RU2181701C2 - Biological preparation "avalon" for cleaning environmental objects to remove petroleum and petroleum derivatives, and method of preparation thereof - Google Patents

Abstract

FIELD: environmental safety. SUBSTANCE: preparation contains porous substrate and strains of destroying microorganisms immobilized in substrate pores and selected for the types of pollutants. Porous substrate is, in particular, foamed glasslike metaphosphates with variable constitution and biodestructor is represented by oil-oxidizing microorganisms. Cleaning procedure includes depositing a sorbent on a surface to be cleaned, after which preparation of invention is applied. EFFECT: essentially simplified transportation of cleaning means to polluted area and enhanced cleaning effect because of higher biological activity of oil-oxidizing microorganisms. 2 cl, 2 tbl, 4 ex

Description

 The present invention, called by the authors "AVALON", relates to means of combating pollution of environmental objects.

 A variety of biological products based on oil-oxidizing microorganisms and their associations are known for purifying the environment from oil and oil products (RF patents 2053205, 1996, 2138451, 1999).

 The closest to the proposed biological product "AVALON" on the set of essential features is the material for biological treatment of waters contaminated with phenol and phenolic compounds "IPK-F" according to the patent of the Russian Federation 2107665, taken by us as a prototype. The prototype contains a porous carrier and biodestructors artificially immobilized into the pores of the carrier.

 The disadvantage of this material is the use of a passive carrier of bacteria that does not provide microorganisms with sources of mineral nutrition, which reduces the oil-oxidizing activity of strains of destructors and reduces the duration of storage of the biological product without loss of quality.

 The aim of the present invention is to develop such a preparation in which optimal conditions are provided for the immobilization and vital activity of microorganism cells on the porous structure for a long time, and their oil-oxidizing activity also increases.

 This goal is achieved by the fact that foamed supercooled quenched melts of metaphosphates of variable composition are used as a carrier, including potassium, magnesium, calcium, sodium, boron, silicon, and trace elements that affect the dissolution rate of the foamed solid carrier in aqueous solutions. As biodestructors, microorganism strains selected to the type of pollution are used. The structure of the foamed glassy metaphosphate carrier provides optimal conditions for the immobilization and vital activity of microorganism cells on the porous structure. At the same time, the conditions for their aeration are significantly improved, and the area of cell nutrition increases. Foamed glassy metaphosphates, being a carrier for oil-oxidizing bacteria, at the same time provide their mineral nutrition. Due to these new properties, there is a significant increase in the biological activity of the drug in relation to hydrocarbons.

 The proposed biological product "AVALON" for cleaning environmental objects from industrial pollution, as well as the prototype, contains a porous carrier and biodestructors artificially immobilized in the pores of the carrier.

The biological product is characterized in that as a porous carrier it contains foamed glassy metaphosphates of variable composition, providing optimal conditions for the immobilization and vital activity of microorganism cells on a porous structure with the following structural formula:
A _m • B _n • [PO ₄ ] _p • Z • X • H ₂ O
where m, n, p are prime integers in the range: m = 2-4; n is 2-6; p = 6-12,
A - monovalent cations of the first group,
In - divalent cations of the second group of the periodic system,
Z - doping trace elements [Zn, Mn, Fe, Cu, Co, Ni, Mo, V, Se, Ag at a concentration of 0.01 ÷ 0.3% each] and
X - additives [SiO ₃ , B ₂ O ₃ , Al ₂ O ₃ , V ₂ O ₅ , SO ₃ , SeO ₃ ], affecting the kinetics of dissolution in the following ratio of components,% mass: KRO ₃ - 10.0 ÷ 35, 0% NARO ₃ - 4 ÷ 8%; Mg (PO ₃ ) ₂ - 4.0 ÷ 18.0%; Ca (PO ₃ ) ₂ - 20.0 ÷ 60.0%;
Z (PO ₃ ) ₂ - 1 ÷ 3%;
X - SiO ₂ - 1.0 ÷ 15.0%; In ₂ About ₃ - 1.0 ÷ 5.0%; Al ₂ O ₃ - 0.1 ÷ 5.0%; V ₂ O ₅ - 0.1 ÷ 0.3%; SO ₃ - 0.2 ÷ 2.0%; SeО ₃ - 0.1 ÷ 0.3%,
and as biodestructors it contains the following strains of microorganisms: Serratia marcescens PL-1, Pseudomonas fluorescens biovar II 10-1, Acidovorax delafieldii 3-1.

The chemical formula of the carrier with the wt.% Is given below:
KPO ₃ - 10 ÷ 35%
NaPO ₃ - 4 ÷ 8%
Mg (PO ₃ ) ₂ - 4 ÷ 18%
Ca (PO ₃ ) ₂ - 20 ÷ 60%
Zn (PO ₃ ) ₂ - 0.1 ÷ 0.8%
Mn (PO ₃ ) ₂ - 0.1 ÷ 0.8%
Fe (PO ₃ ) ₂ - 0.1 ÷ 0.8%
Cu (PO ₃ ) ₂ - 0.1 ÷ 0.8%
Co (PO ₃ ) ₂ - 0.1 ÷ 0.8%
Ni (PO ₃ ) ₂ - 0.1 ÷ 0.8%
Na ₂ B ₄ O ₇ - 0.5 ÷ 5.0%
K ₂ MoO ₄ - 0.1 ÷ 0.3%
K ₂ SeO ₄ - 0.1 ÷ 0.5%
Na ₂ SO ₄ - 0.1 ÷ 1.0%
AgJ - 0.01 ÷ 0.1%
CaV ₂ O ₆ - 0.1 ÷ 0.5%
Na ₂ SiO ₃ - 1.0 ÷ 15.0%
Al ₂ O ₃ - 0.1 ÷ 5.0%
The closest to the proposed method for the preparation of the biological product "AVALON" on the set of essential features is the method according to the patent of the Russian Federation 2116145, which carry out the deep cultivation of strains of destructors on a liquid nutrient medium, prepare a sterile carrier and carry out surface cultivation of microorganisms on the carrier.

 The known method does not allow to obtain a biological product with high destructive activity and a high titer of viable cells.

In the proposed method for producing a biological product for purification of environmental objects from oil and oil products, including deep cultivation of biodestructors on a liquid nutrient medium, preparation of a sterile carrier and surface cultivation of biodestructors on a carrier, it is proposed to use foamed glassy metaphosphates of variable composition providing optimal conditions for immobilization and vital activity of microorganism cells on a porous structure in the process of their surface rest cultivation with the following structural formula:
A _m • B _n • [PO ₄ ] _p • Z • X • H ₂ O
where m, n, p are prime integers with an arbitrary value of p are within the relationship: (m + n) / p = 0.5 ÷ 1.0,
A - monovalent cations of the first group,
In - divalent cations of the second group of the periodic system,
Z - doping trace elements [Zn, Mn, Fe, Cu, Co, Ni, Mo, V, Se, Ag at a concentration of 0.01 ÷ 0.3% each] and
X - additives [SiO ₃ , B ₂ O ₃ , Al ₂ O ₃ , V ₂ O ₅ , SO ₃ , SeO ₃ ], affecting the kinetics of dissolution in the following ratio of components,% mass: KRO ₃ - 10.0 ÷ 35, 0% NaPO ₃ - 4 ÷ 8%; Mg (PO ₃ ) ₂ - 4.0 ÷ 18.0%; Ca (PO ₃ ) ₂ - 20.0 ÷ 60.0%;
Z (PO ₃ ) ₂ - 1 ÷ 3%;
X - SiO ₂ - 1.0 ÷ 15.0%; In ₂ About ₃ - 1.0 ÷ 5.0%; Al ₂ O ₃ - 0.1 ÷ 5.0%; V ₂ O ₅ - 0.1 ÷ 0.3%; SO ₃ - 0.2 ÷ 2.0%; SeO ₃ - 0.1 ÷ 0.3%,
and as biodestructors it contains the following strains of microorganisms: Serratia marcescens PL-1, Pseudomonas fluorescens biovar II 10-1, Acidovorax delafieldii 3-1.

 For the industrial implementation of the invention, typical biotechnological equipment and materials are used.

 Consider the application of the proposed solutions in the following examples.

 Example 1

In a first specific example, the biological product contains bacterial strains of Serratia marcescens PL-1, Pseudomonas fluorescens biovar II 10-1. Pure cultures of microorganisms are isolated from soils contaminated with oil products in selective nutrient media containing hydrocarbons of oil products as the sole source of carbon nutrition. The strains are registered in the microorganism collection of the All-Russian Scientific Research Institute of Plant Protection KMZR VIZR. The strains are stored separately on an agarized nutrient medium (MPA or SPA) at a temperature of 0 + 5 ^o C or in a lyophilized state in ampoules.

Strain Serratia marcescens PL-1, straight sticks with a peritrichous arrangement of flagella, size 0.8 x 1.0-1.6. On standard nutrient media (MPA, SPA, Becton Dickinson) forms round colonies of 3 mm or more in diameter, convex, smooth, the edges are even, red, opaque. Gram stain method Gregersen (1978) - gram-negative. Optional anaerobic. Hemoorganotroph. The enzymatic type of metabolism in the test O \ F. Forms acid from lysine and ornithine. Does not ferment arabinose, xylose. The Voges-Proskauer reaction is positive. Test methyl red negative. Disposes of citrate in Simmons medium. Hydrolyzes gelatin. It has lysine and ornithine decarboxylase. Does not have arginine dehydrolase. Does not form hydrogen sulfide, does not destroy urea. Reduces nitrates to form NO ₂ ^- .

The strain Pseudomonas fluorescens biovar II 10-1 is motile with a polar flagellum, slightly curved rods 0.8-0.9 x 1.1-3.0 in size. On standard nutrient media (MPA, SPA, Becton Dickinson) forms round colonies of 2 mm or more in diameter, convex, smooth, the edges are even, colorless, opaque. Gram stain method Gregersen (1978) - gram-negative. Aerobe. It is able to grow under anaerobic conditions in the presence of nitrates in a nutrient medium. Hemoorganotroph. The oxidative type of metabolism in the O \ F test. Oxidase and catalase are positive. Forms a fluorescent pigment. It grows at +4 ^o С, does not grow at +41 ^o С. It forms levan from sucrose. It has arginine dehydrolase, gelatinase - dilutes gelatin, is capable of denitrification. Starch does not hydrolyze. It utilizes glucose, trehalose, inositol, arginine.

 To obtain seed, the strains were grown separately; microorganisms were co-cultured in the fermenter.

For the cultivation of microorganisms used nutrient medium of the following composition (g / l):
K ₂ NRA ₄ - 1,0
NaNO ₃ - 3.0
KCl - 0.5
MgSO ₄ - 0.3
FeCl ₃ • 6H ₂ O - 0.05
NH ₄ NO ₃ - 2.0
Water - Up to 1 L
Cornmeal - 5.0
Sucrose - 20.0
The cultivation process was carried out in a fermenter at T = 20 ^o C, pH 6.5-7.0 for 24-36 hours under aerobic conditions. As an inductor, 0.02% crude oil was used. After that, immobilization of microorganisms on a porous carrier is carried out by mixing it with a culture fluid containing microorganisms with a titer of 2-5.10 ⁹ cells / ml.

Surface cultivation on a porous support was carried out at 20 ^{° C.} for 72 hours. The titer of a biological product 10 ¹² viable cells in 1 g

Example 2
In the second example, the biological product contains strains of Acidovorax delafieldii 3-1 and Serratia marcescens PL-1.

 Strain Acidovorax delafieldii 3-1 - mobile with polar flagella rods of size 0.6 x 0.8-2.5, located singly and in pairs. On standard nutrient media (MPA, SPA, Becton Dickinson) forms round colonies of 2 mm or more in diameter, convex, smooth, the edges are even, colorless, shiny, opaque.

Gram stain method Gregersen (1978) - gram-negative. Aerobe. Hemoorganotroph. It does not need growth factors. Oxidase and catalase are positive. It grows at +4 ^o С, does not grow at +41 ^o С. Not capable of denitrification. Does not form levan from sucrose. Gelatin does not thin. Starch does not hydrolyze. It uses glucose, glutamate, xylose, ribose, malonate as the sole carbon source, does not use trehalose, inositol, rhamnose and sucrose.

 Deep cultivation of microorganisms was carried out on the same nutrient medium as in the first example.

Surface cultivation on a porous support was carried out at 20 ^{° C.} for 96 hours. The titer of a biological product is 9.10 ¹¹ viable cells in 1 g.

Example 3
In the third example, the Avalon biopreparation samples obtained as described above were used to clean soil contaminated with fuel oil or crude oil. 5 g of sorbent based on foamed metaphosphates were added to the vessels containing 1 kg of soil, the soil was thoroughly mixed, and then the AVALON biological product was added to the vessels in an amount of 1 g, periodically loosening and watering the soil. The sorbent and biological product were not added to the control vessels. The oil content was determined by IR spectrophotometry on the device AN-2 (see table. 1)
where the composition of the sorbent AVA-7:
KRO ₃ - 31.0%
NARO ₃ - 4.0%
Mg (PO ₃ ) ₂ - 8.0%
Ca (PO ₃ ) ₂ - 48.0%
SiO ₂ - 4.4%
In ₂ About ₃ - 3.0%
SO ₃ - 0.2%
V ₂ About ₅ - 0.2%
ZO - Zn, Mn, Fe, Cu, Mo, Co, - 0.2% of each Σ = 1.2% wt. for synthesis
The pore size of the sorbent is not limited, and the density is selected less than 1 g / cm ³ to ensure its retention on the surface of the water.

 After 28 days, the biological activity was 75-90% in various experimental variants. In the control, the content of petroleum products decreased slightly.

Example 4
In plastic cuvettes 21 x 34 cm in size, filled with water (V = 3 L), 2 ml of a mixture of oil and fuel oil was added in a 2: 1 ratio. A sorbent based on foamed metaphosphates in an amount of 3 g was sequentially applied to the water surface, followed by the AVALON biological product in an amount of 1 g. A mixture of oil and fuel oil without sorbent and biological product was introduced into the control vessels.

3-4 days after applying AVALON to a water surface contaminated with fuel oil, a bacterial film formed around the particles of the biological product, which indicates that it is a source of nutrition for microorganisms. After 1 month, a rainbow film was completely absent on the surface of the water. 2 months after the start of the experiment, the aqueous phase was separated from the precipitate by filtration through a paper filter, then the concentration of oil products in the precipitate and in water was determined by IR spectrophotometry on an AN-2 instrument. The results of the study are shown in table 2,
where the composition of the sorbent AVA-8:
KRO ₃ - 33.0%
NARO ₃ - 1.0%
Mg (PO ₃ ) ₂ - 12.3%
Ca (PO ₃ ) ₂ - 44.0%
SiO ₂ - 5.2%
In ₂ About ₃ - 2.0%
SO ₃ - 0.2%
SeO ₃ - 0.2%
ZO - Zn, Mn, Fe, Cu, Mo, Co, Ni, each Σ = 1.2% wt. for synthesis
Analysis of the results showed that in all variants of the experiment, the process of biodegradation of oil products was actively passed (79.6-94.9%).

As a result of the experiments, we can state:
- an increase in the duration of stay of oil-oxidizing microorganisms in the environment by 3-4 times, which ensures an active process of biodegradation without additional introduction of a biological product;
- providing indigenous microflora with mineral nutrition, which increases the efficiency of cleaning environmental objects from technogenic pollution and the restoration of biocenotic links disturbed as a result of these technogenic pollution.

Claims (2)

1. Biological product for cleaning environmental objects from oil and oil products, including a porous carrier and biodestructors, artificially immobilized in the pores of the carrier and selected to the type of impurities, characterized in that as a porous carrier it contains foamed glassy metaphosphates of variable composition, providing optimal conditions for immobilization and vital activity of microorganism cells on a porous structure with the following structural formula:
A _m • B _n • [PO ₄ ] _p • Z • X • H ₂ O,
where m, n, p are prime integers in the range: m = 2-4; n is 2-6; p = 6-12;
A - monovalent cations of the first group;
B - divalent cations of the second group of the Periodic system;
Z - doping trace elements [Zn, Mn, Fe, Cu, Co, Ni, Mo, V, Se, Ag at a concentration of 0.01-0.3% each];
X - additives [SiO ₃ , B ₂ O ₃ , Al ₂ O ₃ , V ₂ O ₅ , SO ₃ , SeO ₃ ], affecting the kinetics of dissolution in the following ratio of components, wt. %:
CRO ₃ 10.0-35.0%; NARO ₃ 4-8%; Mg (PO ₃ ) ₂ 4.0-18.0%; Ca (PO ₃ ) ₂ 20.0-60.0%; Z (PO ₃ ) ₂ 1-3%;
X is SiO ₂ 1.0-15.0%; B ₂ O ₃ 1.0-5.0%; Al ₂ O ₃ 0.1-5.0%; V ₂ O ₅ 0.1-0.3%; SO ₃ 0.2-2.0%; SeO ₃ 0.1-0.3%,
and as biodestructors it contains the following strains of microorganisms: Serratia marcescens PL-1, Pseudomonas fluorescens biovar II 10-1, Acidovorax delafieldii 3-1. 2. A method of obtaining a biological product for cleaning environmental objects from oil and oil products, including the deep cultivation of biodestructors on a liquid nutrient medium, the preparation of a sterile carrier and the surface cultivation of biodestructors on a carrier, characterized in that foamed glassy metaphosphates of variable composition are used as a carrier, providing optimal conditions for immobilization and vital activity of microorganism cells on a porous structure in the process of their surface ultivirovaniya the following structural formula:
A _m • B _n • [PO ₄ ] _p • Z • X • H ₂ O
where m, n, p are prime integers in the range: m = 2-4; n is 2-6; p = 6-12;
A - monovalent cations of the first group;
B - divalent cations of the second group of the Periodic system;
Z - doping trace elements [Zn, Mn, Fe, Cu, Co, Ni, Mo, V, Se, Ag at a concentration of 0.01-0.3% each];
X - additives [SiO ₃ , B ₂ O ₃ , Al ₂ O ₃ , V ₂ O ₅ , SO ₃ , SeO ₃ ], affecting the kinetics of dissolution in the following ratio of components, wt. %:
CRO ₃ 10.0-35.0%; NARO ₃ 4-8%; Mg (PO ₃ ) ₂ 4.0-18.0%; Ca (PO ₃ ) ₂ 20.0-60.0%; Z (PO ₃ ) ₂ 1-3%;
X is SiO ₂ 1.0-15.0%; B ₂ O ₃ 1.0-5.0%; Al ₂ O ₃ 0.1-5.0%; V ₂ O ₅ 0.1-0.3%; SO ₃ 0.2-2.0%; SeO ₃ 0.1-0.3%,
and as biodestructors it contains the following strains of microorganisms: Serratia marcescens PL-1, Pseudomonas fluorescens biovar II 10-1, Acidovorax delafieldii 3-1.

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