces sp. High-concentration endosulfan resolution of strain MJM14747 Ether Alpha Diol Beta Sulfate Total
(ng/ uL ) Endosulfan degradation rate ( % ) 0 days 1.1 135.0 0.1 35.1 0.048 171.3 - 5 days 0.5 59.4 0.1 25.4 0.048 85.4 50.1% 10 days 0.2 35.0 0.9 24.7 0.091 60.8 64.5% 15th 0.2 18.6 0.3 16.6 0.520 36.2 78.9% 20 days 0.1 13.5 0.5 15.0 0.504 29.7 82.7%

Streptomyces sp. High-concentration endosulfan resolution of strain MJM14850 Ether Alpha Diol Beta Sulfate Total
(ng/ uL ) Endosulfan degradation rate ( % ) 0 days 1.1 135.0 0.1 35.1 0.048 171.3 - 5 days 0.3 95.1 0.1 29.6 0.000 125.1 27.0% 10 days 0.1 80.7 1.2 31.3 0.097 113.5 33.8% 15th 0.4 58.1 0.5 24.3 0.115 83.4 51.3% 20 days 0.2 35.6 1.0 21.0 0.118 57.9 66.2%

As shown in FIG. 1 and Tables 1 and 2, the results of the high concentration endosulfan (100 μg/g) resolution test for a total of 20 days, Phanerochaete chrysosporium Y-2 showed a final degradation rate of 72.4%, whereas the Trichosporo n loubieri Y1-A strain showed a degradation rate of 23.4%. On the other hand, as shown in FIG. 2 and Tables 5 to 7, Streptomyces sp. High-concentration endosulfan (100μg/g) resolution test results for 3 species, Streptomyces sp. The MJM14747 strain showed the final degradation rate of 82.7% and was the best, followed by Streptomyces sp. The MJM14731 strain had a degradation rate of 75.5%, Streptomyces sp. MJM14850 strain showed a degradation rate of 66.5%.

Among the components of endosulfan, alpha, which had the highest concentration, showed a sharp slope during the experiment, and decomposition proceeded, whereas for components below 40ng/uL, the decomposition curve including beta was gentle or insignificant. It was confirmed that the cause of the increase or decrease of ether, diol, and sulfate content during the decomposition process was the degradation product of alpha.

After 15 to 20 days of the experiment, the decomposition rate was 60 to 80%, and most of the alpha, the main substance, was decomposed. In the case of beta, the decrease in the decomposition rate seems relatively small because the concentration was low from the start, but the decomposition will continue over time. was judged to be

Example 7: Low Concentration endosulfan Microbial treatment experiments in soil (single microorganisms)

Phanerochaete showing excellent endosulfan degradation in Example 6 chrysosporium Y-2 and Streptomyces sp. A low-concentration endosulfan degradation test was performed with the MJM14747 strain.

The experiment was conducted in the same manner as in Example 6, but the input amount was adjusted so that the final concentration of endosulfan in the soil was low (20 μg/g), and the results are shown in Tables 8 and 9, and FIG. 3 below.

Figure 3 shows the time-dependent change of low-concentration endosulfan according to the microbial treatment of the present invention, (a) is Phanerochaete chrysosporium Y2 strain, (b) Streptomyces sp. It is the result of treatment of strain MJM14747.

Phanerochaete Low-concentration endosulfan degradation of chrysosporium Y-2 strain Ether Alpha Diol Beta Sulfate Total
(ng/ uL ) Endosulfan degradation rate ( % ) 0 days 1.5 10.6 0.3 10.3 0.05 22.7 - 10 days 1.0 6.9 0.8 10.5 0.14 19.3 15.0% 20 days 1.1 4.9 1.0 10.2 0.10 17.2 24.1% 30 days 0.7 5.0 0.5 7.4 0.08 13.7 39.8% 40 days 0.5 3.3 0.3 6.0 0.07 10.3 54.8%

Streptomyces sp. Low-concentration endosulfan degradation of strain MJM14747 Ether Alpha Diol Beta Sulfate Total
(ng/ uL ) Endosulfan degradation rate ( % ) 0 days 1.5 10.6 0.3 10.3 0.050 22.7 - 10 days 1.2 8.3 0.8 10.2 0.158 20.7 8.9% 20 days 1.1 7.6 0.9 10.0 0.101 19.7 13.1% 30 days 0.8 5.3 0.4 7.3 0.046 13.8 39.1% 40 days 0.7 5.3 0.3 7.2 0.040 13.5 40.3%

In the high concentration (100 μg / g) experiment of Example 6 described above, Streptomyces sp. The strain showed a higher endosulfan degradation rate than the Phanerochaete chrysosporium Y2 strain, but as shown in Tables 8 and 9 above, the Phanerochaete chrysosporium Y2 strain showed a degradation rate of 54.8%, while the Streptomyces sp. MJM14747 strain was 40.3%. This is a result contrary to the high-concentration endosulfan (100 μg/g) experiment of Example 6 described above, and these results were obtained from Streptomyces sp. The strain Phanerochaete chrysosporium It is estimated that the resistance to the toxicity of endosulfan at a high concentration of 100 μg/g is stronger than that of Phanerochaete . chrysosporium It was judged that the decomposition rate was accelerated by

Example 8: Low Concentration endosulfan Microbial treatment experiments in soil (complex microorganisms)

Phanerochaete chrysosporium Y2 strain, Streptomyces sp. The endosulfan degradation ability of complex microorganisms compared to single microorganisms of the MJM14747 strain was tested.

It was carried out in the same manner as in Example 7, but the microorganisms were mixed at a wet weight ratio of 30:70, 50:50, and 70:30 to prepare and experiment a complex microorganism, and the results are shown in Tables 10 to 12 and FIG. 4 below. indicated.

Figure 4 Phanerochaete chrysosporium Y2: Streptomyces sp. It shows the change and degradation rate of high concentration endosulfan with time according to the complex microbial treatment of the MJM14747 strain, (a) is a 30:70 blend, (b) is a 50:50 blend, (c) is a 70:30 blend treatment result, ( d) shows the decomposition rate over time of single microorganisms and complex microorganisms.

Low-concentration endosulfan degradation of complex microorganisms ( P. chrysosporium : S. sp.=30:70) P. chrysosporium : S. sp .(14747) = 30:70 ( wet weight ) Ether Alpha Diol Beta Sulfate Total (ng/uL) Degradation rate ( % ) 0 days 1.5 10.6 0.3 10.3 0.050 22.7 - 10 days 1.2 8.4 1.0 10.3 0.137 21.1 7.1% 20 days 1.1 8.1 0.5 9.9 0.089 19.6 13.7% 30 days 1.0 6.0 0.5 8.5 0.049 16.0 29.5% 40 days 0.6 3.6 0.2 6.7 0.057 11.3 50.3%

Low-concentration endosulfan degradation of complex microorganisms ( P. chrysosporium : S. sp. = 50:50) P. chrysosporium : S. sp .(14747) = 30:70 ( wet weight ) Ether Alpha Diol Beta Sulfate Total (ng/uL) Degradation rate ( % ) 0 days 1.5 10.6 0.3 10.3 0.050 22.7 - 10 days 1.3 7.2 1.2 10.1 0.124 19.9 12.4% 20 days 1.2 5.9 0.4 10.2 0.081 17.7 21.8% 30 days 1.1 5.5 0.5 9.2 0.105 16.4 27.9% 40 days 0.4 2.0 0.2 4.9 0.158 7.7 66.3%

Low-concentration endosulfan degradation of complex microorganisms ( P. chrysosporium : S. sp.=70:30) P. chrysosporium : S. sp .(14747) = 30:70 ( wet weight ) Ether Alpha Diol Beta Sulfate Total (ng/uL) Degradation rate ( % ) 0 days 1.5 10.6 0.3 10.3 0.050 22.7 - 10 days 1.1 8.3 1.2 10.0 0.140 20.8 8.6% 20 days 1.2 5.3 0.4 9.7 0.081 16.7 26.4% 30 days 1.0 5.2 0.5 8.8 0.110 15.7 28.9% 40 days 0.4 1.7 0.2 4.2 0.075 6.6 70. 9%

As shown in Tables 10 to 12, Figure 4 above, P. chrysosporium : S. sp. As a result of experimenting with endosulfan degradation by mixing strains in a certain ratio, P. chrysosporium : S. sp. = 70: 30 showed the best resolution under the experimental condition. In particular, the alpha and beta degradation rates were improved by about 17 to 18% compared to when the microorganisms were treated alone. In the high-concentration endosulfan experiment, it was difficult to confirm the change in the slope of the degradation graph over time because the beta concentration was too low compared to the alpha. In particular, it was confirmed that the decomposition rate significantly increased after 30 days.

Example 8: Endosulfan decomposing microorganisms carrier and storage stability

Phanerochaete chrysosporium Y-2: Streptomyces sp. Carrierization and storage stability were tested for single microorganisms of MJM14747 and complex microorganisms mixed in a ratio of 7:3 on a wet weight basis.

The liquid culture medium was tested to observe the change in the biomass of microorganisms according to the storage period, sawdust was used as a microbial carrier to be treated in the field, and soil (loess) was used to visually confirm the settlement of the mixed microorganisms.

Sawdust and loess were autoclaved for 121°C/30min and then left for 24 hours, followed by intermittent sterilization by repeating the sterilization process three times, and finally dried at 65°C in a dryer for 24 hours before use in the experiment.

The three experimental groups of liquid, ocher and sawdust of microorganisms were cultured for a total of 45 days at 30° C. and 60% RH, and sampled at 15-day intervals to check the biomass and spore numbers of microorganisms in Tables 14 to 16, FIGS. 5 and 6 shows. In the case of the culture medium, biomass (dry weight) was measured, and in the case of a soil sample, it was difficult to accurately observe the spores due to soil particles, so it could not be calculated.

5 is a photograph showing the change of state according to the lapse of the storage period in the conditions of sawdust or soil of each microorganism.

6 is a graph showing changes in biomass according to the lapse of a storage period in the conditions of a culture solution (liquid phase) or sawdust of each microorganism.

Treatment conditions for complex microorganisms Phanerochaete chrysosporium Y2 Streptomyces sp 14747 P : S = 7:3 ( wet weight )
P : S = 5.6 : 4.4 ( dry weight ) liquid measure
(mL) wet weight
(mg) dry weight
(mg) liquid measure
(mL) wet weight
(mg) dry weight
(mg) liquid measure
(mL) wet weight
(mg) dry weight
(mg) culture medium 2.0 65.4 4.2 2.0 45.0 2.5 P: 1.23
S: 0.77 P: 40.2
S: 17.3 P: 2.58
S: 1.92 Sawdust (0.5g) 0.6 19.6 1.3 0.6 27.0 1.5 P: 0.37S: 0.23 P: 12.1
S: 5.2 P: 0.77
S: 0.58 Loess (1.0g) 0.6 19.6 1.3 0.6 27.0 1.5 P: 0.37S: 0.23 P: 12.1
S: 5.2 P: 0.77
S: 0.58

(P: Phanerochaete chrysosporium Y2, S: Streptomyces sp. MJM 14747)

Biomass (dry weight) and number of spores of microorganisms according to carrier type and storage period Phanerochaete chrysosporium Y2 0 days 15th 30 days 45 days of culture biomass
(mg/100mL) 210
(100%) 315
(150%) 219
(104.3%) 166
(79.0%) Sawdust (spore/g) 0 8.0×10 ⁶ 6.5×10 ⁶ 5.5×10 ⁶

Biomass (dry weight) and number of spores of microorganisms according to carrier type and storage period Streptomyces sp . MJM 14747 0 days 15th 30 days 45 days of culture biomass
(mg/100mL) 125
(100%) 67
(53.6%) 43
(34.4%) 39
(31.2%) Sawdust (spore/g) 0 4.0×10 ⁶ 3.5×10 ⁶ 1.0×10 ⁶

Biomass (dry weight) and number of spores of complex microorganisms according to carrier type and storage period P : S = 7:3 ( wet weight )
P : S = 5.6 : 4.4 ( dry weight ) 0 days 15th 30 days 45 days of culture biomass
(mg/100mL) 129(P)
+ 96(S)
=225 (100%) 196
(87.1%) 127
(56.4%) 118
(52.4%) Sawdust (spore/g) 0 5.3×10 ⁶ (P)
1.0×10 ⁶ (S) 5.0×10 ⁶ (P)
2.0×10 ⁶ (S) 5.3×10 ⁶ (P)
1.5×10 ⁶ (S)

(P: Phanerochaete chrysosporium Y-2, S: Streptomyces sp. MJM 14747)

As shown in Figure 6 (a), Tables 14 to 16, the result of confirming the biomass change according to the storage period of the liquid complex microorganism Phanerochaete In the case of chrysosporium Y-2, the biomass increased up to 150% until the 15th day of culture, then slowly decreased to 104.3% at the 30th day and 79% at the 45th day, and was relatively stable. But Streptomyces sp. decreased to a level of 53.6% on the 15th day, and showed a moderate decrease rate after 30 days to 34.4% and 31.2% on the 30th and 45th days, respectively. The storage stability of these complex microorganisms decreased to 87.1% on the 15th day, and decreased to 56.4% and 52.4% on the 30th and 45th days, respectively. However, as it was confirmed that the stability of the fungus among complex microorganisms was large, the reason for the mild decrease after 30 days was judged to be the effect of the fungal biomass, and the actual actinomycete biomass was expected to be reduced not significantly different from the storage alone.

6 (b), as shown in Tables 14 to 16, in the case of mold, spores could not be identified immediately after culturing, 8.0×10 ⁶ spore/g on the 15th day, and 6.5×10 ⁶ spore/g on the 30th day g and the 45th day, the decrease was very low with 5.5×10 ⁶ spore/g. Actinomycetes also did not detect spores immediately after culturing, and decreased to 4.0×10 ⁶ spore/g on the 15th day, 3.5×10 ⁶ spore/g on the 30th day, and 1.0×10 ⁶ spore/g on the 45th day. The change in the number of spores when mold and actinomycetes were mixed at 7:3 was 5.3×10 ⁶ spore/g on the 15th day, 5.0×10 ⁶ spore/g on the 30th day, and 5.3×10 on the 45th day. It was maintained at an almost constant level at ⁶ spore/g. Under the same conditions, the change in the number of Actinomycetes spores was 1.0×10 ⁶ spore/g on the 15th day, 2.0×10 ⁶ spore/g on the 30th day, and 1.5×10 ⁶ spore/g on the 45th day. there was no The reason for the difference in the number of spores when mold and actinomycetes are inoculated on sawdust alone is 7:3 and when the two microorganisms are mixed is because the absolute amounts are different, 7 for mold and 3 for actinomycetes based on 10 alone inoculation. was judged to be

In terms of storage stability of mold and actinomycete spores, it was confirmed that the experimental group to which a complex microorganism was applied rather than a single inoculation to sawdust as a single or complex group also had superior spore stability. The endosulfan decomposition performance was also excellent in the complex microorganism, so it was judged to be an ideal result.

On the other hand, the definition of microbial preparations (fertilizers) is “fertilizers manufactured using living microorganisms as the main raw material”, and the form is largely divided into three types: liquid, powder, and granular. The number of guaranteed microorganisms is defined as 1.0×10 ⁵ ~ 1.0×10 ⁶ CFU/mL or g or more for bacteria and yeast, and 1.0×10 ⁵ spore form unit/mL or more for fungi. Therefore, the mold and actinomycetes used in this experiment are all 10 ⁶ spore form unit/g or more, so they satisfy the regulations more than 10 times.

The validity period is differentiated according to the type of microbial preparation because there is a difference in the stability of the number of viable microorganisms. For liquid microorganisms, it is 6 months from the date of manufacture, and for powdery and granular microorganisms is 12 months from the date of manufacture. It was judged that the microbial stability could be maintained in the mid- to long-term if the complex microbial prototype was refrigerated or stored in a cool place after being supported by sawdust.

Example 9: Field Soil Experiment

(1) In situ soil test design

The field soil test was conducted by requesting field soil in a plastic house located in Hyosan-ri, Dogok-myeon, Hwasun-gun, Jeollanam-do.

The size of the test cloth used for the soil test is a control group and two experimental groups, each with an area of 3m×3m (9m ² ) and plowed to a depth of 30cm, and then treated with endosulfan emulsion. The bulk density of the test soil was used as data to calculate the concentration of endosulfan to be treated at 1.05 g/cm ³ . After diluting 150mL of 35% emulsion in 20L water to the control and experimental groups, each was homogenized while spraying the soil of the experimental area, and the process of mixing the soil was repeated several times for good homogenization. The concentration of endosulfan in the soil before tanning was found to be 0.4 ppm.

(2) Complex microbial carrier

Phanerochaete cultured in the same manner as in Examples 1 and 4 chrysosporium Y-2, S. sp. 12 kg of sterilized sawdust was uniformly mixed in the complex microbial cell prepared by centrifugation to support the MJM14747 microorganisms in sawdust. The filtrate other than the microorganisms absorbed in the sawdust was removed and left at a temperature of 25-28° C. for 4 weeks to induce natural drying and spore formation of microorganisms. The amount of sawdust used in this example corresponds to about 2.2% when calculated as the volume ratio of the soil treated in the field soil test (3 m × 3 m × 0.3 m).

(3) treatment of complex microorganisms

The composition of the control group and the experimental group and the microbial treatment process are shown in FIG. 7 . In the control group, 12 kg of sterilized sawdust was mixed, and in the experimental group, 12 kg of sawdust loaded with complex microorganisms were mixed. The process of mixing thoroughly with a small tiller was repeated so that the soil, sawdust, and emulsion could be uniformly mixed. After the sawdust mixing operation, the soil was spread flat again, and 50 L of water was additionally sprayed thereon to have an appropriate moisture content for microbial growth.

(4) Test cell management

After inoculation with complex microorganisms, the soil of the control and experimental groups was overturned to a depth of 30 cm every 30 days to evenly distribute the distribution of microorganisms inside and outside the soil, and at the same time, aeration was given to help the growth of microorganisms. Except for the summer rainy season during the experiment period, July-October, water evaporation proceeded rapidly on sunny days, so about 50 L of water was sprayed every 2-3 days, and the moisture within 20 cm from the soil surface was 70-80 % was maintained. The temperature of the soil during the experiment period was at least 28°C, at maximum 53°C, and on average 37°C. On days when there was a lot of sunlight, both sides of the greenhouse were opened to ventilate, and a shade film was installed at the top to prevent rapid temperature rise.

(4) In situ soil analysis before experiment (soil pH and particle size)

5 g of soil was placed in a 50 mL beaker, 25 mL of distilled water was added, and left for 1 hour while stirring with a glass rod, and then measured with a pH meter. Soil particle size is KS A 5101 standard mesh No. 4, No. 8, No. 16, No. 30, No. 50, No. 100 was used and the respective reference dimensions were 4.75 mm, 2.36 mm, 1.18 mm, 600 μm, 300 μm, and 150 μm. A certain amount of dried soil sample was weighed and sieved sequentially starting with the largest size, and the distribution ratio for each soil particle size was calculated as the ratio of each weight to the total weight.

The soil particle size distribution ratio before the start of the field test is shown in FIG. 8 . Fine sand, silt, and clay with a particle size of 0.15 mm or less accounted for 41%, followed by medium sand and coarse sand with a particle size of 0.15-1.18 mm at 48% and very coarse sand over 2.0 mm The corresponding part accounted for 11%. The composition of such soil particles was judged to be close to that of sandy soil. Sandy soil has a high ratio of quartz and sand, so it has low water holding capacity, but it has good permeability, so minerals are easily ^leached , and it ^has good air permeability. The pH of the soil was 6.83, showing weak acidity, which was also suitable for the growth of actinomycetes and fungi.

(5) Endosulfan analysis

Soil samples were mixed by collecting about 100 g each at a depth of 10 cm from 5 points using a sampler. Then, about 500 g of it was dried in the shade, filtered through a 2 mm sieve, and used as an analysis sample.

For the analysis of endosulfan in soil, Restek's QuEChERS kit (Pennsylvania, USA) was used, and according to the AOAC 2007 method ^{16 )} , magnesium sulfate 20 g, sodium chloride 5 g, magnesium sulfate 20 g, sodium chloride 5 g and After adding 5 g of sodium citrate, 150 mL of acetone was added, and extraction was performed twice for 2 hours. After filtration, the acetone extract was concentrated under reduced pressure at 40°C, and 4 mL of acetonitrile was added to dissolve it. Take 1.5 mL of this, put it in a 2 mL QuEChERS dSPE tube containing 150 mg of magnesium sulfte and 50 mg of primary secondary amine (PSA), shake it for 5 minutes using a Vortex mixer, and then centrifuge (3000 rpm/10 min). , The supernatant was filtered with a syringe filter (0.22 μm) and analyzed using gas chromatography (GC, Agilent Technologies, Santa Clara, USA) GC-FID, and the results are shown in FIGS. 9 to 11 .

Figure 9 shows the time-dependent endosulfan change of the control in the field soil test.

Figure 10 shows the time-dependent endosulfan change of complex microorganisms in the field soil test.

11 shows the decomposition rate (removal rate) of endosulfan by time of the complex microorganism and the control in the field soil test.

The removal rate of endosulfan in the control group was 54.4% during a total of 100 days of experimentation. Among the two isomers, α-endosulfan showed a removal rate of 50.9% at 60 days, and β-endosulfan showed a removal rate of 22.9%. In general, it is reported that the average half-life of endosulfan in soil is 50 days, α-endosulfan has a half-life of 35 days, and β-endosulfan has a half-life of 150 days. From the results of this field experiment, the half-life of α-endosulfan of the control was estimated to be about 60 days and β-endosulfan was estimated to be around 150 days, and it was confirmed that the decomposition rate was relatively slower than the reported average half-life, which was already in the control soil. It was judged to be the effect of endosulfan decomposition by soil microorganisms settled in On the other hand, in the case of the experimental group treated with the complex microorganisms, the removal rate of 89.1% on the 100th day was almost twice as high as that of the control group. On the 60th day of the experiment, α-endosulfan showed a very high removal rate of 95.5%, and β-endosulfan was also as high as 73.1%. The expected half-lives of the two isomers were estimated to be 20 days and 50 days, respectively, and it was confirmed that the decomposition rate was about 3 times faster than that of the control not treated with the complex microorganism.

It is known that the toxicity of endosulfan sulfate generated during the decomposition of endosulfan is greater than that of α-endosulfan, and the rate of decomposition is also very slow. In this field test result, the concentration of endosulfan sulfate increased from 30 days elapsed in both the control and experimental groups, but the degradation rate was insignificant until 100 days elapsed in the control group. After 60 days, the decomposition rate continued to increase, and at the time of 100 days, it was confirmed that about 48% was removed compared to the maximum concentration. On the other hand, endosulfan ether, which has been reported to have little toxicity, decreased very slowly over time in the control group, and was decomposed by about 30% at the 100th day. In the experimental group, endosulfan ether showed a faster decrease than the control group, and the removal rate was 71% at the 100th day. As other endosulfan degradation products, endosulfan diol was insignificant at a very low concentration, and endosulfan lactone was not detected.

The change in soil pH during the experimental period is shown in FIG. 12 . The pH of the untreated soil before the start of the experiment was 6.83, which was slightly acidic, but after treatment with the endosulfan emulsion and complex microorganisms, the pH of the control and experimental groups slightly increased to 7.04 and 7.09, respectively. Both the control group and the experimental group maintained the pH level of 6.9 until day 15-60, but after 60 days, the control group maintained a similar level until day 100, whereas the pH of the experimental group gradually decreased to 6.75 on day 90 and 6.72 on day 100 before the start of the experiment. The pH of the soil decreased slightly. The reason that the pH of the experimental group soil decreased compared to the pre-experimental soil was due to the complex microorganisms P. chrysosporium Y-2 and S . sp MJM14747 is judged to be due to the organic acid produced while proliferating after settling in the soil as shown in FIG. 13, and both strains were reported to have good growth in acidic conditions of pH 5-6.

Korea Microorganism Conservation Center (Overseas) KCCM12440P 20051219 Agricultural Biotechnology Research Institute KACC81078 20181113

Claims (8)

Phanerochaete chrysosporium ( Phanerochaete chrysosporium ) Microorganisms and Streptomyces sp. Microorganisms for decomposing organic chlorine-based pesticides comprising one or more microorganisms selected from the group consisting of microorganisms as an active ingredient. According to claim 1,
The Panerokite Chrysosporium microbes are Panerokite Chrysosporium Y-2 microorganism, a microorganism preparation for decomposing organic chlorine-based pesticides. According to claim 1,
The Streptomyces genus sp.) The microorganism is one or more microorganisms selected from the group consisting of Streptomyces genus MJM14731, Streptomyces genus MJM14747, Streptomyces genus MJM14850 microorganisms, microbial preparations for decomposing organic chlorine-based pesticides. According to claim 1,
The microorganism is Panerokite A microorganism preparation for decomposing organic chlorine-based pesticides , which is a complex microorganism of Chrysosporium Y-2 microorganism and Streptomyces genus MJM14747 . According to claim 1,
The microorganism is Panerokite A microorganism preparation for decomposing organic chlorine-based pesticides , which is a complex microorganism of Chrysosporium Y-2 microorganism and Streptomyces genus MJM14731 . According to claim 1,
The microbial composition further comprises a carrier, microbial preparation for decomposing organic chlorine-based pesticides. 5. The method of claim 4,
The carrier is sawdust or rice husk, a microorganism preparation for decomposing organic chlorine-based pesticides. The organic chlorine-based pesticides are aldrin, endrin, dieldrin, toxaphene, chlordane, heptachlor, mirex, hexachlorobenzene (HCB), dichloro diphenyl trichloro ethane (DDT), α-hexachlorocyclohexane (α-HCH), β-HCH, pentachlorobenzene (PeCB) and At least one selected from the group consisting of endosulfan, a microorganism preparation for decomposing organic chlorine-based pesticides.

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