Isolation, characterization and identification of pesticide degrading bacteria from contaminated soil for bioremediation
Akshita Mehta1 · Kamal Kumar Bhardwaj1 · Mayorin Shaiza1 · Reena Gupta1
Received: 15 June 2020 / Accepted: 8 March 2021 © Akadémiai Kiadó Zrt. 2021
Abstract
In this study, malathion and chlorpyrifos degrading bacteria were isolated from agricultural soil samples taken from the Himachal region in India. A total of 52 organisms were isolated which were further screened for their efficiency for chlor- pyrifos and malathion degradation. Screening was done by checking the growth on Nutrient Agar, Mineral Salt Medium and MacConkey agar plates containing chlorpyrifos and malathion; 37 isolates showed growth in these. Biomass assay and minimum inhibitory concentration (MIC) determination were carried out for the selection of most efficient bacterial isolates. Out of the seven isolates which showed good biomass assay and MIC, only three isolates (PDM-2, PDM-15 and PDM-20) were selected for further studies. These were characterized by various biochemical tests, Gram staining, indole test, methyl red test, Voges-Proskauer test, citrate utilization test and carbohydrate fermentation test. Out of three isolates, PDM-15 showed good resistance against the antibiotics such as erythromycin, chloramphenicol, ampicillin and penicillin and identified as Kocuria assamensis. Degradation of 71.3% of chlorpyrifos and 85% of malathion was observed by the gas chromatography. Therefore, the Kocuria assamensis can be used in the bioremediation of pesticide-contaminated soil.
Keywords Bioremediation · Pesticides · Kocuria assamensis · Malathion · Chlorpyrifos
Introduction
Bioremediation is considered as the most reliable, effective and less expensive environmental cleanup and pollution control process that is currently being investigated for use on a wide variety of chemicals (Aruna et al. 2014; Rashmi and Joseph 2015; Bacosa et al. 2018). Different types of pesticides are used in agricultural fields and are considered beneficial in augmenting crop yields but the excessive and indiscriminate use of pesticides can lead to microbial imbal- ance, environmental pollution and health hazards (Kohler and Triebskorn 2013). So there is a need to develop a rapid
and efficient degradation method to eliminate or minimize the concentrations of pesticides in the environment. To treat the soils contaminated with hazardous materials, a variety of physical and chemical methods are available but many of them do not actually destroy the hazardous compounds but are bound in a modified matrix or transferred from one phase to another (Xenia and Refugio 2016).
Microbes are very vital for the bioremediation of pes- ticides. The phenomenon of biotransformation is common and sometimes very essential for the survival of microor- ganisms responsible for the biodegradation of applied pesti- cides. Chlorpyrifos and malathion both are organophosphate pesticides which are broad spectrum. It has been reported
*
[email protected] Akshita Mehta [email protected]
Kamal Kumar Bhardwaj [email protected]
Mayorin Shaiza [email protected]
earlier that Kocuria sp. is capable of degrading chlorpyrifos (Neti and Zakkula 2013). Considerable amount of research has been done on malathion degradation by bacteria (Singh et al. 2012; Geed et al. 2016) and fungi isolated from differ- ent sources (Abd El-Ghany and Masmali, 2016).
The aim of this work was to identify the potential micro- bial strain able to degrade and utilize pesticides from the contaminated soil.
1 Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla, HP 171 005, India
Vol.:(0123456789)
Materials and methods
Chemicals and pesticides used
All the chemicals used in present investigation were of ana- lytical grade and high purity either procured from Sigma Aldrich (USA) or HIMEDIA (Mumbai, India). The com- mercial-grade pesticides namely chlorpyrifos and malathion were obtained from Ajay Enterprises, Theog, Shimla.
Collection of soil samples
Eleven soil samples were taken from different pesticide- contaminated agricultural fields of Himachal Pradesh.
Agar well diffusion method
Agar well diffusion assay was conducted as described by Naphade et al. (2012) to evaluate the antibacterial activity.
Minimum inhibitory concentration (MIC)
MIC was performed by the methods of Jain et al. (2009). Dilution method was used to determine the MIC of chlor- pyrifos by using sterile microtitre plate of 96 wells. Each well was filled with 100 µl of nutrient broth. Then, 100 µl of 100% chlorpyrifos was added to the first row except in the positive and negative control. Twofold serial dilution was achieved in each row by transferring 100 µl test solu- tion from first well to the next and similarly in subsequent wells so that each well has 100 µl of test solution in serially descending concentrations. 10 µl of different bacterial cul- tures was added in each row one by one. One entire column with test bacteria had 100 µl antibiotics, i.e. negative control instead of chlorpyrifos. Next to negative control column was positive control column without antibiotic and chlorpyri- fos. After this, 10 µl of Resazurin dye (oxidation–reduction indicator) was added to each well. It was used for evalua- tion of cell growth. The microtitre plate was covered with an aluminium foil and incubated at 37 °C for 24 h. Colour changes from purple to pink or colourless were taken as positive indicating the growth of bacteria in the presence of that particular concentration of chlorpyrifos.
Biomass assay for different isolates
Biomass assay was performed to check the growth of pesti- cide degrading bacteria by the method of Kannan and Vani- tha (2004) and Yang et al. (2008). Degradation analyses were performed in 250 ml conical flasks containing 50 ml of MSM medium and 50 µl malathion. Media were inoculated
with different bacterial isolates and incubated in an orbital shaker (150 rpm) at 30 °C for 16 days. An uninoculated flask was used as control. For growth study analysis, 1 ml culture was withdrawn at regular interval of 48 h for 16 con- tinuous days. The biomass concentration was estimated by optical density measurement at 595 nm using UV–Visible spectrophotometer.
Biochemical characterization of pesticides tolerating bacteria
The isolated positive colonies were identified by Gram stain- ing reaction and biochemical tests like indole test, methyl red test, Vogues Proskauer test, citrate test, catalase test, and also the fermentation test for sugars (lactose, maltose, inositol, mannitol, galactose, sorbitol) was done as per the procedure described in Bergey’s manual.
Effect of environmental conditions on growth of selected bacterial isolates
Antibiotic sensitivity test by disc diffusion method
and effect of metal ions and salts on growth of selected bacterial isolates
Selected bacterial isolates were tested for their sensitivity to different antibiotics by means of Kirby–Bauer disc diffusion method (Khan and Malik 2001). Metal ions and salts used in this study were NH4+, Hg2+, Mg2+, Fe2+ and Na+. Negative control, nutrient broth containing cypermethrin and mala- thion was also run parallel (Naphade et al. 2012).
Identification of pesticide degrading bacterial isolate
The 16S rRNA gene sequence method was carried out to identify the pesticide degrading bacterial isolate. The 16S rDNA sequence was used to carry out basic local align- ment search tool (BLAST) alignment search tool of NCBI Genbank database. Based on maximum identity score first fifteen sequences were selected and aligned using multiple alignment software program ClustalW. Distance matrix was generated using ribosomal database project (RDP) database, and the Phylogenetic tree was constructed using MEGA7.
Analysis of pesticides by gas chromatography
In 250 ml of Erlenmeyer flasks, test solutions of chlorpyri- fos and malathion were prepared separately by adding 50 µl of chlorpyrifos and 50 µl of malathion in 50 ml of mineral salt medium (MSM) and inoculated with bacterial isolate and incubated at 37 °C at 150 rpm in an incubator shaker. Both chlorpyifos and malathion (2 µl) test samples were
withdrawn at regular intervals of 48 h parallel with control samples (2 µl). The samples were analysed by using gas chromatography (GC) (Thabit and EL-Naggar 2013).
The results are replicates of three observations.
Results and discussion
Agar well diffusion method (evaluation of pesticide tolerance capacity)
The pesticides contain phosphate group which is easily degraded by bacterial strains thereby indicating their phos- phate solubilizing capability. Out of 52 bacterial isolates,
only 37 were found to be resistant against all the pesticides (chlorpyrifos and malathion).
Minimum inhibitory concentration (MIC)
Out of eight, only five isolates, i.e. PDM-7, PDM-9, PDM- 15, PDM-20 and PDM-33 were capable of tolerating chlor- pyrifos efficiently (Fig. 1a). Colour changes from purple to pink or colourless were taken as positive indicating the growth of bacteria in the presence of that particular concen- tration of chlorpyrifos. Malathion was efficiently tolerated by seven isolates (PDM-2, PDM-9, PDM-10, PDM-11, PDM- 15, PDM-20 and PDM-33) out of eight isolates (Fig. 1b). In a previous study, the bacterial strains CPI 2(51.33%), CPI 6 (67.44%), CPI 9 (59.09%), CPI 10 (53.90%) and CPI 15 (65.34%) gave the highest degradation percentage of chlor- pyrifos (John et al. 2016).
Biomass assay for different isolates
The degradation of chlorpyrifos and malathion was per- formed under the aerobic condition for each strain for a period of 24 days in mineral salt medium and nutrient medium. Cell growth was measured by taking O.D. at 595 nm at a periodic interval of 2 days. For most of the iso- lates, the bacterial growth was increased rapidly after second day of incubation. Out of seven, three isolates showed good biomass production and used pesticides as a carbon source.
Characterization and identification of pesticide tolerating bacteria
Morphological characterization
Some of the morphological characteristics of selected bac- terial isolates are given in Table 1. Three morphologically distinguishable bacterial colonies were observed on nutrient agar plate. It was found that the isolates PDM-2, PDM-15
Fig. 1 a MIC for selected eight bacterial isolates against varying con- centrations of chlorpyrifos. b MIC for selected eight bacterial isolates against varying concentrations of malathion
and PDM-20 were Gram-negative rod, Gram-positive cocci and Gram-negative rod, respectively.
Table 1 Morphological characteristic of selected bacterial isolates
Characteristic Size
PDM-2 2 mm
PDM-15
2mm
PDM-20
3mm
Shape Circular Circular Circular
Colour Off white Off white Cream
Elevation Flat Flat Flat
Margin Entire Entire Entire
Opacity Opaque Translucent Translucent
Consistency Smooth Smooth Smooth
Gram nature Gram-negative rod Gram-positive cocci Gram-negative rod
Biochemical characterization
Results of various biochemical tests of selected bacterial iso- lates are shown in Table 2. Isolate PDM-15 was found to be Voges-Proskauer- positive, sucrose- positive, whereas isolate PDM-20 was negative for Voges-Proskauer and sucrose.
Effect of environmental conditions on growth of selected bacterial isolates
Antibiotic sensitivity test by disc diffusion method
PDM-2 showed sensitivity against Gentamicin (GEN10), Chloramphenicol (C30), Streptomycin (S10), Penicillin (P10), Tetracycline (TE30), Erythromycin (E15) and Ampicillin (AMP10). PDM-15 showed resistance towards Erythromycin (E15), Penicillin(P10), Ampicillin (AMP10), Chloramphenicol (C30) and showed sensitivity against Gentamicin (GEN10), Tetracycline (TE30) and Streptomycin (S10), whereas PDM- 20 showed resistance towards Penicillin (P10), Erythromy- cin (E15) and Ampicillin (AMP10) and sensitivity against Gentamicin (GEN10), Chloramphenicol (C30), Streptomycin (S10) and Tetracycline (TE30).
In a recent study, Xanthomonas maltophilia AC1 and Aci- netobacter sp. were resistant to Ampicillin (AMP), Carbeni- cil in (CAR), Linomycin (MY) and sensitive to Chloram- phenicol (C). However, they exhibited different responses to Fusidic acid (FD) and Clarithromycin (CLR); Xanthomonas maltophilia AC1 was sensitive to these two antibiotics but Acinetobacter sp.was resistant to Clarithromycin (CLR) (Jabeen et al. 2017).
In a previous report, Kocuria mariana from patient under- going peritoneal dialysis showed susceptibility to penicillin, ampicillin, ampicillin-sulbactam, gentamicin, cephalothin, ciprofloxacin, moxifloxacin, erythromycin, clindamycin,
vancomycin, chloramphenicol, tetracycline and rifampin but resistance to trimethoprim-sulfamethoxazole (Lee et al. 2009). Similarly, in a previous finding, Kocuria rhizophila from a patient suffering from methylmalonic aciduria was found to be susceptible in vitro to a wide range of antibi- otics, including all-lactams, macrolides, glycopeptides and quinolones tested with the exception of norfloxacin to which it was resistant (Becker et al. 2008). With the disc diffusion method, Kocuria rhizophila was found resistant to cipro- floxacin, intermediate to erythromycin and susceptible to penicillin, gentamicin, amikacin, tobramycin, tetracycline, vancomycin and teicoplanin (Moissenet et al. 2012).
Effect of metal ions and salts on growth of selected bacterial isolates
Effect of metal ions and salts on growth of selected bacte- rial isolates was determined in the form of turbidity. The growth of bacterial isolate PDM-15 was enhanced by Hg2+ and Mg2+ to the extent of 55.52–95.29% while that of iso- late PDM-20 by 53.2% by Hg2+. This might be due to the fact that they have used metal ions for energy and growth purpose and also play an important role in the biogeochemi- cal cycling of metal ions. In a recent study, Xanthomonas maltophilia AC1 and Acinetobacter sp. were resistant to all heavy metal salts used in the experiment (CuSO4, MnSO4, ZnSO4, NiCl2, CoSO4, Na2SO4, K2Cr2O7) except Xan- thomonas sp. B-B2 that was sensitive to ZnSO4 and K2CrO7 (Jabeen et al. 2017). In a previous study, optimum growth of Staphylococcus sp. DAB-1 W and Kocuria sp. DAB-1Y which were capable of degrading lindane was observed in the presence of NaCl (Kumar et al. 2016).
Identification of bacterial isolate
The culture labelled as PDM-15 was identified as Kocuria
Table 2 Biochemical characterization of selected bacterial
assamensis accession number MH538968 from Xcelris Genomics, Ahmedabad, Gujarat, India.
Isolates test Indole
Methyl red Voges-Proskauer Citrate
Catalase Sucrose Lactose Maltose Mannitol Inositol Sorbitol Galactose
PDM-2
-
-
+
+
+
+
+
+
+
+
+
+
PDM-15
-
-
+
+
+
+
+
+
+
+
+
+
PDM-20
-
-
-
+
+
-
+
-
+
-
+
+Chloramphenicol
The Culture, which was labelled as PDB15, showed similarity with Kocuria assamensis strain R27 (Accession Number: JN700107.1) based on nucleotide homology and phylogenetic analysis.
Analysis of chlorpyrifos and malathion degradation by Kocuria assamensis
Chlorpyrifos and malathion degradation as monitored by gas chromatography (GC) is demonstrated in Fig. 2a, b and Fig. 3a, b. The maximum degradation of chlorpyrifos was observed after 12 days and malathion after 10 days of incuba- tion with Kocuria assamensis. The residual chlorpyrifos and malathion was calculated on the basis of area occupied in the
(-) negative test, (+) positive test gas chromatogram obtained under GC. 71.3% degradation of
Fig. 2 a Gas chromatogram of standard chlorpyrifos. b Gas chromatogram showing chlor- pyrifos degradation by Kocuria assamensis
chlorpyrifos and 85% degradation of malathion were obtained. In a previous study, the degradation of malathion by Azos- pirillum lipoferum was found to be 74.4% using GC (Kanade et al. 2012). As reported earlier, there was 30.78% degradation of chlorpyrifos by Kocuria kristinae in the medium (Hamsa- vathani et al. 2017).
Conclusion for future biology
Nowadays, the problem of pesticides pollution is increas- ing and it causes the environmental hazard. Microorgan- isms are essential for bioremediation of environmental
Fig. 3 a Gas chromatogram of standard malathion. b Gas
chromatogram showing mala- thion degradation by Kocuria assamensis
pollutants because they play a considerable role in the degradation of insecticides. The strain with good degra- dation capability, i.e. Kocuria assamensis was isolated from agricultural soil of different regions of Himachal Pradesh. It has shown that this strain was able to grow in the presence of chlorpyrifos and malathion and it utilized these pesticides as carbon source. Results obtained in this study have shown that the isolate PDM-15 was found to be Gram-positive coccus and was identified as Kocuria assamensis which was capable of degrading pesticides and tolerating antibiotics as well as metal ions.
Acknowledgements The financial support from Department of Bio- technology, Ministry of Science and Technology, Govt. of India, to Department of Biotechnology, Himachal Pradesh University, Shimla (India), is thankfully acknowledged. Fellowship granted to Ms. Aksh- ita Mehta from Department of Environment, Science and Technology (DEST) Himachal Pradesh in the form of Project Fellow is also thank- fully acknowledged.
Declaration
Conflict of interest The authors declare that there is no conflict of in- terests regarding the publication of this article.
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