BIOLOGICAL PRODUCT FOR CLEARING OF WATER, INDUSTRIAL
WASTEWATER AND SOIL FROM CHEMICALS, WHICH ARE RESISTANT TO
DEGRADATION AND METHOD FOR USING THE SAME
Technical Field
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.
Background Art
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 (patent US 4804629),
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 patent US 4803166; 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 (patent US
4891320).
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.
Patent US 6632363 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).
Disclosure of Invention
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.
Brief Description of Drawings
Fig. 1 is a plasmid profile of strains: 2 - 9 Pseudomonas putida VKPM B-10997, 10 - 17
Rhodococcus erhytropolis VKPM Ac- 1882
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-a-propionic acid (Dichloroprop, 2,4-DR), 2-methyl-4-chlorophenoxy-apropionic
acid (Mekoprop, 2M-4HP, MSRR), 2,4,5-trichlorophenoxy-a-propionic acid
(2,4,5-TP, Silvex), 2,4-dichlorophenoxy-a-oil (2,4-DV), methyl- [l-[(butylamino)
carbonyl]-lH-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, l,l-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 Dplasmids
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 1) or compost (5 g/10 1), suspension periodically
is stirred for aeration and microbial activation within 1-3 hours before use (watering the
soil by watering-can (10-20 1/10 m ) 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 5x10 CFU/g of dry product after addition of carrier- sorbent, and other
additives (the source concentrate of 10 10 11 and at least 5x10 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 culturemorphological
features for this kind. Rods, the size of which is 0.2-0.5 at 1.5-3 mih, 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, a-ketoglutarate, DL-a-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 mih, the length - 3-8 mih. 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-a-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 culturemorphological
features for this kind. Motile rods with polar located flagella. The size of
the cells is 1-4 on 1.3-5.4 mih. 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, a-ketoglutarate, DL-a-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.
Examples of implementation of the invention
Example 1
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 Fig. 1 Plasmid profile
of strains: 2 - 9 Pseudomonas putida VKPM B-10997, 10 -17 Rhodococcus erhytropolis
VKPM Ac- 1882).
Example 2
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"
Example 3
Bioproduct "PHENOX", its form - a dry powder. Dry biomass of cells (concentrate) -
Bacillus subtilis VKPM B-10999 no less than 5xl0 9 CFU/g, Pseudomonas putida VKPM
B-10997 at least 5xl0 9 CFU/g and Rhodococcus erythropolis VKPM Ac-1882 at least
5xl0 CFU/g in the ratio (1-2): 1:(l-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 10 10 CFU/g.
Example 4
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 5xl0 CFU/g.
Example 5
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 5x10 CFU/g.
Example 6
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/1 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 10 CFU/ml.
The method of use of the offered product is explained by the following examples:
Example 7
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 5xl0 10 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/1 of an active ingredient. In addition in the aquatic medium additives of mineral
salts were added (g/1): Na2HP0 4.7H20 - 6.4; KH2P0 4 - 1.5; NaCl 0.25 g/1; NH4C 1 0.5 g/1.
For the control the samples of water contaminated with imidacloprid at concentrations of 1
mg/L and 2 mg/1 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%.
Table 1
Dynamics of the content of imidacloprid in water when using the product "PHENOX"
The variation The content of imidacloprid, mg/1
Oday 1st day 3rd day 7th day 10th day
The control - imidacloprid 1
1.00 0.98 0.98 0.94 0.96
kg/ml
The control - imidacloprid 2
2.00 2.09 2.01 1.93 1.95
kg/ml
Biological product +
1.00 0.94 0.81 0.48 0.28
imidacloprid 1 kg/ml
Biological product +
2.00 1.57 1.15 0.83 0.64
imidacloprid 2 kg/ml
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/1 at the 10th day was 0.28 mg/1, (72%
less). At contamination of water at a dose of 2 mg/1 introduction of the biological product
reduces the concentration of imidacloprid to 0.64 mg/1, (68% less). These data indicate that
the biological product "PHENOX" allows purification of water from imidacloprid.
Example 8
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 5xl0 9 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/1). In
the aquatic media additionally mineral nitrogen-phosphorus fertilizer in an amount of 0.5
g/1 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%.
Table 2
Dynamics of the content of phenol after treatment of water with the product "PHENOX"
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).
Example 9
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/1).
Table 3
Dynamics of the content of dichlorophenol after treatment of water with the product
"PHENOX"
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).
Example 10
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/1).
Table 4
Dynamics of the content of 4-chlorophenoxy acetic acid after treatment of water with the
product "PHENOX"
Incubation time The concentration of
4-CPAA, mg/1
0 days 100.0
1 day 96.3
3 days 95.8
5 days 94.1
7 days 92.5
9 days 90.0
11 days 68.7
13 days 63.9
15 days 62.0
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).
Example 1 1
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/1).
Table 5
Dynamics of the content of 2,4-dichlorofenoxyacetic acid after treatment of water with the
product "PHENOX"
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%.
Example 12
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/1).
Table 6
Dynamics of the content of 2,4,5-trichlorofenoxyacetic acid after treatment of water with
the product "PHENOX"
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 3 d day reduces for 15%, to
the 8th day for 45% compared with the initial level (Table 6).
Example 13
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 5xl0 CFU/g and the introduction
of vermicompost - 0.1%).
Table 7
Dynamics of the content of phenol after treatment of water with the product "PHENOX"
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).
Example 14
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 10 1 1 CFU/g and water
contaminated with 2,4-dichlorofenoxyacetic acid, 2,4-D (the content of 2,4-D was 100
mg/1).
Table 8
Dynamics of the content of 2,4-dichlorofenoxyacetic acid after treatment of water with the
product "PHENOX"
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).
Example 15
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/1).
Table 9
Dynamics of the content of dichlorophenol in the treatment of water with the product
"PHENOX"
When using the product 'PHENOX' for the purification of water from dichlorophenol the
concentration of 2,4-DCP in 22 days drops drops to 53% from the starting value (Table 9).
Example 16
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/1).
Table 10
Dynamics of the content of 2,4,5-trichlorofenoxyacetic acid in the treatment of water with
the product "PHENOX"
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).
Example 17
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 -
2xl0 9 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 1of 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 m 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).
Table 1 1
Dynamics of the decontamination of wastewater from phenols using biological product
"PHENOX"
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).
Example 18
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".
Table 12
Dynamics of the decontamination of industrial wastewater from phenols by using the
biological product PHENOX
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
3-7 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).
Example 19
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 l x
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 (K2HP0 4 - 0.05%, NH4C 1 -
0.05%, CaC0 3 - 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 repeted 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/1 in control variation up to 1915
mg/ml in the variation with the biological product).
Example 20
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 3x10 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 exceedes 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.
Table 13
Dynamics of the number of strains of the product "PHENOX" introduced to the
contaminated by pesticide 2,4-D leached top soil
Example 2 1
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%.
Table 13*
Dynamics of the decontamination of soil from 2,4,5-T using the biological product
"PHENOX"
The degree of soil decontamination from 2,4,5-T by using the biological product
'PHENOX' in 14 days was 15%, in 2 days - 27.4%, and in 3 days - 31.9%:and reached
66.5% by the 48 h 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*).
Example 22
Use of the product THENOX' 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 (1x3 m) was arranged, delimited by
wooden partitions with height of 20 cm at 3 sites lxl 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 5 cm. A a height of 60
cm from the ground plots were covered by non- oven 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 - 00 g/m2 and organic fertilizers based
2
on peat - 400 g/m and mixed thoroughly. Product "PHENOX" in the form of dry powder
composition: bacterial cells - 10%, vermicompost - 1%, kaolin - 89% with a titre of 5xl0 9
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/m by sprinkling with a watering can. The surface of the plot was further
watered by 5 litres/m 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%.
Example 23
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/m 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%.
Example 24
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 m (size of 66 m x 8 m) was
marked, delimited in areas of 170 m by protective zones of 0.5 m. The experiment was
repeated 3 times. In contaminated soil vermicompost in the amount of 100 g/m , mineral
nitrogen and phosphorus fertilizer at the rate of 10 g/m were introduced. The product
'PHENOX' in the form of dry powder composition: bacterial cells - 10%, vermicompost -
1%, kaolin - 89% with a titre of 5xl0 9 CFU/g (according to Example 4) in an amount of
2
10 g/m 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 1/m . 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%.
Example 25
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.
Table 14
Fungicidal activity of bacterial strains of the product
- There is no inhibition area
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
Example 26
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 5xl0 9 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 5
The coefficient of variation of data was no higher than 5%.
Table 15
Growth of radish plants when using the biological product "PHENOX"
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 8 1 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).
Example 26
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.
Table 16
Growth parameters of cabbage variety 'Slava' when using the product
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 3 1 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'.
Example 27
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.
Table 17
Growth parameters of the red clover when using the biological product "PHENOX"
Parameter
The variation of treatment Length of the Length of the Germination, Dry weight, g
root, mm seedling, mm %
The control (water) 18 30 88 0.07
The control (IBA) 23 33 9 1 0.09
The product dilution 1:10 34 40 84 0.12
The product dilution 1:100 24 33 100 0.15
The product dilution 1:1000 25 36 96 0.12
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.
Example 27
Use of the product to stimulate the cucumber plant growth. Experiment was conducted as
in the Example 26. The seeds of cucumber variety 'Russkaya Rubashka F were treated
by the dilutions of the product. The length of root and seedling, germination and plant dry
weight was determined after 6 days.
Table 18
Growth parameters of cucumber plants when using the biological product "PHENOX"
Aqueous suspensions of the product have a stimulating effect on the growth of cucumber
variety Russkaya Rubashka Fl. 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 4 1 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.
Example 27
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.
Table 19
Germination parameters of beet seed when using the biological product "PHENOX"
The product at a dilution of 1:100 increases germination of beet seeds by 7% - from 92 to
99% (Table 19).
Example 28
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.
Table 20
Growth parameters of onion plants when using the biological product "PHENOX"
Parameter
The variation of treatment Length of the Length of the Dry weight,
root, mm seedling, mm g
The control (water) 30 57 0.19
The control (IBA) 33 6 1 0.21
The product dilution 1:100 34 69 0.28
The product dilution 1:1000 37 62 0.25
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).
Example 29
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.
Table 2 1
Growth parameters of carrot plants when using the biological product "PHENOX"
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).
Sources of Information
1. Feodorov A. Yu., Volchenko E.V., Korzhenevich V. I., Singircev I.N., Krestyaninov
V.Yu. Polysubstratnyj shtamm-destruktor komponentov stochnih vod proizvodstva fenola.
// Prikladnaya biohimiya I mikrobiologiya. - 1993. - 1.29, N5. - p.716-722.
2. Feodorov A. Yu., Ignatov O. V., Korzhenevich V. I., Shub G. M. Effect of
immobilization in agar gel on the microbial degradation of xenobiotics. //6th European
Congr. Biotechnology (ECB6), Firenze, Italy, June 13-17,1993. - Firenze, 1993. - Abstrs.
Books, V.2. -TU2I2.
3. Korzhenevich V.I., Volchenko E.V., Singircev I.N., Feodorov A.Yu., Shoob G.M.
Microbial treatment of phenolic wastes. - In: Environmental Biotechnol.: Principles and
Applications. - M. Moo-Young et al. (eds.), Kluwer Academic Publishers, 1995. -P.498-
503.
4. Singircev I.N., Volchenko E.V., Korzhenevich V.I., Gumenyuk A.P., Feodorov A.Yu.
Microbal degradation of components of phenolic waste waters. // Prikladnaya biohimiya I
mikrobiologiya. - 2000. - 1.36, N2. - p.178-188.
5. Authors' certificate No. SU 1597346; C02F3/34, C12N1/20; Strain Pseudomonas
pseudoalcaligenes used for treatment of wastes from aromatic compounds /Mironov A.D.,
Korzhenevich V. I., Barkovskiy A.L. -OG No. 37, 1990.
6. Mironov A.D., Krestyaninov V.Yu., Korzhenevich V. I., Yevtushenko I.Ya., Barkovskiy
A.L. Destrukciya 2-nitrobenzoynoy kisloty i drugih aromaticheskih sejedineniy shtammom
Pseudomonas pseudoalcaligenes. // Prikladnaya biohimiya I mikrobiologiya. - 1991. - 1.27,
N4. - p.571-576.
7. Giardina M.C., Giardi M.T., Filacchione G. Atrazine metabolism by Nocardia:
elucidation of the initial pathway and synthesis of potential metabolites. // Agric. Biol.
Chem. - 1982. - Vol. 46. - P.1439-1445/
8. Blaszak M., Pelech R., Graczyk P. Screening of Microorganisms for Biodegradation of
Simazine Pollution (Obsolete Pesticide Azotop 50 WP) // Water Air Soil Pollut. - 201 1.
Vol. 220. P. 373-385.
9. Sajjaphan K, Heepngoen P, Sadowsky MJ, Boonkerd N.J Arthrobacter sp. strain KUOOl
isolated from a Thai soil degrades atrazine in the presence of inorganic nitrogen sources //
Microbiol. Biotechnol. - 2010. Vol. 20. - P. 602-608.
Print Out (Original in Electronic Form)
(This sheet is not part of and does not count as a sheet of the international application)
Print Out (Original in Electronic Form)
RO/ EP (This sheet is not part of and does not count as a sheet of the international application)
3 The indications made below relate to
the deposited microorganism(s) or
other biological material referred to in
the description on:
3-1 page 4
3-2 line 4-5
3-3 Identification of deposit
3-3-1 Name of depositary institution VKPM Russian National Collection of
Industrial Microorganisms
3-3-2 Address of depositary institution FGUP GosNII Genetika, 1 Dorozhny proezd,
1 , Moscow 1 1 75 45 , Russian Federation
3-3-3 Date of deposit 0 6 April 2 0 1 1 ( 0 6 . 0 4 . 2 0 1 1 )
3-3-4 Accession Number VKPM B-1 0 9 97
3-4 Additional Indications Pseudomonas putida
3-5 Designated States for Which All designations
Indications are Made
FOR RECEIVING OFFICE USE ONLY
0-4 This form was received with the
international application: yes
(yes or no)
0-4-1 Authorized officer
Wallenti n, Marko
FOR INTERNATIONAL BUREAU USE ONLY
0-5 This form was received by the
international Bureau on:
0-5-1 Authorized officer
33

CLAIMS
1. An association of strains of Pseudomonas putida deposited in Russian National
Collection of Industrial Microorganisms (VKPM) under the accession number B-10997,
Bacillus subtilis deposited in Russian National Collection of Industrial Microorganisms
(VKPM) under the accession number B-10999 and Rhodococcus erythropolis deposited in
Russian National Collection of Industrial Microorganisms (VKPM) under the accession
number Ac- 1882.
2. A product containing the association strains of Pseudomonas putida VKPM B-
10997, Bacillus subtilis VKPM B-10999 and Rhodococcus erythropolis VKPM Ac- 1882.
3. The product according to claim 2 comprising the association of strains
Pseudomonas putida VKPM B-10997, Bacillus subtilis VKPM B-10999 and Rhodococcus
erythropolis VKPM Ac- 1882 in a weight ratio of (1-2):(1-2):1.
4. The product according to claim 2 or 3 further comprising a sorbent or being
immobilized on the sorbent and optionally mineral, organic and/or stimulating additives.
5. Use of the product according to any one of claims 2-4 for the purification of
ground, soil, water or industrial wastewater from stable to degradation pesticides or oil by
introduction of the product according to any one of claims 2-4 in an effective amount in
contaminated soil, ground, water or wastewater.
6. The use of the product according to claim 5, wherein stable to degradation
pesticides are being selected from the group consisting of chlorophenoxy acetic acids, such
as 2,4-dichlorofenoxyacetic acid (2,4-D), trichlorofenoxyacetic acid (2,4,5-T),
chlorofenoxyacetic acid (CPAA), fenoxyacetic acid (PAA), and 2,4-dichlorophenol and
phenol imidacloprid.
7. Use of the product according to any one of claims 2-4 for stimulating growth
activity of plants and/or as fungicidal preparation.
8. The use of the product according to claim 7, wherein dry composition of the said
product is being introduced into the ground during ploughing, tillage or seeding,
preferably, in an amount of 10 kg per 1 ha of land.
9. The use of the product according to claim 7, wherein the said product in the form
of an aqueous solution is being introduced into the ground by irrigation.
10. The use of the product according to claim 7, wherein introduction of the said
product into the soil is carried out by introducing into the soil seeds treated with the
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suspension of the said product, optionally, with addition of nutrients, such as
vermicompost.