RU2302303C2 - Biological preparation for cleaning environments polluted by crude oil and petroleum products - Google Patents

Abstract

FIELD: environmental pollution elimination.

SUBSTANCE: biological preparation comprises consortium of bacterial cultures: Acidovorax delafieldii "VKPM V-8359 (NT-4)", Pseudomonas fluorescens biovar "VKPM V-8360 (NT-6)", Citobacter amalonaticus "VKPM V-8361 (Yan-3-2)", Pseudomonas fluorescens "VKPM V-8362 (Yan-8-2)", Serratia marcescens "VKPM V-8363 (Yan-10-2)" with titers of live cells at least 10¹⁰.

EFFECT: increased destructive ability regarding crude oil and petroleum products.

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Description

The invention relates to a means of combating pollution of environmental objects with oil and oil products and can be used, for example, in the elimination of oil pollution.

Since over 200 thousand km of oil trunk pipelines and 350 thousand km of oil production pipelines are operated in Russia, up to 40 thousand accidents occur annually on in-field oil pipelines. According to Greenpeace experts, emergency oil losses in Russia reach 25 million tons annually. In oil-contaminated soils, the activity of biota is suppressed, trophic bonds, physico-chemical and biological properties are broken.

Spilled oil is adsorbed by the soil and in the bulk is localized in its upper layer. Volatile oil fractions are removed from the surface due to evaporation, and heavy oils, resins and asphaltenes practically do not weather and slowly seep into the soil. In this regard, the task is to develop highly effective technologies for cleaning and restoring the environment contaminated with oil and oil products.

Modern oil-oxidizing preparations are, as a rule, freeze-dried biomass of active strains of microorganisms, mainly bacteria, in a mixture with nitrogen-phosphorus compounds. Sometimes a neutral sorbent is included in the preparation.

Bacteria of the genus Pseudomonas (Muracami A., Suzuki K., J. Oceanogr. Soc. Jap., 1985, v.41, No. 5, pp337-344; Balashova N.V., Kosheleva I. A., Filonov A.E. et al. Strain Pseudomonas putida BS 3701 - phenanthrene and naphthalene destructor. - Microbiology, 1997, v.66, No. 4, pp. 488-493). A mixed culture of microorganisms is also used, for example, Flavobacterium and Pseudomonas (Horowitz A., Atlas RM - Biodeterior. Proc. 4 ^th Int. Bioterior. Symp., Berlin, Londjn, - 1980, p.15-30). An analysis of the literature indicates the advisability of using groups of microorganisms for the disposal of oil and oil products. The American company "Polybac Corporation" produces drugs "Phenobac" and "Petrobac" (Stoff C.V. - Chemical proceeding, 1982, No. 12 (Dec.), p.11). The preparations contain mutant strains of bacteria. Also known is the preparation "Hydro Tank" (ibid.).

The closest analogue to the claimed biological product is a biological product of the company "Polyinform" (RF patent No. 2138451, C02F 3/34, 1997). Biological product includes aerobic oil-oxidizing bacteria, mineral additives and filler. As an aerobic oil-oxidizing bacteria, there is a consortium of mesophilic bacterial strains of Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus species PI Ku-1 and a consortium of psychrophilic bacterial strains Pseudomonas fluorescens PI LBh-Xon Pasomonas Pomonas Pomonas Pomonas Pomonidae Pomonidae Pomonidae PI LBH-7. As mineral additives use sources of nitrogen, phosphorus, potassium. Sterile peat is used as a filler. The disadvantage of a biological product is the complex (mesophilic and psychrophilic strains of bacteria) composition of microorganisms.

The main objective of the authors of the present invention was to obtain a biological product with a high destructive ability to utilize hard-to-oxidize fractions of oil and oil products, storage-stable, non-toxic, non-carcinogenic, non-toxic and fire hazardous compounds when in contact with air and water.

Pure crops included in the claimed biological product isolated from oil-contaminated soils. The strains are deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), FSUE GosNIIgenetiki.

The strain Acidovorax delafieldii NT-1 (VKPM B-8358) is mobile sticks with polar flagging of 0.6-0.8 × 2.0 μm in size, located singly and in pairs. Gram stain is negative. On standard nutrient media forms round colonies 2 mm in diameter. Convex, smooth, shiny, the edges are even, colorless, opaque. Aerob, chemorganotroph. Does not need growth factors. The oxidase test is positive. Catalase test is positive. Does not form a fluorescent pigment. It grows at 4 ° C, does not grow at 41 ° C. It does not have the ability to denitrify. Gelatin does not hydrolyze. Starch does not hydrolyze. It uses glucose, glutamate, xylose, ribose, malonate, and hydrocarbons as the sole carbon source. Does not use trehalose, inositol, rhamnose and sucrose.

Strain Burkholderia caryophylli NT-4 (VKPM B-8359) - movable straight sticks 0.8-0.9 × 0.9-2.0; 1.5 × 1.5 μm, located singly and in clusters. Gram stain is negative. On standard nutrient media forms round colonies 2 mm in diameter. Convex, smooth, shiny, the edges are even, white, opaque. Aerob, chemorganotroph. Does not need growth factors. The oxidase test is positive. Catalase test is positive. Does not form a fluorescent pigment. It does not grow at 4 ° C, grows at 41 ° C. It has the ability to denitrify with the formation of nitrogen. Gelatin does not hydrolyze. Starch does not hydrolyze. It uses glucose, glutamate, ethanol, succinate, xylose, mannitol, and hydrocarbons as the sole carbon source.

The strain Pseudomonas fluorescens biovar II NT-6 (VKPM B-8360) - sticks are straight, small, single, slightly curved, have polar flagella, do not form spores, sizes 0.8-0.9 × 1.1-3.0 μm . Gram stain is negative. On standard media, it forms round colonies with a diameter of 2 mm or more, convex, smooth, shiny, the edges are even, colorless, opaque. Aerob is able to grow under anaerobic conditions in the presence of nitrates in the medium. Hemorganotroph. Oxidative type of metabolism. It grows at 4 ° C, does not grow at 41 ° C. The strain is catalase-positive, oxidase-positive. It forms a fluorescent pigment. Forms arginine dehydrolase. Hydrolyzes gelatin. Forms Levan from sucrose. Does not hydrolyze starch. Capable of denitrification. Uses glucose, trehalose, arginine, inositol, hydrocarbons as a growth source.

The strain Cifrobacter amalonaticus Jan-3-2 (VKPM B-8361) - sticks straight with rounded ends, single and pair-lying, size 0.7-0.8 × 1.2-2.0 microns. Gram stain is negative. The colonies on nutrient agar are round, convex with an eminence in the center, 3-5 mm in diameter, smooth, shiny, the edges are even, translucent, unpigmented. Enzymatic metabolism. The strain is catalase-positive, oxidase-negative. Optional anaerobic. Possesses ornithine dehydrolase, but not lysine and arginine. Does not utilize citrate and urea. Does not form hydrogen sulfide. Indole forms. The Voges-Proskauer reaction is negative. Does not dilute gelatin. It forms acid from glucose, mannitol, sorbitol, ramnose, amygdalin, arabinose. Does not form acid from inositol, sucrose and melibiosis.

The strain Pseudomonas fluorescens Jan-8-2 (VKPM B-8362) - rods are movable straight lines with rounded ends, singly and paired, size 0.7-0.8 × 2.5-3.5 microns. Gram stain is negative. Colonies on nutrient agar are round, convex 2-3 mm in diameter, smooth, shiny, finely serrated edge, translucent, unpigmented. Oxidative metabolism. The strain is catalase-positive, oxidase-positive. Aerob is able to grow under anaerobic conditions, using nitrate as the final electron acceptor. It has denitrification and does not need growth factors. It grows at 4 ° C, does not grow at 41 ° C. Possesses ornithine dehydrolase, but not lysine and arginine. Disposes of citrate. Does not destroy urea. Does not form hydrogen sulfide. Indole forms. The Voges-Proskauer reaction is negative. Does not dilute gelatin. It forms acid from glucose, mannitol, sorbitol, ramnose, amygdalin, arabinose. Does not form acid from inositol, sucrose and melibiosis.

The strain of Serratia marcescens Jan-10-2 (VKPM B-8363) - sticks straight, size 0.8-0.9 × 1.2-2.4 microns. Gram stain is negative. Colonies on nutrient agar are round, convex, 3-5 mm in diameter, smooth, shiny, the edges are rugged, opaque, red. Enzymatic metabolism. The strain is catalase-positive, oxidase-negative. Optional anaerobic. Possesses ornithine and lysine, but not arginine dehydrolase. Disposes of citrate. Does not destroy urea. Does not form hydrogen sulfide. The Voges-Proskauer reaction is positive. It has gelatinase. It forms acid from glucose, sucrose, mannitol, inositol, sorbitol, amygdalin. It does not form melibiosis, rhamnose. It has nitrate reduction.

To obtain the drug, separate cultivation of the strains was carried out according to standard methods.

The resulting preparations after adding preservatives were stored in the refrigerator at a temperature of 4-6 ° C.

Immediately prior to testing, six preparations, each of which contained one strain of a microorganism-destructor, were mixed in equal amounts.

The biological product was tested on model plants and production conditions.

The results of experiments on model plants in order to determine the destructive ability of a biological product are shown in table 1.

In the first experiment, 500 g of soil contaminated with fuel oil was placed in 1-liter plastic vessels. According to gas chromatography-mass spectrometric analysis, the content of hard-oxidizing heavy fractions was 78%. 10 ml of a laboratory sample of a biological product previously diluted with tap water 10 times were introduced into the soil. As sources of nitrogen, phosphorus and potassium, the calculated amounts of urea, double superphosphate and potassium chloride were added; Tween-80 was used as a surfactant. The soil in the vessels was periodically moistened, preventing drying out, mixing every other day. In the control vessels, the soil was not treated with anything. Not moistened, not mixed.

In the second experiment (conditions are similar to those of experiment 1), soil contaminated with hydrocarbons with a content of light fractions of 70-90%, as well as heavy metals in concentrations 10-50 times higher than the maximum permissible concentrations were used. The initial concentration of hydrocarbons is 21.5 g / kg.

Table 1 Experience option Sample The concentration of hydrocarbons, g / kg The average concentration of hydrocarbons, g / kg The decrease in the concentration of hydrocarbons,% one Dates 01.01 01/29 13.02 03.03 03/23 01.01 01/29 13.02 03.03 03/23 01.01 01/29 13.02 03.03 03/23 Source soil 29.9 30.0 30,0 The control 28.8 27,2 30,4 22.0 24.8 26,4 23,2 19.6 21.6 16,4 27.6 25,2 25.0 21.8 20.6 8.0 16,0 16.7 27.3 31.3 27.8 27.8 25.1 21.9 20.7 Biological product 12.0 9.2 7.2 6.8 5,6 14.6 10.0 7.0 6.4 5,4 51.3 66.0 76.7 87.7 82.0 17,2 11.2 6.8 6.0 5.2 2 Dates 01/31 13.02 03.03 03/23 04/25 01/31 13.02 03.03 03/23 04/25 01/31 13.02 03.03 03/23 04/25 Source soil 18.8 21.5 25.0 The control 18.8 14,4 10.0 10,4 10,2 19.2 15.6 12.0 10.3 10.0 10.7 27.4 44,2 52.1 50,0 19.6 16.8 14.0 10,2 9.8 Biological product 7.6 10.0 10.0 8.4 3,1 8.2 9.6 10.8 8.0 2,8 61.9 55.3 49.8 62.8 86.0 8.8 9.2 11.6 7.6 2,5

Claims (1)

A biological product for cleaning environmental objects from oil and oil products, including a consortium of aerobic bacterial cultures, characterized in that Acidovorax delafieldii VKPM B-8358 (NT-1), Burkholderia caryophylli VKPM B-8359 (NT-4) are used as a consortium of bacterial cultures , Pseudomonas fluorescens biovar VKPM B-8360 (II NT-6), Citrobacter amalonaticus VKPM B-8361 (Jan-3-2), Pseudomonas fluorescens VKPM B-8362 (Jan-8-2), Serratia marcescens VKPM B-8363 ( Jan-10-2) with a titer of living cells of at least 10 ¹⁰ .