Patent Application
20140329677
The invention relates to a novel biological product for purification of water, soil and industrial wastewater contaminated with chemicals resistant to degradation categorized as hazardous, such as the widely used in agriculture pesticides.
Biological preparations—biodestruktors and their use for the treatment of soil and ground from oil and petroleum products containing Bacillus brevis and Arthrobacter species are described in the patents RU 2323970 and RU 2237711. The patent RU 2086667 describes a consortium comprising the microorganisms Pseudomonas putida and Bacillus subtilis.
There are also known biological preparations “Roder” based on strains Rhodococcus ruber VKM Ac-1513D and Rhodococcus erythropolis VKM Ac-1514D, “Lenoil”, “Devoroil”, “Ekobel” based on strains of bacteria Pseudomonas and yeast, which are effectively used to remove aliphatic fractions of oil.
There is known decomposition of chlorinated aromatic pesticides (2,4-dichlorofenoxyacetic acid and 2,4,5-trichlorofenoxyacetic acid) using bacteria Pseudomonas pseudoalcaligenes strain NRRL B-18087 (U.S. Pat. No. 4,804,629), detoxification of organophosphorus pesticide residues and their toxic metabolites—by a strain of bacteria Agrobacterium radiobacter (author's certificate SU 1250572). Author's Certificate SU 1560487 describes a method for destruction of the pesticide 3,4-dichloranilide using algae Chlorella vulgaris BKM-A-10 and Scenedesmus acuminalus UA-2-7a. Decomposition of 2-chlorobenzoic acid using bacteria Pseudomonas putida YNK-1 is being described in U.S. Pat. No. 4,803,166; phosphate contaminants and chlorinated hydrocarbons—by microorganisms Moraxella and Arthrobacter alone or in combination—in patent DE 3729127.
There is known degradation of compounds such as DDT, polychlorphenols, benzopyrenes, dioxines using basidiomycete Phanerochate chrysosporium (U.S. Pat. No. 4,891,320).
Strain Pseudomonas putida—106 is an active destructor of dimethylphenylcarbinol and phenol [1, 2], the bacteria Pseudomonas pseudoacaligenes destroys the aromatic and heterocyclic compounds, most often found in wastewater [3, 4, 5], and the strain of Pseudomonas pseudoacaligenes destroys the aromatic compounds in the solid and liquid medium.
Some museum strains of Nocardia, Arthrobacter, Micromonospora [7, 8, 9] have capacity for destruction of chlorotriazine derivatives, such as simazine and atrazine. U.S. Pat. No. 6,632,363 describes a composition comprising a hydrophobic carrier, and the bacteria Bacillus subtilis, and the method of its use for improving the quality of water containing, for example, pesticides.
Ksenofontova O. et al describes as the most active degraders, strains of aerobic bacteria from the genera Bacillus and Pseudomonas, which destroy the following pesticides: chlorothiazide, juglone, semiquinone, nitrolon, kartocid and karate (“Evolution of soil microorganisms under the influence of pesticides”. Proceedings of the Saratov State University, Scientif. Section “Chemistry and Biological Ecology”, 2007, vol. 7, No. 2, p. 66).
Patent RU 2410170 describes a method for purification of contaminated soil from organic compounds, including pesticides, for example, dichlorobiphenyl (DHB), by adding a sorbent, which is of glauconite breed, pretreated at 200-300° C. and strain of bacteria of the genus Rhodococcus.
In all these methods of detoxification of toxic chemicals (toxic substances) for the decomposition of specific chemicals by certain kinds of microorganisms are offered. Mostly there is described the decomposition of pesticides in the aquatic media by microorganisms utilizing xenobiotics as a sole source of carbon and energy, or there is required the additional use of sorbents. A significant drawback of all these cultures is their narrow specificity for a particular substrate, and that they do not have preparative form and are not used in practice for the treatment of water, industrial wastewater and soil contaminated by hard-degradable chemicals such as pesticides.
Patent RU 2093478 describes the association of bacterial strains Pseudomonas putida VKM 1301, Bacillus subtilis VKM B-1742D and Rhodococcus erythropolis VKM Ac-1339D in the ratio of 1:1:1 for purification of water and soil from oil and petroleum products and polymer additives contained in the drilling mud and its use. However, the use of this association for the purification of water, soil and industrial waste water contaminated with persistent against degradation chemicals such as pesticides, have not previously been known.
There is known biological product “Biava” (patent RU 2248255), which improves soil fertility and stimulates the natural microflora of the medium. Biological product contains amylolytic, proteolytic and nitrogen-fixing microorganisms such as bacteria genus Pseudomonas, Bacillus. The disadvantage of this product is its complex composition, comprising more than 25 types of different organisms not adapted for the associative interaction, which is complicated for production and for maintenance of the strains. The use of a biological product “Biava” by its introduction into the soil is also described in the said patent. This product is not a stimulant of plant growth and has no ability to degradation of persistent chemicals such as pesticides or herbicides from a group of chlorophenoxy acetic acid: 2,4-dichlorofenoxyacetic acid (2,4-D), trichlorophenoxy acetic acid (2,4,5,-T), phenol, 2,4-dichlorophenol, CPAA and other chemical compounds that are described below. These compounds are among the most consistent and persistent in the environment. Intake of these chemicals by e.g. a human can cause serious diseases of organs, tissues, and nervous system. Their presence in food is considered inadmissible (see, e.g. Tinsley, I. Chemical Concepts in Pollutant Behavior. Transl. from English. M.: Mir, 1992, p. 281).
The objective of the present invention is to provide a new biological product for treatment (detoxification) of soil, water and industrial wastewater contaminated with persistent chemicals such as pesticides, phenols, as well as to provide the method of use of the product. The product “PHENOX” of the present invention improves the purification of the environment from toxic substances, and as a consequence, improves the quality of consumer products grown in the treated soil. In addition the microbial degradation of toxic substances to non-hazardous compounds carried out by bacterial cells occurs in the soil. Biological product shows high efficiency in the treatment of industrial wastewater.
The present invention refers to the bioproduct (“PHENOX”) for treatment of ground, soil, industrial wastewater from chemicals resistant to degradation such as pesticides. The offered bioproduct is an association of new strains of bacteria Pseudomonas putida, Bacillus subtilis and Rhodococcus erythropolis in a mass ratio of (1-2):(1-2):(1), which may further comprise a sorbent or to be immobilized on a sorbent, and mineral, organic or stimulating additives.
The new strain Pseudomonas putida was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under the accession number B-10997 (SEQ ID No. 3).
The new strain Bacillus subtilis was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under the accession number B-10999 (SEQ ID No. 1).
The new strain Rhodococcus erythropolis was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under the accession number Ac-1882 (SEQ ID No. 2).
The difference of the proposed product “PHENOX” compared with the prior art solutions is the specified association of strains of bacteria and the given preferable mass ratio.
The product is particularly effective when used for the decomposition of persistent pesticides, selected from the group of chlorofenoxyacetic acids such as 2,4-dichlorofenoxyacetic acid (2,4-D), trichlorofenoxyacetic acid (2,4,5-T), 2,4-dichlorophenoxy-α-propionic acid (Dichloroprop, 2,4-DR), 2-methyl-4-chlorophenoxy-α-propionic acid (Mekoprop, 2M-4HP, MSRR), 2,4,5-trichlorophenoxy-α-propionic acid (2,4,5-TP, Silvex), 2,4-dichlorophenoxy-α-oil (2,4-DV), methyl-[1-[(butylamino) carbonyl]-1H-benzimidazole-2-yl]carbamate and the product on its basis (benomyl, carbendazin, benazol); imidor, wherein the active substance is neonicotinoid imidacloprid; zontran, wherein the active substance is metribuzin, HCCH (hexachlorocyclohexane, hexachloran hexatox, dolmix, sineks) CPAA (chlorofenoxyacetic acid), PAA (fenoxyacetic acid), hexachlorophenol, 1,1-di(4′-chlorophenyl)-2,2-dichloroethane (DDT) and 2,4-dichlorophenol and phenols. Particularly the product is effective when used for the decomposition of persistent pesticides from the group of chlorofenoxyacetic acids, such as—2,4-dichlorofenoxyacetic acid (2,4-D), trichlorofenoxyacetic acid (2,4,5-T), chlorofenoxyacetic acid (CPAA), fenoxyacetic acid (PAA), as well as 2,4-dichlorophenol and phenol, and imidacloprid.
The product of the present invention unlike to the previously known products has additional growth stimulating effect on seed germination and growth of cultivated plants, and also has fungicidal activity.
The invention comprises also a method for use of the offered biological product “PHENOX”. Method consists of introducing of an effective amount of the product in contaminated ground, soil or wastewater.
Preferably, the product is introduced in an amount of 10 kg of dry matter per 1 ha of land during ploughing at an ambient temperature, preferably at a temperature of 15°-35° C. The preferred methods of administration are in the form of an aqueous solution by irrigation. The product can also be introduced to the ground by seeds treated with the product suspension with addition of biogenetic elements, such as vermicompost, or the introduction of a dry formulation of the product in the form of powder into the ground during ploughing or sowing.
The product can be introduced into the soil also by sowing seeds treated with the product suspension with the addition of additives, such as vermicompost. The product is diluted with water in the container by addition of vermicompost, nitrogen and phosphorus fertilizers.
The introduction of a dry formulation of the product in the form of powder in the ground during of ploughing or seeding is possible. The product “PHENOX” proposed according to the invention has a stimulating effect on plant growth, and strains of the product—good ability for adaptation to soil, which leads to an efficient detoxification of the environment. Strains of Pseudomonas putida and Rhodococcus erythropolis carry D-plasmids of the degradation and are able to transmit the property of destruction of pesticides and other harmful substances to indigenous bacterial populations in soil, water, wastewater, thereby increasing clearing from pollutants. The advantage of using this product is the decomposition of pesticides in soil for up to 86% and in liquid medium up to 99%.
The active basis of the offered product is the association of soil bacterial strains Pseudomonas putida, Bacillus subtilis, Rhodococcus erythropolis. All these cultures have polisubstratum specificity simultaneously to multiple xenobiotics: chlorofenoxyacetic acids, such as—2,4-dichlorofenoxyacetic acid (2,4-D), trichlorofenoxyacetic acid (2,4,5-T), phenol, 2,4-dichlorophenol, CPAA and other specified in the examples. Their mixture ensures enhanced purification function of the environment from toxic compounds. As sorbents kaolin and finely crushed peat are used also serving as an organic additive; as organo-mineral and growth stimulating supplements vermicompost, its extract and chitin containing substrates. Before use dry powder of the product is mixed well in the container (bucket, jar) with warm water (25-30° C.) until a homogeneous suspension. 10 g of the product is stirred into 10 liters of water. It is recommended to add azophoska-ammonium nitrate based compound fertilizer (5 g/10 l) or compost (5 g/10 l), suspension periodically is stirred for aeration and microbial activation within 1-3 hours before use (watering the soil by watering-can (10-20 l/10 m2) or treatment of seeds 100-150 ml/100 g).
The biological product is obtained by a separate submerged cultivation, usually within 20-30 hours 3 strains of the said bacteria on the standard biotechnology equipment. Seeds are being obtained by growing strains in synthetic medium with the addition of pesticides and this material is being used for seeding large volume of fermenters.
Accumilated dry biomass of cells of the strains Pseudomonas putida, Bacillus subtilis, Rhodococcus erythropolis is mixed in a ratio of (1-2):(1-2):1. The total titre presents at least 5×108 CFU/g of dry product after addition of carrier-sorbent, and other additives (the source concentrate of 1010-11 and at least 5×108 CFU/mL liquid product). For the industrial production and packaging of the proposed product the standard biotechnological and packaging equipment and materials are used.
The offered bioproduct (“PHENOX”) eliminates penetration into plants, vegetables and berries plant protection chemicals, stimulates seed germination and plant growth, inhibits growth of fungal phytopathogens.
“PHENOX” is used for treating of contaminated by chemicals such as pesticides, phenol, possibly oil—water, industrial wastewater, soil and plant seeds. The strains contained in “PHENOX” are new.
The strain Bacillus subtilis B-10999 is isolated by the enrichment culture method from soil samples from the industrial zone of enterprises of chemical industry of the South Ural. The strain Bacillus subtilis was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under the number B-10999. The strain of Bacillus subtilis is identified on the basis of nucleotide sequence analysis of 16S rRNA gene (SEQ. ID No. 1). The nucleotide sequence (length not less than 500 base pairs) of DNA fragment encoding the 16S rRNA gene, has similarity with the declared type of 99%.
Culture-Morphological Features.
The strain Bacillus subtilis has typical culture-morphological features for this kind. Rods, the size of which is 0.2-0.5 at 1.5-3 μm, single, often in pairs. They form oval and round endospores. The flagella are placed over the entire cell's surface. When grown on meat-peptone broth the culture forms developed matte film, the media, however, remains clear. When shaking in liquid medium, the complete dispersion of biomass does not occur. Colonies are dry, finely wrinkled, with wavy edge. On slices of potato growing is abundant in the form of colonies of a cream colour smooth, strongly big size plicate. Gram-positive.
Physiological and Biochemical Features.
The strain Bacillus subtilis grows well on glucose-peptone medium, LB agar and broth, glucose-mineral medium at a temperature of 28° C. It grows in media containing L-proline, DL-leucine, α-ketoglutarate, DL-α-alanine, L-glutamine, D(+)-xylose, L-asparagine, chitin, DL-serine, glucose, phenol and chlorophenoxy carbolic acids as the sole source of carbon. The strain Bacillus subtilis can be stored in a lyophilized state. Checking of the viability of the strain is carried out by planting of it on glucose-peptone agar, M9 and LB—once in 12 months. When it is stored on slants of these media at 5° C.—replanting is carried out every 3 months. The strain Bacillus subtilis is a non-pathogenic and non-toxic micro-organism. Intravenously infected white mice do not show zoo pathogenic properties.
The strain Rhodococcus erythropolis Ac-1882 is isolated by the enrichment culture method from soil samples from the industrial zone of enterprises of chemical industry of the South Ural. The strain Rhodococcus erythropolis was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under the number Ac-1882. The strain Rhodococcus erythropolis is identified on the basis of nucleotide sequence analysis of 16S rRNA gene (SEQ. ID No. 2). The nucleotide sequence (length not less than 500 base pairs) of DNA fragment encoding the 16S rRNA gene, has similarity with the declared type of 99%.
Culture-Morphological Features.
The strain Rhodococcus erythropolis has typical culture-morphological features for this kind. Cells rod-shaped, rarely branched, the diameter 0.6-0.8 μm, the length—3-8 μm. Cells were moderately polymorphic, often arranged in a V-shape, have an explicit cycle of development coccus-rod-coccus: at the age of 24-36 hours rod-shaped cells begin to shorten and the share of coccoid and oval cells increase. Grown-up colonies are pigmented, orange coloured. The consistency of colonies is paste-like. Gram-positive.
Physiological and Biochemical Features.
The strain Rhodococcus erythropolis grows well on glucose-peptone medium, LB agar and broth, glucose-mineral medium at a temperature of 28° C. It grows in media containing L-proline, D-arabinose, DL-α-alanine, L-glutamine, D(+)-xylose, L-asparagine, DL-serine, casein, glucose, phenol and chlorophenoxy carbolic acids as the sole source of carbon. The strain Rhodococcus erythropolis may be stored in a lyophilized state. Checking of the viability of the strain is carried out by planting of it on glucose-peptone agar, M9 and LB—once in 12 months. When it is stored on slants of these media at 5° C.—replanting is carried every 3 months. The strain Rhodococcus erythropolis is a non-pathogenic and non-toxic micro-organism. Intravenously infected white mice do not show zoo pathogenic properties.
The strain Pseudomonas putida B-10997 is isolated by the enrichment culture method from soil samples from the industrial zone of enterprises of chemical industry of the South Ural. The strain Pseudomonas putida was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under the number B-10997. The strain Pseudomonas putida is identified on the basis of nucleotide sequence analysis of 16S rRNA gene (SEQ. ID No. 3). The nucleotide sequence (length not less than 500 base pairs) of DNA fragment encoding the 16S rRNA gene, has similarity with the declared type of 99%.
Culture-Morphological Features.
The strain Pseudomonas putida has typical culture-morphological features for this kind. Motile rods with polar located flagella. The size of the cells is 1-4 on 1.3-5.4 μm. Colonies on meat-peptone agar are grayish coloured, within the colonies and at their reverse side there is a reddish-brownish pigment. Spores do not occur. The consistency of the colonies is viscid. Gram-negative.
Physiological and Biochemical Features.
The strain Pseudomonas putida grows well on glucose-peptone medium, LB agar and broth, glucose-mineral medium at a temperature of 28° C. Grows in media containing DL-tyrosine, DL-tryptophan, D(+)-xylose, D(−)-mannitol, L-glutamic acid, DL-leucine, α-ketoglutarate, DL-α-alanine, D-ribose, L(+)-arabinose, L-glutamine, cellulose, L-asparagine, chitin, DL-serine, pectin, glucose, casein, and phenol and chlorophenoxy carbolic acids as sole source of carbon. The strain Pseudomonas putida can be stored in a lyophilized state. Checking of the viability of the strain is carried out by planting of it on glucose-peptone agar, M9 and LB—once in 12 months. When it is stored on slants of these media at 5° C.—replanting is carried out every 3 months. The strain Pseudomonas putida is a non-pathogenic and non-toxic micro-organism. Intravenously infected rabbits do not show zoo pathogenic properties.
Identification of plasmid degradation of pesticides in the strains. Destructive properties of bacteria can be determined by extrachromosomal elements [Don, Pemberton, 1985; Ghosaletal., 1985]. These elements—plasmids were found in the strains Pseudomonas putida VKPM B-10997 and Rhodococcus erhytropolis VKPM Ac-1882. They were isolated by alkaline lysis method (Maniatis T., Fritsch, E., and Sambrook, J. Methods of genetic engineering. Molecular cloning: Trans. from English./Ed. AA Baev.—M.: Mir, 1984. p. 480) from the cells of these strains and then plasmid products were fractionated in agarose gel under standard conditions (Maniatis et al., 1984)—(see
Compatibility of strains contained in the product. The absence of an antagonistic activity between strains of the bacteria Bacillus subtilis VKPM B-10999, Rhodococcus erythropolis VKPM Ac-1882 and Pseudomonas putida VKPM B-10997 of the product was determined by the growth of strains in the contact lines in the media and by the method of agar blocks. On the LB agar medium the stroke of strain B. subtilis VKPM B-10999 was applied and perpendicular to it strokes of strains Rhodococcus erythropolis VKPM Ac-1882 and Pseudomonas putida VKPM B-10997 were applied. On the next cup on nutrient medium the stroke Rhodococcus erythropolis Ac-1882 was applied and perpendicular to it strains Bacillus subtilis B-10999 and Pseudomonas putida B-10997 were applied. On the third cup on medium the stroke of Pseudomonas putida B-10997 was applied and perpendicular to it the strains Bacillus subtilis B-10999 and Rhodococcus erythropolis Ac-1882 were applied. In the regions of contact lines inhibiting the growth of any of the strains was not observed. When the experiment was carried out using agar blocks, on the lawn of one of the strains on LB medium blocks of 3-days cultures of the other two strains were laid. No experiments showed inhibiting the growth of strains. This indicated the absence of antagonistic interactions between the strains and their possible effective joint application.
Examples of compositions of the biological product “PHENOX”
Bioproduct “PHENOX”, its form—a dry powder. Dry biomass of cells (concentrate)—Bacillus subtilis VKPM B-10999 no less than 5×109 CFU/g, Pseudomonas putida VKPM B-10997 at least 5×109 CFU/g and Rhodococcus erythropolis VKPM Ac-1882 at least 5×108 CFU/g in the ratio (1-2):1:(1-2) (part of mass—76.5-90%); without or with the introduction of sorbent—finely milled peat—1-10% and/or stimulating additives, also containing mineral salts and trace elements—vermicompost—0.1-1%; water, not more than 10.0%; the total titre of living cells for at least 1010 CFU/g.
Bioproduct “PHENOX”, in the form of a dry powder. Dry biomass of cells—Bacillus subtilis VKPM B-10999, Pseudomonas putida VKPM B-10997 and Rhodococcus erythropolis Ac-1882 in the ratio 1:1:1—5-30%; sorbent—finely milled peat-1-10% and/or stimulating supplement containing also mineral salts and trace elements—vermicompost—0.1-1%; water, not more than 10.0%; sorbent and filler kaolin—up to 100%; the total titre of viable cells for at least 5×108 CFU/g.
Bioproduct “PHENOX”, in the form of a dry powder. Dry biomass of cells—Bacillus subtilis VKPM B-10999, Rhodococcus erythropolis Ac-1882 and Pseudomonas putida VKPM B-10997 in the ratio 1:1:1 in total—5-30%; water, not more than 10.0%; kaolin—up to 100%; the total titre of viable cells for at least 5×108 CFU/g.
Bioproduct “PHENOX” in liquod form: cells of bacteria Bacillus subtilis VKPM B-10999, Rhodococcus erythropolis Ac-1882 and Pseudomonas putida VKPM B-10997 in the ratio 1:1:1 (total titre—109 CFU/ml) in culture medium without additives or with addition of biologically active substances (extract of compost or vermicompost—1-5 ml/l or finely milled compost or vermicompost 0.1% and/or chitin or chitosan or chitin containing raw materials—0.05-0.4%). Possible to store at 0-8° C. for at least 3 months, at temperatures of 18-25° C. up to 15 days. When stored at temperatures of 18-25° C. it is allowable to decrease the number of viable cells to a titre of 107 CFU/ml.
The method of use of the offered product is explained by the following examples:
Use of the product “PHENOX” for treatment of water from imidacloprid. The product “PHENOX” in the form of a dry powder in the following composition: concentrate of the bacterial cells in ratio of 1:2:1 with a total titre 5×1010 CFU/g (the composition of the product according to Example 1) in the amount of 100 mg was introduced in 200 ml of tap water contaminated with a pesticide (insecticide) imidacloprid at concentrations of 1 mg/L and 2 mg/l of an active ingredient. In addition in the aquatic medium additives of mineral salts were added (g/l): Na2HPO4.7H2O—6.4; KH2PO4—1.5; NaCl 0.25 g/l; NH4Cl 0.5 g/l. For the control the samples of water contaminated with imidacloprid at concentrations of 1 mg/L and 2 mg/l with mineral salts without biological product were used. The samples were incubated at ambient temperature with constant stirring in a shaker (120 rpm). The experiment was repeated 3 times. The content of imidacloprid in water was determined by standard chromatographic methods at the 1st, 3rd, 5th, 7th and 10th day (Rudakov et al, 2004). The coefficient of variation of data was no higher than 5%.
The use of a biological product leads to a significant reduction of the content of imidacloprid in contaminated water. Reducing of the concentration of pesticide at the starting contamination of water by imidacloprid 1 mg/l at the 10th day was 0.28 mg/l, (72% less). At contamination of water at a dose of 2 mg/l introduction of the biological product reduces the concentration of imidacloprid to 0.64 mg/l, (68% less). These data indicate that the biological product “PHENOX” allows purification of water from imidacloprid.
The use of a biological product “PHENOX” for the purification of water from phenol. Biopproduct “PHENOX” in the liquid form of the following composition: bacterial cells at a ratio of 1:1:1 with a titre of 5×109 CFU/g (a product composition according to Example 1, without addition of stimulating ingredients) in the amount of 0.01% of the volume was introduced into the water contaminated with phenol (the content of phenols is 100 mg/l). In the aquatic media additionally mineral nitrogen-phosphorus fertilizer in an amount of 0.5 g/l was introduced. For aeration and mixing air was being periodically supplied by a pump in the container with contaminated water. The temperature of water was maintained about 22-25° C. The determination of phenol in the water was conducted by a standard photometric method (Rudakov et al, 2004). The experiment was repeated 3 times. The coefficient of variation of data was no higher than 7%.
When using the product “PHENOX” the concentration of phenol in the water during the first day decreases by 27%, and till the 3rd day—by 33% (Table 2).
Use of the product ‘PHENOX’ for the purification of water from dichlorophenol. The experiment was carried out according to Example 8, but using the ratio of strains of 2:1:1 and water contaminated with dichlorophenol 2,4-DCP (the content of dichlorophenol was 100 mg/l).
When using the product “PHENOX” for the purification of water from dichlorophenol the concentration of 2,4-DCP during the 1st day of incubation decreases at 70% (Table 3).
Use of the product ‘PHENOX’ for the purification of water from 4-chlorophenoxy acetic acid. The experiment was carried out according to Example 8, but using the ratio of strains 1:2:1 and water contaminated with 4-4-chlorophenoxy acetic acid 4-CPAA (the content of 4-CPAA was 100 mg/l).
The use of the product ‘PHENOX’ for the purification of water from 4-chlorophenoxy acetic acid by the 9th day reduces the concentration of 4-CPAA in water for about 10%, and by the 15th day—for 38.0% (Table 4).
Use of the product ‘PHENOX’ for the purification of water from 2,4-dichlorofenoxyacetic acid. The experiment was carried out according to Example 8, but using water contaminated with 2,4-dichlorofenoxyacetic acid 2,4-D (the content of 2,4-D was 100 mg/l).
When using the product ‘PHENOX’ for the purification of water from 2,4-dichlorofenoxyacetic acid the content of 2,4-D till the 3rd day gradually falls for 11%, till the 9th day for 30% and the 15th day for 35%.
Use of the product ‘PHENOX’ for the purification of water from 2,4,5-trichlorofenoxyacetic acid. The experiment was carried out according to Example 8, but using water contaminated with 2,4,5-trichlorofenoxyacetic acid 2,4,5-T (the content of 2,4,5-T was 100 mg/l).
When using the product ‘PHENOX’ for the purification of water from 2,4,5-trichlorofenoxyacetic acid the concentration of 2,4,5-T to the 3rd day reduces for 15%, to the 8th day for 45% compared with the initial level (Table 6).
Use of the product ‘PHENOX’ for the purification of water from phenol. The experiment was carried out according to Example 12, but using the product ‘PHENOX’ in the form of a dry powder (according to Example 3, with a titre of 5×1011 CFU/g and the introduction of vermicompost—0.1%).
When using the product ‘PHENOX’ in the form of a dry powder with catalytic additive for the purification of water from phenol its content reduces by the end of the 1st day for 30%, by the end of the 3rd day for 55% compared with the initial level (Table 7).
Use of the product ‘PHENOX’ for the purification of water from 2,4-Dichlorofenoxyacetic acid. Experiment was carried out according to Example 13, but using the product according to Example 3, without vermicompost with a titre of 1011 CFU/g and water contaminated with 2,4-dichlorofenoxyacetic acid, 2,4-D (the content of 2,4-D was 100 mg/l).
When using the product ‘PHENOX’ for the purification of water from 2,4-dichlorofenoxyacetic acid leads to a gradual decrease of the concentration of 2,4-D—on the 10th day it drops to 59% from the starting value (Table 8).
Use of the product ‘PHENOX’ for the purification of water from dichlorophenol. The experiment was carried out according to Example 13, but using water contaminated with dichlorophenol 2,4-DCP (the content of dichlorophenol was 100 mg/l).
When using the product ‘PHENOX’ for the purification of water from dichlorophenol the concentration of 2,4-DCP in 22 days drops to 53% from the starting value (Table 9).
Use of the product ‘PHENOX’ for the purification of water from 2,4,5-trichlorofenoxyacetic acid. The experiment was carried out according to Example 8, but using the product with stimulant additives and titres 109 CFU/g and water contaminated with 2,4,5-trichlorofenoxyacetic acid 2,4,5-T (the content of 2,4,5-T was 100 mg/l).
The content of 2,4,5-T in water consistently reduces after treatment by ‘PHENOX’—till the 2nd day for 30%, till the 12th day for 69%, and till the 28th day for 84% (Table 10).
Use of the bioproduct ‘PHENOX’ for the purification of wastewater from phenols. Bioproduct ‘PHENOX’ in the form of a dry powder of the following composition: dry biomass of cells—Bacillus subtilis VKPM B-10999, Rhodococcus erythropolis Ac-1882 and Pseudomonas putida VKPM B-10997 in a ratio 1:1:1 in the amount—10%, vermicompost—1%, water not more than 10%, kaolin—up to 100%, titre of alive cells—2×109 CFU/g (prepared according to Example 4). Before using a dry powder of the product was mixed well in the container (bucket, jar) in warm water (25-30° C.) until obtaining a homogeneous suspension. In the container having a working volume, equipped with a stirrer or a device for bubbling air, 1.0 kg of the biological product is introduced, 0.5 kg of azophoska and 200 l of warm technical water was added. To activate the microorganisms the suspension was stirred or perflated with air for 24 hours. Then a working suspension of the product was prepared and 10 kg of fertilizer (azophoska) and 1 kg of lime was added to the wastewater having volume of 10 m3 in the container. The container with wastewater was perflated by air daily. Wastewater pollution of “Ufahimprom” by phenolic compounds was 30.4 mg/liter. The efficiency of the treatment of wastewater from phenols by biological product ‘PHENOX’ was evaluated without additives and with addition of mineral fertilizers in the wastewater (Table 11).
The degree of purification of wastewater from phenols on the second day after the introduction of the biological product ‘PHENOX’ reached 66.4%, and on the 5th day was for more than 99%. Effectiveness of the use of the biological product ‘PHENOX’ for the treatment from phenols increases by the addition of mineral salts in the wastewater, already on the 2nd day the degree of purification of wastewater from phenol reached 89.5%, and on the 7th day—wastewater was released from phenols by more than 99.997% (Table 11).
Use of the biological product ‘PHENOX’ for the treatment of industrial wastewaters from phenols. Treatment of industrial wastewaters was carried out according to the previous example, without addition of fertilizer, but using wastewater of LLC “Novo-Ufimsky NPZ” and JSC “Dubitel”.
The degree of purification of wastewater from phenols of the petrochemical enterprise on the first day after the introduction of biological product ‘PHENOX’ was 66.7% and on the 3-7th day reaches 87.5%. The efficiency of use of the biological product ‘PHENOX’ for the purification of wastewater of tanning industry from phenol during 3-7 days is 79.4% (Table 12).
Use of the product ‘PHENOX’ for the destruction of oil in an aqueous medium. The product in the form of dry powder (composition according to Example 3) with a titre of 1×1010 CFU/g in the amount of 100 mg was introduced in 200 ml of water, contaminated with crude oil (at a concentration of 1%) with addition of salts (K2HPO4—0.05%, NH4Cl—0.05%, CaCO3—0.001%). The oil-contaminated water after inoculation of product and mineral salts of nitrogen, phosphorus, potassium and lime was incubated for 10 days at ambient temperature under static conditions with daily stirring. Control was made by variation without introduction of the product. The experiment was repeated for 3 times. On the third day dark flakes in the volume of water and smaller than in the control variation stains of oil on the surface were observed. On the 5th day in the variation with the product a small amount of oil stains on the surface of water was observed, about for a half the film of oil was destroyed on the walls of the flask. By the 10th day of the experiment a small amount of oil on the surface of water and on the walls of the container was observed. Mass concentration of oil in the samples was measured on the 10th day in the concentratometer KH-2 (manufacturing company LLC PEP SIBEKOPRIBOR, Novosibirsk) by the spectrophotometric method in terms of selective absorption of petroleum products in the infrared area of spectrum at a wavelength of 3.42 micrometers [The method of measurement of mass concentration of NP in the samples of drinking water, natural and wastewater by infrared spectrophotometry PND F 14.1:2:4.168-2000. Novosibirsk, 1998, 17 p.]. The coefficient of the variation of data—no more than 5%. Use of the product reduces the amount of oil in water for 28% (from 2652 mg/l in control variation up to 1915 mg/ml in the variation with the biological product).
Adatation of bacterial strains of the product in the soil. Dynamics of the population of bacterial strains of the product in the soil was studied in model experiments. A suspension of cells in the amount of 3×108 cells per 1 g of soil was placed in glass flask with 50 g of leached top soil. The experiment included 3 variations: without xenobiotic (the control) and 2,4-D at concentrations of 100 and 10 000 MPC. MPC for the 2,4-D in soils—0.1 mg/kg. Soil moisture was 60% of full capacity. Periodically the strains of the product ‘PHENOX’ were isolated on minimal salt agar medium M9 with the addition of 2,4-D as the sole source of carbon and energy. The number of colony forming units (CFU) of bacterial strains in the control soil increased within first 30 days, and then there was a decline of population density of these bacteria (Table 13). In polluted 2,4-D the density of the bacterial population was higher in the first 2.5 weeks after the introduction than in the controls, and then it gradually decreased. In the most polluted soil bacterial population of ‘PHENOX’ sharply increases and 10-50 times exceeds the population density of these strains in the control. This rise of the number of bacteria ‘PHENOX’ is due to using xenobiotic as a source of nutrition. Reducing the number of bacteria ‘PHENOX’ in the soil in this variation occurs after 30 days. By day 52nd the density of the population of introduced strains in all variants was at a similar level, indicating a decrease of the concentration of 2,4-D in contaminated soils, as well as possible involvement in the degradation of pesticides of other microorganisms.
A good adaptation of the microorganisms to soils was revealed. Similar data were obtained in experiments with other types of soils—gray forest, podsol soil and xenobiotics in three concentrations of 100 MPC, 1000 MPC, 10 000 MPC. MPC for the 2,4-D in soils is 0.1 mg/kg. This shows the possibility of the use of the product of the present invention in these types of soils.
Use of a biological product “PHENOX” for the treatment of soil contaminated with pesticide 2,4,5-T (trichlorofenoxyacetic acid). Soil (100 g) of arable horizon was sieved through a sieve of 2 mm diameter and placed in glass flasks for 500 ml. 2,4,5-T was introduced in soil in the amount of 100 mg/g and mixed thoroughly. Bioproduct ‘PHENOX’ with a titre of 109 CFU/g (according to Example 5) was inoculated into a bottle with the soil, moistened to 60% of full capacity and also mixed. Flasks were closed with cotton plugs with parafilm and incubated at constant humidity and temperature of 25° C. in a thermostat. Determination of 2,4,5-T in soil was carried out by the standard method (The method of pesticides measurement in soil, M.E., Medicine, 1984,—p. 256 p.). The experiment was repeated 3 times. The coefficient of variation of data was no higher than 8%.
The degree of soil decontamination from 2,4,5-T by using the biological product ‘PHENOX’ in 14 days was 15%, in 21 days—27.4%, and in 30 days—31.9%: and reached 66.5% by the 48th day. The rate of decomposition of 2,4,5-T increases significantly after 30 days by incubation of soil with biological product ‘PHENOX’ (Table 13*).
Use of the product ‘PHENOX’ for the disintegration of pesticide (fungicide) TMTD in soil. The venue of the experiment: concrete ground, adjacent to the warehouse for storage of pesticides (The region of Altai, Biysk city, 4 Prigorodnaya Street). In summer time on an open concrete ground a suit plot area of 3 m2 (1×3 m) was arranged, delimited by wooden partitions with height of 20 cm at 3 sites 1×1 m. The bottom of the plots was spread with plastic film to prevent the spread of pollutants from the soil to concrete ground. Plots were filled with a layer of top soil with thickness of 15 cm. At a height of 60 cm from the ground plots were covered by non-woven covering material (spunbond). In the soil the pesticide (fungicide) tetramethylthiuram disulfide (TMTD) at the rate of 50 g/m2 was introduced, vermicompost at the amount 50 g/m2, mineral nitrogen and phosphorus fertilizer enriched with micronutrients—100 g/m2 and organic fertilizers based on peat—400 g/m2 and mixed thoroughly. Product “PHENOX” in the form of dry powder composition: bacterial cells—10%, vermicompost—1%, kaolin—89% with a titre of 5×109 CFU/g (according to example 4) of 300 g was dissolved in 15 liters of water (25-30° C.) and incubated 24 hours with periodic agitation to improve aeration and activate micro-organisms. The suspension of biological product was applied to a surface of the plot at the rate 5 litres/m2 by sprinkling with a watering can. The surface of the plot was further watered by 5 litres/m2 of soil. Soil contaminated with TMTD, after treatment with the product “PHENOX” was loosened to improve aeration and mulched by a thin layer of sawdust. If necessary, and in case of no rain the soil in the plot was occasionally loosened and moistened up to 60-70% of full capacity. To evaluate the effectiveness of soil decontamination from TMTD by the method of sample middling, samples were taken each 1.5 and 3 months. The quantitative content of TMTD in the samples was determined chromatographically. The coefficient of variation of data was not higher than 5%. 1.5 months after the treatment with the product “PHENOX” the content of TMTD in soil reduced by 53%, in 3 months—by 75%.
Use of the product ‘PHENOX’ for the decomposition of the pesticide (herbicide) dichlorolurea in soil. The experiment was conducting similar to the Example 22. The pesticide (herbicide) dihlorolurea was introduced into the soil at the rate of 50 g/m2 1.5 months after the treatment with the product “PHENOX” the content of tetramethylthiuram disulfide TMTD in soil reduced by 59%, in 3 months—by 78%.
Use of the product ‘PHENOX’ for the purification the soil from chlorinated aromatic compounds. Venue of the experiment: Rostovskaya Oblast, Salski region. Characteristics of the plot: land for agricultural purposes, contaminated with chlorinated aromatic compounds. In summer time in an open air a plot area of 530 m2 (size of 66 m×8 m) was marked, delimited in areas of 170 m2 by protective zones of 0.5 m. The experiment was repeated 3 times. In contaminated soil vermicompost in the amount of 100 g/m2, mineral nitrogen and phosphorus fertilizer at the rate of 10 g/m2 were introduced. The product ‘PHENOX’ in the form of dry powder composition: bacterial cells—10%, vermicompost—1%, kaolin—89% with a titre of 5×109 CFU/g (according to Example 4) in an amount of 10 g/m2 to obtain a working solution was dissolved in a container and mixed well in warm water (25-30° C.) until a homogeneous suspension. Working suspension of the product “PHENOX” was applied to the surface of the soil at the rate of 10 l/m2. The application was carried out by sprinkling with any provided for this purpose machines and devices. The treatment was performed at an average daily soil temperatures at least +5° C. and not above +30° C. The introduced biologic product and fertilizers were worked in the upper layer of the contaminated soil by plowing, digging, cultivation or other methods of tillage. The soil periodically was loosened to improve aeration and humidified up to 60-70% of full capacity (if necessary, in case of the absence of rain). After two weeks the soil at the site was plowed and humidified. To evaluate the effectiveness of soil decontamination from chlorinated aromatic compounds by the method of sample middling, samples were taken every 1.5 and 3 months. The quantitative content of aromatic compounds in the samples was determined by the standard method (Rudakov et al, 2004). The coefficient of variation of data was not higher than 9%. 1.5 months after the treatment with the product ‘PHENOX’ the content of aromatic compounds in soil reduced by 35%, in 3 months—by 68%.
Assessment of fungicidal activity of the product (in the ratio 1:1:1). The fungicidal activity of bacterial strains B. subtilis VKPM B-10999, Rhodococcus erythropolis VKPM Ac-1882 and Pseudomonas putida VKPM B-10997 of the product was determined by the standard method of agar blocks (Egorov, 1976). Petri dish with a solid nutrient medium was sown with phytopathogenic fungus lawn culture. Strains of fungi was used, that caused destruction of the roots, wilting, mottling, root rot—Fusarium oxysporum, Alternaria tennuisima, Alternaria alternata, and gray mold on leaves and fruits of plants—Botrytis cinerea, rotting of agricultural products—Geotrichum candidum. Each culture was sown into 2 cups of Czapek's medium and on its surface the blocks of bacterial strains were placed, cut out of 5-days lawn of bacteria on standard LB-agar. 4 blocks of the bacterial strain sown by phytopathogenic fungus on each medium were placed. Cups were incubated for 2 days at 5° C. (for the diffusion of antibiotic substances in the agar), and then—at 25° C. and the existence of zones of growth of the inhibition of fungi was established.
Fusarium
Alternaria
Alternaria
Botrytis
Geotrichum
oxysporum
alternata
tennuisima
cinerea
candidum
Bacillus subtilis
Rhodococcus
erythropolis
Pseudomonas
putida
Fungicidal activity against all tested in the experiment of phytopathogenic fungi is established for the strain Pseudomonas putida B-10997, and for the strains of B. subtilis B-10999, Rhodococcus erythropolis Ac-1882—to fungus causing predominantly root infections and decay (Table 14). That is the bacterial product has fungicidal activity to pathogens that determines various fungal diseases of plants.
The plant growth stimulating activity of the product
Use of the product to stimulate plant growth of radish. The product was in the form of dry powder of the following composition: bacterial cells—10% (dry biomass of cells Bacillus subtilis VKPM B-10999, Pseudomonas putida VKPM B-10997 and Rhodococcus erythropolis Ac-1882 in the ratio 1:1:1), kaolin—89% with a titre of 5×109 CFU/g was dissolved in 10 ml of distilled water (dilution 1:10) and then sequential dilutions of 1:100 and 1:1000 were prepared. Radish seeds of the variety of ‘Rose-red’ with a white tip were spread on filter paper in Petri dishes for 25 seeds in each Petri dish. The experiment was repeated 4 times. Seeds were treated with the product suspension in an amount of 2 ml for a Petri dish by using an automatic dispenser. Control variations were the seeds germinated in distilled water and seeds treated with 0.05% solution of indole-butyric acid (IBA)—the stimulatator of the growth of plants of auxin type. Petri dishes with treated seeds were incubated under natural light and at ambient temperature in a humid chamber. The length of roots, the length of shoots and the dry weight of plants was determined at the 5th day. The coefficient of variation of data was no higher than 5%.
Suspension of the product stimulates the growth of the red radish and accumulation of biomass. The maximum effect was observed from the aqueous suspension of the product of 1:100. The product at a dilution of 1:100 increased the length of the 5 days radish roots from 69 to 81 mm (17%), length of seedling from 30 to 42 mm (40%) and dry weight of plants by 37%, from 0.27 to 0.37 g (Table 16).
Use of the product ‘PHENOX’ to stimulate plant growth of cabbage. The experiment was conducted similarly to the Example 25 (the content of the product: dry biomass of cells—Bacillus subtilis VKPM B-10999, Pseudomonas putida VKPM B-10997 and Rhodococcus erythropolis Ac-1882 in the ratio 1:1:1—10%; sorbent—finely milled peat—89%, vermicompost—1%). Seeds of cabbage variety ‘Slava’ were treated by the dilutions of the product. The length of roots and shoots of plants were determined after 5 days.
Aqueous suspension of the product (at all tested dilutions) increases the length of roots up to 50%—from 46 to 63-69 mm and length of shoots by 19%—from 31 to 37 mm at a dilution of 1:100 and 1:1000 (Table 16). The results indicate a strong stimulating effect of the product on the growth of roots of cabbage variety ‘Slava’.
Use of the product to stimulate the clover seed germination and plant growth. The experiment of conducted as in the Example 26. The red clover seeds were treated with the dilutions of the product. After 5 days the length of the root and the length of the seedling, germination and dry weight of plants were determined.
The product at a dilution of 1:10 increases the linear dimensions of the plant—root length by 89% (from 18 to 34 mm) and length of the seedling by 33% (from 30 to 40 mm). The product at a dilution of 1:100 enables improvement of germination by 12%—from 88 to 100% and the increase of dry weight by 114%—from 0.07 to 0.15 g (Table 17). Thus, the product has a strong stimulatory effect on root growth and increase of plant mass of clover.
Use of the product to stimulate the cucumber plant growth. Experiment was conducted as in the Example 26. The seeds of cucumber variety ‘Rausskaya Rubashka F1’ were treated by the dilutions of the product. The length of root and seedling, germination and plant dry weight was determined after 6 days.
Aqueous suspensions of the product have a stimulating effect on the growth of cucumber variety Russkaya Rubashka F1. For example, in an aqueous dilution of 1:10 product increases the length of the root at 61%—from 46 to 74 mm, the length of the seedling at 24%—from 33 to 41 mm, the germination at 16%—from 84 to 100% and dry weight of plants by 37%—from 0.46 to 0.63 g (Table 18). The greatest effect of the product is on root growth of cucumber.
Use of the product to stimulate the beet seed germination. The experiment was conducted similarly to the Example 26. Beet seed variety ‘Detroit’ was treated by the dilutions of the product. Seed germination was determined on the 7th day.
The product at a dilution of 1:100 increases germination of beet seeds by 7%—from 92 to 99% (Table 19).
Use of the product to stimulate the growth of onions plants. The experiment was conducted similarly to the Example 27. Onion seeds of variety ‘Karmen’ were treated with the preparation at dilutions of 1:100 and 1:1000. The length of root and seedling, germination and plant dry weight is determined after 10 days.
The product at a dilution of 1:1000 increases the length of the root by 23% from 30 to 37 mm, the length of the seedling by 9% from 57 to 62 mm and the increase of plant dry weight by 32%, from 0.19 to 0.25 g (Table 20).
Use of the product to stimulate the carrot plant growth. The experiment was conducted similarly to the Example 28. Carrot seeds of variety ‘Krasniy Velikan’ were treated with the dilutions of the product. The dry weight of plants was determined after 10 days.
The product at the dilution of 1:1000 increased the dry weight of seedlings of carrots by 67%—from 0.03 to 0.05 g (Table 21).
The presence of pesticides in soil, water and industrial wastewater was tested using the gas-chromatic method for the determining of pesticides described by Rudakov O. B. and others (Sputnik khromotografista.—Voronezh: Vodoley, 2004.—p. 528).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011128996 | Jul 2011 | RU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/052089 | 2/8/2012 | WO | 00 | 1/8/2014 |
