RU2041172C1 - Method for cleaning soil from oil and oil products - Google Patents

Abstract

FIELD: oil industry. SUBSTANCE: method involves introduction into soil of a bacterial culture mixed with mineral fertilizer. The bacterial culture is the strain Mycobacterium Sp-5 kV in the amount of 15·10⁶-15·10⁷ cells per 1 g of oil products. The bacterial culture is injected in a liquid state or on sterile organic substrate in the form of a bacterial preparation with a bacteria content of 10¹⁰-10¹¹ cells per 1 g (or 1 mm) of the preparation. If necessary, the soil is limed in advance to pH 6.0-6.5 and enriched with organic fertilizer. EFFECT: recultivation of oil-polluted soil. 3 cl, 1 dwg, 5 tbl

Description

 The invention relates to the oil industry, agriculture and the environment and can be used for biological reclamation of lands contaminated with oil and oil products as a result of accidents and operational losses during production, transportation and, most importantly, human activities. Oil and, practically, all oil products have a sharp depression of the functional activity of soil flora and fauna, thereby excluding the soil as an object of environmental management. Restoring the natural fertility of oil-contaminated soils requires significantly more time and effort than other technogenic pollution. Numerous research and development has been devoted to this problem; Among the latter, the most effective way is the artificial introduction of specially selected strains of microorganisms into the soil to be cleaned, capable of more or less actively oxidizing (assimilating) the oil itself or its processed products diesel fuel, kerosene, etc. Such microorganisms are introduced into the soil as part of other ingredients; strains of microorganisms, their number, composition of related ingredients, form of the drug, manufacturing technology in most cases belong to the category of know-how.

 Known bacterial preparation "Putidul", designed to clean oil-contaminated land and water bodies. The drug "Putidule", taken as a prototype, is made on the basis of the bacterial culture of the natural strain Pseudomonas putuda-36 [1, 2] The drug indicates its high biological effectiveness. However, it has a number of significant drawbacks.

1. Preparation "Putidoyl" is a dry bacterial mass and to its preliminary "revival" requires time-consuming set of activities performed in field conditions: heating of large quantities of water (5m ³⁾ to a temperature of 18-28 ^{° C} (in practice, up to 28 ^C. ), providing aeration for 12-16 hours, mixing.

 2. In addition, the low effectiveness of the bacterial drug Putidul in microbial self-cleaning processes has been shown. In addition, it has a depressing effect on the natural microbial biocenosis (see Novikov Yu.V. Komzolova N.V. "Study of the bacterial drug Putidul" intended for cleaning water bodies from oil. "Water resources 1992 N 2 p.121-123 )

 The aim of the invention is the creation of the bacterial drug "Naftox", devoid of the above disadvantages of the prototype and is able to actively oxidize oil and oil products, thus creating the prerequisites for the use of cleaned soils for economic purposes and to improve the ecological environment. The invention provides some parameters of practical methods of using the Naftox preparation.

 The goal is achieved by using Mycobacterium Sp-5kB strain, isolated from natural sources and characterized by a high degree of utilization of oil and oil products, for cleaning soils from oil pollution.

 The comparative rate of utilization of oil, diesel fuel and aviation kerosene by strains Ps.putida-36 and Mycobacterium Sp-5kB is given in table. 1.

 The genus Mycobacterium has several advantages compared with other microorganisms in the process of microbiological degradation of oil in natural conditions. Microorganisms of this kind, with high oxidative activity, have the ability to accumulate hydrocarbons in cells with simultaneous emulsification in the case of cultivation in aqueous media. This statement is probably also valid for media such as soil solution.

 Cultural and morphological features of the selected strain are as follows. Small, thick, slightly curved sticks with rounded edges, do not form spores and capsules. When cultivated on a rocking chair or in a fermenter, it forms cocciform elements. Dimensions 2.5 x 0.2 microns. Gram-positive (stained fuzzy). Aerobes. Colonies on the surface of the agar are orange-pink, dry, with uneven edges; the core of the colony is brighter. During deep seeding of agar media, surface colonies are pinkish in the shape of asterisks. Acid and alcohol resistant. Gelatin is not liquefied.

 Mycobacteria are ubiquitous in areas of oil production and refining. 13 types of mycobacteria capable of utilizing oil have been identified.

 Mycobacteria is the main microflora of activated sludge from wastewater treatment plants in oil refineries.

 Mycobacteria are able to assimilate not only normal alkanes, but also related components of oil: cyclic compounds phenol, benzoic acid, naphthalene, cyclohexane, higher fatty acids. It is extremely important that these substrates are often more preferred sources of carbon and energy than carbohydrates, such as glucose.

 Mycobacteria are not demanding, unlike pseudomonads or other genera of bacteria, to environmental conditions; they are found not only in the upper horizons of soils, but even at a depth of 25-550 m of clay deposits.

 The considered strain of mycobacteria has been tested by the appropriate medical service and approved for widespread use. The strain Mycobact.Sp-5 kB was deposited at the All-Russian Research Institute of Agricultural Microbiology under N 375 A.

 In the process of this development, studies were conducted on the "survival rate" of different genera and strains of bacteria in soils saturated with oil. It was found that in the majority of bacteria introduced into such soil (various types of pseudomonads, including Ps.putida-36), only about 10% remain in a viable state for no more than 30 days, while at least 50% of the number of introduced mycobacterial cells remained viable within 120 days.

 Oxidation of hydrocarbons in the soil by chemical or biological means can lead to the formation of phytotoxic products with all the negative consequences of the agronomic plan that follow from this. In other words: for biological objects, the initial oil substrates can be less toxic than the intermediate products of their oxidation.

 Comparison of the action of Ps. putida-36 and Mycobacterium-5 kB showed that during cultivation on 2% diesel fuel and kerosene (liquid, aerated cultures) toxic compounds that inhibit plant growth accumulate in the fermentation liquid, and the fermentation liquid of the proposed culture contains significantly less such toxins. than in the prototype culture (see data in Table 2).

 Thus, from the data given in table 2, the advantage of the proposed strain of mycobacteria compared with the culture of the prototype is obvious.

The proposed strain of mycobacteria can be used in the form of the bacterial drug "Naftox". The drug "Naftox" is obtained according to the well-known technological scheme adopted for the production of nitrogen-fixing bacteria preparations (Pozdnyakova A.I., Khotyanovich A.V. et al. Auth. St. N 922104, 1982 N 15). Neutralized to a pH of 6.8-7.0 gamma-sterile peat in plastic bags is inoculated with a liquid culture of oil-oxidizing bacteria with the simultaneous introduction of a carbon source and energy in this case, n-alkanes C ₁₅ -C ₂₀ in an amount of 1-2% by weight of dry peat mix and incubate at 18-20 ^about ; final humidity of Naftoks 55-60% Warranty period of shelf life of Naftoks is 6 months. One gram of the drug contains at least 10 ⁹ -10 ¹⁰ cells of 5KV mycobacteria.

 It is known that the main factor in the population of the soil with introduced microorganisms is the rate of their reproduction in this substrate. However, the survival (preservation) of this strain in soil or other similar substrates, as a rule, does not correlate with the rate of reproduction. Meanwhile, in the case of the microbiological method of cleaning the soil from oil pollution, it is important to maintain the viability of bacteria for the longest possible time.

The drawing shows a comparative characteristic of the considered strains introduced into oil-contaminated soil (15 kg of aviation kerosene in 1 m ^{3 of} soil).

 From the above data it follows that the strains under consideration clearly differ in survival in the soil for a long time: Pseudomonas putida 36 at the beginning more intensively accumulates cells, but their titer rapidly decreases. The decrease in the number of Mycobacterium cells is relatively slow. Undoubtedly, the “attackability” of oil products is determined not only by the specific enzymatic activity of a given strain of microorganisms, but also by their quantity. When developing the Naftox preparation, it was assumed that the effectiveness of the preparation is determined by the number of bacteria in this preparation and their ability to maintain a high and relatively stable titer in soils subjected to biological treatment.

 In addition, Mycobacterium Sp-5KB differs from Pseudomonas-Putida 36 strain, as well as from other strains of this genus, in its high technological effectiveness. The manufacturability of the strain provides a high and stable titer in the preparation and in the soil for a long time, which is fundamental in the industrial production and practical use of a biological product.

In order to determine the minimum amount of oil-oxidizing bacteria necessary for the almost complete oxidation of oil products in the soil, model experiments were performed using diesel fuel and aviation kerosene introduced into the soil, followed by inoculation of such soil with different amounts of oil-oxidizing bacteria. The results are presented in the summary table. 3. The duration of the experiment is 3 months; temperature 20-22 ^{° C;} soil moisture 60% of full moisture capacity; pH 6.5.

Thus, the data obtained show that for the almost complete decomposition of diesel fuel and aviation kerosene in the soil, 10 ⁵ -10 ⁷ cells per gram of soil are sufficient and necessary load of oil-oxidizing bacteria; moreover, with a hydrocarbon content of up to 10 kg / m ³ this value is not less than 10 ⁵ , up to 50 kg / m ³ not less than 10 ⁶ and from 50 to 100 kg / m ³ not less than 10 ⁷ introduced cells per gram of cleaned soil.

 In the practice of cleaning work, it is more rational to operate with the amount of the drug and the load of bacterial cells per unit weight (1 g) of oil products in contaminated soil (Table 4).

 A method of cleaning the soil from oil pollution with the help of bacterial preparations is carried out with the preliminary agrochemical characteristics of the reclaimed areas, including the analysis of the contents of digestible forms of nitrogen, phosphorus, potassium, organic matter (humus), pH, humidity. Based on the data obtained, the necessary mineral and organic fertilizers and lime are additionally added to the soil, and when there is a lack of moisture, watering is carried out. These agrochemical analyzes and measures should be carried out not only with the aim of creating optimal living conditions for autochthonous and introduced microflora, but also for the subsequent successful cultivation of crops in areas cleared of oil.

 Preparations in solid or liquid forms are introduced, as a rule, by pre-mixing them with a filler in fertile (garden) soil or compost. A large volume of the mass of the resulting mixture is more convenient to distribute using appropriate agricultural machines. The use of fertile land or compost as fillers promotes the initial growth of the bacteria of the drug, increasing the titer, which favorably affects the intensity of decomposition of oil and oil products in the soil.

PRI me R 1. On the soil surface, an area of 1 ha containing 100 kg / m ^{3 of} diesel fuel or aviation kerosene, using a manure spreader, spread 100 tons of compost mixture containing 300 kg of Naftox prepared on the basis of sterile peat and containing 10 ¹¹ bacterial cells in one gram. The mixture is prepared separately using mixing devices. The cultivated area is plowed at a depth of 25-30 cm. After one month, deep disking (25-35 cm) is carried out and, if necessary, the area is irrigated with such a quantity of water that the soil moisture is 60% of its full moisture capacity. After watering, after 3-5 days, carry out a reservation; if necessary, this operation is carried out 2-3 times per season, preventing the formation of a crust on the soil surface. In the event that soil cleaning is started in the spring, winter crops are sown in August-September; if the cleaning operation is started in the summer, then sowing is carried out next spring, preferably using fodder herbs (mixture). The degree of purification after carrying out these measures is 18.8 days 46.8%
PRI me R 2. 1 ha of the cultivated area containing 10 kg / m ³ aviation kerosene, sprayed 630 l of an aqueous suspension of the drug "Naftox". For this, 30 L of a liquid preparation containing 10 ¹⁰ cells / ml is pre-mixed with 600 m of water; the treated area is scented to a depth of 20-25 cm and then proceed as described in example 1. Utilization of oil hydrocarbons for the above period is 24%
PRI me R 3. In the soil contaminated with oil (10 kg / m ³ ) only mineral fertilizers in the amount of N ₈₀ P ₇₀ are applied and delivered as described in example 1. The degree of decomposition of petroleum hydrocarbons in 18 days amounted to 12% of the original quantity.

PRI me R 4. The soil contaminated with oil products moisten up to 60% of the full moisture capacity, as in examples 1, 2, 3. The degree of decomposition of oil products was only 3.5%
The results of the above examples are summarized in Table 5, from which it follows that the proposed Mycobacterium-5KB strain and the Naftox biological product developed on its basis are an effective means of accelerated cleaning of soils from oil and oil products, improving the ecological situation and returning lands to agricultural use.

 As a result of targeted microbiological studies, strains of microorganisms using oil and oil products as the sole carbon source were selected from samples taken in areas of long-term anthropogenic pollution by selection.

 Microorganism cultures underwent laboratory testing for their ability to oxidize oil and oil products, as a result of which the most active strain of mycobacteria of 5 KV was displayed. Based on this strain, a preparation technology was developed (commercial name "NAFTOKS") and pilot batches of this preparation in liquid form and on a solid organic carrier substrate were developed.

After a comprehensive laboratory test, the drug was tested in the field at the Ruchi oil depot, contaminated with light oil products and a mixture of oil wastes (SNO), in an amount of 17 to 65 kg / m ^{3 of} soil.

During the tests of the drug, the bacterial load on the soil being cleaned was constant (10 ⁷ cells / 1 g of soil); organic and mineral components introduced into the soil simultaneously with the NAFTOX preparation were variable.

 The test results showed that the introduction of a bacterial culture of 5KV accelerates the degradation of oil pollution. This process is enhanced by mineral and especially organic fertilizers (organic carrier substrate). For 48 days of cleaning, a decrease in the level of contamination by 63.5% was recorded, whereas in the control variant it was only 12%. Studies also showed that the strains used as a biological base in the Naftox preparation are completely non-pathogenic (conclusion of the microbiology laboratory VNITIAF from 05.20.91). Moreover, laboratory studies have shown that the use of the drug "Naftox" to some extent helps to increase the fertility of the cultivated land.

 Thus, the undoubted effectiveness of the bacterial preparation "Naftox" and the promise in using it to accelerate the biological treatment of soils and soils from oil pollution are shown.

Claims (3)

1. METHOD FOR CLEANING SOIL FROM OIL AND OIL PRODUCTS, including introducing a bacterial culture in a mixture with mineral fertilizer, characterized in that a natural strain of Mycobacterium VNIISCHM 375A is used as a bacterial culture in an amount of 15 · 10 ⁶ 15 · 10 ⁷ cells per 1 g of oil products. 2. The method according to claim 1, characterized in that the bacterial culture is applied to the soil in liquid form or on a sterile organic carrier substrate with a bacterial content of 10 ^{1 0} 10 ^{1 1} cells in 1 g (or 1 ml) of the preparation.  3. The method according to claim 1, characterized in that the soil is preliminarily calcified to a pH of 6.0 to 6.5 and organic fertilizers are applied.

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