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The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine
Journal Pre-proof The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine Simranjeet Singh, Vijay Kumar, Niraj Upadhyay, Joginder Singh
PII:
S2213-3437(19)30662-1
DOI:
https://doi.org/10.1016/j.jece.2019.103539
Reference:
JECE 103539
To appear in:
Journal of Environmental Chemical Engineering
Received Date:
6 July 2019
Revised Date:
3 November 2019
Accepted Date:
11 November 2019
Please cite this article as: Singh S, Kumar V, Upadhyay N, Singh J, The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine, Journal of Environmental Chemical Engineering (2019), doi: https://doi.org/10.1016/j.jece.2019.103539
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The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine
Simranjeet Singh1,2,3#, Vijay Kumar4#, Niraj Upadhyay5, Joginder Singh1*
1. Department of Biotechnology, Lovely Professional University, Phagwara, Punjab, India -144002. 2. Punjab Biotechnology Incubators, Mohali (Punjab) -160059, India. 3. Regional Advanced Water Testing laboratory (RAWTL), Mohali (Punjab) -160059,
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India.
4. Regional Ayurveda Research Institute for Drug Development, Madhya Pradesh (India) - 474009.
5. Department of Chemistry, Dr. Harisingh Gour University, Sagar University, Madhya
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contribution
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# equal
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Pradesh (India) - 4740003.
* Corresponding author email: [email protected]
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Graphical Abstract
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Highlights
First time, the degradation kinetics and mechanism of atrazine was thoroughly investigated in the presence & absence of Fe(II), Cu(II) &humic acid (HA).
Based on the kinetic study the half-life period of atrazine decomposition for RK1, RK2 and RK3 alone was 1.97, 2.29 and 3.07 days respectively. In the presence of Cu(II) i.e. RK1/2/3 + Cu(II), the observed decomposition rate was
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10.38, 2.66 and 5.10 days.
In experimental condition with Fe(II) i.e. RK1/2/3 + Fe(II), the observed
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decomposition rate was 3.04, 4.36 and 6.69 days respectively.
With the applications of HA i.e. RK1/2/3 + HA, the observed decomposition rate was
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23.15, 2.84 and 3.71 days respectively.
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The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine
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The biodegradation behaviour of atrazine and kinetic mechanism for biodegradation were thoroughly investigated in the presence and absence of Fe(II), Cu(II) and humic acid (HA).
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The degradation of atrazine into its metabolites was detected through the GC-MS analysis. The observed half-life period of atrazine decomposition for three strains (RK1, RK2 and RK3) was 4.53, 5.29 and 7.07 days respectively. The observed half-life period with the applications of Cu(II) i.e. RK1/2/3 + Cu(II) was 23.90, 6.13 and 11.74 days respectively. In experimental condition where Fe(II) was added i.e. RK1/2/3 + Fe(II), the observed half-life period was 7.00, 10.05 and 15.40 days respectively. With the applications of HA i.e. RK1/2/3 2
+ HA, the observed half-life period was 53.32, 6.54 and 8.56 days respectively. Out of three bacterial strains, Pseudomonas fluorescens (RK2) has shown efficient degradation behaviour in the absence and presence of Fe(II), Cu(II), and HA followed by Azotobacter chroococcum (RK3) and Streptomycetaceae bacterium (RK1). Current study revealed that Pseudomonas fluorescens (RK2) could be used to decompose the atrazine under the environmental stress.
Keywords: Atrazine; kinetic study; bio-decomposition; humic acid; Pseudomonas
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fluorescens.
1. Introduction
Atrazine (6-chloro-4-N-ethyl-2-N-propan-2-yl-1,3,5-triazine-2,4-diamine) is a herbicide,
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white crystalline solid, which is employed to regulate grassy and broadleaf in sugar cane crops, sorghum, corns all over the world [1-4]. It is a triazine herbicide, usually contains
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cyanazine and simazine and applied 70K-90K tonnes annually throughout the globe [3,4]. It
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was extensively used in Unites States for more than over past 35 years on corns, sorghum, wheat, conifers, nuts etc. [3]. In India, 340 tonnes of technical grade atrazine of the technical was consumed and was second most highly consumed pesticide in India [4,5]. It was first
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synthesized and introduced in the 1940s and also used in amalgamation or alone with other herbicides [4-8]. It has a half-life period of 41-231 days [3], and were having sky scraping
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potential to deteriorate the water bodies and agricultural fields [4-7]. It has been banned in
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several countries, because it metabolites/ residues often remain in agricultural fields and water bodies for several years [9-22]. The surpass levels of atrazine in drinking water of Europe and USA is 0.1 and 3 μgL−1 respectively [15-32]. It has found from the various studies that the chlorinated atrazine majorly leads to disruption of endocrine system of humans and mammals [30-41]. Furthermore, it induces reactive oxidative stress resulting in infertility of pigs, amphibians, and rats etc, reduced semen quality and sperm dysfunction
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[12-28]. In females, it disrupts sexual hormones and intrude estrogens receptors for proper dysfunction, instinctive abortion, irregularities in ovarian cycles, developmental births defects etc [22-29]. It is well known that many environmental treatment methods such as adsorption [4251], photo-catalytical degradation [45-51], advanced oxidation process [42-51] have been reported for the removal of environmental pollutants but biodegradation emerged as a most cost-effective as well as the natural remedial method which has the ability to eradicate
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chemicals such as xenobiotic from the ecosystem [35-41]. Microbial population involved in degradation of atrazine and its major metabolites includes B. subtilis HB-6 [19], Arthrobactersp [18], Arthrobactersp [5], E. cloacae strain JS08 [13], Arthrobacter sp. strain
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DAT1 [17], Arthrobacter sp. GZK-1 [6], Arthrobacter sp. T3AB1 [38] Arthrobacter strain DNS 10 [10], Klebsiella sp. A1 Comamonassp.A2 [11], Arthrobacter sp. AD26 [41]. smaltophilia,
Bacillus
licheniformis,
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Stenotrophomona
Bacillus
megaterium,
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Rahnellaaquatilis Umbelopsisisabellina Volutellaciliata, Botrytis cinerea [38-41] isolated worldwide.
Moreover, divalent metal ions, zero valent iron and soil’s humates are used for
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environmental remediation including decontamination of pesticides [26-37]. Divalent metal ions and zero-valent iron facilitated degradation of pesticides, and it is a potential remediation
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strategy for elimination of pesticides. The major reason for the selection of Cu(II) and Fe(II)
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was the multiple applications of both metal ions in the living system and total environment. The iron [Fe(II)] and copper [Cu(II)] are very important micronutrients of soil [30-38]. These ions improve the plant health through complex mechanism. Improper distributions of these ions may alter the crop production and human health too [25-30]. Therefore, we tested the hypothesis “biodegradation of atrazine in the presence and absence of metal ions [Fe (II) and Cu(II)] and humic acid (HA)”. Initially, 35 bacterial strains
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were screened from the agricultural land and out of these three best strains were used for further study.
2. Experimental design 2.1. Sample Collection Soil samples were collected from six different agricultural fields of Kapurthala district Punjab (India). For the removal of large debris and unwanted material all the samples were sieved
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through a 2 mm sieve and homogenized with the help of mortar and pestle and kept at 4°C for further experimentation work. 2.2. Chemicals
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Technical grade Atrazine (98.0%) was obtained from Rallis India Ltd. The analytical reagent (AR) grade chemicals and media were purchased from Hi-media and Sigma Aldrich India.
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2.3. Isolation and characterization of atrazine-degrading bacterium
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Soil samples were collected from agricultural fields of six different villages of Kapurthala, Punjab, India having long history of atrazine usage (Elevation height 225 m, Longitude 75.38 E, and Latitude 31.38 E,). Six villages of district Kapurthala of state Punjab (India) selected
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for study includes: Bholana, Dona, Dhadwandi, Dhariwal, Noorpurdona, Kharsona, Mothanwal, and Tiba.
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Microbial population were obtained by media enrichment technique in which atrazine
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serves as sole source of energy for microbes. About 5 g of soil sample was poured in 500 ml of Erlenmeyer flask having 100 ml of enrichment medium. Enrichment medium composed of 100 mg/L Atrazine and mineral salts medium (K2HPO4 (0.6g), KH2PO4 (2.5 g), MgSO4.H2O (4.03g) and NaCl (0.4g) per litre) with addition of atrazine at 100 mg/L concentration [19,20]. Cultures were incubated at 28C for 4 days on rotatory incubator in dark conditions (150 rpm). After four days, the 1 ml of bacterial culture suspension was transferred to fresh
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mineral salts medium containing atrazine (100 mg/L)under same incubation temperature for 4 days. This was repeated for 5 times with atrazine and thereafter inoculated in mineral salts medium containing atrazine. Three isolates, RK1, RK2, and RK3 were obtained through this technique 2.4. Molecular identification of atrazine degrading bacteria After isolationand purification of selected strains, the16S ribosomal RNA sequencing was conducted at Samved Biotech Pvt. Ltd. located at Ahmadabad, India. For the confirmation
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the identity of three isolated strains, 1.5 kb 16 s rRNA was amplified using the total DNA and sequenced by using the 1492R and 27F universal primers. The phylogenetic analysis and tree constructed as given by Singh et al. 2019 [30-32].
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2.5. Inoculum preparation for atrazine-degradation studies
Isolated strains were cultured in mineral salts medium having 100 mg/L of atrazine in a
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rotatory incubator (120 rpm) at 30oC for 24 hours. The isolated strains were homogenized at
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6500x g for 5-10 min and cell counts were adjusted using 0.5 McFarland standards to 107 cells/ml.
2.6. Optimization of concentrations, pH and temperature effects
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The concentrations of atrazine, humic acid, Fe(II) and Cu(II) as well as the effect of pH (5, 7 and 9) and temperature (10, 20 and 30oC) was optimized. The cell growth of individual
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bacterial strain was checked at 600 nm (O.D. 600) by using the UV-visible spectroscopy.
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2.7. Degradation of atrazine by the isolates in mineral medium and preparation of Inoculum for degradation studies To exploit the capability of the isolates to disintegrate atrazine, isolated strains were grown in broths and suspension culture was inoculated with isolates in each flask to reach a biomass level of OD600 = 0.91. The mineral medium was prepared with three different concentrations of atrazine 100 ppm as the energy source of carbon, carbon and nitrogen and carbon and
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sulphur. The minimal medium will be equipped with pesticides at different concentrations in the absence and presence Fe(II), Cu(II) and HA respectively. At regular period (i.e. at 0, 3, 7, 10, 14 day), 20 ml of the broth were worn-out to determine growth and depletion of atrazine. The growth of bacteria was examined by a UV-Vis spectrophotometer (1800UV Shimazdu) at 600 nm using triple distilled water as control. For the assessment of concentration of residual atrazine, gas chromatography (GCMS) was employed. Flasks containing minimal medium with 100 ppm atrazine served as i.e. Fe(II), Cu(II) and
humic acid on
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controls. To confirm the effect of metal ions
degradation process, above experiment was repeated under different compositions like bacteria + atrazine + metal ion [Fe (II) and Cu(II)] and HA [17,30-32].
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2.7. Analysis of atrazine decomposition by GC-MS
For Gas chromatography (GC-MS) analysis, 100 ml of medium was centrifuged at 9000 X g
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for 20 min keeping 4ºC for clarification and filtration was done with the Whatmann filter
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paper No. 1. The medium was then extracted with addition of ethyl acetate in 1:1 ratio. Extracted organic material was kept for air drying and concentrates upto 0.1 mL. Gas chromatography (GC-MS) equipped with ECD detector and DB5 capillary column of
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dimensions (0.25mm × 0.25μm × 30m) comprising splitless injector which are operated at 70 eV. Helium was employed as a carrier gas with flow rate of 1.5 mL per min. The temperature
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was adjusted to 280°C and 300°C for transfer line and to trap ion respectively. The
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concentration of residual atrazine was assessed on the basis of comparison of peaks in the abiotic control with respect to the samples. For recognition of metabolites, comparison of mass spectra of standards was done with mass spectra of products [33]. To perform the kinetic study, standard plot was prepared and samples were compared with standard plot at regular time interval (at 3rd, 7th, 10th and 14th day). 2.8. Degradation Kinetics
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The decompositions rates of the atrazine were determined by using pseudo-first order kinetics [25-35]. Briefly, Eqs. (1)–(3) represents equations of the variation in the concentration with respect to given time plotted. Linear regression is used by plotting graph with time (in day) against Log Ct (in ppm). d[C]/ dt = −kobs[C] ------- (1); ln[C]/ [C]0 = kobst ------- (2); t1/2= (1/ kobs) × ln2 ------ (3),
[C]0=the initial concentration of atrazine (mg/l)
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where, [C] = concentration of atrazine at time t (mg/l)
kobs= constant for pseudo-first order (day−1) equal to slope of line i.e. kobs= - slope.
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2.9. Statistical Analysis
To avoid any error and to maintain reproducibility of data, all the experiments were
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performed in triplicates. MS Excel worksheets were used for calculation of standard errors
3. Results
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calculation of ANOVA.
SPSS 16 statistical software were used for
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and means in the experimental data and
Enrichments were employed and followed over time, isolating a bacterial culture which has
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the ability to utilize atrazine as carbon and energy source (including nitrogen and carbon
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source), for progression. For one month, enrichment with selective component was repeated, and repeated transferring of samples from culture to fresh minimal medium containing atrazine exhibited the growth of the isolates of pure atrazine degrading strains. Total, eleven strains of bacteria were isolated from soil as they were able to consume atrazine and were designated as from RK1 to RK11. The strain RK1, RK2, and RK3 were found to be the most efficient that were able to utilize atrazine as source of carbon for growth and were selected
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for further study. Phylogenetic position and 16s rDNA analyses have confirmed that isolates strains were Streptomycetaceae bacterium RK1, Pseudomonas fluorescens RK2, and Azotobacter chroococcum RK3 and submitted to NCBI under accession numbers KJ206091.1, KJ466148.1, KJ511860.1 (Supplementary Figure – S1). 3.2. Degrading ability of isolated strains The bio-decomposition of atrazine (100 mg/L) was checked on regular time interval (0th, 3rd, 7th, 10th and 14th) for the 14 days of incubation with RK1 to RK11 strain. However, three
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strains (RK1, RK2 and RK3) have shown significant utilization of atrazine. All the isolated cultures show threshold increase in their growth from 3rd day till 10th day. After 10thdays of incubation, the relative growth of all the isolates was constant (Table – 1).
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In fourteen days experiment, the strain RK1 alone has shown significant biodecomposition as compared to other test conditions i.e. in the existence of Cu(II) and HA,
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except Fe(II) (Figure -1). Results indicated that bacterial strain RK1 can decompose atrazine
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more than 95 % in the presence of iron which is similar to that of without Fe(II). With the addition of Cu(II) and HA, significant (p <0.05)decrease in the atrazine decomposition has been noticed which was 61 and 39 % respectively. The order of bio-decomposition of
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atrazine with various experiments was inoculum + atrazine ~ inoculum + atrazine + Fe(II) > inoculum + atrazine + Cu(II) ≫ inoculum + atrazine + HA (Figure -1).
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The strain RK2 has shown better decomposition rates in 14 days experiments. The
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order of bio-decomposition of atrazine with various experiments was inoculum + atrazine ~ inoculum + atrazine + Cu(II) ~ inoculum + atrazine + HA > inoculum + atrazine + Fe(II) > (Figure -1). After 14 days experiments, more than 87% decomposition was noticed in all experimental conditions with bacterial strain RK2. Here, non-significant (p <0.05) difference were noticed among the various experiment conditions which indicates that RK2 can utilize atrazine under all conditions i.e. even under the stress of Cu(II), Fe(II) and HA.
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Under various test conditions, bacterial strain RK3 has shown more than 77% decomposition of atrazine. The decomposition was 77 and 85% in the presence of Cu(II) and Fe(II). The strain RK3 alone has decomposed near about 95% atrazine which was almost closer to that of in the presence of HA (91%) (Figure -1). The observed bio-decomposition order of atrazine with various experiments was inoculum + atrazine ~ inoculum + atrazine + HA > inoculum + atrazine + Cu(II) ≫ inoculum + atrazine + Fe(II) (Figure -1). Figure – 1 exhibited that in the absence of Fe(II), Cu(II) and HA, all three strains
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have ability to decompose more than 95% of atrazine. In Cu(II), treated groups RK2 (96%) have shown significant decomposition decrease as compared to other two strains RK3 (85%) and RK1 (61%). Applications of Fe(II) have shown 77 to 95% decomposition of atrazine.
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The results were significantly different from each other i.e. 95, 87 and 77% for RK1, RK2 and RK3 respectively. In the presence of HA acid, decomposition rates of RK2 (95%) and
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RK3 (91%) were almost similar to each other but dramatically different for RK1 (39%).
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Based on the present study, to choose the best strain among the RK1, RK2 and RK3 under various conditions, the exact order may be RK2 > RK3 ≫ RK1. 3.3. Degrading kinetics of isolated strains
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The kinetic study of atrazine decomposition including its half-life period (t1/2), equation of line and regression constant r2 is mentioned under Table – 1. The observed half-life value was
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maximum for RK1 strain in the presence HA which was 53.32 days. The second highest
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decomposition rate was also with RK1 strain in Cu(II) treated groups which was 23.90 days. In the presence of Fe(II), RK3 strain has shown third highest decomposition rate that was 15.40 days. Applications of Cu(II) in RK3 inoculum has shown half-life period 5.11 days. In the Fe(II) treated groups, RK2 has decomposed atrazine with an half-life period 10.05 days. The remaining experiments in the absence and presence of Fe(II), Cu(II) and HA have halflife period from 4.53 to 3.72 days (Table – 1). Overall, without applications of Cu(II) Fe(II),
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and HA, RK1 have shown better decomposition rate (4.53 day) as compared to RK2 (5.29 day) and RK3 (7.07 day). Similar trends were noticed with the applications of Fe(II) i.e. the observed half-life period was RK1(7.00) > RK2 (10.05 day) > RK3 (15.40 day). It means, bacterial strain RK1 can decompose atrazine with significant half-life period under the influence of Fe(II). It has been found that RK2 and RK3 (RK2 > RK3) are efficient to degrade atrazine under the treatment of Cu(II), Fe(II), and HA whereas RK1 can do it only in the existence of Fe(II) only (Table – 1).
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3.4. GC-MS analysis and most probable decomposition mechanism To propose the most probable mechanism of atrazine bio-decomposition GC-MS analysis was performed. The GCMS results of bio-decomposition of atrazine at various conditions has
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mentioned under Figure – 2 and supplementary Figures S2-S15. GC-MS study revealed that atrazine was into eleven fragments in the presence and absence of Fe(II), Cu(II) and HA
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(Figure – 3). Pure atrazine was found in GC-MS at RT 19.35 min which has shown
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corresponding peaks (m/z) at 215, 200, 173, 120, 93, 58 and 43. In the absence and presence of Cu(II), Fe(II), and HA, GC-MS has shown two additional peaks (major only) at RT 5.42 and 11.8 min. The corresponding value of mass to charge ratio of metabolites at RT 5.42 min
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was 78, 63 and 45. Similarly, the m/z ratio for peak at RT 11.58 min was 152, 120, 92, 65. It is observed that atrazine was decomposed through the formation of metabolites like N2-
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ethyl-6-hydroxy-N4-isopropyl- 1,3,5-triazine-2,4-diaminium (m/z 200), 6-chloro-N2-ethyl(m/z
173),
N-isopropylammelide
(m/z
170),
4-amino-6-
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1,3,5-triazine-2,4-diamine
(ethylamino)-1,3,5-triazin-2-ol (m/z 155), (methylamino/ethylamino) methanediol (m/z 120), (aminomethylamino) methanediol (m/z 92), aminomethanediol (m/z 63) and 2methylpropane (m/z 58). Based on the GC-MS analysis the most probable mechanism of atrazine decomposition is mentioned under Figure – 3.
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4. Discussion Recently, various techniques gaining attentions to remove the pollutants from the environment including fabricated materials i.e. nanoparticles, carbon nano tubes etc. [42-51]. These fabricated materials are most effective to removes the metal ions [43,46,47], dyes [44,45,50,51] and other pollutants. The biodegradation technique is considered as cost effective and eco-friendly where no chemical method is used. This is the first study on atrazine interactions with soil humic acid and metal ions. The
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optimized concentrations level of the atrazine, humic acid, Fe(II) and Cu(II) were 100, 10, 25 and 15 ppm respectively. At higher concentration, degradation rates were decreased as mentioned in our recent report [28-32]. This may attributed to the toxicity of chemicals at
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higher concentration [28-32. The decrease in the Also, the optimized pH and temperature was 7 and 30oC respectively which was similar to recent reports [6,10,13,17,18,38-41]. Since
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atrazine contains various coordinating sites (N and NH), it is assumed that it will be involved
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in complexation with metal ions such as Fe (II) and Cu(II) [4,10,25,2-32]. Atrazine can form H-bonds with humic substances through NH bond(s) which may be the case with RK1 strain consequently low decomposition rate was noticed. In comparison with atrazine decomposing species
of
Arthrobacter
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microbial
sp.
TES6
(60%),
Arthrobacter
sp.
(90%)
Pseudaminobacter sp. (71%), Arthrobacter sp. strain MCM B-436 (65%), Arthrobacter sp.
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strain HB-5 (70%) B. subtilis Strain HB-6 (55%), Arthrobacter sp. GZK-1 (65%),
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Arthrobactersp E. cloacae strain JS08 (70%), Arthrobacter sp. strain DAT1 (80%), Arthrobacter strain DNS 10 (70%), Comamonas sp. A2 (775), Klebsiella sp. A1 (84%) [5,6,13,16-22], the strains RK1, RK2 and RK3 have shown better degradation of atrazine. De-chlorination, de-alkylation and de-amination are known to be the major routes for atrazine transformation. Hydrolytic de-chlorination is the main mechanism for atrazine degradation using enzyme atrazine chlorohydrolase. The next step is the action of amino-hydrolases
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enzymes which triggers two hydrolytic deamination reactions in which it is further converted into N-ethylammelide [14] or N-isopropylammelide [6]. These N-ethylammelide and Nisopropylammelideare lastly degraded to cyanuric acid [20,41]. Second pathway for degradation of atrazineincludes the N-dealkylation of isopropyl chains and ethyl to di-ethyldi-isopropyl atrazine disopropylatrazine, and
diethylatrazine [16-19].
Further, these
metabolites undergo hydroxylation by forming cyanuric acid [3,4,38]. Though it was the first study of atrazine decomposition in the presence of HA, Fe(II), and Cu(II) so no comparison
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was done with recent studies. Atrazine contain one or more binding sites (most probably NH) and they may interact with essential metal ion and organic matter (Trevisan et al., 2010). Kumar et al, 2016 [4]
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have reported that atrazine complexes with Fe (II) and Cu(II) through the N and NH sites. The complexation of the atrazine is dependent on transport, bioaccumulation and
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bioavailability in the ecosystem [25-31]. Metal ions such as Fe(II) and Cu(II) and humic
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substances form weak complexes with atrazine and therefore have a pessimistic effect on degradation as per as our assumption [29,30,32]. The samples without treatment of metal ions Fe(II), Cu(II) and humic substances exhibits extreme degradation of atrazine as matched to
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others after 14 days, humic acid plays an important role in the treatment of atrazine. By forming weak interactions with metal ions it is supposed that Cu(II) and Fe(II), decreases the
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degradation percentage by showing their toxic effects on bacterial catabolism and humic acid
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[33-37]. Although, humic substances blocks the active sites (like OH, NH and NH2 responsible for H-bonding and weak bondings) of atrazine through weak bonding which results in bacterial catabolism. As the all three bacterial strains exhibits the ability to grow and survive on atrazine alone without any additional source and this make them ideal for biodegradation of atrazine under various conditions.
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5. Conclusion This study systematically explored the degradation of atrazine in presence and absence of Fe(II), Cu(II) and humic acid (HA). Following conclusions were drawn: 1. In the presence of Cu(II) i.e. RK1/2/3 + Cu(II), the observed t1/2 values were 23.90, 6.13 and 11.74 days. 2. In experimental condition with Fe(II) i.e. RK1/2/3 + Fe(II), the observed t1/2 values were 7.00, 10.05 and 15.40 days respectively.
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3. With the applications of HA i.e. RK1/2/3 + HA, the observed t1/2 values were 53.32, 6.54 and 8.56 days respectively.
4. The kinetic study revealed that in the absence of Fe(II), Cu(II) and HA, RK1 (1.97
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day) is the better strain than RK2 (5.29 day) and RK3 (7.07 day).
5. Overall, in the presence of Fe(II), Cu(II) and HA, RK2 is the better strain than RK3
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and RK1.
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6. Current study revealed that Pseudomonas fluorescens (RK2) could be used to
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decompose the atrazine under the environmental stress.
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on Chlorophyll‐a Fluorescence Revisited—Implications in Photosystems Emission and Ecotoxicity Assessment. Photochem. Photobiol. 90 (2014) 107-112. [8] N. Kadian, A. Gupta,S. Satya, R.K. Mehta, A. Malik, Biodegradation of herbicide (atrazine) in contaminated soil using various bioprocessed materials. Bioresour. Technol. 99 (2008) 4642-4647.
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[9] D. Kapoor, S. Singh, V. Kumar, R. Romero, J. Singh, Antioxidant enzymes regulation in plants in reference to reactive oxygen species (ROS) and reactive nitrogen species (RNS).Plant Gene 19 (2019) 100182. [10] S. Kaur, V. Kumar, M. Chawla, L. Cavallo, A. Poater, N. Upadhyay, Pesticides Curbing Soil Fertility: Effect of Complexation of Free Metal Ions. Front. Chem. 5 (2017) 1-9. [11] S. Bhati, V. Kumar, S. Singh, J. Singh, Synthesis, biological activities and docking studies of piperazine incorporated 1, 3, 4-oxadiazole derivatives. J. Mol. Struc. 1191 (2019)
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197-205. [12] M. Silva, M.E. Azenha, M.M. Pereira, H.D. Burrows, A. Fernandes, Immobilization of halogenated porphyrins and their copper complexes in MCM-41: Environmentally friendly
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photocatalysts for the degradation of pesticides. Appl. Catal. B. 100 (2010) 1-9.
[13] R.D.J. Solomon, A. Kumar, V.S. Santhi, Atrazine biodegradation efficiency, metabolite
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Zhe. Uni. Sci. B. 14 (2013) 1162-1172.
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detection, and trzD gene expression by enrichment bacterial cultures from agricultural soil. J.
[14] E. Topp, H. Zhu, S.M. Nour, S. Houot, M. Lewis, D. Cuppels, Characterization of an Atrazine-Degrading Pseudaminobacter sp. Isolated from Canadian and French Agricultural
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Soils. Appl. Environ. Microbiol. 66 (2000) 2773-2782. [15] P.A. Vaishampayan, P.P. Kanekar, P.K. Dhakephalkar, Isolation and characterization of
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Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric
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soil. Int. Biodeter. Biodegrad. 60 (2007) 273-278. [16] J. Wang, L. Zhu, A. Liu, T. Ma, Q. Wang, H. Xie, R. Zhao, Isolation and characterization of an Arthrobacter sp. strain HB-5 that transforms atrazine. Environ. Geochem. Health 33 (2011) 259-266. [17] Q. Wang, S. Xie, Isolation and characterization of a high-efficiency soil atrazinedegrading Arthrobacter sp. strain. Int. Biodeter. Biodegrad.71 (2012) 61-66.
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[18] Q. Wang, S. Xie, R. Hu, Bioaugmentation with Arthrobacter sp. strain DAT1 for remediation of heavily atrazine-contaminated soil. Int. Biodeter. Biodegrad. 77 (2013) 63-67. [19] J. Wang, L. Zhu, Q. Wang, J. Wang, H. Xie, Isolation and characterization of atrazine mineralizing Bacillus subtilis strain HB-6. PloSone 9 (2014) e107270. [20] C. Yang, Y. Li, K. Zhang, X. Wang, C. Ma, H. Tang P. Xu, Atrazine degradation by a simple consortium of Klebsiella sp. A1 and Comamonas sp. A2 in nitrogen enriched medium. Biodegrad. 21 (2010) 97-105.
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[21] Y. Zhang, Z. Jiang, B. Cao, M. Hu, Z. Wang, X. Dong, Metabolic ability and gene characteristics of Arthrobacter sp. strain DNS10, the sole atrazine-degrading strain in a consortium isolated from black soil. Int. Biodeter. Biodegrad. 65 (2011) 1140-1144.
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[22] V. Kumar, S. Singh, R. Singh, N. Upadhyay, J. Singh, P. Pant, R. Singh, B. Shrivastava, A. Singh, V. Subhose, Spectral, structural and energetic study of acephate, glyphosate,
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monocrotophos and phorate: An experimental and computational approach. J. Taibah Uni.
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Sci. 12 (2018) 69-78.
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[24] V. Kumar, S. Singh, R. Singh, N. Upadhyay, J. Singh, Design, synthesis, and characterization of 2,2-bis(2,4-dinitrophenyl)-2-(phosphonatomethylamino)acetate as a
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herbicidal and biological active agent. J. Chem. Biolog. 11 (2017) 1-12.
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[25] V. Kumar, V. Kumar, S. Kaur, S. Singh, N. Upadhyay, Unexpected formation of Nphenyl-thiophosphorohydrazidic
acid
O,S-dimethyl
ester
from
acephate:
chemical
biotechnical and computational study. 3 Biotech 6 (2016) 1-11. [26] V. Kumar, N. Upadhyay, V. Kumar, S. Sharma, A review on sample preparation and chromatographic determination of acephate and methamidophos in different samples. Arab. J. Chem. 8 (2015) 624-631.
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[27] V. Kumar, S. Singh, J. Singh, N. Upadhyay, Potential of plant growth promoting traits by bacteria isolated from heavy metal contaminated soils. Bull. Environ. Contam. Toxicol. 94 (2015) 807-815. [28] G.K. Sidhu, S. Singh, V. Kumar, S. Datta, J. Singh, Environmental toxicity, monitoring and biodegradation of organophosphate pesticides: a review. Crit. Rev. Environ. Sci. Technol. 49 (2019) 1135-1187. [29] S. Singh, V. Kumar, J. Singh, Kinetic study of the biodegradation of glyphosate by
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indigenous soil bacterial isolates in presence of humic acid, Fe(III) and Cu(II) ions. J. Environ. Chem. Eng. 7 (2019) 103098.
[30] S. Singh V. Kumar S. Singh J. Singh, Influence of humic acid, iron and copper on
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microbial degradation of fungicide Carbendazim. Biocatal. Agric. Biotechnol. 20 (2019) 101196.
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[31] S. Singh, V. Kumar, G.K. Sidhu, S. Datta, J. Singh, Plant growth promoting
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rhizobacteria from heavy metal contaminated soil and their plant growth promoting attributes for Pisumsativum L. Biocatal. Agric. Biotechnol. 17 (2019) 665-671. [32] S. Singh, V. Kumar, N. Upadhyay, J. Singh, S. Singla, S. Datta, S. Efficient
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biodegradation of acephate by Pseudomonas pseudoalcaligenes PS-5 in the presence and absence of heavy metal ions [Cu(II) and Fe(III)], and humic acid. 3 Biotech 7 (2017) 262.
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[33] S. Andleeb, Z. Jiang, K. ur-Rehman, E.k. Olajide, Z. Ying, Influence of Soil pH and
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Temperature on Atrazine Bioremediation. J. North Agri. Uni. (English Edition) 23 (2016) 1219.
[34] Y.S. Keum, Q.X. Li, Reduction of nitroaromatic pesticides with zero-valent iron. Chemosphere 54 (2004) 255-263. [35] V. Kumar, S. Singh, A. Singh, V. Subhose, O. Prakash, Assessment of heavy metal ions, essential metal ions, and antioxidant properties of the most common herbal drugs in Indian
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Ayurvedic hospital: For ensuring quality assurance of certain Ayurvedic drugs. Biocatal. Agric. Biotechnol. 18 (2019)101018. [36] V. Kumar, S. Singh, B. Srivastava, R. Bhadouria, R. Singh, Green Synthesis of Silver Nanoparticles Using Leaf Extract of Holopteleaintegrifolia and Preliminary Investigation of Its Antioxidant, Anti-inflammatory, Antidiabetic and Antibacterial Activities. J. Environ. Chem. Eng. 7 (2019) 103094. [37] V. Kumar, M. Chawla, L. Cavallo, A.B. Wani, A. Manhas, S. Kaur, A. Poater, H.
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Chadar, N. Upadhyay, Complexation of trichlorosalicylic acid with alkaline and first row transition metals as a switch for their antibacterial activity. Inorg. Chim. Acta 469 (2018) 379-386.
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[38] C. Liu, F. Yang, X. Lu, F. Huang, L. Liu, C. Yang, Isolation, identification and soil remediation of atrazine-degrading strain T3 AB1. Wei sheng wuxuebao Actamicro biologica
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Sinica 50 (2012) 1642-1650.
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[39] J. Mahía, A. Martín, T. Carballas, M. Díaz-Raviña, Atrazine degradation and enzyme activities in an agricultural soil under two tillage systems. Sci. Total Environ. 378 (2007) 187-194.
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[40] R. Marecik, P. Króliczak, K. Czaczyk, W. Białas, A. Olejnik, P. Cyplik, Atrazine degradation by aerobic microorganisms isolated from the rhizosphere of sweet flag
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(Acoruscalamus L.). Biodegrad. 19 (2008) 293-301.
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[41] L.I. Qingyan, L.I. Ying, Z.H.U. Xikun, C.A. Baoli, Isolation and characterization of atrazine-degrading Arthrobacter sp. AD26 and use of this strain in bioremediation of contaminated soil. J. Env. Sci. 20 (2008) 1226-1230. [42] X. Zhang, Q. Huang, F. Deng, H. Huang, Q. Wan, M. Liu, Y. Wei, Mussel-inspired fabrication of functional materials and their environmental applications: Progress and prospects. Appl. Mater. Tod. 7 (2017) 222-238.
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[43] X. Zhang, Q. Huang, F. Deng, H. Huang, Q. Wan, M. Liu, Y. Wei, Preparation of amine functionalized carbon nanotubes via a bioinspired strategy and their application in Cu 2+ removal. Appl. Surf. Sci. 343 (2015) 19-27. [44] Q. Huang, M. Liu, J. Chen, Q. Wan, J. Tian, L. Huang, R. Jiang, Y. Wen, X. Zhang, Y. Wei, Facile preparation of MoS2 based polymer composites via mussel inspired chemistry and their high efficiency for removal of organic dyes. Appl. Surf. Sci. 419 (2017) 35-44. [45] Q. Huang, M. Liu, J. Chen, Q. Wan, J. Tian, L. Huang, R. Jiang, Y. Wen, X. Zhang, Y.
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Wei, Surface functionalized SiO2 nanoparticles with cationic polymers via the combination of mussel inspired chemistry and surface initiated atom transfer radical polymerization: Characterization and enhanced removal of organic dye. J. Colloid Interface Sci. 499 (2017)
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170-179.
[46] Q. Huang, M. Liu, J. Chen, Q. Wan, J. Tian, L. Huang, R. Jiang, Y. Wen, X. Zhang, Y.
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Wei, Synthesis of polyacrylamide immobilized molybdenum disulfide (MoS2@PDA@PAM)
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composites via mussel-inspired chemistry and surface-initiated atom transfer radical polymerization for removal of copper (II) ions. J. Taiwan Inst. Chem. Eng. 86 (2018)174184.
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[47] Q. Huang, M. Liu, J. Chen, Q. Wan, J. Tian, L. Huang, R. Jiang, Y. Wen, X. Zhang, Y. Wei, Preparation of polyethylene polyamine@tannic acid encapsulated MgAl-layered double
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hydroxide for the efficient removal of copper (II) ions from aqueous solution. J. Taiwan Inst.
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Chem. Eng. 82 (2018) 92-101. [48] Z. Guangjian, C. Tingting, H. Long, L. Meiying, J. Ruming, W. Qing, D. Yanfen, W. Yuanqin, Z. Xiaoyong, W. Yen, Surface modification and drug delivery applications of MoS2 nanosheets with polymers through the combination of mussel inspired chemistry and SETLRP. J. Taiwan Inst. Chem. Eng. 82 (2018) 205-213.
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[49] Y. Liu, H. Huang, D. Gan, L. Guo, M. Liu, J. Chen, F. Deng, N. Zhou, X. Zhang, Y. Wei, A facile strategy for preparation of magnetic graphene oxide composites and their potential for environmental adsorption. Ceram. Int. 44 (2018) 18571-18577. [50] D. Gan, M. Liu, H. Huang, J. Chen, J. Dou,Y. Wen, Q. Huang, Z. Yang, X. Zhang, Y. Wei, Facile preparation of functionalized carbon nanotubes with tannins through musselinspired chemistry and their application in removal of methylene blue. J. Mol. Liq. 271 (2018) 246-253.
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[51] Y. Lei, Y. Cui, Q. Huang, J. Dou, D. Gan, F. Deng, M. Liu, X. Li, X. Zhan, Y. Wei, Facile preparation of sulfonic groups functionalized Mxenes for efficient removal of
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methylene blue. Ceram. Int. 45 (2019) 17653-17661
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Table and Figure Captions Table - 1. Kinetics of biodegradation of atrazine by three bacterial strains in mineral salt medium. Figure – 1. Removal (%) of atrazine after incubation with three strains in mineral salt medium under different conditions. Figure – 2. GC-MS analysis of atrazine 7th day sample in the influence of Cu(II) in the presence of bacterial strain RK2. Figure – 3. Most probable mechanistic pathway of decomposition of atrazine in mineral salt
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ro of
medium.
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Table - 1.Kinetics of biodegradation of atrazine by three bacterial strains in mineral saltmedium. Experimental Conditions
K
t1/2 (days)
Equation of line
r2
Streptomycetaceae bacterium (RK1) Inoculum + atrazine
0.153
4.53a,b
y = -0.153x + 2.154
0.99
Inoculum + atrazine + Cu(II)
0.029
23.90a,b
y = -0.029x + 1.956
0.91
ab
Inoculum + atrazine + Fe(II)
0.099
7.00 ,
y = -0.099x + 2.071
0.99
Inoculum + atrazine + HA
0.013
53.32a,b
y = -0.013x + 1.971
0.99
Pseudomonas fluorescens (RK2) 5.29a,b
y = -0.131x + 2.054
0.98
Inoculum + atrazine + Cu(II)
0.113
ab
6.13 ,
y = -0.113x + 2.131
0.98
Inoculum + atrazine + Fe(II)
0.069
10.05a,b
y = -0.069x + 1.989
0.99
Inoculum + atrazine + HA
0.106
6.54a,b
y = -0.106x + 2.144
0.99
y = -0.098x + 2.039
0.98
y = -0.059x + 1.938
0.94
y = -0.045x + 1.943
0.94
y = -0.081x + 2.066
0.99
Azotobacterchroococcum (RK3) Inoculum + atrazine
7.07a,b
0.098
ab
0.059
11.74 ,
Inoculum + atrazine + Fe(II)
0.045
15.40a,b ab
0.081
8.56 ,
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Inoculum + atrazine + HA
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Inoculum + atrazine + Cu(II)
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0.131
Inoculum + atrazine
a = results were significantly differ (at p <0.05) for three strains at same experimental conditions.
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a* = results were non significantly differ (at p <0.05) for two or more than two strains at same experimental conditions.
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b = results were significantly differ (at p <0.05) for each strain at different four experimental conditions.
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Figure – 1.Removal (%) of atrazine after incubation with three strains in mineral salt medium under different conditions.
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Figure – 2.GC-MS analysis of atrazine 7th day sample in the influence of Cu(II) in the
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presence of bacterial strain RK2.
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Cl
OH
N
OH
N
HN
N
H2 N
HN
N
N
NH
N HN
HN
HN
N2-ethyl-6-hydroxy-N4-isopropyl1,3,5-triazine-2,4-diaminium m/z 199
Atrazine m/z 215
amino((ethylamino)methylamino)methanol m/z 119
OH OH
N OH N
+
HO
NH
2-methylpropane m/z 58
N
HO
OH
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H2 N
N
HN
NH
N
HN
6-chloro-N2-ethyl-1,3,5-triazine-2,4-diamine
N H
m/z 173
N
H2 N
N H
N
HO
4-amino-6-(ethylamino)-1,3,5-triazin-2-ol
N
OH
cyanuric acid m/z 129
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m/z 155
N
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N
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OH N
(aminomethylamino) methanediol m/z 92
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N-isopropylammelide m/z 170
OH
N
NH2
OH
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Figure –3. Most probable decomposition pathway of atrazine.
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(ethylamino)methylamino) methanediol m/z 120
OH OH
HO NH
HO NH2
(methylamino)methanediol m/z 77
aminomethanediol m/z 63
PII:
S2213-3437(19)30662-1
DOI:
https://doi.org/10.1016/j.jece.2019.103539
Reference:
JECE 103539
To appear in:
Journal of Environmental Chemical Engineering
Received Date:
6 July 2019
Revised Date:
3 November 2019
Accepted Date:
11 November 2019
Please cite this article as: Singh S, Kumar V, Upadhyay N, Singh J, The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine, Journal of Environmental Chemical Engineering (2019), doi: https://doi.org/10.1016/j.jece.2019.103539
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The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine
Simranjeet Singh1,2,3#, Vijay Kumar4#, Niraj Upadhyay5, Joginder Singh1*
1. Department of Biotechnology, Lovely Professional University, Phagwara, Punjab, India -144002. 2. Punjab Biotechnology Incubators, Mohali (Punjab) -160059, India. 3. Regional Advanced Water Testing laboratory (RAWTL), Mohali (Punjab) -160059,
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India.
4. Regional Ayurveda Research Institute for Drug Development, Madhya Pradesh (India) - 474009.
5. Department of Chemistry, Dr. Harisingh Gour University, Sagar University, Madhya
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contribution
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# equal
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Pradesh (India) - 4740003.
* Corresponding author email: [email protected]
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Graphical Abstract
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Highlights
First time, the degradation kinetics and mechanism of atrazine was thoroughly investigated in the presence & absence of Fe(II), Cu(II) &humic acid (HA).
Based on the kinetic study the half-life period of atrazine decomposition for RK1, RK2 and RK3 alone was 1.97, 2.29 and 3.07 days respectively. In the presence of Cu(II) i.e. RK1/2/3 + Cu(II), the observed decomposition rate was
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10.38, 2.66 and 5.10 days.
In experimental condition with Fe(II) i.e. RK1/2/3 + Fe(II), the observed
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decomposition rate was 3.04, 4.36 and 6.69 days respectively.
With the applications of HA i.e. RK1/2/3 + HA, the observed decomposition rate was
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23.15, 2.84 and 3.71 days respectively.
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The effects of Fe(II), Cu(II) and humic acid on biodegradation of atrazine
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The biodegradation behaviour of atrazine and kinetic mechanism for biodegradation were thoroughly investigated in the presence and absence of Fe(II), Cu(II) and humic acid (HA).
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The degradation of atrazine into its metabolites was detected through the GC-MS analysis. The observed half-life period of atrazine decomposition for three strains (RK1, RK2 and RK3) was 4.53, 5.29 and 7.07 days respectively. The observed half-life period with the applications of Cu(II) i.e. RK1/2/3 + Cu(II) was 23.90, 6.13 and 11.74 days respectively. In experimental condition where Fe(II) was added i.e. RK1/2/3 + Fe(II), the observed half-life period was 7.00, 10.05 and 15.40 days respectively. With the applications of HA i.e. RK1/2/3 2
+ HA, the observed half-life period was 53.32, 6.54 and 8.56 days respectively. Out of three bacterial strains, Pseudomonas fluorescens (RK2) has shown efficient degradation behaviour in the absence and presence of Fe(II), Cu(II), and HA followed by Azotobacter chroococcum (RK3) and Streptomycetaceae bacterium (RK1). Current study revealed that Pseudomonas fluorescens (RK2) could be used to decompose the atrazine under the environmental stress.
Keywords: Atrazine; kinetic study; bio-decomposition; humic acid; Pseudomonas
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fluorescens.
1. Introduction
Atrazine (6-chloro-4-N-ethyl-2-N-propan-2-yl-1,3,5-triazine-2,4-diamine) is a herbicide,
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white crystalline solid, which is employed to regulate grassy and broadleaf in sugar cane crops, sorghum, corns all over the world [1-4]. It is a triazine herbicide, usually contains
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cyanazine and simazine and applied 70K-90K tonnes annually throughout the globe [3,4]. It
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was extensively used in Unites States for more than over past 35 years on corns, sorghum, wheat, conifers, nuts etc. [3]. In India, 340 tonnes of technical grade atrazine of the technical was consumed and was second most highly consumed pesticide in India [4,5]. It was first
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synthesized and introduced in the 1940s and also used in amalgamation or alone with other herbicides [4-8]. It has a half-life period of 41-231 days [3], and were having sky scraping
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potential to deteriorate the water bodies and agricultural fields [4-7]. It has been banned in
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several countries, because it metabolites/ residues often remain in agricultural fields and water bodies for several years [9-22]. The surpass levels of atrazine in drinking water of Europe and USA is 0.1 and 3 μgL−1 respectively [15-32]. It has found from the various studies that the chlorinated atrazine majorly leads to disruption of endocrine system of humans and mammals [30-41]. Furthermore, it induces reactive oxidative stress resulting in infertility of pigs, amphibians, and rats etc, reduced semen quality and sperm dysfunction
3
[12-28]. In females, it disrupts sexual hormones and intrude estrogens receptors for proper dysfunction, instinctive abortion, irregularities in ovarian cycles, developmental births defects etc [22-29]. It is well known that many environmental treatment methods such as adsorption [4251], photo-catalytical degradation [45-51], advanced oxidation process [42-51] have been reported for the removal of environmental pollutants but biodegradation emerged as a most cost-effective as well as the natural remedial method which has the ability to eradicate
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chemicals such as xenobiotic from the ecosystem [35-41]. Microbial population involved in degradation of atrazine and its major metabolites includes B. subtilis HB-6 [19], Arthrobactersp [18], Arthrobactersp [5], E. cloacae strain JS08 [13], Arthrobacter sp. strain
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DAT1 [17], Arthrobacter sp. GZK-1 [6], Arthrobacter sp. T3AB1 [38] Arthrobacter strain DNS 10 [10], Klebsiella sp. A1 Comamonassp.A2 [11], Arthrobacter sp. AD26 [41]. smaltophilia,
Bacillus
licheniformis,
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Stenotrophomona
Bacillus
megaterium,
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Rahnellaaquatilis Umbelopsisisabellina Volutellaciliata, Botrytis cinerea [38-41] isolated worldwide.
Moreover, divalent metal ions, zero valent iron and soil’s humates are used for
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environmental remediation including decontamination of pesticides [26-37]. Divalent metal ions and zero-valent iron facilitated degradation of pesticides, and it is a potential remediation
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strategy for elimination of pesticides. The major reason for the selection of Cu(II) and Fe(II)
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was the multiple applications of both metal ions in the living system and total environment. The iron [Fe(II)] and copper [Cu(II)] are very important micronutrients of soil [30-38]. These ions improve the plant health through complex mechanism. Improper distributions of these ions may alter the crop production and human health too [25-30]. Therefore, we tested the hypothesis “biodegradation of atrazine in the presence and absence of metal ions [Fe (II) and Cu(II)] and humic acid (HA)”. Initially, 35 bacterial strains
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were screened from the agricultural land and out of these three best strains were used for further study.
2. Experimental design 2.1. Sample Collection Soil samples were collected from six different agricultural fields of Kapurthala district Punjab (India). For the removal of large debris and unwanted material all the samples were sieved
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through a 2 mm sieve and homogenized with the help of mortar and pestle and kept at 4°C for further experimentation work. 2.2. Chemicals
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Technical grade Atrazine (98.0%) was obtained from Rallis India Ltd. The analytical reagent (AR) grade chemicals and media were purchased from Hi-media and Sigma Aldrich India.
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2.3. Isolation and characterization of atrazine-degrading bacterium
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Soil samples were collected from agricultural fields of six different villages of Kapurthala, Punjab, India having long history of atrazine usage (Elevation height 225 m, Longitude 75.38 E, and Latitude 31.38 E,). Six villages of district Kapurthala of state Punjab (India) selected
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for study includes: Bholana, Dona, Dhadwandi, Dhariwal, Noorpurdona, Kharsona, Mothanwal, and Tiba.
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Microbial population were obtained by media enrichment technique in which atrazine
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serves as sole source of energy for microbes. About 5 g of soil sample was poured in 500 ml of Erlenmeyer flask having 100 ml of enrichment medium. Enrichment medium composed of 100 mg/L Atrazine and mineral salts medium (K2HPO4 (0.6g), KH2PO4 (2.5 g), MgSO4.H2O (4.03g) and NaCl (0.4g) per litre) with addition of atrazine at 100 mg/L concentration [19,20]. Cultures were incubated at 28C for 4 days on rotatory incubator in dark conditions (150 rpm). After four days, the 1 ml of bacterial culture suspension was transferred to fresh
5
mineral salts medium containing atrazine (100 mg/L)under same incubation temperature for 4 days. This was repeated for 5 times with atrazine and thereafter inoculated in mineral salts medium containing atrazine. Three isolates, RK1, RK2, and RK3 were obtained through this technique 2.4. Molecular identification of atrazine degrading bacteria After isolationand purification of selected strains, the16S ribosomal RNA sequencing was conducted at Samved Biotech Pvt. Ltd. located at Ahmadabad, India. For the confirmation
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the identity of three isolated strains, 1.5 kb 16 s rRNA was amplified using the total DNA and sequenced by using the 1492R and 27F universal primers. The phylogenetic analysis and tree constructed as given by Singh et al. 2019 [30-32].
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2.5. Inoculum preparation for atrazine-degradation studies
Isolated strains were cultured in mineral salts medium having 100 mg/L of atrazine in a
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rotatory incubator (120 rpm) at 30oC for 24 hours. The isolated strains were homogenized at
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6500x g for 5-10 min and cell counts were adjusted using 0.5 McFarland standards to 107 cells/ml.
2.6. Optimization of concentrations, pH and temperature effects
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The concentrations of atrazine, humic acid, Fe(II) and Cu(II) as well as the effect of pH (5, 7 and 9) and temperature (10, 20 and 30oC) was optimized. The cell growth of individual
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bacterial strain was checked at 600 nm (O.D. 600) by using the UV-visible spectroscopy.
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2.7. Degradation of atrazine by the isolates in mineral medium and preparation of Inoculum for degradation studies To exploit the capability of the isolates to disintegrate atrazine, isolated strains were grown in broths and suspension culture was inoculated with isolates in each flask to reach a biomass level of OD600 = 0.91. The mineral medium was prepared with three different concentrations of atrazine 100 ppm as the energy source of carbon, carbon and nitrogen and carbon and
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sulphur. The minimal medium will be equipped with pesticides at different concentrations in the absence and presence Fe(II), Cu(II) and HA respectively. At regular period (i.e. at 0, 3, 7, 10, 14 day), 20 ml of the broth were worn-out to determine growth and depletion of atrazine. The growth of bacteria was examined by a UV-Vis spectrophotometer (1800UV Shimazdu) at 600 nm using triple distilled water as control. For the assessment of concentration of residual atrazine, gas chromatography (GCMS) was employed. Flasks containing minimal medium with 100 ppm atrazine served as i.e. Fe(II), Cu(II) and
humic acid on
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controls. To confirm the effect of metal ions
degradation process, above experiment was repeated under different compositions like bacteria + atrazine + metal ion [Fe (II) and Cu(II)] and HA [17,30-32].
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2.7. Analysis of atrazine decomposition by GC-MS
For Gas chromatography (GC-MS) analysis, 100 ml of medium was centrifuged at 9000 X g
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for 20 min keeping 4ºC for clarification and filtration was done with the Whatmann filter
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paper No. 1. The medium was then extracted with addition of ethyl acetate in 1:1 ratio. Extracted organic material was kept for air drying and concentrates upto 0.1 mL. Gas chromatography (GC-MS) equipped with ECD detector and DB5 capillary column of
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dimensions (0.25mm × 0.25μm × 30m) comprising splitless injector which are operated at 70 eV. Helium was employed as a carrier gas with flow rate of 1.5 mL per min. The temperature
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was adjusted to 280°C and 300°C for transfer line and to trap ion respectively. The
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concentration of residual atrazine was assessed on the basis of comparison of peaks in the abiotic control with respect to the samples. For recognition of metabolites, comparison of mass spectra of standards was done with mass spectra of products [33]. To perform the kinetic study, standard plot was prepared and samples were compared with standard plot at regular time interval (at 3rd, 7th, 10th and 14th day). 2.8. Degradation Kinetics
7
The decompositions rates of the atrazine were determined by using pseudo-first order kinetics [25-35]. Briefly, Eqs. (1)–(3) represents equations of the variation in the concentration with respect to given time plotted. Linear regression is used by plotting graph with time (in day) against Log Ct (in ppm). d[C]/ dt = −kobs[C] ------- (1); ln[C]/ [C]0 = kobst ------- (2); t1/2= (1/ kobs) × ln2 ------ (3),
[C]0=the initial concentration of atrazine (mg/l)
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where, [C] = concentration of atrazine at time t (mg/l)
kobs= constant for pseudo-first order (day−1) equal to slope of line i.e. kobs= - slope.
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2.9. Statistical Analysis
To avoid any error and to maintain reproducibility of data, all the experiments were
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performed in triplicates. MS Excel worksheets were used for calculation of standard errors
3. Results
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calculation of ANOVA.
SPSS 16 statistical software were used for
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and means in the experimental data and
Enrichments were employed and followed over time, isolating a bacterial culture which has
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the ability to utilize atrazine as carbon and energy source (including nitrogen and carbon
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source), for progression. For one month, enrichment with selective component was repeated, and repeated transferring of samples from culture to fresh minimal medium containing atrazine exhibited the growth of the isolates of pure atrazine degrading strains. Total, eleven strains of bacteria were isolated from soil as they were able to consume atrazine and were designated as from RK1 to RK11. The strain RK1, RK2, and RK3 were found to be the most efficient that were able to utilize atrazine as source of carbon for growth and were selected
8
for further study. Phylogenetic position and 16s rDNA analyses have confirmed that isolates strains were Streptomycetaceae bacterium RK1, Pseudomonas fluorescens RK2, and Azotobacter chroococcum RK3 and submitted to NCBI under accession numbers KJ206091.1, KJ466148.1, KJ511860.1 (Supplementary Figure – S1). 3.2. Degrading ability of isolated strains The bio-decomposition of atrazine (100 mg/L) was checked on regular time interval (0th, 3rd, 7th, 10th and 14th) for the 14 days of incubation with RK1 to RK11 strain. However, three
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strains (RK1, RK2 and RK3) have shown significant utilization of atrazine. All the isolated cultures show threshold increase in their growth from 3rd day till 10th day. After 10thdays of incubation, the relative growth of all the isolates was constant (Table – 1).
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In fourteen days experiment, the strain RK1 alone has shown significant biodecomposition as compared to other test conditions i.e. in the existence of Cu(II) and HA,
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except Fe(II) (Figure -1). Results indicated that bacterial strain RK1 can decompose atrazine
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more than 95 % in the presence of iron which is similar to that of without Fe(II). With the addition of Cu(II) and HA, significant (p <0.05)decrease in the atrazine decomposition has been noticed which was 61 and 39 % respectively. The order of bio-decomposition of
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atrazine with various experiments was inoculum + atrazine ~ inoculum + atrazine + Fe(II) > inoculum + atrazine + Cu(II) ≫ inoculum + atrazine + HA (Figure -1).
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The strain RK2 has shown better decomposition rates in 14 days experiments. The
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order of bio-decomposition of atrazine with various experiments was inoculum + atrazine ~ inoculum + atrazine + Cu(II) ~ inoculum + atrazine + HA > inoculum + atrazine + Fe(II) > (Figure -1). After 14 days experiments, more than 87% decomposition was noticed in all experimental conditions with bacterial strain RK2. Here, non-significant (p <0.05) difference were noticed among the various experiment conditions which indicates that RK2 can utilize atrazine under all conditions i.e. even under the stress of Cu(II), Fe(II) and HA.
9
Under various test conditions, bacterial strain RK3 has shown more than 77% decomposition of atrazine. The decomposition was 77 and 85% in the presence of Cu(II) and Fe(II). The strain RK3 alone has decomposed near about 95% atrazine which was almost closer to that of in the presence of HA (91%) (Figure -1). The observed bio-decomposition order of atrazine with various experiments was inoculum + atrazine ~ inoculum + atrazine + HA > inoculum + atrazine + Cu(II) ≫ inoculum + atrazine + Fe(II) (Figure -1). Figure – 1 exhibited that in the absence of Fe(II), Cu(II) and HA, all three strains
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have ability to decompose more than 95% of atrazine. In Cu(II), treated groups RK2 (96%) have shown significant decomposition decrease as compared to other two strains RK3 (85%) and RK1 (61%). Applications of Fe(II) have shown 77 to 95% decomposition of atrazine.
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The results were significantly different from each other i.e. 95, 87 and 77% for RK1, RK2 and RK3 respectively. In the presence of HA acid, decomposition rates of RK2 (95%) and
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RK3 (91%) were almost similar to each other but dramatically different for RK1 (39%).
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Based on the present study, to choose the best strain among the RK1, RK2 and RK3 under various conditions, the exact order may be RK2 > RK3 ≫ RK1. 3.3. Degrading kinetics of isolated strains
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The kinetic study of atrazine decomposition including its half-life period (t1/2), equation of line and regression constant r2 is mentioned under Table – 1. The observed half-life value was
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maximum for RK1 strain in the presence HA which was 53.32 days. The second highest
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decomposition rate was also with RK1 strain in Cu(II) treated groups which was 23.90 days. In the presence of Fe(II), RK3 strain has shown third highest decomposition rate that was 15.40 days. Applications of Cu(II) in RK3 inoculum has shown half-life period 5.11 days. In the Fe(II) treated groups, RK2 has decomposed atrazine with an half-life period 10.05 days. The remaining experiments in the absence and presence of Fe(II), Cu(II) and HA have halflife period from 4.53 to 3.72 days (Table – 1). Overall, without applications of Cu(II) Fe(II),
10
and HA, RK1 have shown better decomposition rate (4.53 day) as compared to RK2 (5.29 day) and RK3 (7.07 day). Similar trends were noticed with the applications of Fe(II) i.e. the observed half-life period was RK1(7.00) > RK2 (10.05 day) > RK3 (15.40 day). It means, bacterial strain RK1 can decompose atrazine with significant half-life period under the influence of Fe(II). It has been found that RK2 and RK3 (RK2 > RK3) are efficient to degrade atrazine under the treatment of Cu(II), Fe(II), and HA whereas RK1 can do it only in the existence of Fe(II) only (Table – 1).
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3.4. GC-MS analysis and most probable decomposition mechanism To propose the most probable mechanism of atrazine bio-decomposition GC-MS analysis was performed. The GCMS results of bio-decomposition of atrazine at various conditions has
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mentioned under Figure – 2 and supplementary Figures S2-S15. GC-MS study revealed that atrazine was into eleven fragments in the presence and absence of Fe(II), Cu(II) and HA
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(Figure – 3). Pure atrazine was found in GC-MS at RT 19.35 min which has shown
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corresponding peaks (m/z) at 215, 200, 173, 120, 93, 58 and 43. In the absence and presence of Cu(II), Fe(II), and HA, GC-MS has shown two additional peaks (major only) at RT 5.42 and 11.8 min. The corresponding value of mass to charge ratio of metabolites at RT 5.42 min
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was 78, 63 and 45. Similarly, the m/z ratio for peak at RT 11.58 min was 152, 120, 92, 65. It is observed that atrazine was decomposed through the formation of metabolites like N2-
ur
ethyl-6-hydroxy-N4-isopropyl- 1,3,5-triazine-2,4-diaminium (m/z 200), 6-chloro-N2-ethyl(m/z
173),
N-isopropylammelide
(m/z
170),
4-amino-6-
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1,3,5-triazine-2,4-diamine
(ethylamino)-1,3,5-triazin-2-ol (m/z 155), (methylamino/ethylamino) methanediol (m/z 120), (aminomethylamino) methanediol (m/z 92), aminomethanediol (m/z 63) and 2methylpropane (m/z 58). Based on the GC-MS analysis the most probable mechanism of atrazine decomposition is mentioned under Figure – 3.
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4. Discussion Recently, various techniques gaining attentions to remove the pollutants from the environment including fabricated materials i.e. nanoparticles, carbon nano tubes etc. [42-51]. These fabricated materials are most effective to removes the metal ions [43,46,47], dyes [44,45,50,51] and other pollutants. The biodegradation technique is considered as cost effective and eco-friendly where no chemical method is used. This is the first study on atrazine interactions with soil humic acid and metal ions. The
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optimized concentrations level of the atrazine, humic acid, Fe(II) and Cu(II) were 100, 10, 25 and 15 ppm respectively. At higher concentration, degradation rates were decreased as mentioned in our recent report [28-32]. This may attributed to the toxicity of chemicals at
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higher concentration [28-32. The decrease in the Also, the optimized pH and temperature was 7 and 30oC respectively which was similar to recent reports [6,10,13,17,18,38-41]. Since
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atrazine contains various coordinating sites (N and NH), it is assumed that it will be involved
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in complexation with metal ions such as Fe (II) and Cu(II) [4,10,25,2-32]. Atrazine can form H-bonds with humic substances through NH bond(s) which may be the case with RK1 strain consequently low decomposition rate was noticed. In comparison with atrazine decomposing species
of
Arthrobacter
na
microbial
sp.
TES6
(60%),
Arthrobacter
sp.
(90%)
Pseudaminobacter sp. (71%), Arthrobacter sp. strain MCM B-436 (65%), Arthrobacter sp.
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strain HB-5 (70%) B. subtilis Strain HB-6 (55%), Arthrobacter sp. GZK-1 (65%),
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Arthrobactersp E. cloacae strain JS08 (70%), Arthrobacter sp. strain DAT1 (80%), Arthrobacter strain DNS 10 (70%), Comamonas sp. A2 (775), Klebsiella sp. A1 (84%) [5,6,13,16-22], the strains RK1, RK2 and RK3 have shown better degradation of atrazine. De-chlorination, de-alkylation and de-amination are known to be the major routes for atrazine transformation. Hydrolytic de-chlorination is the main mechanism for atrazine degradation using enzyme atrazine chlorohydrolase. The next step is the action of amino-hydrolases
12
enzymes which triggers two hydrolytic deamination reactions in which it is further converted into N-ethylammelide [14] or N-isopropylammelide [6]. These N-ethylammelide and Nisopropylammelideare lastly degraded to cyanuric acid [20,41]. Second pathway for degradation of atrazineincludes the N-dealkylation of isopropyl chains and ethyl to di-ethyldi-isopropyl atrazine disopropylatrazine, and
diethylatrazine [16-19].
Further, these
metabolites undergo hydroxylation by forming cyanuric acid [3,4,38]. Though it was the first study of atrazine decomposition in the presence of HA, Fe(II), and Cu(II) so no comparison
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was done with recent studies. Atrazine contain one or more binding sites (most probably NH) and they may interact with essential metal ion and organic matter (Trevisan et al., 2010). Kumar et al, 2016 [4]
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have reported that atrazine complexes with Fe (II) and Cu(II) through the N and NH sites. The complexation of the atrazine is dependent on transport, bioaccumulation and
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bioavailability in the ecosystem [25-31]. Metal ions such as Fe(II) and Cu(II) and humic
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substances form weak complexes with atrazine and therefore have a pessimistic effect on degradation as per as our assumption [29,30,32]. The samples without treatment of metal ions Fe(II), Cu(II) and humic substances exhibits extreme degradation of atrazine as matched to
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others after 14 days, humic acid plays an important role in the treatment of atrazine. By forming weak interactions with metal ions it is supposed that Cu(II) and Fe(II), decreases the
ur
degradation percentage by showing their toxic effects on bacterial catabolism and humic acid
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[33-37]. Although, humic substances blocks the active sites (like OH, NH and NH2 responsible for H-bonding and weak bondings) of atrazine through weak bonding which results in bacterial catabolism. As the all three bacterial strains exhibits the ability to grow and survive on atrazine alone without any additional source and this make them ideal for biodegradation of atrazine under various conditions.
13
5. Conclusion This study systematically explored the degradation of atrazine in presence and absence of Fe(II), Cu(II) and humic acid (HA). Following conclusions were drawn: 1. In the presence of Cu(II) i.e. RK1/2/3 + Cu(II), the observed t1/2 values were 23.90, 6.13 and 11.74 days. 2. In experimental condition with Fe(II) i.e. RK1/2/3 + Fe(II), the observed t1/2 values were 7.00, 10.05 and 15.40 days respectively.
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3. With the applications of HA i.e. RK1/2/3 + HA, the observed t1/2 values were 53.32, 6.54 and 8.56 days respectively.
4. The kinetic study revealed that in the absence of Fe(II), Cu(II) and HA, RK1 (1.97
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day) is the better strain than RK2 (5.29 day) and RK3 (7.07 day).
5. Overall, in the presence of Fe(II), Cu(II) and HA, RK2 is the better strain than RK3
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and RK1.
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6. Current study revealed that Pseudomonas fluorescens (RK2) could be used to
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decompose the atrazine under the environmental stress.
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[51] Y. Lei, Y. Cui, Q. Huang, J. Dou, D. Gan, F. Deng, M. Liu, X. Li, X. Zhan, Y. Wei, Facile preparation of sulfonic groups functionalized Mxenes for efficient removal of
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na
lP
re
-p
methylene blue. Ceram. Int. 45 (2019) 17653-17661
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Table and Figure Captions Table - 1. Kinetics of biodegradation of atrazine by three bacterial strains in mineral salt medium. Figure – 1. Removal (%) of atrazine after incubation with three strains in mineral salt medium under different conditions. Figure – 2. GC-MS analysis of atrazine 7th day sample in the influence of Cu(II) in the presence of bacterial strain RK2. Figure – 3. Most probable mechanistic pathway of decomposition of atrazine in mineral salt
Jo
ur
na
lP
re
-p
ro of
medium.
22
Table - 1.Kinetics of biodegradation of atrazine by three bacterial strains in mineral saltmedium. Experimental Conditions
K
t1/2 (days)
Equation of line
r2
Streptomycetaceae bacterium (RK1) Inoculum + atrazine
0.153
4.53a,b
y = -0.153x + 2.154
0.99
Inoculum + atrazine + Cu(II)
0.029
23.90a,b
y = -0.029x + 1.956
0.91
ab
Inoculum + atrazine + Fe(II)
0.099
7.00 ,
y = -0.099x + 2.071
0.99
Inoculum + atrazine + HA
0.013
53.32a,b
y = -0.013x + 1.971
0.99
Pseudomonas fluorescens (RK2) 5.29a,b
y = -0.131x + 2.054
0.98
Inoculum + atrazine + Cu(II)
0.113
ab
6.13 ,
y = -0.113x + 2.131
0.98
Inoculum + atrazine + Fe(II)
0.069
10.05a,b
y = -0.069x + 1.989
0.99
Inoculum + atrazine + HA
0.106
6.54a,b
y = -0.106x + 2.144
0.99
y = -0.098x + 2.039
0.98
y = -0.059x + 1.938
0.94
y = -0.045x + 1.943
0.94
y = -0.081x + 2.066
0.99
Azotobacterchroococcum (RK3) Inoculum + atrazine
7.07a,b
0.098
ab
0.059
11.74 ,
Inoculum + atrazine + Fe(II)
0.045
15.40a,b ab
0.081
8.56 ,
re
Inoculum + atrazine + HA
-p
Inoculum + atrazine + Cu(II)
ro of
0.131
Inoculum + atrazine
a = results were significantly differ (at p <0.05) for three strains at same experimental conditions.
lP
a* = results were non significantly differ (at p <0.05) for two or more than two strains at same experimental conditions.
Jo
ur
na
b = results were significantly differ (at p <0.05) for each strain at different four experimental conditions.
23
ro of -p re lP na ur Jo
Figure – 1.Removal (%) of atrazine after incubation with three strains in mineral salt medium under different conditions.
24
ro of -p re lP na
Figure – 2.GC-MS analysis of atrazine 7th day sample in the influence of Cu(II) in the
Jo
ur
presence of bacterial strain RK2.
25
Cl
OH
N
OH
N
HN
N
H2 N
HN
N
N
NH
N HN
HN
HN
N2-ethyl-6-hydroxy-N4-isopropyl1,3,5-triazine-2,4-diaminium m/z 199
Atrazine m/z 215
amino((ethylamino)methylamino)methanol m/z 119
OH OH
N OH N
+
HO
NH
2-methylpropane m/z 58
N
HO
OH
ro of
H2 N
N
HN
NH
N
HN
6-chloro-N2-ethyl-1,3,5-triazine-2,4-diamine
N H
m/z 173
N
H2 N
N H
N
HO
4-amino-6-(ethylamino)-1,3,5-triazin-2-ol
N
OH
cyanuric acid m/z 129
na
m/z 155
N
lP
N
re
OH N
(aminomethylamino) methanediol m/z 92
-p
N-isopropylammelide m/z 170
OH
N
NH2
OH
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ur
Figure –3. Most probable decomposition pathway of atrazine.
26
(ethylamino)methylamino) methanediol m/z 120
OH OH
HO NH
HO NH2
(methylamino)methanediol m/z 77
aminomethanediol m/z 63