WO2011019304A2 - Method for immobilizing cells of microorganisms into a sorbent used for oil pollution cleanup - Google Patents

Abstract

The invention relates to bioengineering concerned with providing means for cleaning up the environment. The invention proposes a novel industrial method for immobilizing cells of microorganisms into a sorbent used for cleaning up oil-contaminated soils and bodies of water. The resultant biosorbent absorbs and decomposes both large spills of petroleum products and thin opalescent films. An industrial method for immobilizing petroleum-degrading microbial biological agents onto hydrophobic oleophilic carrier sorbents has been developed. An aerosol consisting of an oil-based or petroleum-based emulsion having microorganisms in the composition thereof is applied to sorbent particles which are in a suspended state. Said method is universal for different hydrophobic oleophilic sorbents since it can be used for immobilizing any petroleum-degrading biological agents in different hydrophobic oleophilic sorbents. Said method is versatile by virtue of the fact that the process for immobilizing microbial cells contained in oil or in petroleum products into sorbent particles is performed in a chamber. This process is monitored and continued until the required level of biological agent is contained in the sorbent. In a biosorbent which has been produced in accordance with the claimed method, the biological agent is always reliably fixed to the sorbent, which makes it possible to use a biosorbent both for cleaning up soils and for cleaning water.

Description

 The method of immobilization of microorganism cells in a sorbent used to clean oil pollution

 Technical field

 The invention relates to the field of biotechnology, creating tools for cleaning the environment. A new industrial method is proposed for immobilizing microorganism cells into a sorbent used to clean oil-contaminated soils and water areas. The use of biosorbent obtained by the claimed method allows the cleaning of large areas of the water surface, marshy areas and land areas from the effects of oil spills, including thin rainbow films.

 Biosorbent is a sorbent carrier with immobilized microorganisms, oil destructors. Processing the oil slick with a biosorbent blocks its further distribution (the effect of physicochemical booms), which makes it possible to collect more than 90% of this pollutant. Biodegradation practically solves the issue of utilization of sorbents after use, which greatly simplifies their use.

 Now in the world about two hundred different sorbents are produced or used to eliminate oil spills, which are divided into: inorganic, natural organic and organomineral, as well as synthetic. Such a large number of different types of sorbents indicates the dissatisfaction of consumers with the quality of known sorbents and the search for new species that satisfy the “price-ratio” ratio. The quality of sorbents is determined mainly by their capacity with respect to oil, the degree of hydrophobicity (not soaking in water), buoyancy after oil sorption, the possibility of oil desorption and regeneration or utilization of the sorbent.

 State of the art

 The following methods for immobilizing a biogen on a sorbent carrier are available.

 1. Mechanical mixing of sorbent and bioagent.

 The disadvantage of this method is that when applying this sorbent to the surface of the water, the sorbent and the bioagent are separated. The biosorbent obtained in this way is effective only for cleaning the soil.

 2. The method of producing biosorbent described in patent RU (11) 2299181 (13) C2 (51) IPC C02F З / 34 (2006.01) C12N 1/26 (2006.01) Cl 2R 1/77 (2006.01) Cl 2R 1/645

(2006.01) “BIOCOPBEHT FOR CLEANING WATER SURFACE FROM OIL I.

OF PETROLEUM PRODUCTS)) and a similar method for the production of biosorbent described in patent RU (11) 2318736, (13) C2, (51) IPC C02FЗ / 34 (2006.01), C12N1 / 26 (2006.01) "BIOCOPBEHT FOR PURIFICATION OF WATER BASES FROM BASED OIL PRODUCTS BACTERIA AND YEAST MUSHROOMS. ” A method of manufacturing a biosorbent according to RU (11) 2299181 (13) C2 (51) IPC C02F 3/34 (2006.01) C12N 1/26 (2006.01) C12R 7/77 (2006.01) Cl 2R 1/645 (2006.01) "BIOCOPBEHT FOR WATER SURFACE CLEANING FROM OIL AND OIL PRODUCTS)), consists in immobilizing the biomass of the strains of micromycete Fusrium lateritium HK-204 or Gliocladium into a hydrophobic sorbent of oil

deliquesseps HK-205 or Gliocladium delicepseps HK-206 or a consortium of these strains, through the overgrowth of mycelium fungi sorbent placed on a nutrient medium. Next, the resulting preparation is dried. The sorbent is made of a hydrophobic oil sorbent based on peat. The fungal mycelium is 20-50% by dry weight.

 And a similar method for the production of biosorbent described in patent RU (11) 2318736, (13) C2, (51) IPC C02FЗ / 34 (2006.01), C12N1 / 26 (2006.01) “BIOCOPBEHT FOR PURIFICATION OF WATER BODIES FROM OIL PRODUCTS BASED ON STRAUGE WERE WATER YARRY. GIBOB ". This method of biosorbent production involves the immobilization of the yeast fungi Capdida lirolutis, Capdida guilliermopdii, Pischia guilliermopdii and bacteria cultures Rhodossossus eruthorophoris on an oil hydrophobic sorbent. Arthrobaster sp. in an amount of from 10 to 50 percent (by dry weight) by means of sorbent overgrowth with fungus mycelium, followed by air drying.

 A disadvantage of the above described methods for producing biosorbents is that the process of obtaining the above biosorbent is intermittent, depending on the time of sorbent fouling with the fungi. Hydrophobic sorbent overgrowth with biomass requires additional reactors in which special conditions are maintained, additional drying chambers of hundreds of square meters in size, because the above methods for producing biosorbents were carried out in laboratory conditions in a small volume and are not suitable for industrial production.

3. Close to the claimed method is: a method of obtaining a biological product for cleaning the aquatic environment from oil pollution, described in RF patent N ° 2255052 (13) C2 (51) 7 C02FЗ / 34, C12N11 / 04, B01J20 / 22, B01J20 / 30, C12N11 / 04, C12RE01, C02FZ / 34. С 12Nl 1/04, B01J20 / 22 “METHOD FOR CLEANING THE WATER MEDIA FROM POLLUTION OF OIL PRODUCTS AND BIOLOGICAL PRODUCT FOR CLEANING THE WATER MEDIA FROM POLLUTION OF AMI OIL PRODUCTS”. The method for producing a biological product specified in this patent is that the carrier substance, the microorganism growth factor substance, and the biomass of oil-degrading microorganisms immobilized by dispersing the biomass of the oil-degrading microorganisms in the mass of the carrier material with their physical connection with the carrier substance. As a carrier substance, a composition of Ca-alginate was used. gel, n-alkanes with Sn-Sib and substance-growth factor of microorganisms, while the physical connection of oil-degrading microorganisms with the carrier substance is carried out by the formation of droplets from a mixture of suspended biomass with the ingredients of the composition of the carrier substance and polymerization of droplets with the formation of granules in an aqueous solution containing Ca. ions To immobilize oil-degrading microorganisms by dispersing the biomass of oil-degrading microorganisms in the mass of the carrier substance with their physical connection with the carrier substance, prepare a 1% aqueous mixture with sodium alginate (for example, in an amount of 100 ml for the mentioned amount of biomass suspension), mix it thoroughly and keep it in 2-3 hours to complete the absorption by fragments of sodium alginate. Growth factors, for example, corn extract in the amount of 10 g per 100 ml of the mixture, are added to the thus obtained mixture with sodium alginate. The mixture with sodium alginate and suspended biomass in equal volumes is mixed by means of a homogenizer for 2-3 minutes, then pumped through a nozzle into the zone of exposure to the air stream supplied to the ejection area, for example, to form granules from 0.18 to 0.55 mm at a rate of 0.002 m ³ / min. Drops formed by the action of an air jet on a mixture jet fall into an aqueous solution containing Ca ions (for example, a 2% aqueous solution of CaCl2), where, due to the chemical combination of calcium ions with sodium alginate and polymerization during aging (for example, for 30 minutes), the formation of drops of Ca-alginate gel, they acquire the properties of a separate body and become granules. In this case, there is a physical connection of oil-degrading microorganisms with the substance of the granules, and the granules thus obtained are carriers of the dispersed suspended biomass of immobilized oil-degrading microorganisms and where the composition of the carrier is a composition of Ca-alginate gel, n-alkanes with C HC 16 and substances - growth factor. The granules are filtered, washed and placed in saline.

 The disadvantage of this method is that as a result of its use, a biological product is obtained, which is not a biosorbent, has a short residence time in the aquatic environment - it soaks and decomposes.

 There are biological products where the biological agent is prepared in the form of a water-oil suspension, which ensures quick contact of the biological product with oil products and liquids with oleophilic properties, for example, see RF patent N ° 2180276 Cl from

2001.02.19, “OLEOPHILIC BIOLOGICAL PRODUCT USED FOR CLEANING

OIL-CONTAMINATED GOODS. "

The challenge facing the developers of the proposed method is to develop industrially suitable high-performance method, providing the manufacture of high-quality oleophilic biosorbent, incorporating microorganisms - oil destructors, hereinafter - bioagents.

 Disclosure of invention

A method of immobilizing microorganism cells into a hydrophobic oleophilic sorbent used to clean oil contaminants consists in mixing two aerosols until the sorbent is saturated with a bioagent. The first aerosol consists of a gaseous medium in which sorbent particles are suspended. The second aerosol consists of a gaseous medium in which a mixture of either oil or a petroleum product and a culture fluid with a bioagent is suspended. An oil or oil aerosol contains, respectively, an oil or oil base mixed with a culture liquid with microbial oil-destructive cells with a biotiter of 10 ⁹ - 10 ¹⁰ KOE / ml, which ensures the destruction of oil products absorbed subsequently

biosorbent.

 In the chamber create a mixture of sorbent and oil or oil, non-toxic to the used microorganisms (carriers of biomass of microbial cell strain-oil destructors), aerosols.

 By definition, “Aerosol is a dispersed system consisting of small solid or liquid particles suspended in a gaseous medium (usually in air).” (See - Wikipedia).

 To obtain aerosols, standard equipment is used - sprayers of solid and liquid substances, and dispensers are used to set the required volumes.

 Sorbent particles have a size of 1 ÷ 3 mm in diameter.

 The size of the droplets of oil or oil aerosol 15 ÷ 25 microns.

 Drops of oil or oil aerosol fall on the surface and in the pores of the hydrophobic sorbent, providing quick contact and the introduction of microbial cells into the branched structure of the hydrophobic sorbent. In the chamber, the process of immobilization of microbial cells contained in oil or in oil into sorbent particles continues until the bioagent content is at least 10 living cells per 1 g of sorbent.

 As the sorbent, any hydrophobic oleophilic sorbent, for example, peat sorbent or organomineral sorbent, can be used.

 The technical result of the claimed invention is the creation of an industrial method of immobilization of various bioagents on various hydrophobic oleophilic sorbents, in which it is possible to carry out the process of saturation of the sorbent with bioagents to the required level of bioagent in the sorbent.

- This method is universal for various hydrophobic oleophilic sorbents, because can be used to immobilize any oil-degrading bioagents in various hydrophobic oleophilic sorbents. Given result

achieved by applying to the hydrophobic oleophilic sorbent, in suspension, an aerosol consisting of an oil or oil emulsion containing microorganisms-oil destructors.

 The basis of the proposed method is the property of an oleophilic sorbent to sorb oil or oil. Due to the fact that the sorbent has a porous structure with pore sizes from 100 to 300 μm, aerosol droplets, the size of which can be from 15 to 25 μm, fall on the surface of the sorbent particles, partially settle on its surface and partially penetrate inside its pores. Together with the oleophilic base, bioagents-oil destructors fall on the surface of the sorbent and in its pores.

 The particle size of the sorbent may lie in the range from 1 to 3 mm in diameter. The size of various microorganisms-oil destructors can be from 0.9 to 15 microns, which allows them to be in the composition of aerosol drops and enter the pores of the sorbent. When the sorbent is saturated with a bioagent, the particles of the sorbent become slightly heavier. The sorption ability of the obtained biosorbent almost does not decrease compared to the initial sorbent.

 In the biosorbent obtained in accordance with the claimed method, there is a stably reliable fixation of the bioagent on the sorbent, which allows the use of the biosorbent for both soil cleaning and water purification.

 Using biosorbents obtained by the method according to this invention, sorption and utilization of oil and / or oil products from the surface being cleaned are carried out simultaneously.

 Biosorbents obtained by the present method are characterized by high biodegradation activity in the elimination and intense pollution and thin rainbow films.

 The best embodiments of the invention

 The hydrophobic peat sorbent SORBONAFT (TU 0392-001-55763877-2003) was chosen for the production of biosorbent. Sorbent obtained by the method described in the patent of the Russian Federation N ° 2336125. On the basis of the proposed method, a composition of environmentally friendly hydrophobic peat mineral oil sorbent is developed under the name

"Biosorbonaft". It includes an oleophilic hydrophobic sorbent - Copbonft - 99% and a water-oil emulsion of microorganisms-oil destructors - 1%. The maximum absorption capacity of the sorbent for the reference oil product is 650%, the particle size of the bulk sorbent is 1 - 3.0 mm. This sorbent has a porous structure, well adsorbs oil 350 - 800%, diesel 300-350%. As strains that ensure the destruction of petroleum products - one or more strains of microbial cells ZOOGLOEA SP. 14H, ARTNOVASTER SP. 13H, ARTNOVASTER SP. 15H,

BACILLUS SP. ZN, BACILLUS SP. 12 H USED FOR REMEDIATION OF RESERVOIRS AND SOILS CONTAMINATED BY OIL AND OIL PRODUCTS, OR ASSOCIATION OF STRAINS

MICROORGANISMS-DESTRUCTORS ON THEIR BASIS (Patent for the invention of the Russian Federation JVg 2266958).

B ^'as a carrier oil destructors-microbial biomass cells strains selected liquid oil.

 In another embodiment of the biosorbent, oil from the Usinsky field is selected as a carrier of biomass of strains of microbial cells-oil destructors.

 In the third embodiment of the biosorbent, an organomineral sorbent is selected as the sorbent.

 The inventive method allows you to change the components used to obtain biosorbent.

 Industrial applicability

 The method is economical and simple, does not require complex equipment and high costs in mass production. Mixing of aerosols can be carried out by known means — using pneumatic sprayers for a sorbent and a spray for liquids connected to sorbent and liquid dispensers.

 The inventive method is controllable - the process of saturation of the sorbent with nutrients is controllable due to what is performed in the chamber, while the time of mixing the aerosols and the conditions of the saturation process — air flow intensity, temperature, humidity, pressure in the chamber — are first experimentally selected and then kept constantly. Microbial cell saturation of oil or oil products is also a controlled process.

 Mixing in a closed chamber also allows for

ecological purity of this process.

 When oil products are sorbed into a biosorbent, the collected oil products are mixed with the oil or oil present inside the biosorbent. Inside the microbial cells freely move in the oil-oil or oil-oil environment, while part of the microbial cells from the sorbent gets on the spot of spilled oil products and destroys this spot, being outside the biosorbent particle. The biosorbent, which has absorbed the maximum amount of oil products, can sink to the bottom of the reservoir. It is very likely that bioagents within the biosorbent will destroy the sorbed oil product. Left without food, bioagents will die, and a peat layer will remain at the bottom.

Claims

The method of immobilization of microorganism cells in a sorbent used to clean oil contaminants  1. The method of immobilization of microorganism cells in a hydrophobic oleophilic sorbent used to clean oil contaminants, which consists in mixing an aerosol, including a sorbent and aerosol, including a mixture of either oil, or an oil product and a culture fluid with microbial oil-destroying cells until the sorbent is saturated with microbial oil-testing cells .  2. The method of immobilization of microorganism cells in a sorbent used for cleaning oil pollution according to claim 1, characterized in that the mixing is carried out in a chamber.  3. A method for immobilizing microorganism cells into a sorbent used to clean oil contaminants, which differs from the method according to claim 1 in that the particle size of the sorbent is 1 ÷ 3 mm in diameter.  4. The method of immobilization of microorganism cells in a sorbent used to clean oil contaminants according to claim 1, characterized in that a peat sorbent is used as the sorbent.  5. The method of immobilization of microorganism cells in a sorbent used for cleaning oil contaminants according to claim 1, characterized in that an organomineral sorbent is used as a sorbent. 6. A method of immobilizing microorganism cells into a sorbent used for oil pollution purification according to claim 1, characterized in that one or several microbial cell strains are used as strains providing destruction of oil products: with a biotiter of 10 ⁹ - 10 ^l0 KOE / ml, providing destruction petroleum products absorbed in subsequent biosorbent.  7. A method for immobilizing microorganism cells into a sorbent used for oil pollution purification, which differs from the method according to claim 1 in that the droplet size of the oil aerosol is 15–25 μm. 8. 'A method for immobilizing microbial cells into a sorbent used to clean oil contaminants, which differs from the method according to claim 1 in that the process of immobilizing microbial cells contained in oil or oil into sorbent particles in the chamber is continued to a bioagent level of at least 10 ⁹ living cells per 1 g of sorbent.