TECHNICAL FIELD

The present invention relates to obtaining microbial fertilizer by means of different methods by preparing bacteria or bacteria mixtures in the form of a formulation which eliminates phenoxy acid derivative herbicides in soil and in water.

KNOWN STATE OF THE ART

Phenoxy acid derivative herbicides are the sub-group of phenoxy herbicide family and comprise more than 50 compounds [14]. Phenoxy acid derivative herbicides imitate the function of oxy which is a plant growth hormone and they lead to over-growth and eventually to the death of the plant [3].

Today, phenoxy acid derivative herbicides are used in great amounts in agricultural and non- agricultural fields and this leads to residue problems in receiving bodies like soil, underground water and water basins and threatens human and environment health. In soils which become contaminated due to phenoxy acid derivative herbicides, the decay half-life of these chemicals has been reported as approximately 30 days [5]. The researches have shown that only 0.01 -6% of the pesticides affects the targeted pest and the remaining 94-99% of the pesticides ends up as pollutants to the soil and to the other natural eco-systems. Moreover, these chemicals lead to residue on the consumed and exported products and therefore, the exportation of these products is prevented and big damages are given to the producer and to the economy.

The most frequently used method for cleaning the remaining organic pollutants from the soil and from the other receiving bodies is to carry the contaminated soil to predetermined areas, to bury or to burn in bark boilers. However, all of these methods are expensive, difficult and time-consuming. Moreover, these methods are not suitable agricultural fields which are contaminated with pesticides. The most efficient method used for elimination of phenoxy acid derivative herbicides from receiving bodies has been reported as microbial degradation [1 ]. In the literature, various bacteria strains have been reported which are isolated from receiving bodies which become contaminated due to phenoxy acid derivative herbicides and which have the degradation capability of these chemicals [12], [13], [1 1 ].

In the Chinese patent document with number CN103740622 B and existing in the present art, Cupriavidus campinensis BJ71 isolate is described. The invention relates to a strain of degrading herbicide 2,4-dichloro-phenoxy-acetic acid and an application thereof, belonging to the technical fields of environmental engineering and microbial engineering. The strain cupriavidus campinensis of degrading herbicide 2,4-dichlorophenoxyacetic acid is named BJ71 , with collection number of CCTCC (China Center for Type Culture Collection) M2014006. The strain has the capacity of using 2,4-dichlorophenoxyacetic acid as the unique carbon source and energy, and can obviously reduce the concentration of 2,4- dichlorophenoxyacetic acid in the environment. Experiments prove that the strain has more than 92% of 2,4-dichlorophenoxyacetic acid degradation rate in 21 days, and the experiments show that the strain can be applied to bioremediation of 2,4- dichlorophenoxyacetic acid polluted soil. It is observed that the degradation durations mentioned in the patent are insufficient when the dissolving values of said organic contamination in nature are taken into consideration.

Again, in the Chinese patent document with number CN103173377 A, Enterobacter sp. SE08 isolate is described. The invention belongs to the field of bioremediation, and particularly relates to a 2-methyl-4-chlorophenoxyacetic acid herbicide degrading bacterium SE08, and a screening method and application thereof. The 2-methyl-4-chlorophenoxyacetic acid herbicide degrading bacterium SE08 is Enterobacter sp. of which the collection number is CGMCC No.7016 and the collection date is 2012.12.18; and the 2-methyl-4- chlorophenoxyacetic acid herbicide degrading bacterium SE08 is collected at China General Microbiological Culture Collection Center. Thus, it is not known whether said isolate shows activity in receiving bodies.

In the US patent document with number US4804629 A, Pseudomonas pseudoalcaligenes NRRL B-18087 isolate is described. Pseudomonas pseudoalcaligenes strain NRRL B-18087 was isolated as a pure culture from the sewage treatment plant. The strain can utilize chlorinated aromatic compounds as the sole carbon source and degrade both 2,4- dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), as well as other xenobiotic compounds. The statement that the mentioned isolate provides degradation of other xenobiotics is a very indefinite statement since there are thousands of different xenobiotics which are known to leave residue in the nature.

In the US patent document with number US4816403 A, Pseudomonas NRRL B-18086 isolate is described. Pseudomonas strain NRRL B-18086 was isolated as pure culture from the sewage treatment plant. The strain can utilize chlorinated aromatic compounds as the sole carbon source and will degrade both 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5- trichlorophenoxyacetic acid (2,4,5-T), as well as other xenobiotic compounds. The statement that the mentioned isolate provides degradation of other xenobiotics is a very indefinite statement since there are thousands of different xenobiotics which are known to leave residue in the nature.

In the patent document with number US6284514 B1 , Corynebacterium sp. K2-17 isolate is described. The invention relates to the new bacterial strain Corynebacterium sp. K2-17 and to a process for the microbial decontamination of materials polluted with compounds from the production of phenoxy-alkanoic acid herbicides, such as 2,4-dichlorophenoxybutyric acid (DCPB), 4-chloro-2-methylphenoxybutyric acid (MCPB), 2,4-dichlorophenoxyacetic acid (2,4- D), 4-chloro-2-methylphenoxyacetic acid (MCPA), 2,4-dichlorophenol (DCP), and 4-chloro-2- methylphenol (MCP) in a pH range of from slightly acidic to strongly alkaline. It has not been disclosed that the invention is used in agricultural fields and the invention provides decontamination of phenoxy acid derivative herbicides in agricultural soils.

OBJECT OF THE INVENTION In order to eliminate the disadvantages of the known state of the art, an object of the present invention is to provide in situ elimination of pollutants by means of selected and specially developed microorganisms.

Another object of the present invention is to provide chemicals, used in production step, which are environment-friendly and which are not harmful to nature and living organisms.

Another object of the present invention is to have easy usage thanks to the usability of liquid product watering channels, spraying or dripping water systems. Another object of the present invention is provide low-cost usage as the product is more economic and easier to apply when compared with the physicochemical methods. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is the phylogenic tree view based on rRNA genes and by using Neighbor-Joining method.

Figure 2.1 , 2.2 are the degradation graphics of the bio-preparation, which eliminates phenoxy acids, in concentrations of 10 mg/L and 100 mg/L 2,4D.

Figure 3 is the gene sequence view of the Pseudomonas putida NG28 strain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to microbial fertilizer obtained by means of different methods by preparing isolated bacteria or bacteria mixtures, isolated from the soil by means of enrichment method, in the form of a formulation.

The present invention relates to microbial fertilizer which provides decontamination of herbicides which are phenoxy acid derivatives like 2,4-dichloro-phenoxy-acetic acid (2,4D), 4-chloro-2-methyl-phenoxy acetic acid (MCPA), 2-(2,4-dichloro-phenoxy) propaneoic acid (2,4DP),2,4-dichloro-phenol (DCP) and 4-chloro-2-methyl-phenol (MCP) from the soil and from the receiving water bodies and which does not comprise chemical substances that are harmful to environment and which supports plant growth and increases efficiency of macro and micro elements added as fertilizer or already present in soil and does not need an additional agricultural cost. The subject matter microbial fertilizer comprises Pseudomonas putida NG28 strain as the active substance.

A bacterial strain, which belong to Pseudomonas species and eliminate phenoxy acid derivative herbicides from mixed microbial cultures obtained by selective culture enrichment process from soil samples that were collected from Mersin region, have been isolated. Identification of the purified bacteria has been realized by means of polymerase chain reaction (PCR) and 16S rDNA sequencing method. Clean sequences have been obtained after filtering, by using bio-informatics methods, from raw sequencing results and after clipping of the badly read regions. By putting these obtained sequences one below the other, the consensus sequence has been obtained. The consensus sequence is scanned in NCBI databases and the bacteria which they match have been determined. The reference type of the obtained pesticide eliminating isolate, which has been taken to the closest culture, has been determined as

Pseudomonas putida genotype PhyCEm-187 (99% similarity (1416/1418 base pair)). The isolate has been named as Pseudomonas putida NG28. The obtained 16S rRNA sequence has been added to the NCBi Gene Bank (Access number: MF164043).

The first step in degradation of the phenoxy acid type herbicides is the breaking of the ether bonds which results in formation of 4-chloro-2-methy!-pheno! ( CP). This reaction Is started by oxygenases coded by tfdA and tfdAa genes [6], [7]. The remaining degradation steps are coded by tfdB-F genes. The forward enzymatic reactions are completed with Krebs tri- carboxyiic axis cycle [9] coded by chromosomal genes and as the final product, C0 ₂ , H ₂ 0 and chlorine are formed [4]. The bacterial strains, known to degrade phenoxy acid herbicides, have been collected under three groups based on evolutionary and phenotypic characteristics [8], [2]. These groups have been shown in the phylogenic tree based on 16S rRNA genes and obtained by using Neighbor-Joining method (Figure 1 ).

Pseudomonas putida strain is provided in Group 1 among the microorganisms which degrade NG28 phenoxy acid and it comprises plasmid which includes tfdA-f gene sets. In that context, Pseudomonas putida can use NG28 phenoxy acid herbicides as a carbon source and can degrade these chemicals.

The degradation pathway of MCPA which is one of the phenoxy acid type herbicides used frequently has been shown in the figure [9].

TCA cvcie ■— Hooc- ^■ -¾··

iseiorse Pseudornonas putida NG28 strain has been grown in water-based nutrient medium (pH 5.0- 8.0) comprising molasses between 2-10%, yeast extract between 1 -5%, MgCI ₂ between 0.01 - 0.05%, CaC! ₂ between 0.005-0.02%, MnCI ₂ between 0,0005-0.002% and 2,4D between 0.01 - 0.08%. The product obtained as a result of 48 hours fermentation has been formulized by means of mixing 2-8% molasses, 1 -4% glycerol-xanthan gum-ethanol mixture and 1000- 2000 mg/L potassium sorbate. The pH of the final product changes between 4.0 and 7.0.

The efficiency of the prepared formulation has been proven by means of efficiencies of 2,4D (2,4D-dichloro-phenoxy acetic acid) elimination, which is the most frequently found phenoxy acid in the market, in soil samples polluted controllably in laboratory conditions. 50 grams of sterile soil samples (auto-cleaved twice) have been placed into sterile glass bottles, and 2,4D solutions with two different concentrations (10 mg/L; 100 mg/L) have been added thereon. 1 mL bio-preparation has been added to the homogenized samples with the help of a sterile spatula and bio-preparation has not been added to abiotic controls. The samples comprising 10 mg/L 2,4D have been incubated at room temperature for 24 hours and the samples comprising 100 mg/L 2,4D have been incubated at room temperature for 60 hours.

All samples have been dried trice repeatedly. Samples have been taken at specific intervals and 2,4D degradation has been monitored by means of high-performance liquid chromatography (HPLC) system (10). The bio-preparation comprising Pseudornonas putida as active substance has completely 10 mg/L 2,4D pesticide at a duration which is shorter than 24 hours. The lack of degradation in the abiotic controls clearly shows that the degradation process was biotic. The elimination graphics of phenoxy acid elimination bio-preparation in concentrations of 10 mg/L and 100 mg/L 2,4D have been shown in Figures 2.1 and 2.2.

The field tests of the obtained microbial fertilizer have been set up in Adana region. The aim of these tests was to observe the effect of the product in real conditions and to report its effect on agricultural products. At the same time, soil samples have been taken from the field and pesticide analysis have been made and the effect of the product in the field has been certified. The percent average death proportions of the microbial fertilizer against weeds such as Field Bindweed (Convolvulus arvensis), Rough Cocklebur (Xanthium strumarium), Red Rooted Foxtail (Amaranthus retleflexus) and Goosefoot (Chenopodium album) have been observed and it has been determined that when the subject matter microbial fertilizer (2,4-D Amine (200 ml/min) + microbial fertilizer (1000 ml/min)) is used in agricultural fields against 2,4-D Amine (200 ml/min) herbicide, the death proportions of plants decrease by 10.13%. Thus, it has been understood that the residue of 2,4-D Amine drug in soil is reduced. The results are shown in Table 1 .

Table 1 . % effect death reduction proportions of microbial fertilizer against weeds improved 5 against reducing of residue efficiency of 2,4-D Amine drug in Adana (Seyhan-Karayusuflu Village) agricultural fields.

Death Effect % Against Weeds

Field Rough Red Rooted

Repeating Goosefoot

Herbicides Bindweed Cocklebur Foxtail

number (Chenopodium- (Convolvulu- (Xanthium- (Amaranthus- album)

sarvensis) strumarium) retleflexus)

1 95 95 95 95

2 90 90 100 90

2,4-D 3 95 90 90 100

Amine 4 90 95 95 90

(200 5 90 90 100 90

ml/min) 6 95 90 100 95

7 90 90 95 95

Average 92.14 (a) 91.43 (a) 96.43 (a) 93.57 (a) 93.39

1 80 90 90 85

2 75 90 80 90

2,4-D 3 85 80 90 80

A D A D ^Itt e ^r a ^g eea ^r e _r a _g eeavv Amine 4 90 85 80 85

P ⁱ P ⁱ % % ^ttr o ^p o ^r on _r o _p o ^r on

(200 5 90 80 85 80

ml/min) 6 75 70 85 90

+ 7 85 80 90 85

Microbial Average 82.86 (b) 82.14 (b) 85.71 (b) 85.00 (b) 83.93

Fertilizer 2,4-D

(1000 Amine %

ml/min) Effect 10.08 10.16 11.11 9.16 10.13

Reduction

Proportion

Control 1 4 13 14 7

(Average 2 5 7 14 9

Number of 3 6 8 18 13 Plants in 4 4 8 12 9 m ² ) 5 7 1 1 9 1 1

6 8 9 1 1 7

7 4 12 16 14

Average 5.43 9.71 13.43 10.00

In parallel to this, at the end of the test, the soil samples taken from the fields where microbial fertilizer is used and is not used have been sent to an accredited laboratory for detailed analysis. It has been reported by means of detailed pesticide analysis whether the 5 subject matter microbial fertilizer leads to a change in 2,4D amine concentration or not. As a result of the tests where detailed analysis of acidic herbicide concentrations is realized in soil samples, 2,4D concentration has been determined as 0.256 mg/kg in soil samples where 2,4D amine application is realized and where microbial fertilizer is not used. Besides, it has been found that besides the 2,4D application, 2,4 concentration has been found under the 10 detection limit (0.01 mg/kg) in the samples where microbial fertilizer is used. These findings form evidence that the product realizes 2,4D degradation in soil.

As a result of the field tests, it has been observed that the product eliminates the pesticide residues which accumulate in the soil and the product has effect in plant growth and plant 15 resistance. In Antalya region, the favorable effect of the microbial fertilizer on plant growth within 9 days as a result of single-dose application (2 liters per decare of land) applied to pepper (Capsicum) seedlings grown in greenhouse has been apparently observed.

The effect of the obtained microbial fertilizer on the product efficiency has also been 0 observed in pumpkin growing. The effect of microbial fertilizer, applied in single-dose (1 liter per decare of land), on plant growth within 4 days has been detected.

The microbial fertilizer whose active substance is Pseudomonas putida strain functions efficiently under aerobic conditions, between pH 4 and 10 and between temperature values 5 of 10 and 38 ^S C. The product suspension is in concentrated (SC) form and the usage amount changes between 1 to 4 liters per decare in soil.

The subject matter microbial fertilizer provides decontamination of phenoxy acid derivative herbicides like 2,4-dichloro-phenoxy-acetic acid (2,4D), 4-chloro-2-methyl-phenoxy-acetic 0 acid (MCPA), 2-(2,4-dichloro-phenoxy) propaneoic acid (2,4DP),2,4-dichloro-phenol (DCP) and 4-chloro-2-methyl-phenol (MCP) from the soil and from the receiving water bodies within 24 and 48 hours.