KR20000034035A - Bioaugmentation of oil contaminated soil by microbial composition which can decompose hydrocarbons derived from petroleum - Google Patents

Abstract

PURPOSE: A microbial composition for bioaugmentation of oil contaminated soil is produced by optimum fermentation producing the combinations of hydrocarbon decomposing microorganism isolated from oil contaminated soil. Addition of substrates specifically metabolized by microorganism in the composition increases the viability of microorganisms in the contaminated soil and stabilizes and maximizes bioaugmentation. CONSTITUTION: Three hundred of microorganism isolated from oil contaminated soil are screened based on the degradation and emulsification of oil judged from the growth on the plate or liquid culture containing crude oil or gasoline. For the liquid culture test, minimal medium containing 3% of hydrocarbon is used. Acinetobacter sp. W1, Pseudomonas sp. HB26, and Bacillus sp. L11 are selected and cultivated. The culture medium comprises 17-23g/L of glucose, 2-4g/L of yeast extract, 3-4g/L of sodium phosphate, 0.7-1.0g/L of potassium phosphate, 2-4g/L of ammonium sulfate, and very small amount of magnesium sulfate. After the culture, 0.2-0.4% of NaCl, 0.2-0.4% of citrate, 0.15-0.25% of sodium benzoate, and very small amount of sodium phosphate(I, II) are added to preserve the microorganism composition.

Description

Biological Purification of Oil Polluted Soils Using Petroleum Hydrolyzed Microbial Agents

The present invention relates to a biological restoration method of oil-contaminated soil using strain combinations of Acinetobacter sp W1, Pseudomonas sp HB26, and Bacillus sp L11, which are powerful hydrocarbon degradation strains. The present invention relates to a biological purification method of oil contaminated soil using a combination of strains of E. sinobacter species W1, Pseudomonas species HB26 and Bacillus species L11, and a special substrate-containing formulating technique for enhancing adaptability and viability in oil-contaminated soils. .

Biological cleanup of oil-contaminated soils includes soils near reservoirs, soils near oil pipelines, and soils near underground storage tanks such as gas stations. In particular, contaminated soil around underground storage tanks has emerged as one of the most important treatment targets in the United States, and various biological purification methods have been tried. These biological purification methods have the advantages of being environmentally friendly, economical and safe compared to other physical and chemical methods.

Biological treatment of oil contaminated soil and groundwater includes in-situ biological soil purification and Ex situ biological soil purification. In-situ biological purification is a technique used in situations where it is difficult to excavate contaminated soil due to the presence of buildings or underground pipe networks around the contaminated soil, and includes bioventing and air sparging methods. These methods are basically technologies to restore the contaminated area by supplying oxygen and microbial nutrient sources as electron acceptors to activate oil-degrading microorganisms such as soil. On the other hand, Exitu biological purification is a method of excavating and treating the soil, there are methods such as landfilling, composting, biopiles, landfarming.

Landfarming technology is relatively economical over other methods unless it is a heavy burden to transport contaminated soil, but it is also difficult to perform at low temperatures. It is one of the most useful technologies as a means of biologically purifying the contaminated soil of gas station underground storage tanks, which is a problem in Korea recently. Soil cultivation techniques use liquid or solid mineral fertilizers, which aim to promote the growth and activity of petroleum hydrocarbon-degrading microorganisms in the soil. In recent years, attempts have been made to increase the speed of biological purification by adding microbial spawns separated and mass-cultured in laboratories, especially in the US, such as EBT. The bioaugmentation technique by the introduction of microorganisms is a soil cultivation technique. It is one of the important techniques to increase the usefulness of. In particular, biological purification by microbial input is required to perform soil restoration within a short period of time in the presence of low hydrocarbon decomposition strains or difficult to decompose oil contaminants in the contaminated soil, or in the presence of high concentration of pollutants. In order to be an essential skill.

Petroleum-based hydrocarbon-degrading strains that are separated in the laboratory for use in soil purification, along with their ability to decompose to contaminants, are resistant to various environmental conditions such as low or high temperatures, high concentrations of contaminants and heavy metals, And oil pollutants are searched and selected based on the secretion ability of biological surfactants to facilitate degradation.

Currently, some microorganism-related products for the biodegradation of oil-contaminated soils are developed and marketed in some advanced countries. However, due to the lack of technical perfection or economic feasibility, they are not adopted or applied in actual field. Hydrocarbon-decomposed microorganisms isolated from laboratories are less likely to grow high in contaminated soils or compete with indigenous microorganisms if they are less adaptable to the real environment, and consequently fail to accelerate the biodegradation of the contaminants. Because there are many.

An object of the present invention is to provide a microbial spawning agent for biological restoration of oil-contaminated soil by isolating a strong hydrocarbon degradation strain and constructing a combination showing the best performance among these strains to produce an optimum fermentation and formulation. In addition, the purpose of the present invention is to provide a technology for formulating by using the substrate as a nutritional substance specifically preferred by each microorganism to increase the field adaptability of the combination of microorganisms, as a result, to obtain a stable and maximized efficiency of biological purification.

1 is a graph showing the microbial state of untreated soil.

2 is a graph showing the microbial state analysis of soil to which 0.1% NH ₄ Cl and potassium phosphate buffer solution (10 mM) were added.

Figure 3 is a graph showing the microbiological analysis in contaminated soil inoculated with the microbial agent of the present invention.

4 is a graph showing the reduction rate of BTEX (benzene, toluene, ethylbenzene, xylene).

5 is a graph showing the reduction rate of TPH (total petroleum hydrocarbon).

The present invention relates to a method for the isolation, production, combination and formulation of microorganisms having good hydrocarbon degradability, and a method for preparing special nutrients to maximize the activity of these microorganisms and take advantage in contaminated soil environments, and formulated microorganisms. And biological restoration method of oil polluted soil using special nutritional substance

That is, the present invention relates to a method for biological purification of oil contaminated soil by soil cultivation using a microorganism prepared by combining microbial strains such as strain Esinetobacter W1, Pseudomonas HB26, and Bacillus L11.

At this time, 300 kinds of dominant species microorganisms collected from oil-contaminated soils are purely separated and cultured in solid and liquid medium including crude oil and diesel oil, and then strains having high decomposition rate and emulsifying activity for crude oil and diesel oil are selected, Esinetobacter, characterized in that the solid plate medium containing a variety of hydrocarbons, such as crude oil and diesel, is selected for the separation of strains having a large diameter of the transparent ring and a high growth rate and crude oil conversion rate in a liquid medium containing a minimum medium and 3% hydrocarbons. The present invention provides a method for separating microbial strains such as W1, Pseudomonas HB26, and Bacillus L11, and provides microbial strains such as Ecinetobacter W1, Pseudomonas HB26, and Bacillus L11 that are separated according to the above method.

In addition, in the composition of the medium for increasing the growth rate of the strain, glucose 17-23 g / L, yeast extract 2-4 g / L, sodium hydrogen phosphate 3-4 g / L, potassium dihydrogen phosphate 0.7-1.0 g / It provides a culture medium for strain culture, characterized in that it contains a trace component such as L, ammonium sulfate 2-4 g / L, traces of magnesium sulfate, the microorganism for long-term preservation after culturing the microorganism strain in the medium To provide a microbial agent prepared by mixing the strain 0.2 to 0.4% sodium chloride, 0.2 to 0.4% citric acid, 0.15 to 0.25% sodium benzoate and a small amount of potassium dihydrogen phosphate, potassium dihydrogen phosphate and the like.

Hereinafter, the present invention will be described in more detail.

1. Isolation and Screening of Petroleum Hydrolyzed Microorganisms

Over 300 kinds of dominant species microorganisms collected from 20 oil-contaminated soils in Korea were purely isolated and cultured in solid and liquid media of Table 1 including crude oil and diesel oil, respectively, and strains having high decomposition rate and emulsifying activity for crude oil and diesel oil. Was searched.

Badge composition Badge composition Liquid medium (g / L) Solid Medium (g / L) KH ₂ PO ₄ 1.5 Nutrient agar Na ₂ HPO ₄ 4.5 MgSO _{4 7} H ₂ O 0.1 NH ₄ Cl 0.3 Various hydrocarbons such as crude oil or diesel 10 10 Agar 15

The solid plate medium containing various hydrocarbons such as crude oil and diesel oil was selected to have a large transparent ring diameter and a high growth rate and a high crude oil conversion rate in a liquid medium containing 3% of hydrocarbons in the minimum medium. In order to measure the emulsifying activity of each strain, the culture medium of Table 2 was used for each strain cultured at 30 ℃ for 3 days.

Microbial Growth Medium for Emulsification Activity Badge composition Content (g / L) KH ₂ PO ₄ 1.5 Na ₂ HPO ₄ 4.5 MgSO _{4 7} H ₂ O 0.1 NH ₄ Cl 0.3 glucose 10

In addition, the emulsion activity was measured by adding 0.1 ml of the culture solution to a test tube containing 7 ml of Tris-HCl buffer (pH 7.2) and 0.1 ml of crude oil. After shaking for 1 hour at 25 ° C and 60 rpm, the absorbance was measured at OD 600 to measure 0.1 OD (optical). density) was 1 unit. The number of cells was measured and counted by diluting the culture medium and the soil sample to a final cell concentration of 10 ⁴ to 10 ⁷ CFU / ml, smearing 0.1ml each on the nutrient agar to form colonies between 40 and 400.

Growth Rate, Emulsification Activity and Conversion Rate of Hydrocarbon Degrading Strains Strain Growth rate (CFU / ml) Emulsifying Activity (unit / ml) Oil conversion rate (%) Growth on Soil Flats crude oil Via crude oil Via crude oil Via crude oil Via Wl 2.5 × 10 ⁸ 3.5 × 10 ⁸ 51.2 45.4 72% 67% + + Lll 2.2 × 10 ⁶ 1.0 × 10 ⁶ 250.4 201.6 45% 55% - - HB26 1.5 × 10 ⁵ 1.9 × 10 ⁸ 102.6 130.4 68% 70% - + HB62 2.1 × 10 ⁷ 1.8 × 10 ⁷ 30.4 25.2 58% 45% - - DUG 19 5.4 × 10 ⁸ 2.1 × 10 ⁸ 150.1 201.8 68% 67% - - RP6 3.0 × 10 ⁸ 1.5 × 10 ⁸ 120.2 130.2 65% 66% - -

* +: Transparent ring formation

* KH ₂ PO ₄ 1.5 g / L, Na ₂ HPO ₄ 4.5 g / L, MgSO ₄ 7H ₂ O 0.1 g / L

NH ₄ Cl 0.3 g / L, oil 3% at 30 ° C for 3 days

Cell growth, emulsification activity and oil conversion rate of purely isolated strains are shown in Table 3. The majority of bacteria did not grow well in medium containing 3% crude oil or diesel oil or diesel, while the selected strains showed good growth rates. In addition, the emulsification activity and oil conversion rate did not necessarily coincide with each other, and even though no clear ring was formed in the crude oil and the oil-containing solid medium, the bacteria having excellent oil degradability existed in the liquid medium. Of these, W1 and HB26, which are excellent in crude oil and diesel oil, and L11, which have excellent emulsifying activity, were selected. L11 showed low degradation rate for crude oil and diesel oil, but showed high resolution for vegetable hydrocarbons such as palm oil and edible oil, and showed good emulsification activity due to secretion of biological surfactant.

2. Identification of Strong Hydrocarbon Strains

Identification of the final screened strain was carried out using the BIOLOG, Inc. Automated Bacterial Identification System.

Three strains W1, HB26, and L11 were identified, and W1 was found to be of the genus Ecinetobacter. The strain was named as acinetobacter W1, and HB26 was identified as Pseudomonas genus, which was named Pseudomonas HB26. L11 was identified as Bacillus and named Bacillus L11.

3. Measurement of Growth Rate and Degradation Rate in Oil Polluted Soils Using a Hydrocarbon Degrading Strain Combination

(1) genetic labeling of microorganisms

In order to test the adaptability of Echinetobacter W1 and Pseudomonas HB26, which are powerful hydrocarbon-degrading strains, in real soils, two strains were genetically labeled to measure growth in soil. In the case of Ecinetobacter, 0.1 ml suspension was plated on nutrient agar and incubated for 2 to 3 hours at 30 ° C. Then, 0.2ml of the antibiotic solution prepared for each concentration was poured into the center and dried at 30 ° C for 24 hours. The colonies shown later were selected and used as mutants with resistance to the antibiotics.

Rifampicin solutions of 0.2,0.5 and 1.0 mg / ml of rifampicin solution were used to select antibiotic resistant strains of Ecinetobacter, and in the case of Pseudomonas, resistant strains were selected using rifampicin solution in the same manner. However, Pseudomonas is resistant to both rifampicin and nalidic acid by selecting resistant strains using 0.2, 0.5, 1.0 mg / ml of nalidixic acid solution in the same way as rifampicin. Mutants were made and used as genetic marker strains.

(2) Measurement of growth rate and decomposition rate of petroleum hydrocarbon in oil contaminated soil

Experiments on a laboratory scale were conducted to test the growth rate of the genetically labeled strains in oil contaminated soil and the resolution of petroleum-based total hydrocarbons. Transfer lightly contaminated soil samples (pollution concentration 1,500ppm) from the gas station to the test reactor (20cm × 15cm × 10cm) 2kg each and flip them twice a week while maintaining at room temperature so that the water content in the soil is 15-20%. Was done. The two genetically labeled strains were incubated at 30 ° C. in nutrient broth for 2 days, and then inoculated to 1 × 10 ⁶ CFU per gram of final soil. In addition, inorganic salts NH ₄ Cl (0.1% final) and potassium phosphate buffer (potassium phosphate buffer) was added to the contaminated soil to a final concentration of 10mM.

Total petroleum hydrocarbon (TPH) was quantified by adding anhydrous sodium sulfate solution to 30 g of soil, extracting for 16 hours from 100 m of dichloromethane / acetone 1: 1 solution, and analyzing the extract by GC / FID.

BTEX (benzene, toluene, ethylbenzene, xylene) was analyzed by GC / FID after supernatant of 15 g of soil was immersed in 10 ml of methanol.

Total bacteria were counted in nutrient plate medium, Esinetobacter W1 was added to 0.02mg / ml of Rifampicin, and 0.02mg / ml of Rifampicin and 0.02mg / ml of Nalidic Acid in Pseudomonas HB26. Count in media.

The analysis of total petroleum hydrocarbon decomposition strains was performed using KH ₂ PO ₄ 1.5 g / L, Na ₂ HPO ₄ 4.5 g / L MgSO ₄ 7H ₂ O 0.1 g / L, NH ₄ Cl 0.3 g / L, CaCl ₂ · 2H ₂ O 0.01 g / L, MnSO ₄ .2H ₂ O 0.01 g / L, diesel oil 20 g / ml, and agar 15 g / L were counted in a flat medium.

The pH was measured by mixing well with 1 (soil): 5 (distilled water) and standing for 5 minutes and measuring with a pH meter.

Water content was measured by putting 10 g of soil in an evaporating dish and evaporating at 120 deg. C for 4 hours in a desiccator.

4. Formulation of lycolytic microorganisms using the strain

(1) fermentation method

High concentration fermentation production medium of Ecinetobacter sp W1 and Pseudomonas sp HB26 Na ₂ HPO ₄ · 12H ₂ O 3.32 g KH ₂ PO ₄ 0.83 g (NH ₄ ) ₂ SO ₄ 3 g Yeast extract 3 g Glucose 20 g MgSO _{4 7} H ₂ O 0.4g Trace ingredients 2ml / L Trace ingredient composition (per 1 liter) H ₃ BO ₃ 0.3 g CoSO ₄ · 7H ₂ O 0.2 g ZnSO ₄ · 7H ₂ O 0.1g MnCl ₂ 4H ₂ O 0.03 g NaMoO ₄ 2H ₂ 0 0.03 g NiCl ₂ · 6H ₂ O 0.02 g CuSO ₄ · 5H ₂ O 0.01 g Na-citrate 5 g FeSO ₄ 7H ₂ O 20 g CaCl ₂ · 2H ₂ O 10 g

When fermentation was carried out at 30 ° C. for 48 hours while maintaining the pH of 7.0 in a 500 L fermenter, the composition of Table 4 was 5 × 10 ⁹ CFU / ml for Ecinetobacter W1 and 1 × 10 ¹⁰ CFU / ml for Pseudomonas HB26. High concentration cultures were possible.

(1) Liquidification and Preservation Methods of Fermented Petroleum Hydrocarbon Degrading Strains

The microorganisms fermented and produced in (1) were liquefied by the following formulated composition to prevent contamination, to commercialize and to preserve for a long time.

Liquid formulation additive Amount NaCl 0.3% Citric acid 0.3% Sodium benzoate 0.2% Spices 0.01% K ₂ HPO ₄ 0.107% KH ₂ PO ₄ 0.052% UV protectant 3ml / ton

The change in viable cell number with storage time is shown in Table 6.

As shown in Table 6, more than 80% viable cell count was maintained for up to 3 months, and no contamination by other microorganisms was observed.

Storage period Viable Count (CFU / ml) first 3.75 × 10 ⁹ 1 month 3.12 × 10 ⁹ 2 months 3.10 × 10 ⁹ 3 months 3.01 × 10 ⁹

5. Special formulation method using microbe-specific substrate

In order to increase the adaptability of the prepared oil-degrading microorganisms, by adding a specific carbon source, which is best used by each strain as a nutrient substance, together with the nutrient, it can be predominantly at a high density at the beginning of the inoculation. The efficiency of the solution could be increased.

Petroleum Hydrocarbon Degradation Promoting Nutrients Component (%: Oil Pollution Soil Standard) K ₂ HPO ₄ 0.0428% KH ₂ PO ₄ 0.0296% UREA 0.02% Ammonium Sulfate 0.0035% Ammonium Nitrate 0.019% Sodium hexaphosphate 0.0001%

The above composition is an inorganic salt added to promote the growth of microorganisms in the biological restoration of oil contaminated soil. The addition of lactic acid and tween80, which act as specific carbon sources for Pseudomonas HB26 and Ecinetobacter W1, increased the initial density of soil application.

Example 1 Preparation of Microbial Preparation

Next, inoculate the oil-degrading microbial agent to 1 × 10 ⁶ CFU / g in 1kg of oil-contaminated soil, and add the inorganic salt composition and lactic acid of Table 7 by the following concentrations, and then incubate at 30 ° C. for 24 hours. This is the result of measuring the viable cell count of microorganism.

As can be seen in Table 8, the number of viable bacteria of Pseudomonas HB26 increased significantly when the specific substrate lactic acid was added.

Growth Rate of Pseudomonas HB26 Viable Cells in Oil-Contaminated Soils by Lactic Acid Addition Lactate added amount Viable Count (CFU / ml) 0.1% 1.1 × 10 ⁹ 0.01% 5.3 × 10 ⁸ 0.001% 1.9 × 10 ⁸ No treatment 5.8 × 10 ⁷

Example 2 Preparation of Microbial Preparation

It measures in the same manner as in Example 1. As shown in Table 9, as the concentration of Tween 80 increases, the number of viable bacteria of Ecinetobacter W1 increases.

Growth Rate of E. cobacter sp W1 in Oil-Contaminated Soil by Tween 80 Addition Tween 80 addition amount Viable Count (CFU / ml) 0.1% 7.8 × 10 ⁸ 0.01% 5.9 × 10 ⁸ 0.001% 1.8 × 10 ⁸ No treatment 4.3 × 10 ⁷

Example 3 Field Application

1. Biological restoration by soil cultivation using the microbial agent

The microbial agent and the activation promoter were prepared to attempt the biological restoration of oil contaminated soil by landfarming method at the pollution site. Refined oil and kerosene using three microorganisms such as Ecinetobacter W1, Pseudomonas HB26, and Bacillus L11 by stacking soil at a height of 1m on the 1700m ² site. The main ingredient was applied to soil contaminated with 1900 ppm TPH.

Application Method 1. Site Construction

Sites for the treatment of contaminated soil are to be flattened and a leachate collection pipe is installed to collect the leachate by inclining to the central part of the entire site. Lay sand on top of it 20 cm high. In order to prevent the loss of the contaminated soil, the outer part of the site was formed with a non-polluted defense wall of more than 1m height and the inner wall was waterproofed with 0.5 to 3mm vinyl to prevent the outflow of soil and oil.

Application Method 2. Pretreatment of Polluted Soil

Before stacking the contaminated soil on the composition site, 100mm or more coarse stones mixed in the soil were separated using a 100mm sieve.

Application Method 3. Flipping Equipment

The equipment for flipping contaminated soil and mixing microorganisms was carried out using single plows, four plows and roundabouts loaded on the tractor. Single blade plow is a plow with one blade that can be turned over to a depth of 600 mm to 1000 mm deep. The four-blade plow is a four-blade plow that can be turned over to a depth of 400 mm to 600 mm in soil depth. The rotary used equipment to flip the soil and crush the soil location within 400mm of soil depth.

Application Method 4. Microorganism Input Equipment

Microorganisms and supplements were fed to contaminated soil using a sprayer self-assembled with an agricultural sprayer on a 5-horsepower engine.

Application Method 5. Microorganism Input Method for Contaminated Soil

The microorganisms were administered to add 1 × 10 ⁵ to 1 × 10 ⁷ CFU of oil-degrading microorganisms per gram of soil. The amount of microbial nutrient added was 1.2 kg per ton of soil. The timing and frequency of administration of microorganisms and supplements were injected three times at a weekly interval.

Microorganisms were introduced at the same time flipping using the flip equipment of the application method 3 above.

The microorganisms are fed and inverted evenly with half of the initial microorganisms and nutrients on the soil surface, and then flipped once with a four-day plow. After agitating and crushing the soil with a single blade plow, the remaining 1/2 of the microorganism and the auxiliary was added to each other and then crushed once for each four-day plow, rotary, four plow and rotary. The addition of the remaining secondary tertiary microorganisms and nutrients was carried out in the same manner.

Method of application 6. Inversion of soil

After the microorganisms were added, the soil flipping operation was repeated once every two days. The method of flipping was performed once in the order of rotary, four-day plow, rotary, one-day plow, rotary, four-day plow and rotary.

Application method 7. Soil pollution analysis and microorganism analysis

Analysis of TPH and BTEX concentrations of contaminated soils, analysis of microorganisms of contaminated soils, and analysis of pH and water content were carried out at the intervals of one week.

Application Result 1. Pollution concentration

Change in pollution concentration time BTEX (ppm) TPH (ppm) Early 30 1,500 7 days later 19 1,200 14 days later 0.001 or less 657 21 days later 0.001 or less 495 28 days later 0.001 or less below 10

Application Result 2. Microbial Change

Microbial changes time Total microbial count (CFU / g-soil) Oil Degradation Microbial Water (CFU / g-soil) Early 3.75 × 10 ⁵ 10 ⁴ or less 7 days later 2.00 × 10 ⁶ 7.50 × 10 ⁴ 14 days later 5.00 × 10 ⁷ 2.00 × 10 ⁶ 21 days later 1.30 × 10 ⁸ 7.90 × 10 ⁵ 28 days later 4.80 × 10 ⁶ 5.90 × 10 ⁵

Application Result 3. Change in moisture content

time Moisture content (%) Early 20.10 7 days later 19.86 14 days later 15.35 21 days later 14.71 28 days later 14.21

Application Result 4. Change of pH

pH change time pH Early 8.50 7 days later 8.44 14 days later 8.60 21 days later 8.56 28 days later 8.50

The effect of the present invention is to provide a microbial spawning agent for biological restoration of oil-contaminated soils by isolating strong hydrocarbon degradation strains, constructing a combination that shows the best performance among these strains, and optimally producing and formulating them. By providing a technology for formulating by using the preferred substrate as a nutritional substance, it is possible to increase the field adaptability of the microbial combination as a result, it is possible to obtain a stable and maximized efficiency of biological purification.

Claims (5)

Biological Purification of Oil Contaminated Soils by Soil Cultivation Using Microbial Agents Combining Microbial Strains Such as Echinetobacter W1, Pseudomonas HB26, and Bacillus L11 Over 300 kinds of dominant species microorganisms collected from oil-contaminated soils are purely isolated and cultured in solid and liquid mediums, including crude oil and diesel oil, and the strains with high decomposition rate and emulsifying activity for crude oil and diesel oil are selected and crude oil. Esinetobacter W1, characterized in that the solid plate medium containing a variety of hydrocarbons, including diesel and diesel, is selected for the separation of strains having a large diameter and a high growth rate and a high crude conversion rate in a liquid medium containing a minimum medium and 3% hydrocarbons. , Isolation method of microorganisms such as Pseudomonas HB26, Bacillus L11 Microorganism strains such as Ecinetobacter W1, Pseudomonas HB26, Bacillus L11 isolated according to the method of claim 2 In the composition of the medium for increasing the growth rate of the strain of claim 3, glucose 17 to 23 g / L, yeast extract 2 to 4 g / L, sodium hydrogen phosphate 3 to 4 g / L, potassium dihydrogen phosphate 0.7 to 1.0 g / L, medium for strain culture according to claim 3, characterized in that it contains trace components such as ammonium sulfate 2-4 g / L, traces of magnesium sulfate After culturing the microorganism strain of claim 3 in the medium of claim 4, 0.2 to 0.4% sodium chloride, 0.2 to 0.4% citric acid, 0.15 to 0.25% sodium benzoate and traces of potassium dihydrogen phosphate Microbial agent prepared by mixing potassium hydrogen