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Microbiology and Biotechnology Letters

Research Article(보문)

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Microbial Biotechnology  |  Cell Culture and Biomedical Engineering

Microbiol. Biotechnol. Lett. 2024; 52(1): 44-54

https://doi.org/10.48022/mbl.2312.12004

Received: December 6, 2023; Revised: February 1, 2024; Accepted: February 6, 2024

Isolation, Identification and Use of Bacterial Strain Ochrobactrum intermedium PDB-3 for Degradation of the Pesticide Chlorpyrifos

Diyorbek Kosimov1,2*, Lyudmila Zaynitdinova1, Aziza Mavjudova1, Muzaffar Muminov3, and Oybek Shukurov4

1Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan, A. Kadyri Str. 7B, 100128, Tashkent, Uzbekistan
2National University of Uzbekistan named after Mirzo Ulugbek, Tashkent 100174, Uzbekistan
3Center for Advanced Technologies under the Ministry of Higher Education, Science and Innovations of Uzbekistan
4Institute of Fundamental and Applied Research at the National Research University TIIAMEKori Niyoziy 39, Tashkent 100000, Uzbekistan

Correspondence to :
Diyorbek Qosimov,        diyor-qosimov91@mail.ru

Abstract

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One of the serious modern environmental problems is pollution caused by highly toxic pesticides. Only small amounts of applied pesticides reach their target, and the rest ends up in soil and water. Chlorpyrifos is a toxic, broad-spectrum organophosphate insecticide. In humans, chlorpyrifos inhibits acetylcholinesterase (AChE) in the peripheral and central nervous system, and particularly in children, small amounts of this pesticide cause neurotoxic damage. As the toxic effects of chlorpyrifos and its persistence in the environment require its removal from contaminated sites, it is essential to study the biological diversity of chlorpyrifos-degrading microorganisms. In this study, we sought to determine the chlorpyrifos-degrading ability of the bacterial strain Ochrobactrum intermedium PDB-3. This strain was isolated from soil contaminated with various pesticides and identified as PDB-3 based on morpho-cultural characteristics, MALDI-TOF MS, and 16S rRNA. Studies were conducted for 30 days in sterile soils containing initial concentrations of 50, 75, 100, and 125 mg/kg of chlorpyrifos. To determine the degradation of chlorpyrifos, a liquid culture of the strain was added to the soil at three optical densities: 0, and after 24 and 48 h (OD = 0.03, 0.2 and 0.32). Using GX-MS, we determined that chlorpyrifos was converted to 3,5,6-trichloro-2-pyridinol (TCP). We also found that with increasing optical density, rapid degradation of the initial concentration of chlorpyrifos occurred. Sterile soil without strain PDB-3 was used as a control sample.

Keywords: Ochrobactrum intermedium, chlorpyrifos, bioremediation, biodegradation, optical density, pesticide

Graphical Abstract


Introduction

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Pesticides have been used for a long time to combat various pests of agricultural plants, which, on the one hand, leads to increased productivity, but on the other, has a negative impact on the environment [1]. These chemicals are divided into several classes and include organochlorines, organophosphates, pyrethroids, carbamates, triazines, and neonicotinoids [2−6]. Currently, organophosphate (OP) pesticides are most often used in agriculture [7]. OPs are esters of phosphoric acid. This group of pesticides affects various insects, animals and even humans by reducing or completely inhibiting the activity of the enzyme acetylcholinesterase, which is necessary for the functioning of the nervous system [3]. OP pesticides are used against crop pests due to their broad spectrum of action and high efficiency [8, 9]. Statistics show that OP pesticides rank first, accounting for 38%, among all pesticides used worldwide [10]. Excessive and widespread use [11−13], as well as the accumulation of such highly toxic chemicals in the environment, represent a serious threat to living organisms [14, 15]. Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate) [16], a widely used broad-spectrum organophosphorus pesticide [17], is used in agriculture to protect peaches, apples [18], grain, and vegetable crops from various types of pests [19]. Its half-life in soil ranges from 60 to 120 days [20]. The use of this insecticide on a large scale leads to contamination of soil, water and other environmental components [20, 21]. In addition, its remains have been found in various ecological systems [22]. Leaching of the applied pesticide may lead to contamination of surface/ground water, ultimately wreaking adverse effects on biological systems [23]. Therefore, early identification and subsequent disinfection and detoxification of contaminated environments is crucial. It is also important to note that approximately 0.1% of the applied chlorpyrifos reaches the target organism (pest), and the rest enters the soil and water, being distributed in the environment [24].

Organophosphorus pesticides can be removed from the environment or decontaminated using physical, chemical and biological methods. The most effective and environmentally safe among these is the biological method [25]. Bioremediation, a technique that involves harnessing the power of microorganisms to detoxify and decompose pollutants, has received increased attention as an effective biotechnological approach to clean up contaminated environments [26]. Microorganisms in nature can decompose pesticide residues, and this method is low cost and environmentally friendly [27]. Among microorganisms capable of degrading pesticides, bacteria and fungi play the main role [28]. Bacteria such as Enterobacter spp., Klebsiella spp., Alcaligenes faecalis [29], Ochabactrum sp. JAS2 [30, 31], Bacillus subtilis, Y242 [32], Bacillus spp. [33], Bacillus subtilis NJ11 [34], Pseudomonas aeruginosa [35], and Pseudomonas putida [36] are capable of degrading the pesticide chlorpyrifos.

Therefore, our objective in this work was to search, isolate and characterize a bacterial strain capable of actively degrading chlorpyrifos.

Materials and Methods

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Collection and Characterization of Soils

Soil samples were taken from a farmer's field (Uzbekistan), where pesticides such as chlorpyrifos, cypermethrin, and others have been used for many years. The soil was taken from a depth of 0−15 cm. The obtained samples were cleaned of large inclusions before drying in the laboratory. Then, the soil was sifted through a stainless steel sieve (diameter 2 mm) and characterized by physicochemical parameters. Prior to analysis, the soil was placed in special bags and sterilized by autoclaving for 30 min at 121℃.

Samples and Reagents

The chlorpyrifos standard (97% purity) was obtained from Pioneer international Ltd., China. Chromatographic- grade acetone and all other chemicals and reagents used were analytical grade and commercially available. The initial solutions were carried out on the basis of acetone. We used a Mettler Toledo pH meter. For the isolation of microorganisms, the following nutrient media were used: MPA, MPB (manufactured by HiMedia Pvt. Ltd., India), as well as mineral salt medium (MSM) (pH 6.8−7.0) containing (g/l): K2HPO4 - 1.5; KH2PO4 - 0.5; NaCl 0.5; (NH4)2SO4 - 0.5; MgSO4·7H2O, -0.2 and 1 ml of a solution of trace elements according to Fedorov. The microelement solution consisted of (g/l) H3BO3 - 5.0; Na2MoO4·2H2O - 5.0; MnSO4·4H2O - 3.0; KI, 0.5; NaBr, 0.5; ZnSO4·7H2O - 0.2; and Al2(SO4)3· 18H2O - 0.3.

Isolation of Bacteria Resistant to Chlorpyrifos

In laboratory conditions, 1 kg of soil from the farmer's field was additionally contaminated with chlorpyrifos and kept at 30℃ for a month. After a month, 10 g of soil samples were added to Erlenmeyer flasks (250 ml) containing nutrient broth with the addition of chlorpyrifos and incubated for 48 h on a rotary shaker at 150 rpm and 30℃. Dilutions were then prepared and added to nutrient agar plates containing 50 mg/l chlorpyrifos. After 3 days, the grown colonies were counted and individual isolates were subcultured into dishes and incubated for another 5 days at 28℃. Individual colonies were subcultured in dishes with a mineral salt medium with the addition of higher concentrations of chlorpyrifos (up to 100 mg/l). The resulting isolates were maintained at 4℃ and subcultured every month.

Identification of Bacteria

Identification of bacteria isolated from individual colonies formed on nutrient agar with added chlorpyrifos was carried out by studying the morphological and cultural properties. Analysis using MALDI-TOF MS was then conducted, and the nucleotide sequence of 16S rRNA was determined.

DNA Extraction. First, a nutrient broth was prepared, to which a bacterial culture was then added and grown at a temperature of 30℃ for 12 h. Next, bacterial DNA was isolated from the bacterial culture grown in 10 ml nutrient broth using the RIBO-prep (InterLabServis, Russia) reagent kit. DNA extraction was performed according to the manufacturer's protocol. Extracted DNA samples were analyzed in 0.9% agarose using a spectrophotometer, and DNA samples were stored at -20℃. Amplification of the 16S rRNA gene was carried out using the PCR method. The 16S rRNA gene was selected for molecular-genetic identification of bacterial cultures. The following primers were used to amplify the 16S gene (Table 1).

Table 1 . Primers used for16S rRNA PDB-3 sequencing.

NameSequenceReference
27F5'-AGAGTTTGATCCTGGCTCAG-3'37
1492R5'-GGTTACCTTGTTACGACTT-3'37


The total volume of the amplification reaction was 20 μl, and a ready-made lyophilized PCR core kit (Isogene, Russia) was used. Primers (5 pmol/μl) were added at 2 μl, free nucleotides at 2.5 μl, and 2 μl of DNA at 20 ng/μl. Enzyme and free nucleotides were in the ready-made kit and placed separately in each test tube in a lyophilized form. Then, 10 μl of buffer and up to 20 μl of deionized water were added. The reaction program was carried out as follows: initial denaturation 95℃ - 5 min, 95℃ - 20 s, for 35 cycles, 57℃ - 20 s, 72℃ - 40 s, and final denaturation 72℃ - 3 min. The PCR product was analyzed by 2% agarose gel electrophoresis. The PCR product was purified using the GFX™ PCR DNA and Gel Band Purification Kit and its concentration was measured on a Qubit 2.0 (Invitrogen, USA) apparatus. The purified PCR product was subjected to a sequencing reaction using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA). The sequence product was purified using the BigDye XTerminator Purification Kit and loaded into the sequencer. Sequencing reactions and sequencing product purification procedures were performed according to Kit instructions. The resulting sequence product was processed in the Codon Code Aligner software and compared (BLAST) to the NCBI database. The resulting sequence was deposited in the international NCBI database and registered with the corresponding number (GenBank: OL587509.1).

Inoculum Preparation

For soil inoculation, the culture was grown in mineral saline medium containing chlorpyrifos for 48 h. The control was the medium with chlorpyrifos without culture. Growth activity was checked by optical density using a spectrophotometer (UV-5100B, UV-VIS Spectrophotometer) [38−40]. To determine the efficiency of decomposition of the initial concentration of chlorpyrifos, a liquid culture of the strain was added to the soil in three optical densities formed at 0, and after 24 and 48 h.

Biodegradation of Chlorpyrifos in Soil

Studies on the biodegradation of chlorpyrifos with the isolated strain PDB-3 were carried out in sterile soils. Soil samples (100 g) were spiked with chlorpyrifos to final concentrations of 50, 75, 100, and 125 mg/kg by adding an acetone-based chlorpyrifos solution. Initially, the prepared solution was added to a small part (15−20 g) of the soil, which was then mixed with the remaining amount of soil after the solvent had evaporated. Soil samples were inoculated and incubated at 30℃. Tests were performed in triplicate. The control was uninoculated sterile soil with chlorpyrifos. The duration of the experiment was 30 days.

Method of Gas Chromatographic Analysis

The study was performed on an Agilent 8890B Split/ Splitless Gas Chromatograph used with an Agilent 5977B Series GC/MSD in SIM, SCAN, and Electron Impact (EI) Ionization modes. The following are given as the conditions for the gas chromatograph analysis: Analytical column HP-5ms Ultra Inert, 30 m × 250 μm × 0.25 μm, Injection volume 1 μl, Injection mode, Split-free Evaporator temperature 280℃, UI Liner, splitless, single taper, fiberglass spray gasket gold-plated, Ultra Inert with washer carrier gas: hydrogen, constant flow = 1.2 ml/min, thermostat program 60℃ for 1 min, then 40℃/min to 170℃, then 10℃/min to 310℃, then hold for 2 min. The temperature in the transport line is 280℃. MS conditions: Delay to eliminate solvent effects 3.5 min, Data collection mode SIM, SCAN, amplification factor 1.00, source temperature 250℃, quadrupole temperature 150℃.

Statistical Analyses

Statistical analysis and exponential curve fitting were performed using Origin 8.6 software (Microcal Software Inc., USA). Results were expressed as mean ± SE, and one-way ANOVA was performed to determine their statistical significance.

Results and Discussion

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Isolation of Bacteria Resistant to Chlorpyrifos

To isolate chlorpyrifos-resistant microorganisms, we used soil from a farmer's field that had been treated for a number of years with the pesticides chlorpyrifos, cypermethrin, and others (Fig. 1A). Under laboratory conditions, soil samples were additionally contaminated with chlorpyrifos at a rate of 10−50 mg/kg soil (Fig. 1B). The treated samples were kept in a thermostat for a month at 30℃ for the adaptation of microorganisms to the pesticide. A month later, samples (10 g of soil) were added to Erlenmeyer flasks containing nutrient broth with chlorpyrifos and incubated on a rotary shaker at 150 rpm at 30℃ for 48 h. As a result of such passages, isolates resistant to these concentrations of the pesticide were isolated. A total of 17 isolates were isolated that were capable of growing and multiplying on a medium containing chlorpyrifos. Isolates Nos. 3, 6, 11, 16 showed good growth at chlorpyrifos concentrations of up to 50 mg/kg. In the next series of experiments, the concentration of chlorpyrifos was increased to 100 mg/l. Based on the results of studying the resistance of the isolated microorganisms to chlorpyrifos, isolate “No. 3” was selected, which showed good and stable growth (cell titer up to 107 CFU/g) at a concentration of 100 mg/l (Fig. 1C). This isolate was also introduced into MSM medium with chlorpyrifos (Fig. 1D). A further increase in the concentration of chlorpyrifos to 150 mg/l had a toxic effect on cell growth.

Figure 1.Isolation of microorganisms from pesticide-contaminated soil. (A) Farmer's field where chlorpyrifos has been used for many years, (B) Contaminated soil, (C)-Isolation of a strain from soil, (D) Growth in MSM medium with chlorpyrifos.

Isolate No.3 was then investigated to determine its morpho-cultural properties, which revealed rod-shaped single cells with a size of 1.2−2.0 μm. On MPA the isolate forms smooth convex, shiny colonies, is white-gray in color, and 2−2.5 mm in diameter. It is gram-negative, does not form spores, and grows well at 30−35℃ and pH 6.9−7.2. (Fig. 2).

Figure 2.Initial strain identification. (A) Strain purification (B) Gram stain.

MALDI-TOF MS analysis was also carried out, and in accordance with the results obtained, this strain was tentatively assigned to the species O. intermedium. For more accurate identification, the nucleotide sequence of 16S rRNA was determined. The results showed 100% similarity to the species O. intermedium (Fig. 3).

Figure 3.Phylogenetic tree of the Ochrobactrum intermedium strain PDB-3.

Table 2 . Scientific classification of Ochrobactrum intermedium.

KingdomTypeClassOrderFamilyGenusView
BacteriaProteobacteriaAlpha ProteobacteriaRhizobialesBrucellaceaeOchrobactrumOchrobactrum intermedium


16S rRNA gene sequence data of O. intermedium strain PDB-3 were processed using Unigene Ver35 and Chromas software, and low-confidence peaks were removed. The DNA sequences were then BLASTed against the NCBI nucleotide database. Highly similar DNA sequences were aligned using the MegaX program and the ClustalW algorithm. The phylogenetic tree was constructed using the neighbor-joining algorithm in the MegaX program.

Thus, strain O. intermedium PDB-3 showed very close similarity (99.6%) to strains of O. intermedium. This indicates that the strain under study actually belongs to the phylum O. intermedium. The strain was registered in the National Center for Biotechnology Information (NCBI) database: GenBank: OL587509.1. This species of bacteria was first described in 1998 [41]. Table No. 2 shows the scientific classification of O. intermedium.

Based on recent genome comparison studies, the genus Ochrobactrum was reclassified and its species included in the genus Brucella [42, 43]. O. intermedium degrades many pollutants [44], including pesticides [45, 46]. For example, O. intermedium SP9 degraded the pesticide cypermethrin to 69.1% within 8 days [47]. Moreover, Ochrobactrum anthropi is used for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) [48], and O. anthropi strain L1-W successfully degraded the air pollutant Di-2-ethylhexyl phthalate (DEHP) [49].

Growing O. intermedium for Soil Application

The culture was grown in 100 ml of mineral salt medium containing chlorpyrifos. The colonies were incubated on a rotary shaker at 30℃ and 150 rpm. The control was a non-inoculated medium with chlorpyrifos. The growth of the culture was monitored by optical density (on a spectrophotometer at a wavelength of 600 nm). Five milliliter aliquots were removed from the growing culture broth at 12-h intervals. As the data showed, the maximum growth of the O. intermedium PDB-3 strain was observed after 48 h. In this case, the optical density was 0.32. To determine the potential of the strain, the culture was added to the soil with an optical density forming the initial hour (OD = 0.03), after 24 (OD = 0.2) and after 48 (OD = 0.32) hours (Fig. 4).

Figure 4.Growth of culture within 48 h.

Biodegradation of Chlorpyrifos in Soil

Initially, in laboratory conditions, the soil was dried and all unnecessary parts (stones, plant debris) were removed. The soil was then sterilized in an autoclave at 121℃ for 45 min. After preparing acetone-based solutions of chlorpyrifos, they were added to a small part of the soil (approximately 20% of the total soil mass), after thoroughly mixing the soil with the solution, mixed with the remaining amount of soil. Then, the biomass culture formed within 48 h was added. Inoculation of the strain into the soil was carried out at three optical densities (OD = 0.03; OD = 0.2; OD = 0.32). The experiments lasted 30 days. For the reproduction and development of microorganisms, it is necessary to maintain humidity, because when the humidity decreases, the growth and reproduction of microorganisms slows down, which negatively affects the process of biodegradation. To maintain humidity (about 70%), the test soil samples were irrigated with sterile water every 3 days. Soil samples for chromatographic analysis were taken at intervals of 5 days (0, 5, 10, 15, 20, 25, 30). The results of GC-MS analysis showed the presence of chlorpyrifos with a peak appearing in soil samples at a retention time of 10.410 min (Fig. 5A). Over time, this peak decreases gradually. Initially, the rate of decomposition was somewhat slower, then with an increase in microbial biomass in the soil environment, starting from 10−15 days, the degradation of the pesticide accelerated. On day 15 of the study, a new peak appeared at a retention time of 9.648 min corresponding to TCP (Fig. 5B). The metabolite obtained as a result of the decomposition of chlorpyrifos is a representative of organochlorine pollutants; its halflife in the natural environment ranges from 65 to 360 days [50]. The formation and accumulation of TCP in soil can limit the proliferation of microorganisms and also inhibit the degradation of chlorpyrifos [51]. GC-MS results showed that TCP residues were undetectable at the end of our studies and degraded to an unknown polar metabolite (Fig. 5C).

Figure 5.GC-MS chromatograms obtained from the degradation of chlorpyrifos. (A) Chlorpyrifos, (B) 3,5,6-trichloro-2-pyridinol (TCP), (C) Decrease in initial concentration of chlorpyrifos.

Inoculation of PDB-3 into the soil at different optical densities showed the effectiveness of using the strain in the process of degrading chlorpyrifos. For example, the percentage of pesticide degradation was 88.7% (50 mg/ kg) when bacteria were introduced at OD = 0.03. Further results are as follows: 88.4; 80.8 and 54.4% (initial 75, 100 and 125 mg/kg). The introduction of the strain at OD = 0.2 showed 91.6, 91.3, 84.8 and 72%, respectively. An increase in OP to 0.32 had a positive effect on the degradation process and was 97.4, 94.8, 92.5, and 83.6%, respectively. The maximum degradation of chlorpyrifos was observed when the PDB-3 strain was introduced at OP = 0.32, which showed the possibility of further use of this strain in the process of bioremediation of soils contaminated with the pesticide chlorpyrifos. In addition, the degradation of the total mass of the pesticide decreases with a decrease in the optical density of the strain. The experiment lasted 30 days. From the 10th day of the study, an acceleration of the degradation process was detected. We found that in the control variant, the initial concentration of chlorpyrifos in all variants remained practically unchanged and the percentage of degradation was 15.2% (50 mg/kg); 13.3% (75 mg/kg); 10.7% (100 mg/kg), and 10% (125 mg/kg). There is evidence in the literature that in the process of pesticide degradation, a special role belongs to soil microorganisms, but there are also other factors that influence the degradation of pesticides, such as soil type [52], humidity [53], soil pH [54, 55], and temperature [56, 57]. One of these factors may have contributed to some of the decrease in the initial chlorpyrifos concentration in the non-inoculated control soil. A dynamic quantitative analysis of the strain was also carried out on the formation of biomass during the degradation of chlorpyrifos. General microbial analysis showed that in the first week of research, the PDB-3 strain adapted to the food source, so the degradation of the pesticide occurred somewhat more slowly. Then, starting from the 10th day of the study, an increase in the formation of the total biomass of the PDB-3 strain was observed as shown in Fig. 6. (The results are given in 10-6 CFU/g).

Figure 6.Bacterial analysis of the studied soil during the degradation of chlorpyrifos. (A) 5th day, (B) 10th day, (C) 15th day.

This, in turn, had a positive effect on the degradation of the initial amount of pesticide by the bacterial culture. After 25 days and at the end of the experiment, we found that the total biomass of bacteria decreased, which, in turn, may indicate that the source of nutrients in the medium decreased. Figure 7 shows data on determining the degradation of the residual concentration of chlorpyrifos when adding the O. intermedium strain PDB-3 in three optical densities.

Figure 7.Determination of biodegradation of chlorpyrifos. (A) 50 mg/kg, (b) 75 mg/kg, (C) 100 mg/kg, (D) 125 mg/kg.

The degradation of chlorpyrifos in soil is influenced not only by moisture, soil pH, and temperature, but also by its initial concentration [58]. According to our findings, degradation is faster in small concentrations, and increasing the initial amount may slow down the biodegradation process. In the degradation of chlorpyrifos, microbial consortia are more effective than a single strain. The researchers formed the ECO-M bacterial consortium by isolating bacterial strains that degrade chlorpyrifos. With the help of this consortium, 100% degradation of chlorpyrifos was achieved within 6 days, at an initial concentration of 50 m/l. Furthermore, on the basis of ECO-M, chlorpyrifos was converted to 3,5,6-tri-chloro-2-pyridinol (TCP) and 2-hydroxypyridine [58]. In the presence of a mixture of two fungi (C1 and C3), the researchers were able to reduce the half-life of chlorpyrifos to 13.6 days in vitro, which was 231 days in the control. This fungal consortium showed 98.4% degradation of chlorpyrifos (600 mg/l) within 30 days under Czapek dox medium conditions [60]. It was reported that, within 35 days, chlorpyrifos at a concentration of 12 mg/kg was successfully degraded to 79.5% in soil [60]. Bacillus pumilus strain C2A1 showed 97% degradation of 50 mg/ kg chlorpyrifos in 45 days [61]. It is reported that higher concentrations have a negative effect on the degradation rate, whereas at low concentrations, the biodegradation rate becomes rapid [62]. The main metabolite of chlorpyrifos degradation is 3,5,6-trichloro-2-pyridinol (TCP) and diethylthiophosphate (DETP), which are formed as a result of the degradation of chlorpyrifos by soil microorganisms. Further degradation of these products results in the formation of non-toxic metabolites, such as CO2, H2O, and NH3 [63]. Scientists observed the transient accumulation of 3,5,6-trichloro-2-pyridinol (TCP) during the biodegradation of chlorpyrifos based on the new fungal strain Cladosporium cladosporioides Hu-01. However, this intermediate product did not accumulate in the medium and quickly disappeared. This strain provides a metabolic pathway for the complete detoxification of chlorpyrifos and its hydrolysis product TCP [50].

In the 1970s, after the ban on the production and use of organochlorine pesticides (due to their high toxic effects), the amount of organophosphorus pesticides increased significantly, especially in developing countries [64]. The insecticide chlorpyrifos has been widely used worldwide since the 1960s in agriculture to control crops such as cotton, grain plants, vegetables, and fruits. It is moderately toxic, has neurotoxic and immunotoxic properties and is harmful to both animals and humans. This insecticide causes a reduction in the population of bacteria, fungi and actinomycetes in the soil and inhibits nitrogen mineralization in the soil [65].

Acknowledgments

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We would like to thank the staff of the Institute of Biorganic Chemistry of the Academy of Sciences of the Republic of Uzbekistan and the Center for Advanced Technologies for their contribution and assistance to this article.

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