Consorcium of strains of microorganisms acinetobacter sp. and ochrobacterium sp., used for purification of water and soil from oil and oil products

FIELD: chemistry.

SUBSTANCE: invention relates to microbiological industry and can be used in biological purification of water and soil from oil and oil products. Claimed is consortium of strains of microorganisms Acinetobacter sp. VKM B-2753D and Ochrobacterium sp. VKM B-2754D, possessing nitrogenase activity.

EFFECT: consortium is capable of atmospheric nitrogen fixation and possesses high utilising ability with respect to oil and oil products with their high content in substrate.

1 tbl, 3 ex

 

The invention relates to the microbiological industry, the new consortium of strains of bacteria-destructors of oil and oil products, with nitrogenase activity (ability to fix atmospheric nitrogen), and can be used for cleanup of soils and water contaminated by oil and oil products in a shorter period.

Known strain of Pseudomonas putida 36 having the ability to biodegradation of oil and oil products [1].

The disadvantage of this strain is that it grows and utilizes the oil very slowly and only at high temperature (28-42°C). On the 30th day of Pseudomonas putida 36 utilizes 68-74% of the oil.

Known strain of Bacillus species 739 [2] and obtained on its basis of bacterial drug "Basicity" [3], used for remediation of soils polluted by oil and oil products.

The disadvantage of this strain is its low efficiency, especially in conditions of high content of oil and oil products in the hydrocarbon substrate.

The closest technical solution is a product for cleaning soil and water from oil and oil products, comprising a consortium of two bacterial strains of Bacillus brevis IB DT-1 and Arthrobacter species IB DT-3 [4] (prototype).

The disadvantage of this drug is its inability to fixation of atmospheric nitrogen, which is very important when disposing pleva�of erodov, contaminating soil and water, as excess oil and petroleum products affect the ratio of carbon:nitrogen in the system and this requires making additional source of nitrogen for the successful utilization of hydrocarbons.

An object of the invention is the obtaining of a consortium of strains of bacteria capable of fixing atmospheric nitrogen and transfer it in an easily accessible form and have high recycling capacity in relation to oil and oil products in a wide range of content in the hydrocarbon substrate in a shorter period.

The resulting consortium of bacterial strains isolated from grey forest soil, artificially contaminated with diesel fuel, and is in the Collection of microorganisms of the Institute of biology, Ufa scientific center, Russian Academy of Sciences.

Received the consortium consists of two strains of microorganisms, each of which is deposited in all-Russian collection of microorganisms and characterized by the following features.

Characterization of a strain of Acinetobacter sp. NO. IB DT-5.1/1 (VKM B - 2753D).

Gram-negative stationary short thick sticks, there are coccoid single or dual, multiply by dividing: the constriction and septum, catalanopolonesa, oxidationsmittel, indole, hydrogen sulfide does not form; does not dilute gelatin; milk does not change, poddala�Ivan doesn; nitrate does not recover. Sources of carbon assimilates glucose, sucrose, galactose, mannose, raffinoses, starch, sorbitol, glycerin. The IPA forms white smooth shiny colonies, profile evenly towering, smooth edge, diameter 2.0-2.5 mm. is Able to oxidize the hydrocarbons. Grows within temperature range 6-30°C, at a pH of 5.8-7.8. Non-pathogenic. Stored on the shoals MPA and in lyophilized state at a temperature of +4°C.

Characterization of strain Ochrobactrum sp. NO. IB DT-5.3/2 (VKM B - 2754D).

Gram-negative pleomorphic single sticks are shortened with age up to cocci. On dense nutrient media to form circular flat smooth matte color opaque colonies on the edges is less dense than in the center. Saprophyte. Aerobe. Catalanopolonesa, oxidization. The oxidative metabolism type. Well assimilates sucrose, fructose, maltose, lactose, xylose, mannitol, mannose, arabinose, L-rhamnose, glycerol. When grown on glucose forms a gas. Learns asparagine, arginine, alanine; weakly absorbs valine, lysine. Consume citrate as the sole carbon source. Does not hydrolyze gelatin. Starch decompose. Indole not formed. The reaction of Voges-Proskauer negative. The lack of growth on the medium with 6% NaCl. Oxidize oil hydrocarbons. Grows within temperature range 6-30°C, with pH of 5.8-7.8. Non-pathogenic. Stored on the shoals MPA and in lyophilized state at a temperature of +4°C.

Example 1. The consortium of strains of Acinetobacter sp. No. IB DT-5.1/1 and Ochrobactrum sp. No. IB DT-5.3/2 after fermentation in a nutrient medium Ashby of the following composition, g/l: mannitol - 20; NaCl and 0.2; CaCO3- 5,0; K2SO4- 0,1; K2HPO4To 0.2; MgSO4·7H2O - 0,2; distilled water - 1000 ml, containing sources of nitrogen, capable of fixing atmospheric nitrogen in the amount of 0.18 µg N2/ml/h. Nitrogenase activity was determined by acetylene method chromatographic [5]. The consortium according to the prior art is not capable of fixing atmospheric nitrogen.

Example 2. The consortium of strains of Acinetobacter sp. No. IB DT-5.1/1 and Ochrobactrum sp. No. IB DT-5.3/2 is grown on a nutrient medium of the following composition, g/l: Na2CO3To 0.1; CaCl2- 0,01; MnSO4·7H2O - 0,02; FeSO4·7H2O - 0,02; NaH2PO4- 1,5; K2HPO4To 1.0; MgSO4·7H2O - 0,2; NH4NO3- 2,0; distilled water - 1000 ml.

Studied destructively activity of the consortium of strains when grown on mineral medium of the above composition with the introduction as the sole source of carbon diesel fuel. The medium is poured into 100 ml flasks with a volume of 250 ml and sterilized at 1 ATM for 30 minutes. Seeded 2% bacterial suspension, diesel fuel contribute �terylene 1; 5; 10; 15 wt.%. The cultivation is carried out stationary at room temperature, providing aeration by forced flow of air by means of a microprocessor (air consumption 0.2 l/min). In the form of control put in the same flask with the addition of 100 ml of medium, an appropriate number of petroleum products without bacteria. In parallel, put the same flask with medium, oil cells and a consortium of two bacterial strains of Bacillus brevis IB DT-1 and Arthrobacter species IB DT-3 for comparison utilizing their abilities in the same conditions. At the end of the experiment (7 days) culture fluid was extracted with hexane, biomass cells are removed, the remaining oil is dried to constant weight. Compare "dry" weight balance of oil products in the control and experimental samples, find the percentage of utilization of petroleum products. Comparative data of utilization of petroleum products are presented in table.1.

Example 3. As in example 2, only in a nutrient medium lacking a nitrogen source NH4NO3. Comparative data of utilization of petroleum products are presented in table.1.

Thus, in table the results of the experiments indicate a high degree of utilization of petroleum products proposed by a consortium of microorganisms strains of Acinetobacter sp. No. IB DT-5.1/1 and Ochrobactrum sp. NO. IB DT-5.3/2. Advantages of�Twomey of the consortium is its effectiveness in a wide range of oil in the hydrocarbon-containing substrate even in the absence of nitrogen at the expense of the capacity for fixing atmospheric nitrogen (nitrogenase activity).

Table 1
Recycling rate (wt.%) of petroleum products by microorganisms in the presence of a source of nitrogen in the environment
StrainThe presence ( + ) absence (-) of the source of nitrogen in the environment1,05,010,015,0
A consortium of Acinetobacter sp. No. IB DT-5.1/1 and Ochrobactrum sp. No. IB DT-5.3/2+93,8093,7562,1856,14
-93,8093,7062,1055,89
The consortium of Bacillus brevis IB DT-1 and Arthrobacter species IB DT-3 (prototype)+93,8093,7059,4254,40
-40,1830,56Bed (19.2418,88

The list of references

1. Dyadechko V. N., Tolstokorova, L. E., Gashev S. N. and etc On biological remediation of oil contaminated sandy soil of the Middle Ob region // soil science. - 1990, No. 9. - P. 148-151.

2. Copyright certificate of the USSR No. 1743019, CL C12N 1/20, 1989.

3. RF patent №2077397, CL. SC 3/32, 1997.

4. RF patent №2232806, CL C12N 1/20, 2004.

5. Umarov M. M. Associative nitrogen fixation. - M.: Izd-vo Mosk. University press, 1986. - 136 p.

The consortium of strains of microorganisms Acinetobacter sp. ECM IN-2753D and Ochrobactrum sp. ECM IN-2754D with nitrogenase activity, used for purification of water and soil from oil and oil products.



 

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