SCREENING OF PGPR TRAITS AND MONOCROTOPHOS PESTICIDE DEGRADATION PROPERTIES OF ACTINOBACTERIAL ISOLATES

SCREENING OF PGPR TRAITS AND MONOCROTOPHOS PESTICIDE DEGRADATION PROPERTIES OF ACTINOBACTERIAL ISOLATES

Ankita Rani *, Abhishek Mathur and Sanjay Gupta

Department of Biotechnology, School of Biosciences, Swami Rama Himalayan University, Dehradun, Uttarakhand, India.

ABSTRACT: Bioremediation of organic compounds and heavy metals has been recognized to be a successful and effective technique in rhizoremediation of soil toxicity using the beneficial microbes which produce different metabolites and enzymes to degrade the toxic compounds/ pesticides available in the soil. In the present study, actinobacterial isolates were explored and identified for bioremediation of soil toxicity available in the soil. The actinobacterial isolates were utilized for the promising PGPR traits and ability to degrade the monocrotophos pesticide residues available in the soil. The “opd” gene of interest responsible for monocrotophos pesticide degradation trait was isolated and cloned in vector DNA to produce recombinant DNA. The recombinant DNA was transformed in E. coli cells to produce multiple copies in E. coli cells resulting in recombinant (transformed) and non-recombinant (non-transformed) colonies. The transformed E. coli cells were inoculated in nutrient broth having pesticide concentration. The transformed cells degraded the pesticide, and the HPLC method determined the derivatives produced. The formulations based on actinobacterial isolates were tested, and field applications were done to determine the reduced soil toxicity, if any, observed.

Keywords: Actinobacterial isolates, PGPR traits, Monocrotophos pesticides, Bioremediation, Rhizoremediation, Reduction in soil toxicity, Recombinant cells, Least toxic pesticide derivatives

INTRODUCTION: Bioremediation is also used to convert hazardous substances into less toxic components. Microorganisms have a vital role in the breakdown and mineralization of these contaminants. Bioremediation as sustainable technology is significant in examining and reducing manmade chemicals released into the environment. Bioremediation is the microorganisms used to attain the function of bioremediation. Bioremediation technology has been commonly categorized as ex-situ and in-situ bioremediation.

In situ bioremediation comprises the treatment and elimination of the contaminated material at the site, whereas, in ex-situ, the contaminated are treated somewhere else. bioventing, bioleaching, biostimulation, land farming, composting, bioaugmentation, rhizofilteration, and phytoremediation are a few examples of bioremediation technologies ^1-5. Biodegradation and bioremediation are similar methods up to an extent since both of these approaches employ microorganisms for the alteration or breakdown of pesticides.

The only difference between these two is that biodegradation is a natural process whereas bioremediation is considered technology. Various aspects are limiting factors for pesticide degrading microorganisms, such as pH, temperature, nutrients, water potential and a number of metabolites or pesticide content in soil ^6-8. Actinobacterial isolates produce non-antibiotic molecules which exhibit bioactivities, such as immuno-suppressors, phytotoxins, bio-pesticides, Nano-particles, probiotics, enzyme inhibitors and different enzymes and proteins that are involved in the degradation of complex polymers and biomolecules ^9-12, that single isolates can mineralize a limited number of these xenobiotic pesticides. Still, consortia of bacteria are mostly required for complete degradation of the process. Members of this group of gram-positive bacteria have been found to degrade pesticides with widely different chemical structures, including organochlorines, s-triazines, triazinones, carbamates, organophosphates, organ phosphonates, acetanilides and sulfonylureas. Single isolates can mineralize a limited number of these xenobiotic pesticides, but consortia of bacteria are often required for complete degradation ^13-15.

MATERIALS AND METHODS:

Screening of Isolates for PGPR Traits: The actinobacterial isolates were screened for PGPR traits ^16-20.

A. IAA Production: Indole acetic acid (IAA) production was quantitatively estimated by Salkowski method. The actinobacterial cultures were grown on Luria broth liquid medium at 36±2 °C. Fifty milliliter of Luria Bertani (LB) broth containing 0.1% DL tryptophan were inoculated with 500 µl of 48 h old actinobacterial cultures and incubated in refrigerated incubator shaker at 30±0.1°C at 180 rpm for 48 h in dark. Fully grown bacterial cultures were centrifuged at 10,000 rpm for 10 minutes at 4°C. Estimation of IAA production in the supernatants was done using a colorimetric assay. One milliliter (1 ml) of supernatant was mixed with 100 ml of 10 mM orthophosphoric acid and 2 ml of the Salkowski reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35% HCIO4) at 28±2 ^oC for 30 minutes. The development of pink colour in test tubes at the end of the incubation indicated IAA production. The pink colour absorbance measured the quantification of IAA at 530 nm after 30 minutes in UV/VIS spectrophotometer. The results were tabulated as Higher IAA producers (+++); Medium IAA producers (++), and Lowest IAA producers (+).

B. Phosphate Solubilization: A loop full of isolated pure fresh actinobacterial cultures was streaked on the Centre of agar plates modified with Pikovskaya agar with insoluble tricalcium phosphate (TCP) and incubated for 120 h at 28±2°C 18. The halo zone around the bacterial colonies indicated positive phosphate solubilization ability (Pikovskaya, 1948). The results were tabulated as Higher phosphate solubilizers (+++); Medium phosphate solubilizers (++), and Lowest phosphate solubilizers (+).

C. Siderophores and HCN Production: Qualitative estimation of siderophore production by the actinobacteria isolates was determined by adopting the modified chrome azurol sulphonate (CAS) assay method. Production of siderophore was determined by developing an orange halo zone around the actinobacterial colonies. In addition, all the bacterial isolates were screened for HCN production. The Colour change of the filter paper from deep yellow to reddish-brown colour indicated the production of HCN. The results were tabulated as Higher Siderophores and HCN producers (+++); Medium Siderophores and HCN producers (++) and Lower Siderophores and HCN producers (+).

D. Catalase Activity: Actinobacterial cultures were grown in a nutrient agar medium for 48 h at 28°C. The 48-hour-old bacterial colonies were added with 2-3 drops of hydrogen peroxide (3%) on a clean glass slide and mixed using a sterile toothpick. Oxygen evolution as effervescence indicated catalase activity (Rorth and Jensen, 1967). The results were tabulated as Higher Catalase producers (+++); Medium Catalase producers (++), and Lower Catalase producers (+).

Screening of Actinobacterial Isolates for Monocrotophos Pesticides Degradation:

Primary Screening:

Determination of Zone of Clearance of Pesticide Degradation: The screening of actinobacterial isolates for monocrotophos pesticide degradation was performed as per the method described. The growth of isolated actinobacterial cultures were inoculated separately in 100 ml Mineral Salt Media (MSM) enriched with an addition of 25 ppm MCP pesticide. These samples were incubated on a rotary shaker (150 rpm) at 30°C for 7 days. The growth curve for the actinobacterial isolates was determined via optical density/absorbance using UV- spectrophotometer at various time intervals. After which one, the cultures were frequently transferred every 3-4 days or until increased turbidity was evidenced.

After 3-4 times of repeated sub-culturing, 0.1 ml culture broth was pipette and introduced in wells punctured within pesticide agar plates. Cultures were incubated at 30°C for 5-8 days. Pesticide degrading isolates were then screened, which developed a clear zone around the wells determining clearance ²¹.

Secondary Screening:

Determination of Pesticide Degradation by Culture Streak Technique: The pesticide degradation was observed on pesticide-enriched agar by streaking the actinobacterial cultures; the pesticide degradation ability of the actinobacterial isolates was determined as a clearance zone.

Expression of Genes of Promising Isolates of Actinobacteria in E. coli Cells: Escherichia coli was utilized as one of the organisms of choice to produce recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols for the high-level production of multiple functional proteins. In the present investigation, the genes for monocrotophos pesticide (MCP) degradation were isolated from promising actinobacterial isolates, fused with vector DNA and transformed into E. coli cells. The recombinant E. coli cells were further utilized for the biodegradation of monocrotophos pesticides (MCP). The enzymes for degradation of monocrotophos pesticides were identified as phosphodiesterase (PTE) or organophosphorus hydrolase (OPH) specifically encoded by the opd gene. This gene has been Expression in genes of promising isolates of Actinobacteria in E. coli cells.

IMAGE 1: REPRESENTATION OF THE RELATIONSHIPS BETWEEN PESTICIDES, MICROBIAL COMMUNITIES AND THE DISCOVERY OF NEW BIODEGRADATION PROCESSES OMICS = HIGH THROUGHPUT-BASED CHARACTERIZATION OF BIOMOLECULES CHARACTERISTIC OF BIOPROCESSES; DNA, GENOMICS; MRNA, TRANSCRIPTOMICS; PROTEIN, PROTEOMICS; METABOLITES, METABOLOMICS

Biodegradation of Monocrotophos Pesticides: The concentrations of monocrotophos pesticide (MCP) at 10 µg/ml were utilized in nutrient broth in different sets, followed by sterilization and inoculation of the E. coli cells and incubation of the broth for 48 hours duration. The degradation of MCP pesticide in the broth culture was determined using TLC and HPLC, as described by Chao et al., 2009. In brief, 4 mL of filtrate was extracted with ethyl acetate from a 5-mL aliquot of culture supernatant. All experimental MCP concentrations were determined at 276 nm by HPLC (PerkinElmer 200 series, CT, USA). The solution filtrate (10 μL) was separated and added with methanol (1:1). Ten, it went across 0.45- µm PVDF filter and injected into the HPLC column (C18, 250×4.6 mm, 5μm, Phenomenex, CA, USA). The 10 μL filtered sample was injected into the HPLC column, and the 0.5% acetic acid and methanol (1:4 v/v) were used as eluent at 1 mL/min flow rate.

Formulations of Actinobacterial Strains: The formulations were prepared as per the following modified methods ²².

1. Alginate-Kaolin Based Granular Formulation (AL-KAO Granular Formulation): Sodium alginate solution was prepared by dissolving 20 g of dry sodium alginate in a minimum volume (10 ml) of sterile distilled water. This mixture was poured into 1 L of swirling, warm, distilled water and allowed to mix on the stirrer for 30 min until a homogeneous suspension was obtained. The alginate solution was sterilized for 15 min at 121 °C and 101 kPa. Twenty grams of the potent dried Actinobacterial isolate propagules were mixed thoroughly with 20 g of previously sterilized kaolin (aluminum silicate), and the mixture was added in small portions (2 g) into 1 L of swirling, sterile distilled water supplemented with 4 drops of Tween 20. The alginate-kaolin-actinobacterial mixture was allowed to swirl in the stirrer until ready for mixing with the sodium alginate solution. A droplet-forming device was constructed by attaching a 1-L reagent bottle with a spout at the bottom to a T-valve outlet system. The entire device was sterilized for 15 min at 121 °C and 101 kPa before use. The sodium alginate solution and kaolin mixture were added to the reagent bottle in 1:1 ratio and stirred continuously. At the same time, the suspension was allowed to drip through an Eppendorf pipette tip, attached to the T- 10 valve, into a sterile solution of 0.1 M CaCl₂. The resulting alginate-kaolin beads were then allowed to stand in a fresh 0.1 M CaCl₂ solution for 30 min, filtered through a sterile cheese cloth, and washed at least three times with sterile distilled water. The beads were lyophilized at -70 °C, and their dry weight was recorded. This lyophilized granular formulation of mycobacterium (hereafter referred to as beads) was stored in the dark at 4 °C.

2. Flour-Kaolin Based Granular Formulation (FLO-KAO Granular Formulation): The flour and kaolin material were sterilized separately for 15 min at 121 °C and 101 kPa. Upon cooling, 200g of flour, 4 g of kaolin, and 20 g of the inoculum were mixed thoroughly and sieved through a sterile 250 µm pore screen to obtain a homogeneous mixture of the three components. To this mixture, 180 ml sterile distilled water supplemented with 2 drops of Tween 20 was added slowly and mixed thoroughly until a dough was formed. The flakes were lyophilized at -70°C. The lyophilized formulations in liquid hereafter referred to as granules) were stored in the dark at 4°C.

3. Calcium carbonate-CMC Based Powder Formulation (CC-CMC Powder Formulation): The calcium carbonate and CMC material was sterilized separately for 15 min at 121 °C and 101 kPa. Upon cooling, CMC was mixed with 20 g of the inoculum thoroughly and sieved through a sterile 250 µm pore screen to obtain a homogeneous mixture. For this mixture (10 Kg) produced with CMC, the quantity of calcium carbonate was mixed in 90 Kg concentration. The lyophilized formulations in liquid, referred to as powder, were stored in the dark at 4 °C. The material was further utilized to check the shelf life of isolates and the application of powder in the bioremediation of soil toxicity.

RESULTS AND DISCUSSION: As per the studies performed, 120 soil samples were collected from different field areas of the Uttarakhand region (Tehri-Garhwal, Chamoli, Srinagar, Uttarkashi, and Haridwar) having dominant usage of monocrotophos pesticides. Amongst these samples, a total of 280 microbes was isolated; out of which 24 isolates of Actinobacteria (8.57%) were isolated. The actinobacteria isolates were screened on specific agar media and characterized by morphological colony appearance and staining procedures. The actinobacteria isolates were categorized based on a) type of pigment production and colony and color. These actinobacterial isolates were further screened for their identification by molecular. The results revealed the strains of the genera viz. Micromonospora (65%), Actinomycetes (25%) and Streptomyces (10%) (Data not shown). The isolates of each of the mentioned genera were screened for PGPR assays Table 1.

TABLE 1: PGPR ASSAYS OF ACTINOBACTERIAL ISOLATES

 *+++, Dominant producer, ++, Medium producedr, +, Lowest Producer, –, Negligible producer

TABLE 2: PERCENTAGE DEGRADATION OF ACTINOBACTERIAL ISOLATES

FIG. 1: PERCENT DEGRADATION RATE OF ACTINOBACTERIAL ISOLATES

The degradation rate (%) of the actinobacterial isolates was calculated based on no. of strains of the respective genera which showed a zone of clearance. The results are shown in Table 2 and Fig. 1, Fig. 2 and Fig. 3. The isolated opd gene of interest was isolated and cloned in vector DNA to produce recombinant DNA Fig. 4A, Fig. 4B. The recombinant DNA was transformed in E. coli cells to produce multiple copies in E. coli cells resulting in recombinant (transformed) and non-recombinant (non-transformed) colonies. The transformed E. coli cells were inoculated in nutrient broth having pesticide concentration.

FIG. 2: PRIMARY SCREENING: DETERMINATION OF ZONE OF CLEARANCE OF PESTICIDE DEGRADATION

FIG. 3: SECONDARY SCREENING- DETERMINATION OF PESTICIDE DEGRADATION BY CULTURE STREAK TECHNIQUE

FIG. 4(A): PREPARATION OF RECOMBINANT DNA AND TRANSFORMATION IN E. COLI CELLS

FIG. 4(B): SCREENING OF RECOMBINANT AND NON- RECOMBINANT COLONIES (BLUE COLONIES WERE REGARDED AS NON-RECOMBINANT COLONIES WHILE WHITE COLONIES WERE REPRESENTED AS RECOMBINANT COLONIES)

DETECTOR A CH1 276NM

FIG. 5(A): HPLC CHROMATOGRAM OF ORIGINAL MCP AVAILABLE IN NUTRIENT BROTH

DETECTOR A CH1 276NM

FIG. 5(B): HPLC CHROMATOGRAM OF DEGRADATIVE MCP RESIDUES AVAILABLE IN NUTRIENT BROTH AFTER BIODEGRADATION

The transformed cells degraded the pesticide and derivatives produced were determined by HPLC method Fig. 5A, Fig. 5B. Three formulations of actinobacterial strain viz., Alginate-Kaolin Based Granular Formulation (AL-KAO granular formulation), Flour-Kaolin Based Granular Formulation (FLO-KAO granular formulation), Calcium carbonate-CMC Based Powder Formulation (CC-CMC powder formulation) were prepared and utilized in field trials to study the reduction in soil toxicity. The results revealed that, when applied to monocrotophos enriched soil, FLO-KAO granular formulation revealed 62% degradation in toxicity Table 3, Fig. 6.

TABLE 3: FIELD APPLICATION TRIALS OF FORMULATIONS

FIG. 6: GRAPHICAL REPRESENTATION OF FIELD APPLICATION TRIALS OF FORMULATIONS

Funding Information: No Funding Agency acknowledged.

ACKNOWLEDGMENT: The authors acknowledge the help obtained from the college management for providing research facilities and support for performing this study.

CONFLICTS OF INTEREST: The authors stated no Conflict of Interest for the publication of this research article.

REFERENCES:

1. Navarrete IA, Tee KA, Unson JR and Hallare AV: Organochlorine pesticide residues in surface water and groundwater along Pampanga River, Philippines. Environmental Monitoring and Assessment 2018; 190(5): 1-8.
2. Health implications from monocrotophos use: a review of the evidence in India 2013.
3. Lake IR, Hooper L, Abdelhamid A, Bentham G, Boxall AB, Draper A, Fairweather-Tait S, Hulme M, Hunter PR, Nichols G and Waldron KW: Climate change and food security: health impacts in developed countries. Env Health Perspectives 2012; 120 (11): 1520-6.
4. Ashesh A, Singh S, Devi NL and Yadav IC: Organochlorine pesticides in multi-environmental matrices of India: A comprehensive review on characteristics, occurrence, and analytical methods. Microchemical Journal 2022; 16: 107306.
5. Abioye OP, Ijah UJ, Aransiola SA, Auta SH and Ojeba MI: Bioremediation of Toxic Pesticides in Soil Using Microbial Products. In Mycoremediation and Environmental Sustainability Springer Cham 2021; 1-34.
6. Government of India, Ministry of Home Affairs. 10. Accidental deaths and suicides in India 2007. New Delhi: National Crime Records Bureau, 2008. (http://ncrb.nic.in/ADSI2007/home.htm - accessed 02 February 2009).
7. Tang W, Ji H and Hou X: Research progress of microbial degradation of organophosphorus pesticides. Progress in Applied Microbiology 2018; 17: 1(1).
8. Desjardins D, Brereton NJ, Marchand L, Brisson J, Pitre FE and Labrecque M: Complementarity of three distinctive phytoremediation crops for multiple-trace element contaminated soil. Science of the Total Environment 2018; 610: 1428-38.
9. Wu H, Lai C, Zeng G, Liang J, Chen J, Xu J, Dai J, Li X, Liu J, Chen M and Lu L: The interactions of composting and biochar and their implications for soil amendment and pollution remediation: a review. Critical Reviews in Biotechnology 2017; 37(6): 754-64.
10. Aparicio J, Solá MZ, Benimeli CS, Amoroso MJ and Polti MA: Versatility of Streptomyces sp. M7 to bioremediate soils co-contaminated with Cr (VI) and lindane. Ecotoxicology and Environmental Safety 2015; 1(116): 34-9
11. Desoky ES, Saad AM, El-Saadony MT, Merwad AR and Rady MM: Plant growth-promoting rhizobacteria: Potential improvement in antioxidant defense system and suppression of oxidative stress for alleviating salinity stress in Triticum aestivum (L.) plants. Biocatalysis and Agricultural Biotechnology 2020; 30: 101878.
12. Piechulla B, Lemfack MC and Kai M: Effects of discrete bioactive microbial volatiles on plants and fungi. Plant, Cell & Environment 2017; 40(10): 2042-67.
13. Subramani R and Sipkema D: Marine rare actinomycetes: a promising source of structurally diverse and unique novel natural products. Marine Drugs 2019; 17(5): 249.
14. Sharma S, Fulke AB and Chaubey A: Bioprospection of marine actinomycetes: recent advances, challenges and future perspectives. Acta Oce Sinica 2019; 38(6): 1-7.
15. Satish GP, Ashokrao DM and Arun SK: Microbial degradation of pesticide: a review. African Journal of Microbiology Research 2017; 11(24): 992-1012.
16. Kaur R and Goyal D: Toxicity and degradation of the insecticide monocrotophos. Environmental Chemistry Letters 2019; 17(3): 1299-324.
17. Alvarez A, Saez JM, Costa JS, Colin VL, Fuentes MS, Cuozzo SA, Benimeli CS, Polti MA and Amoroso MJ: Actinobacteria: current research and perspectives for bioremediation of pesticides and heavy metals. Chemosphere 2017; 166: 41-62.
18. Srinivasulu M, Nilanjan PC, Chakravarthi BV, Jayabaskaran C, Jaffer MG, Naga RM, Manjunatha B, Darwin RO, Juan OT and Rangaswamy V: Biodegradation of monocrotophos by bacteria isolated from soil. African Journal of Biotechnology 2017; 16(9): 408-17.
19. Kaur R and Goyal D: Toxicity and degradation of the insecticide monocrotophos. Environmental Chemistry Letters 2019; 17(3): 1299-324.
20. Alvarez A, Saez JM, Costa JSD, Colin VL, Fuentes MS, Cuozzo SA and Benimeli CS: Actinobacteria: Current research and perspectives for bioremediation of pesticides and heavy metals. Chemosphere 2017; 166: 41-62.
21. Salaria N, Sharma S and Sharma S: Actinomycetes: Potential and Applications. Int J Allied Pract Resear Rev 2017; 32-43.
22. Gaur N, Narasimhulu K and Pydi Setty Y: Recent advances in the bio-remediation of persistent organic pollutants and its effect on environment. Journal of Cleaner Production 2018; 198: 1602-31.
23. Chakravarthi BK, Naravaneni R, Philip GH and Redddy CS: Investigation of monocrotophos toxic effects on human lymphocytes at cytogenetic level. African Journal of Biotechnology 2009; 8(10).
24. Balakrishnan SL and Rao PV: Monocrotophos degradation potential of bacterial isolates isolated from agricultural soils of Visakhapatnam Dist. J Pure Appl Microbiol 2019; 13(1): 393-402.
25. Singh BK and Walker A: Microbial degradation of organophosphorus compounds. FEMS Microbiology Reviews 2006; 30(3): 428-71.
26. Baghel PS and Bhawana P: Isolation of microorganism for bioremediation of monocrotophos pesticide. International Journal of Current Microbiology and Applied Sciences 2013; 2(11): 202-5.
27. Buvaneswari G, Thenmozhi R, Nagasathya A, Thajuddin N and Kumar P: GC-MS and molecular analyses of monocrotophos biodegradation by selected bacterial isolates. African Journal of Microbiology Research 2018; 12(3): 52-61.
28. Jia KZ, Cui ZL, He J, Guo P and Li SP: Isolation and characterization of a denitrifying monocrotophos-degrading Paracoccus sp. M-1. FEMS Microbiology Letters 2006; 263(2): 155-62.
    

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    How to cite this article:

    Rani A, Mathur A and Gupta S: Screening of PGPR traits and monocrotophos pesticide degradation properties of actinobacterial isolates. Int J Pharm Sci & Res 2023; 14(2): 731-39. doi: 10.13040/IJPSR.0975-8232.14(2).731-39.

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