RU2181768C2 - Consortium of bacterium erwinia species and arthtrobacter simples as destructor of petroleum products and organic substrate in vegetable raw and animal husbandry waste - Google Patents

Abstract

FIELD: microbiology, biotechnology, agriculture, ecology. SUBSTANCE: invention proposes a consortium of bacterium Erwinia species GSB-TB 15/99 B and Arthtobacter simplex GSB-TB 16/99 B taken in the ratio from 0.1:1 to 100:1. This consortium is effective in preparing fertilizer by means of aerobic and anaerobic cattle manure, avian dung processing and in treatment of subjects from petroleum products. Consortium shows favorably on soil and ecologically important biocenosis. Invention can be used for decomposition of petroleum products and organic substrate in vegetable raw and animal husbandry waste. EFFECT: enhanced efficiency of consortium.

Description

 The invention relates to the field of microbiology and can be used in biotechnology and agriculture for the decomposition of petroleum products and organic substrates in plant materials and animal waste, in particular during aerobic and anaerobic processing of livestock manure, bird droppings, as well as in any areas of human activity for cleaning objects from petroleum products.

 Known strains of microorganisms - destructors of petroleum products used to clean natural and artificial objects from oil and petroleum products.

 For example, the bacterial strain Pseudomonas putida-36 (SU, copyright certificate 1076446) is known that is used to purify water and soil and allows to utilize up to 75% of oil when the oil content to the volume of contaminated substrate (soil, water) is up to 10%. A disadvantage of the known strain is the cleaning of only slightly contaminated objects.

 Known strains of microorganisms used in biotechnology, agriculture for the decomposition of organic substrates in plant materials.

For example, strains of thermophilic bacteria of the genus Lactodicterium or Streptodacterium and mesophilic bacteria of the genus Streptococcus, Azotobacterium, Pseudomonas (GB, patent 2026087) are known that are used to decompose manure in a medium containing also carbon sources, mineral acids and proteins, at temperatures of 5-35 ^o С within 7-14 days. A disadvantage of the known strains is their applicability only for highly diluted manure for a long period of time.

 Known bacterial strain Erwinia species VKPM B-7005 (RU, patent 2103359), capable of decomposing organic substrates in plant materials under aerobic and anaerobic conditions with high speed, oil and oil products in the process of cleaning various objects from these substances, hydrocarbon substrate with the release of a large amount gaseous products.

The specified strain of Erwinia species VKPM B-7005 with a periodic method of cultivation under aerobic conditions at a temperature of 32-36 ^o C and a pH of 5-7 is able to completely utilize contamination with oil products for 36-48 hours. The strain is also able to decompose the organic substrate in plant materials, suitable for aerobic and anaerobic processing of animal manure and bird droppings. On a carbon substrate, it is able to synthesize low molecular weight organic acids, aldehydes and alcohols.

 The disadvantage of this strain should be recognized as its unsuitability for cleaning heavily contaminated areas, since, as mentioned above, the strain synthesizes low molecular weight organic acids, aldehydes and alcohols on a carbon substrate, which, when accumulated in the medium, inhibit the process of further utilization of the carbon substrate.

 The technical problem solved by the present invention is to develop a consortium of microorganisms that is highly active in the decomposition of oil and oil products and organic substrate in plant materials and animal waste at a high concentration of organic substrate.

 The technical result obtained as a result of the implementation of the invention consists in providing the possibility of accelerated and more complete degradation of oil and oil products at the treatment facilities, as well as the decomposition of the organic substrate in plant materials with a high content of the specified organic substrate.

 To achieve the specified technical result, it was proposed to use a consortium of bacteria Erwinia species GSB-TB 15/99 B and Arthrobacter simplex GSB-TB 16/99 B, taken in a ratio of 0.1: 1 to 100: 1, used as a degrader of petroleum products and organic substrate in plant materials and livestock and poultry waste.

 The essence of the present invention consists in the isolation from the natural environment (soil mixture) of a natural bacterial strain Arthrobacter simplex and, based on it, the selection of a new strain of this culture, as well as the selection of a new strain of Erwinia species, leading the pedigree of the strain isolated from the natural environment (oil rock (core ) Of the Khadyzhenskoye field), under conditions of co-cultivation in order to obtain a highly active consortium of microorganisms in the decomposition of petroleum products and organic substrates in plant materials and waste livestock farming with a high concentration of organic substrate.

 The selection of a new strain of Arthrobacter simplex was carried out from a soil mixture sample taken near an oil well at the border of oil-contaminated soil and soil covered with vegetation.

Spent the cultivation of a natural mixture of soil microorganisms for 1 day at a temperature of 32 ^o C on a medium (A) containing, g:
The culture fluid of the fermentation process of microorganisms Erwinia sp. - 100
Soil sample - 1
To obtain the culture fluid used in medium (A), the Erwinia species VKPM B-7005 collection strain was grown under aerobic conditions for 2 days at a temperature of 32 ^° C and pH 7.0 on medium (B) containing, g:
Wednesday 9 with m / e "according to Alikhanyan" GUP "GosNIIsintezbelok" - 100
Oil - 1
Corn Extract - 0.1
After completion of the Erwinia species growing process, the culture fluid was separated from the main biomass by centrifugation and sterilized.

From a culture suspension of Erwinia sp. Did sowing on a solid nutrient medium (B) of the following composition, g:
The culture fluid of the fermentation process of microorganisms Erwinia sp. - 100
Meat peptide broth - 1
Agar - 1.5
Another culture identified as Arthrobacter simplex was grown on medium “A” and plated on medium “B”. The cycle was repeated until a pure bacterial culture was obtained.

 Determination of the systematic position of the selected strain, carried out in accordance with the "Determination of bacteria" (Bergey'S Manual of Systematic Bacteriology - Baltimore; Hong Kong; London; Sydney - 1984, 1986, 1989, v.1, 2, 3, 4) showed its belonging to Arthrobacter simplex.

 The selection of new strains of Erwinia species GSB-TB 15/99 B and Arthrobacter simplex GSB-TB 16/99 B was carried out by using a large number of co-cultivation cycles of Erwinia sp. VKPM B-7005 and the natural strain Arthrobacter sim. on medium "B" and plating a suspension of two microorganisms on solid media, and in each subsequent cycle of co-culture relative to the initial inoculum, the proportion of Arthrobacter sim was gradually reduced. from the initial ratio of Erwinia sp .: Arthrobacter sim. = 1: 1 to 100: 1, and the amount of substrate (oil) in medium "B" was increased from 1 to 10 wt.%.

 Determining the degree of pathogenicity of a consortium of new selected strains of bacteria Erwinia species GSB-TB 15/99 B and Arthrobacter simplex HSB-TB 16/99 B confirmed the absence of pathogenicity.

 Cultural and morphological characters.

 Cultural and morphological characteristics of the strain Erwinia species GSB-TB 15/99 B.

 Erwinia species GSB-TB 15/99 B forms shiny colonies on meat and peptone agar with glucose and yeast extract of 1-3 mm from white (young culture) to cream-colored roundish with a smooth edge. Cells are short, (0.7-1.0 • 1.0-2.5 microns), mobile sticks. Under adverse conditions and with aging, the culture forms capsules.

 Cultural and morphological characteristics of the Arthrobacter simplex strain GSB-TB 16/99 B.

 Arthrobacter simplex GSB-TB 16/99 B forms, on meat and peptone agar with glucose and yeast extract, 1-3 mm matte wrinkled cream colonies of a rounded shape with a smooth or scalloped edge. Young culture - sticks (0.7-1.0 • 1.0-2.5 microns) of various shapes: straight, curved, can form v-shaped figures. Under adverse conditions and with aging, the culture forms capsules.

 Physiological and biochemical signs of the consortium.

 The consortium of bacteria Erwinia species GSB-TB 15/99 B and Arthrobacter simplex GSB-TB 16/99 B, taken in a ratio of 10-100: 1, is able to decompose or transform organic substances contained in oil and oil products or plant materials and animal waste and poultry farming. At the same time, the consortium is able to utilize a larger amount of organic substrate in a batch process, a pure culture of Erwinia species GSB-TB 15/99 B, due to the development of Arthrobacter simplex bacteria HSB-TB 16/99 B on Erwinia metabolic products. in the inoculum choose Erw.:Arthr.=10:1. The ratio of cultures in the inoculum is selected specifically for each process.

 It is preferable to store and store each culture of the consortium separately.

The temperature range (28-32 ^o C), as well as the indicator of the environment (pH 5-7) for both cultures are the same.

 Erwinia sp. - optional anaerobic, Arthrobacter sim. aerobic culture, able to work with limited oxygen concentration.

 In the process of decomposition of the organic substrate, the ratio of cultures varies depending on the nature of the carbon substrate and the duration of the process.

Both cultures are stored in test tubes at 40 ^o C under oil on meat and peptone agar with 2% glucose and 0.1% corn extract, pH 7.0.

The cultures are propagated in test tubes at 30 ^{° C.} on meat peptone agar with 2% glucose and 0.1% corn extract, pH 7.0.

 For training Arthrobacter simp. during storage and propagation, at least 25% of the culture fluid of the Erwinia sp. accumulation process is added to all nutrient media.

 The consortium can be used as follows.

Example 1 (control). An area of soil with a degree of contamination of 45-63% (near-wellbore area) is treated with a suspension of Erwinia sp. VKPM B-7005. Bacterial biomass is grown in a fermenter at a temperature of 32 ^o C and a pH of 7.0 in a nutrient medium of the following composition, g / l: oil - 40.0, molasses - 10.0, sodium chloride - 20.0, phosphoric acid - 3 , 0, ammonium hydroxide - 4.0, potassium chloride - 0.8, magnesium sulphate - 0.3 and trace elements. After biomass accumulation, it is concentrated by centrifugation and canned. To prepare a working suspension of microorganisms, immediately before treatment, the condensed biomass is diluted in river water to a cell concentration of about 10 ⁸ cells / ml with the addition of mineral fertilizer (ammofoski) based on the concentration of nitrogen, phosphorus and potassium, g / l: 2.0, 1 , 5 and 0.7, respectively. An awning was stretched over the area to be cleaned to prevent the washing off of microorganisms by rain. The site is dried naturally until a crust forms on top, then gently moisten the soil evenly with a suspension of microorganisms at the rate of 0.5 l / m ² . Within 7 days, the site is daily watered at the rate of 1 l / m ² . Then the soil is moistened again with a suspension of microorganisms in an amount of 0.25 l / m ² with continued watering. After 7 days, the soil is again moistened with a suspension of microorganisms and watering is continued for another 7 days. Throughout the entire soil cleaning process, only the upper moist soil layer is carefully loosened. As moisture penetrates inside, the thickness of the loosened layer is increased. After 21 days of treatment, the residual degree of contamination was 24%.

Example 2. A similar plot of soil was treated with a suspension of a consortium of bacteria Erwinia species GSB-TB 15/99 B and Arthrobacter simplex HSB-TB 16/99 B. Biomass Erwinia species GBS-TB 15/99 B is grown in a fermenter at a temperature of 32 ^o C and pH medium - 7.0 in the nutrient medium of the composition, g / l: oil - 40.0, molasses - 10.0, sodium chloride - 20.0, phosphoric acid - 3.0, ammonium hydroxide - 4.0, potassium chloride - 0.8, magnesium sulphate seven-water - 0.3 and trace elements. After biomass accumulation, it is concentrated by centrifugation and canned. The biomass Arthrobacter simplex GSB-TB 16/99 B is grown in a fermenter at a temperature of 32 ^o C and a pH of 7.0 in a nutrient medium composition, g / l: culture fluid of the fermentation process of microorganisms Erwinia sp. - 50, Wednesday 9 with m / s "according to Alikhanyan" State Unitary Enterprise "State Research Institute of Synthesis Proteins" - 50; molasses - 1.5. After biomass accumulation, it is concentrated by centrifugation and canned. To prepare a working suspension of microorganisms, immediately before treatment, the condensed biomass of Erwinia sp. and Arthrobacter simp., taken in a ratio of 100: 1, respectively, are diluted in river water to a total cell concentration of 10 ⁸ cells / ml with the addition of mineral fertilizer (ammofoski) based on the concentration of nitrogen, phosphorus and potassium, g / l: 2, 0, 1.5 and 0.7, respectively. Next, the processing of the site is carried out, as in example 1. The final residual degree of contamination is 6%. This indicator is 4 times less than in example 1, i.e. the degree of purification is 4 times higher.

Example 3. Carry out the process of obtaining biofertilizer from waste forestry and agriculture. A mixture of sawdust, shavings, bark, leaves, stems and roots, as well as paper. Dried, crushed, steamed, poured with stocks of livestock and poultry complexes, acidified with sulfuric acid to pH 2-4, incubated for a day. It is alkalinized with an ammonia solution to pH 5-7 and a suspension of a consortium of microorganisms Erwinia species GSB-TB 15/99 B and Arthrobacter simplex HSB-TB 16/99 B taken in a ratio of 10: 1, respectively, in an amount of 5 vol.% To the volume of treated material at a concentration of cells in suspension of 10 ⁸ cells / ml The resulting mass is treated for 3 days at a temperature of 28-30 ^o C in the apparatus with mechanical stirring and air supply of 0.2-0.5 rpm • min. After processing, the mass contains, by weight. % of solids: total nitrogen - not less than 4, total phosphorus - not less than 1.5, total potassium - not less than 1.0, total carbon - not less than 40. Moisture content - 50-70%. The resulting mass is used as an independent fertilizer.

 The above examples confirm the receipt of the specified technical result.

Claims (1)

 A consortium of bacterial strains Erwinia species GSB-TB 15/99 B and Arthrobacter simplex GSB-TB 16/99 B, taken in a ratio of 0.1: 1 to 100: 1, is a degrader of oil products and organic substrate in plant materials and animal waste and poultry farming.