KR100440864B1 - Bacterial preparations comprising new Acinetobacter strain, and biological purification method of oil-spoiled environment using them - Google Patents

Abstract

The present invention relates to a microbial preparation comprising the new strain IN-24 strain of the genus Acinetobacter as an active ingredient and a method for biological purification of an oil contaminated environment using the same.

Description

Bacterial preparations comprising new Acinetobacter strain, and biological purification method of oil-spoiled environment using them}

The present invention relates to a microbial preparation comprising the new strain IN-24 strain of the genus Acinetobacter as an active ingredient and a method for biological purification of an oil contaminated environment using the same.

The mass production and exposure of natural compounds derived from petroleum compounds has threatened the destruction of ecosystems and human health due to their toxicity and carcinogenicity. Among the methods used to restore the environment contaminated by oil, such as petroleum, the biological purification method is economical because it does not require additional equipment installation compared to the physical purification method, and it is added compared to the chemical purification method that distributes chemicals in large quantities. It is attracting attention because it is less likely to cause pollution and is environmentally friendly.

The biological purification method uses microorganisms capable of breaking down oil, and the strains used for environmental purification are separated and mass-cultured based on the growth ability and activity in the field application along with the excellent decomposition ability for oil. Directly or formulated into a contaminated environment. The method using microorganisms accelerates the rate of biological purification in a short time period by accelerating the rate of biological purification in the case of contamination with oil that is difficult to decompose naturally, in an environment in which there is a small amount of organisms capable of decomposing oil, or when a large amount of oil spills. This is a useful technique for getting

Therefore, in the past, strains having oil-degrading ability have been isolated, and preparations using them have been developed. However, due to technical perfection or economical problems, they are not often applied in actual field.

Microorganisms separated from the laboratory are often inadequate in real environment, and when introduced into a contaminated environment, microorganisms are difficult to dominate and compete with existing microbial communities. In addition, most microbial preparations tend to have low oil degradation efficiency in high concentrations of oil contaminated sites, unlike the experimental conditions in the laboratory, because the nutritional substances or oxygen necessary for the growth of microorganisms are not properly supplied. When using a combination of microorganisms of several genera to complement the oil degradation ability, the microbiological ecology between the different microorganisms may not be complementary to each other, the degradation of each degradation ability may occur. On the other hand, rarely well-established assays for side effects that can occur when microorganisms or agents are applied to the real environment, namely, toxicity to other organisms and the surrounding environment, are often relying solely on the oil-degrading capacity found in the laboratory. Can cause additional pollution and disrupt ecosystem balance.

Therefore, the invention of the microbial agent which is not only excellent in oil decomposition ability, adaptability and activity in the field, and does not affect the ecosystem and its use remains to be sought.

In the present invention, in order to achieve the above object is to isolate the microorganisms having excellent oil decomposition ability, the production capacity and activity is maximized at the time of field injection, and to prepare a microbial preparation that does not adversely affect other organisms and the surrounding environment, and this as oil It is intended to provide a method that can be practically applied to contaminated environments.

The present invention relates to a microbial agent comprising the strain IN-24 of the genus Acinetobacter as an active ingredient, and a method for biological purification of an oil contaminated environment using the same.

The IN-24 strain of the present invention is obtained as a mutant strain of the IN-2 strain of the genus Acinetobacter.

IN-2 strains were nominated by the All Union Scientific Research Institute of Genetics and Selection of Commercial Microorganisms. A microorganism belonging to the genus Acinetobacter, deposited under the number ВКПМ B-5971, registered in Russian patent 2064017. The IN-2 strain has excellent ability to biochemically oxidize petroleum and is not toxic to surrounding organisms and the environment, and has been usefully prepared as a microbial agent for treating in an oil contaminated environment.

In order to further maximize the excellent environmental purification effect of the IN-2 strain, research has been continued to find a variant strain derived from the IN-2 strain exhibiting superior efficacy than the original strain. The IN-24 strain of the present invention is the strain finally selected from among the variant strains of the IN-2 strain through this study, the separation method is as follows.

A microbial formulation consisting of 65% dried nutrient cells of IN-2, 20% diammonium phosphate, 5% sodium chloride and 10% moisture is diluted to an appropriate concentration and treated in oil-contaminated soil. Several kinds of mutant strains derived from the IN-2 strain exist in the suspension obtained by partially extracting soil in which oil decomposition is actively occurring and washing with distilled water. The suspension is inoculated in beef extract agar (beaf-extract agar) medium and the microbial colonies obtained by culturing are transferred to a liquid medium containing oil, respectively. The obtained culture broth was again plated on a beef extract agar medium, and strains showing the best characteristics in terms of the ability to biochemically oxidize oil, cell division ability and thermal stability were selected. The isolated IN-24 strain was deposited on July 25, 2001 to the Russian Collection of Microorganisms (VKM) under the Budapest Treaty under the number VKM-V2266D.

The IN-24 strain of the present invention is a rod-type Gram-negative bacterium, which can grow at a temperature of 4-50 ° C. and a wide range of pH 6.0-8.0, and the optimum growth condition is ( 30 ± 1) ° C, pH 7.0 ± 0.2. The colony is grayish white round and has a slightly convex shape as a whole. Biochemical features include aerobic bacteria, negative for oxidase, positive for catalase, and weak for proteolytic enzymes. Glucose, ribose, xylose and arabinose may be used as the carbon source, and ammonium may be grown as the nitrogen source.

IN-24 strain of the present invention has better oil degradation rate and cell division ability than the original strain IN-2 strain. Therefore, the microbial preparations containing the IN-24 strain of the present invention as an active ingredient are used in the oil-contaminated environment, that is, the coastal and factory sites contaminated with oil, storage facilities and oil pipelines, gas stations and oil storage tanks. It can be usefully used to clean the environment, such as near restaurants to use.

The microbial preparation of the present invention consists of 55 to 67% dried feeder cells of IN-24 strain, 18 to 30% diammonium phosphate, 5% sodium chloride and 10% moisture. Sodium chloride, which acts as a preservative, does not lose its activity even during long-term preservation, and diammonium phosphate, widely used as a fertilizer, provides conditions such as nutrients and pH at concentrations that can maximize the degradation activity of microorganisms when applied to actual sites. .

The microbial preparation of the present invention contains at least 1 × 10 ⁸ live bacteria per gram, and the biochemical oxidation capacity for oil is at least 75%. Optimum conditions when using the microbial preparation of the present invention are (24 ± 5) ° C, pH 6.0-8.0. Since the microbial agent of the present invention is not toxic to other organisms and the surrounding environment, it can be used for environmental purification without additional pollution while balancing the ecosystem. The microbial agent of the present invention can be used for reservoirs contaminated with oil, seawater, soil, plant areas and storage rooms.

When the microbial preparation of the present invention is used in practice, the preparation is diluted in a diluent for melting the preparation in which mineral fertilizer is dissolved in water so as to be 0.03 to 0.05%. The amount of the agent to be diluted may be about 3 to 15 kg / ㏊ depending on the degree of contamination and the type of the agent to be diluted. After diluting the agent, the product is ventilated for about 20 to 30 minutes to sufficiently supply oxygen for the activity of the microorganism. If continuous ventilation is not possible, ventilate at intervals of 1.5 to 2.5 hours. After that, the activation process must be performed at 23 to 32 ° C. for 4 to 6 hours, and when activated at a lower temperature, 16 to 20 hours are required.

When treating the activated preparation dilution, it is possible to maximize the biological environment restoration effect by treating the fertilizer diluent such as nitric acid or phosphoric acid together. Fertilizers are used to stimulate biocommunity and to formulate the optimum conditions for the preparation of the preparation. The fertilizer is dissolved in water to a concentration of 0.07 to 0.1%. After the preparation and fertilizer treatment, the soil is changed and allowed to be well ventilated to further increase the rate of purification. The fertilizer diluent is then added at a concentration of 0.5 to 1.0 ℓ / m2 once every 10 to 15 days, so that the conditions such as nutrients, pH, dissolved oxygen, etc., which can activate the oil-degrading ability of microorganisms, are optimally maintained. The efficiency can be maximized.

After about two weeks of treatment of the microbial preparation of the present invention, the oil shows signs of decomposition, and in the case of soil, it turns brown after 7 to 10 days, bubbles are formed, and the viscosity of the soil changes. This is an indicator that the biochemical oxidation of petroleum is active. If no such signs of degradation appear, the microbial formulation composition is treated again. In areas that are constantly contaminated with petroleum, microbial formulations are processed once a month.

The treatment target, technology, usage and precautions according to the characteristics of each environment are as follows.

Treatment target Treatment technology Formulation Dosage Fertilizer Usage Precautions One Oil-contaminated reservoir Spray a dilution of the formulation onto the water using a fire truck 2.0-5.5㎏ / ㏊ 2-3㎏ / ㏊ Petroleum bed 10 mm 2 Oil-Contaminated Swamp The treatment method is like a reservoir 2.0-10㎏ / ㏊ 2-3㎏ / ㏊ 3 Oil-polluted land If the soil depth is less than 15 cm, it is better to change the ground. Spray formulation dilution at 0.5-1.5 L / m ^2. If contaminated soil depth is more than 15 cm, remove contaminated soil and transfer to cleanup site. The formulation is processed every 7-10 days 3.0-15㎏ / ㏊ 3-5 kg / ㏊ Temperature 10 ℃ or above, Oil concentration 10% or below 4 Oil contaminated sewage from industrial enterprises It can be accelerated by ventilation and after 5-7 days cleanup is complete. 1.5-2.0 g / kg 0.002-0.003% Sewage temperature 16-42 ℃ 5 Sewage from oil refinery Purification of the reservoir tank is carried out in the same way as for natural reservoirs. Purification of the flowing water is sprayed with an appropriate amount of the formulation into the sewage reservoir tank and left at 12-16 hours at 16-42 ° C. 2.0-5.5㎏ / ㏊ 2.0-3.0㎏ / ㏊ 6 Containers with oil and petroleum products Rinse the walls of the container with the preparation diluent, add an appropriate amount and leave until the contaminants are completely decomposed. It usually takes 3-7 days. 1.5-2.0 g / kg 0.5-0.7㎏ / ㎥

Hereinafter, a method of isolating the strain of the present invention and preparing a microbial agent composition and using it for environmental purification will be described in detail, but the present invention is not limited thereto.

Example 1 Isolation of IN-24 Strain

In order to isolate the strains with the best oil resolution and cell division among the mutant strains of the IN-2 strain of the genus Acinetobacter, 65% of dried nutrient cells of IN-2, 20% of diammonium phosphate, 5% of sodium chloride and moisture The microbial formulation consisting of 10% was diluted to a concentration of 0.03% and treated with oil contaminated soil. A part of the soil in which oil decomposition was actively occurring was collected, and 10 g of the soil was washed with 90 ml of distilled water for 10 minutes. 1 ml of the suspension thus obtained was inoculated in beef extract agar medium and incubated at 30.0 ± 2 ° C. for 48 hours to separate microbial colonies. After each colony was transferred to a liquid medium containing oil and incubated for 72 hours, the obtained culture solution was plated on a beef extract agar medium to obtain an isolated microbial colony.

After inoculation, all colonies were transferred to a medium, and then strains were selected that exhibited excellent properties such as the ability to biochemically oxidize oil, cell division ability, and thermal stability. Again, the screening was repeated to find the strain with the best activity and named this strain IN-24. IN-24 strain of the present invention was deposited on July 25, 2001 as the number VKM-V2266D to the Russian Collection of Microorganisms (VKM), an international depository institution under the Budapest Treaty.

Example 2 Measurement of Final Cell Production of IN-24 Strain

In order to confirm that the IN-24 strain has a high cell division capacity to cover the number of cells required for the preparation of microbial preparations, the cell number and dry biomass by culturing the IN-24 strain and IN-2 strain of the present invention Was measured. First, 200 ml of the culture solution of each cell shaken at 28 rpm at 300 rpm was inoculated into 15 L fermentation medium, and fermented under continuous stirring at a temperature of 28 to 30 ° C. and a pressure of 0.5 ati. The fermentation medium contained 1.1 to 1.8% of the total reducing substance to measure the biochemical oxidation ability of the bacteria. Near the end of the fermentation, the number of cells produced by each strain was measured. The microorganisms which finished fermentation were dried by powder to determine the dry biomass. The cell numbers and dry biomass of each strain are shown in Table 2.

Strain Cell count / ml Dry biomass IN-2 4.5 × 10 ⁹ 65 5.6 × 10 ⁹ 76 6.8 × 10 ⁹ 82 IN-24 3.1 × 10 ¹⁰ 98 1.5 × 10 ¹⁰ 91 2.0 × 10 ¹⁰ 96

As shown in Table 2, the IN-24 strain had a much higher cell number after culturing than the IN-2 strain, and the dry biomass was also high. Taken together, the IN-24 strain increases the final cell production by 50% compared to the IN-2 strain.

Example 3 Checking Oil Degradation Ability

In order to confirm that the oil-degrading ability of the IN-24 strain was superior to that of the derived strain IN-2, the IN-24 strain and the IN-2 strain were respectively subjected to a temperature of (29.0 ± 1) ° C in a beef extract agar medium. Incubate for 24 hours. The grown bacteria were washed with physiological saline and the number of cells was measured so that 1 × 10 ⁷ cells per ml were present. 1 ml of the cell solution, 1 ml of 7% diammonium phosphate, and 0.5 ml of diesel oil were added to 100 ml of distilled water, and cultured for 120 hours at a temperature of (30.0 ± 2) ° C. At certain times, the culture was passed through a separatory funnel and left until phase separation. The procedure was repeated until the liquid became clear, and then the extract was slightly warmed to remove chloroform. The dried extract thus obtained was further dissolved by adding 100 to 200 µl of chloroform. Among them, 1-2 µl was injected into a chromatograph with a syringe. The analysis was performed in a temperature range of 80 ° C to 300 ° C under conditions of increasing 6 ° C per minute, and the results were shown in Table 3 by calculating the degree of petroleum oxidation.

Strain Diesel oil decomposition rate (%) 36 hours 48 hours 72 hours 96 hours IN-2 18 39 58 84 IN-24 34 52 71 92

As shown in Table 3, IN-24 was much superior to the diesel oil decomposition rate compared to its derived strain IN-2.

Example 4 Application of IN-24 Strain to Oil Contaminated Soil

In order to actually apply the IN-24 strain to the oil-contaminated environment and to confirm the result, the IN-24 strain of the present invention and the IN-2 strain derived therefrom were treated to 0.8 kPa of oil-contaminated soil, respectively. Diammonium phosphate was treated together at a concentration of 0.5% to increase the efficiency in the treatment. Quantitative analysis of the difference in the amount of oil extracted from the soil at certain time periods, and the results are shown in Table 4.

Strain Oil degradation rate in soil (%) 30 days 60 days IN-2 44.12 77.89 IN-24 47.5 94.21

As shown in Table 4, IN-24 was superior to the oil-purified soil purification effect compared to its derived strain IN-2.

Example 5 Preparation of Microbial Formulation Using IN-24 Strain as an Active Ingredient

A microbial formulation composition containing the IN-24 strain as an active ingredient was prepared according to the composition ratio of dried feeder cells 65%, diammonium phosphate 20%, sodium chloride 5%, and water 10% of the IN-24 strain.

Example 6 Toxicity Test of IN-24 Strains and Microbial Agents Containing IN-24 Strains as Active Ingredients

To assess the adverse effects of IN-24 strains and microbial preparations comprising IN-24 strains as active ingredients, toxicity to other organisms and the surrounding environment was examined.

In lethality experiments on warm-blooded animals, the median lethal dose (LD ₅₀ ) measured by intraperitoneal injection of bacteria and agents into white rats was greater than 10 ⁹ bacterial cells. The microbial preparation contains about 10 ⁸ live bacteria and is safe for warm-blooded animals such as rats.

To determine the effect of the IN-24 strain on the body organs of warm-blooded animals, the strain was injected into the blood and visceral organs of rats. After 30 days of observation, they did not metastasize to other organs and showed very low mortality, indicating little toxicity. Additives included in the formulation showed little toxicity such as carcinogenicity and body accumulation.

The IN-24 strain and the microbial preparations containing the IN-24 strain as active ingredients have not been shown to affect the natural microflora of the surrounding environment, i.e., the surrounding microbial community. Up to 2000 mg of sugar did not have an algicidal effect, plankton organisms up to 2000 mg per liter, 4.7 mg per liter, underwater beetles up to 50 mg per liter, sturgeon eggs up to 176.52 mg per liter, sturgeon fish up to 60.11 mg per liter The annual carp showed up to 50 mg / L resistance. Since the maximum allowable concentration when applying the microbial agent containing the IN-24 strain to the environment is 0.5 mg / l, it can be seen that the microbial agent composed of the IN-24 strain will be harmless to other organisms and the surrounding environment. .

Since the microbial agent comprising the IN-24 strain of the present invention as an active ingredient has a high oxidative activity against oil, there is an effect of efficiently purifying the oil-contaminated environment.

Since the microbial agent containing the IN-24 strain of the present invention as an active ingredient has little side effects on the surrounding ecosystem, it can be expected to effectively clean the environment without fear of additional pollution while maintaining the balance of the ecosystem.

Claims (6)

IN-24 strain of the genus Acinetobacter. The genus IN-24 strain according to claim 1, characterized in that Accession No. VKM-V2266D. The microorganism preparation according to claim 1 or 2, wherein the strain IN-24 of the genus Acinetobacter is an active ingredient. The microorganism for oil pollution environmental purification according to claim 3, comprising 55 to 67% of dried feeder cells of the strain IN-24 of the genus Acinetobacter, 18 to 30% of diammonium phosphate, 5% of sodium chloride, and 10% of moisture. Formulation. A method of purifying an oil contaminated environment using the microbial agent of claim 4. The method of claim 5, wherein the microbial agent is dissolved in 0.03 to 0.05% mineral fertilizer diluent and ventilated for 20 to 30 minutes, followed by an activation process at 23 to 32 ° C. for 4 to 6 hours to purify the oil contaminated environment. .