DE19652580A1 - Bacterial mixture for regenerating crude oil contaminated earth and water - Google Patents

Abstract

Bacterial mixture for regenerating earth and waters contaminated by crude oil and/or oil products comprises 6.0-13.0 % dry biomass of the bacterium "Bacillus Megateria-1 BD", 14.0 - 25.0 % dry "Biosurfactant BS-4" and 62.0-80.0 % of a porous powder with a particle size of 100-2000 micron.

Description

The present invention relates to the microbiological Purification of water and petroleum by petroleum products have been contaminated with the help of a bacterial Mixture, which is featured for these specific purposes will hit.

The contamination of water and soil is there takes care of where oil is extracted, transported and processed becomes. For the restoration of the state of the earth and the waters before pollution are so far physi calic, chemical and bacteriological methods applied. The biological method turns out to be the economy most and has the least negative effects the environment.

The previously known possibilities of regeneration through Degradation of petroleum products in petroleum and water, which are contaminated by oil and oil products, using biolo gisch acting agents are burdened with the disadvantage that  the natural degradation processes with insufficient speed expiration. The natural process of regeneration can be 0.5 to 10 years and depends heavily on the climatic, physical, chemical and biological Conditions at the place of contamination. There are some Methods for intensifying the regeneration of water and known earths. They include, according to US Patent 4727031 aeration, the introduction of nitrates and Hydrogen peroxide, according to SU patent 1158258 bring organic ingredients and according to US Patent 4727031 the introduction of inorganic substances into water and Earth materials.

It is known that the most effective stimulation of Re generation process by introducing bacterial Substances in water and soil is reached. Are known Compositions which are mainly based on EP patent 0977422; U.S. Patent 4,822,490; US Patent No. 4521515 and DE Patent 38 11 856 bacterial strains of the type "Pseudomonas" ver turn. However, bacteria of the type "Pseudomonas" ecologically seen bacteria of the R strategy (Andrews J.H., Harris R.F. and K - selection and microbial ecology / Advences in microbial ecology [ed. K.C. Marshall], v. 9 - N.Y. a London; Plenum Press, 1986, p. 99-147). It is known, that bacteria of the R strategy under extreme conditions (osmotic pressure, lack of food, etc.) die quickly. Therefore it is cheaper to use Mikroor in the regeneration process to use mechanisms under the most diverse order global conditions remain stable. These are microorganisms of the spore-forming type. The farthest are spores in nature spreading bacteria of the Bacillus type. they are suitable candidates for use in cleaning ver unpolluted water and soil.  

Furthermore, according to US Patent 4822490 and DE Patent 38 11 856 Compositions of bacterial substances known from Carriers of bacterial cells exist. For the production of the Emulsion and for more effective irrigation of the soil are the bacterial mixture according to DE Patent 37 33 341 added surface-active or foam-forming components mixes. However, this has a very disadvantageous effect proposed mixture of substances that contained therein surface-active and foam-forming substances often for Humans and other biological formations are toxic. The Problem of the regeneration of soils and water, which were contaminated by oil and oil products, using of microbiological processes, is still after known prior art has not yet been satisfactorily resolved and therefore requires new, more effective methods and Medium.

The object of the present invention is a Mixture of substances using petroleum and / or petroleum products of degrading bacteria for the regeneration of Crude oil and oil products contaminated soil and water, which guarantees the highest possible degree of cleaning, remains stable under different environmental conditions, simple and is safe to use and easy to handle can be proposed and a method of manufacture position to develop.

This object was achieved by the provision of a bacterial mixture for the purification of soils and water contaminated by petroleum and oil products, in which, according to the invention, the bacterial strain "Bacillus Megateria-1 BD" and biogenetic components, which consist of a Mixture of the "Biosurfaktant BS-4" exist and a multiple porous powder with a grain size of 100 to 2000 microns in the following percentage composition

• - dry biomass of the bacteria "Megateria-1 BD" 6.0-13.0%
• - dry "biosurfactant-BS-4" 14.0-25.0%
• - porous powder 62.0-80.0%

be used.

"Biosurfactant BS-4" is obtained by hydrolysis of the microbiomass with a 2.8-5.6 percent potassium hydrooxide solution at 20-30 ° C won over the course of 10-24 hours. On finally the solution is added with the addition of phosphoric acid neutralized and with porous powder and the bacteria suspension mixed. The resulting mixture can be used both as dry powder, as well as being used as a suspension. The mixture mentioned ensures maximum cleaning of the water and soil of oil and oil products.

The use of the bacteria has proven to be extremely favorable strain "Bacillus Megateria-1 BD", which consists of 2.6 trillion of living cells per gram of biomass. The dry matter consists of endospores and dry, living cells. Biosurfaktant has emulsion and foam-forming properties which favor the cleaning of the water and the bottom of petroleum products. The share of "Biosurfactant BS-4" in the mixture mentioned is 14.0-25.0%. This proportion proves to be optimal since less than 14% the emulsifying and foam-forming effect is insufficient and the development of the bacteria in more than 25% "Megateria-1 BD" heavily braked at the point of pollution becomes.  

The mixture also contains fine-pored powder of grain size from 100 to 2000 microns. This size has proven to be effective proven because with a smaller grain size the ventilation of the Soil and with larger grain size the ability of Penetration into the floor to be cleaned is insufficient. The Powder must be porous in order for the bacteria to settle in the natural contact zone with the oil under air to guarantee. The use of powder is particularly suitable made of porous glass.

The task also posed, a process for manufacturing The position of the bacterial mixture was developed resolved that a method was found that was based on the Cultivation of the bacteria "Bacillus Megateria-1 BD", the Production of the "biosurfactant BS-4" and from the following preparation of the mixture.

To produce the biomass, which is stable to changes in the environmental conditions, the cultivation process is continued until the ratio between spores and active cells of the bacillus is 0.6 to 0.8. At the same time, 2.0 to 5.0 mass percent K ₂ HPO _{4 is} added to the bacterial suspension.

"Biosurfactant BS-4" is obtained by hydrolysis of the microbiomass with a 2.8-5.6 percent potassium hydrooxide solution at 20-30 ° C won over the course of 10-24 hours. Subsequently the solution is neutralized with the addition of phosphoric acid and mixed with porous powder and the bacterial suspension. Then the powder with the bacterial cells is removed filtered and at a temperature of 30-50 ° C to one Moisture content of 5-8% dried.  

The use of the mixture according to the invention improves the ongoing process of regeneration by oil and oil products polluted soil and water by 15-30% compared to the use of one in U.S. Patent 4,822,490 written bacterial mixture and enables the conversion of hydrocarbons in ecologically neutral components, resulting in a significant improvement in environmental conditions leads in the treated area.

The mixture of bacteria described here is for people Animals and plants harmless and works both in sweet and also in salt water and in soil. One particularly stands out the property to be emphasized is the stability towards chemical Contamination of the water or the soil - also with toxic substances - and stability against sudden change in environmental conditions.

These and other advantages of this invention will become apparent further described below. The bacterial strain "Bacillus Megateria-1 BD "was from the earth in the area alongside a gas processing plant in Ukraine.

The bacterial strain "Bacillus Megateria-1 BD" is characterized as follows:

• - Diameter of the cells after one day of growth in one Nutrient solution 1.1-1.5-2.0-5.0 micron;
• - Predominant shape of the cells - rod-shaped;
• - Gram positive - endospore-forming strain.

The bacterial cultures grow on a nutrient medium protruding and form circular, colorless bulges a diameter of 2-4 mm. The trunk is aerobic and propagates at temperatures of 3-40 ° C, the opti  male temperature is 30 ° C. It doesn't turn sour Reaction with glucose, arabinose and xylose. Reaction with following hydrocarbons as a food base is after shown: hexadecane, toluene, petrol, naphthalene, paraffin, Kerosene and asphalt. The catalase is positive. The reaction according to Foks-Proskauer is negative. A lecitinous activity is missing. The cells are starch-releasing. The oppression by Nitrate is not available. The strain is able to Glycol, ethylene and dietyl glycol to grow. He doesn't need one special nutrients and is able to grow where Hydrocarbons are present, d. that is, when the mineral water consumption not more than 70 grams per liter is. The parameters of growth at five-day inten sive cultivation in connection with different coals Hydrogen are shown in Table 1.

Growth parameters of the Bacillus "Megateria-1 BD" bacteria

Growth parameters of the Bacillus "Megateria-1 BD" bacteria

Dry cells retain their properties over a long period of time Viability and ability to hydrocarbon too oxidize.

The bacterial strain mentioned here is one of the harmless, non-pathogenic strains. Tests with white mice, to which these bacteria had been administered orally and by injection, proved its complete harmlessness. White mice weighing 18-20 grams, which received 0.5 × 10 ⁶ or 1 × 10 ⁶ living cells of the bacterium Bacillus "Megateria-1 BD" orally and 0.3 to 1 ml filtrate of the living cultures by injection , showed neither clinical illnesses nor changes of the internal organs within 15 days. The clinical observation of the scientific staff who worked directly with the bacteria also showed no abnormalities and the complete absence of allergic symptoms.

The strain was cultivated under laboratory conditions on a substrate with a pH of 7.2 and the following composition

• - hydrocarbon. . . 8 gr / l; NH ₄ NO ₃ . . . 1 gr / l; MgCl ₂ . . . 0.1 gr / l; KH ₂ PO ₄ . . . 3.0 gr / l; K ₂ HPO ₅ . . . 7 gr / l.

For the industrial production of biomass consisting of Cells of the "Bacillus Megateria-1 BD" strain become one Nutrient solution uses which nitrogen, phosphorus and potassium and contains hydrocarbon. The cultivation takes place aerobic at a temperature of 30 ° C. Among these be conditions the bacteria grow and multiply vigorously  and are stable during drying and storage. The so won Biomass can be used both as a powder and as a liquid solution be used.

In this invention is the use of the bacterial strain in combination with "Biosurfaktant BS-4" and porous powder been proposed. "Biosurfaktant BS-4" is published by Hy Drolysis of the microbiomass with a 2.8 - 5.6 percent Potassium hydrooxide solution at 20-30 ° C in the course of 10-24 Hours won. Then the solution is added neutralized by phosphoric acid up to an FH value of 7.0. "Biosurfaktant BS-4" is a mixture consisting of un toxic peptides, lipids and others on themselves dissolving biological components. The mixture has emul yawing and foam-forming properties that the bak serial conversion of petroleum and oil products in the mineral and stimulate in the water.

The application of the porous powder in this mixture causes better contact of the bacteria with the oil and the air and increases the air supply to the bacterial cells in the Water and earth. The bacterial described here The mixture is a dark gray powder. It can be both as Powder can also be used as a solution. The advantage this product and its manufacture consists in the Increasing the efficiency of oil regeneration and oil products contaminated soil and water. The Mixture is against extreme environmental changes stable conditions and does not require any special bearings conditions.

The mixture can be successful in combating ver impurities in both fresh and sea water or at  the cleaning of industrial waste water or areas with Contamination of the soil due to oil or oil products be set.

The cheapest is the use of the substance, consisting of the bacterial strain "Megateria-1 BD" and the mixture "Biosur faktant BS-4" and the porous powder with a grain size of 100-2000 microns in the following percentage composition:

• - dry biomass of the bacteria "Megateria-1 BD" 6.0-13.0%
• - dry "biosurfactant-BS-4" 14.0-25.0%
• - porous powder - 62.0-80.0%.

The mixture is mixed with the "biosurfactant" the porous powder and subsequent mixing with the Cell solution made. The preparation is a dark gray Powder. It is recommended to dissolve the powder in water and to use in this form. The proposed mixture is compared to other preparations due to its low Consumption of bacterial components particularly effective because their activity even under extreme environmental conditions to keep.

The advantages of this invention will become apparent from the below described examples particularly clearly. Examples 1 and 2 illustrate the manufacture of a stationary bio component. Example 3 describes the preparation of the "Biosurfactant BS-4". Example 4 illustrates the preparation the bacterial mixture and, finally, Examples 5 to 7 illustrate the application of the cell suspension of the Bacteria "Bacillus Megateria-1 BD", mixture of these suspensions sion with "Biosurfaktant BS-4", the pure "Biosurfaktant BS-4", dry bacterial cells attached to particles of the  porous powder, the bacterial mixture described and a control mixture for regeneration by petroleum and Oil products from polluted waters and soil.

example 1

The production of the biocomponent is illustrated in this example. The bacterial strain "Bacillus Megateria-1 BD" is produced in a nutrient solution in the presence of hydrocarbons (petroleum) according to the method of deep cultivation in the fermenter with a capacity of 5 liters. The nutrient solution consists of: petroleum (raw). . . 8.0 g; NH ₄ NO ₃ . . . 1 g; MgCl ₂ . . . 0.1 g; KH ₂ PO ₄ . . . 3 g; K ₂ HPO ₄ . . . 7 g. The pH is 7.2. The ratio between the substances supplied and the capacity of the container of the fermenter is 1:10. The cultivation is carried out with aeration (air consumption 0.5 l / h) and a temperature of 30 ° C. The concentration of the dry biomass is 5.3 g / l and 8.4% endospores after 48 hours of cultivation and 12.8 g / l and 17.2% endospores after 120 hours of cultivation. The biomass produced is then centrifuged to a concentration of 100 g / l and then mixed with the porous powder with a grain size of 250-2000 microns in a ratio of 1:12 for one hour and then filtered.

The powder with the attached bacterial cells is at a temperature of 30 ° C to a humidity of 3% with an air consumption of 0.5 l / h. dried. The number of living cells after two minutes of exposure to ultrasound is 6.8 × 10 ¹⁰ per g dry powder.

Example 2

The biomass and the dry mixture of the biomass and the glass powder are produced analogously to the technology from Example 1 with the following exceptions. Kerosene is used instead of crude oil. After 48 hours of cultivation in the fermenter, 4 g of crude oil, 5 g of K ₂ HPO ₄ and 45 g of K ₂ HPO _{4 are} added. The concentration of the dry biomass after 48 hours of cultivation is 8.1 g / l with 7.6% endospores. The dry matter concentration after 120 hours is 17.5 g / l at 21% endospores. The biomass produced is then centrifuged to a concentration of 100 g / l and then mixed with the porous powder with a grain size of 250-2000 microns in a ratio of 1:12 for one hour and then filtered.

The powder with the attached bacterial cells is at a temperature of 30 ° C to a humidity of 3% with an air consumption of 0.5 l / h. dried. After two minutes of ultrasound exposure, the number of living cells was 7.3 × 10 ¹⁰ / g of the dry powder.

Example 3

This example illustrates the production of the "Biosurfak tant BS-4 "from microbiomass (waste) of a municipal waste case removal operation. The microbiological mass will with 5-7 percent potassium hydrooxide in a ratio of 1: 1 mixed and after 24 hours at a temperature of 21-28 ° C. with phosphoric acid up to pH 7.0 neutralized. The values obtained are in Table 2 listed.  

Result of the production of "Biosurfaktant BS-4"

Result of the production of "Biosurfaktant BS-4"

The emulsifiability of "Biosurfaktant BS-4" in kerosene - Water was 56%; the surface tension of the solution - 47.48 dynes / cm.

Example 4

In this example, the preparation of the bacterial mixture described is explained. The solution of the "Biosurfaktant BS-4" with its dry constituents is prepared as described in Example 3 and mixed with the porous glass powder in a ratio of 11: 1 kg for one hour. The powder is then filtered and mixed with the suspension analogously to Example 1. After two hours, filter again and the filtrate at 30 ° C in an air stream with an air consumption of 1.1 l / h. dried to a residual moisture of 3%. The number of living cells is determined after two minutes of exposure to ultrasound with 8.6 × 10 ¹⁰ / g dry matter.

Example 5

This example uses a natural Suspension of the cells "Megateria-1 BD" and a mixture of these Suspension with "biosurfactant BS-4" explained. An earth substance Sample weighing 0.5 kg was mixed with 100 ml crude oil  contaminated and then sprinkled with 50 ml of suspension and kept at a temperature of 24 ° C for 5 weeks. The initial concentration of petroleum in the petroleum sample was 18 200 mg / kg. The results are in Table 3 listed.

Results of the attempt to restore the soil Crude oil pollution

Results of the attempt to restore the soil Crude oil pollution

From Table 3 it can be seen that the sprinkling of ver unpurified soil sample with a cell suspension in combination nation with "biosurfactant BS-4" to an 89.1 percent Cleaning after 35 days. At the same time only achieved a purification of 70.6% with the cell solution.

Example 6

This example uses dry biomass the bacteria of the strain "Bacillus Megateria-1 BD" stored on the porous glass powder as in Example 1 (Recipe 1); Example 2 (Recipe 2); Example 3 (Recipe 3); Example 4 (recipe 4) and as for the control mixture U.S. Patent No. 4,822,490, industrially herge poses.

Mixes according to recipes 1; 2; 3, the control mixture and the mixture described here became  Regeneration of water and soil contaminated by Raw oil applied at 30 ° C.

The liquid nutrient solution for this goal consisted of

• - a mixture of black earth and crude oil in the ratio 10: 1 = 100 g;
• - recipe 1; 2 and 3 or control mixture and described Mixture = 5 g;
• - water = 1000 g.

The results are shown in Table 4.

Results of experiments to test recipes 1-3 and of the blends in the regeneration of crude oil contaminated water and soil

Results of experiments to test recipes 1-3 and of the blends in the regeneration of crude oil contaminated water and soil

From Table 4 it can be seen that the one described here Blend is able to remove 91.2% of the petroleum. in the In contrast, the cleaning effect of Control mix 75.6%.  

The use of the mixture described here is guaranteed practically a 100 percent regeneration by petroleum and oil products contaminated surfaces.

Example 7

In this example, the application of the invention proposed mixture according to recipe 4 and according to the U.S. Patent 4,822,490 industrial control mix in real environmental conditions comparatively Darge poses. The experiment was explained as in example 4, carried out, but after 5 days the earth and Centrifuged water sample and in the air at a tempera dried from 50 ° C to a residual moisture of 3%. Thus, the drying out of the earth was natural Conditions simulated. Then the earth became he re-moistened.

The results obtained are in Table 5 shown.

Results of the investigation Petroleum contaminated earth materials - before and after drying the soil -

Results of the investigation Petroleum contaminated earth materials - before and after drying the soil -

As can be seen from the results of this table, it works the drying out of the soil when using the control preparations negatively affect the cleaning of the floor. In which The mixture provided according to the invention remains the cleaning effect still in satisfactory relations.

The mixture provided according to the invention described here can effectively clean industrial scale soiled by petroleum and oil products and Waters are used. The mixture can both in its dry form, as well as in an aqueous solution are applied, the solution then being sprayed on the surface to be cleaned is applied. The type of Spray device will not have any special requirements posed.

Claims (4)

1. Bacterial mixture for the regeneration of soils and waters contaminated by petroleum and / or oil products, characterized in that the mixture consists of the bacterium "Bacillus Megateria-1 BD" as an active component and biogenic components, essentially a mixture of "Biosurfaktant BS -4 "and porous powder with a grain size of 100-2000 microns in the following percentage composition:

• - dry biomass of the bacteria "Megateria-1 BD" 6.0-13.0%
• - dry "biosurfactant BS-4" 14.0-25.0%
• - porous powder 62.0-80.0%.

2. Mixture according to claim 1, characterized in that the 2.2 billion cells living in this mixture per gram of dry matter. 3. Mixture according to claim 1 and 2, characterized in that the "biosurfactant BS-4" contained in this mixture Hydrolysis of the biomass with 2.8-5.6% potassium peroxide within from 10-24 hours at a temperature of 30 ° C and then neutralizing the resulting solution with Ortophosphoric acid is made.   4. Mixture according to claim 1, characterized in that this mixture cultures of "Bacillus Megateria-1 BD" as well a mixture of a bacterial suspension and porous powder, which processes with a solution of "Biosurfaktant BS-4" was included.