RU2077578C1 - Strain of bacterium mycobacterium species "иж-4" used for water and soil treatment from oil and petroleum products - Google Patents

Abstract

FIELD: microbiology, ecology. SUBSTANCE: invention relates to new strain of bacterium Mycobacterium species "ИЖ-4" isolated from the accumulative culture of microorganism-destructors of petroleum products. Strain is obtained from silt sample from Arkhangelsk region bulk oil-plant. Strain utilizes petroleum products as a single carbon source at broad temperature range and for relatively short times. Preparation can be used for petroleum products treatment in sewage equipments and for biological sewage treatment. EFFECT: isolation of strain indicated above, enhanced efficacy of treatment. 3 tbl

Description

 The invention relates to the field of microbiology and relates to the production of a new strain of bacteria that can be used to purify water and soil from oil and oil products.

 A known strain of Pseudomonas aerugenosa PAO containing plasmids Cam, Oct, Nah, Sal, which utilize individual hydrocarbons from the oil (US patent N 3813316, CL 195-28, 1974). Its disadvantage is the low rate of utilization of crude oil.

 The known strain Pseudomonas aerugenosa 121, capable of utilizing octane (collection of the Museum of the Laboratory of Extrachromosome Heredity, IBPM Academy of Sciences of the USSR, 1976).

 The disadvantage of the strain is the low speed and a narrow range of recyclable oil products.

The Pseudomonas putida 36 strain (ac CCCP N 1076446, C 12 N 15/00, 1984), which has high biodegradation characteristics of oil and oil products, is widely used. However, the disadvantage of this strain is that it grows and utilizes petroleum products only at a relatively high temperature (28-30 ^o C). The percentage of oil utilization in Pseudomonas putida 36 is 68 74 on the 30th day. The disadvantage of this strain is that at 4 ^o C it does not grow at all and utilizes oil only at high temperatures.

 The objective of the invention is to obtain a strain of bacteria with high utilizing ability in relation to oil and oil products in a wide temperature range in a relatively short time.

 The obtained strain of Mycobacterium sp IL 4 was isolated from a sludge sample contaminated with oil products of the village. Izhma of the Arkhangelsk region.

 The strain was deposited by the Central collection of microorganisms of the State concern "Biological product", collection number CCM V-60B.

 The resulting strain has the following morphological-cultural and physiological-biochemical characteristics. Gram-positive straight, curved sticks 0.2 0.6 x 1.0 10 microns, acid and alkali-resistant, motionless.

 On meat-peptone agar (MPA), after 48 hours, it gives small, convex, round, with an even edge, fine-grained orange colony. According to the consistency of the colony, they are pasty, not transparent, shiny, easily removed by loop from agar.

 In meat and peptone broth (BCH) it grows in the form of sediment at the bottom, a slight turbidity of the medium, with the formation of a parietal ring.

 The culture grows on a mineral medium, using oil and oil products as a carbon source.

 Physiological and biochemical properties.

 Carbohydrates do not oxidize or ferment except glucose. Uses citrates and malonates as a carbon source. Attitude to nitrogen sources: nitrates does not restore, denitrification is absent, phenylalonines do not deaminate. It does not need additional growth factors. It does not have protolytic activity. Catalytic, oxidase-negative, has a weak urease activity.

Optional aerobic grows in the temperature range from 4 ^o C to 40 ^o C optimum 28 ^o C, optimum pH 7.0.

 The strain is not virulent, not toxic, tested on white mice.

Storage conditions in lyophilized form in ampoules at 4 ^o C. The strain can be maintained by regular reseeding (1 time in 2 weeks) on mineral medium 8E with oil and oil products, as well as in a complete medium.

Example 1. The strain Mycobacterium sp IL 4 is grown in 250 ml flasks containing 50 ml of sterile mineral medium. The media is prepared using tap water, using disubstituted ammonium phosphate at a concentration of 0.75 g / l as a mineral base. Sterile oil and petroleum products are applied strictly by weight with an accuracy of up to 0.05 mg. The concentration of the hydrocarbon substrate is 1%. The medium is infected with a two-day agar culture at the rate of 10 ⁷ cells / ml.

An uninfected flask with medium and oil is used as a control. The flasks are exposed at a temperature of 10 ^o C and 26 ^o C for 7 days on a rocking chair (80 rpm). At the same time, they put the same flask with the medium and petroleum products and Pseudomonas putida 36 cells to compare their utilizing ability under the same conditions. After 7 days, the fraction of the utilized substrate is determined by the gravimetric method after extraction of oil products with eloroform in the experimental and control flasks.

 The results are presented in table 1.

As can be seen from table 1, the utilizing ability of the strain Mycobacterium sp IL 4 in many petroleum products is higher than that of Pseudomonas putida 36 at a temperature of 26 ^o C. And at low temperatures, Pseudomonas putida 36 does not utilize oil and fuel oil at all.

Example 2. The strain Mycodasterium sp IL 4 is grown in 100 ml flasks containing 50 ml of sterile mineral medium. The medium is prepared in fresh and sea water using disubstituted ammonium phosphate at a concentration of 0.75 g / l as a mineral base. Sterile oil (specific gravity 888) and fuel oil are added strictly by weight with an accuracy of 0.05 mg. The concentration of the hydrocarbon substrate varies from 0.5 to 1.0%. The medium is infected with a two-day agar culture at the rate of 10 ⁷ cells / ml. An uninfected flask with medium and oil is used as a control. The flasks are exposed at a temperature of 10 ^o C and 25 ^o C for 7 to 14 days in a hospital. The fraction of the utilized substrate is determined by the gravimetric method after oil extraction with chloroform in the experimental and control flasks.

 The results of experiments on cleaning the surface of the water from oil pollution are presented in table 2.

Example 3. The strain Mycobasterium sp IL 4 is grown in 500 ml cylinders containing 400 ml of sterile mineral medium (tap water with the addition of 0.75 g / l of disubstituted ammonium phosphate). Sterile petroleum products are added by weight. The medium is infected with a two-day culture previously grown on a mineral medium with 1% oil. Using the AEN-3 aquarium compressor, air is supplied to the cylinders at a rate of 0.1 l / min. As a control, uninfected cylinders with medium and oil products are used. The cylinders were exposed at room temperature 20-22 ^o C for two weeks. The fraction of the utilized substrate is determined by the gravimetric method after extraction of petroleum products with chloroform in the experimental and control cylinders.

 The results are presented in table 3.

The data in table 3 show that over two weeks of incubation, the amount of oil products utilized by the strain Mycobacterium sp IL 4 reaches 67 - 80%
Thus, the advantage of the proposed strain is that it grows even on a depleted environment, while not reducing the ability to utilize petroleum products, which allows it to be used for biological treatment of water and soil from petroleum products.

Claims (1)

1 The bacterial strain Mycobacterium species TSKMK "B" TSKM V-60 B, used to purify water and soil from oil and oil products.

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