Atrazine degradation by Bacillus safensis strain BUK_BCH_BTE6 isolated from agricultural land in northwestern Nigeria

Atrazine herbicide is known to disrupt the endocrine system and is potentially carcinogenic. Its continual application leads to high residue levels in soil, causing water pollution, which when consumed is associated with devastating health effects. This research reported the isolation and characterization of a new bacterial strains from active agricultural soil with the potential to biodegrade atrazine as a sole carbon source. An enrichment method was utilized to isolate the bacteria (A1, A2, B1, B2, C1 and C2) on mineral salt media (MSM) following serial dilution. Six isolates were screened for their tolerance to various concentrations of atrazine (500 to 1500 mg∙L ‐1 ), and only isolate B1 tolerated up to 1500 mg∙L ‐1 atrazine. The isolate was identified molecularly as Bacillus safensis strain BUK_BCH_BTE6 based on 16S rRNA gene sequencing and molecular phylogenetic analysis. Characterization of the isolate based on the effects of temperature, pH, substrate concentration, incubation time, inoculum size, and heavy metals revealed optimum growth and atrazine degradation at 35 °C, a pH of 7.5, 400 mg∙L ‐1 , at 48 h, and inoculum size of 600 µL, respectively. The growth of the isolate was inhibited by 2 ppm Hg, Cd, Cr, Pb, Ar, and Ni, while Fe, Cu, and Zn stimulated it. GC‐MS analysis revealed a degradation efficiency of 88.85% within 120 h, while metabolites such as desethyldeisopropylatrazine, deisopropylatrazine, N‐ethylammelide, and cyanuric acid were also detected. This isolate is a highly atrazine‐tolerant and efficient atrazine degrader that could be employed for bioremediation of atrazine‐polluted sites.


Introduction
The rise in population and the need for better agricultural production in addition to industrial advances and urban ization were the major sources of environmental contami nation we are facing today (Ali et al. 2020).Increased in dustrialization, wars, use of intensive metals and organic substances have leveraged these problems (Benjamin et al. 2019).Modern agriculture depends on herbicides to eradi cate weeds to maximize crop yield.Thus, these herbicides are of economic importance to the increasing world popu lation (Singh and Singh 2016).
Atrazine (1chloro3ethylamino5isopropylamino 2, 4, 6 triazine) is an herbicide of the triazine class with molecular formula C 8 H 14 ClN 5 (He et al. 2019).It is often present on surface and groundwater, with a halflife of 13-261 days (Li et al. 2019).It acts against broadleafed weeds and some grass weeds that inhibit growth of crops, also restrain some perennial weeds (He et al. 2019).It is the most widely used herbicide worldwide on crops such as sorghum (Sorghum bicolor), maize (Zea mays) and sugarcane (Saccharum officinarum) because of its low cost and effectiveness (Shamsedini et al. 2015).
Several tons of herbicides are applied every year in agriculture, causing harm to nontarget organisms.They can reach water through runoff from treated plant and soil and are able to leach down the soil to contaminate groundwater or if immobile, they remain on the top soil where it accumulates as a toxin and affect microorganisms (Warsi et al. 2017).The application and continuous use of atrazine increases its residue in soil, causing water pollu tion.Atrazine monitoring in southwestern Nigeria, reveals higher concentrations of atrazine and its chlorostriazine metabolites in handdug wells than in borehole and stream water, with concentrations ranging between 0.01 mg•L 1 and 0.08 mg.L 1 .Hazard index values associated with in gestion and dermal for both children and adults were be low the acceptable limit of 2.0 µg•L 1 in the US and above 0.1 µg•L 1 in Europe.Atrazine concentration as low as 0.02 mg•L 1 appears to trigger defense mechanisms capa Muhammad et al. Indonesian Journal of Biotechnology 28(3), 2023, 127-136 ble of protecting the structural integrity of the brain.These concentrations may not pose any threat to brain function, but concern should be raised at 0.08 mg•L 1 (Owagbori aye et al. 2022).Atrazine herbicide has negative effect on plant photosynthesis, microorganism and human health as it is considered an endocrine disruptor and carcino gen.This necessitates the need for its remediation.The conventional physicochemical approaches for remediating soil have some drawbacks such as high costs, formation of secondary pollutant and damage to soil fauna, all of which fail to completely remove atrazine (OluArotiowa et al. 2019).Thus, innovative techniques are critically needed (OluArotiowa et al. 2019).Bioremediation is considered a harmless, efficient, and economical biotechnological ap proach for the removal of atrazine in polluted areas.To date, report on the isolation and characterization of indige nous bacteria with atrazinedegrading capability in Nige ria is limited and perhaps lacking from the northwestern part of the country despite the extensive utilization of this herbicide in agricultural activity.

Sample collection
A random sampling technique was used to select three agricultural farmland from which soil samples were col lected in Kura Local Government Area of Kano, North western Nigeria, located at 11°46´N, 8°49´E and 12°01´N, 8°29´E (Umar et al. 2020).Soil samples were collected at the depth of 5-10 cm below the topsoil using a ster ile scoop after removal of the top layer of the soil up to 1-5 cm.The samples in sterile containers were trans ported to the Microbiology laboratory at Bayero Univer sity Kano for analyses.The samples were dried, homoge nized and passed through a 2 mm sieve.Analytical grade atrazine with 90% purity was procured from Emerald Sci entific, USA.All experiments involving microorganisms were done in a class II biosafety cabinet.

Enrichment and isolation of atrazine-degrading bacteria
The bacteria were isolated using culture enrichment method in a mineral salt medium comprising 0.
of NaCl, and 1 m•L 1 and a solution of trace element concentrate was prepared ac cording to Sawangjit (2016) (Macwilliams and Liao 2016).Into 250 mL Erlenmeyer flasks containing 100 mL of MSM which was supplemented with 100 mg•L 1 atrazine, 10 g of soil sample was inoculated and homogenized.The flasks were then incubated at 37 °C under orbital agita tion at 150 rpm.After 7 d, 10 mL aliquot of each culture was transferred to the new MSM broth (100 mL).Sub sequently, each culture was serially diluted from 10 1 to 10 6 in sterile distilled water and proper dilutions of 1×10 6 were plated on MSM agar, supplemented with 0.1 mL of atrazine (100 mg•L 1 ).The Petri dish was then incubated for 48 h at 37 °C.Bacterial colonies grown were isolated according to their morphology, and the selected morpho types were purified by repeated streaking on MSM agar (Muhammad et al. 2021).The isolates were then grown on the stock culture of nutrient agar and incubated at 37 °C for 24-48 h.Bacterial isolates were then stored refrig erated at -20 °C for further studies.

Screening of atrazine-tolerant isolate
A total of six bacterial isolates (A1, A2, B1, B2, C1 and C2) were isolated based on their morphology after cul ture enrichment and tested for their ability to tolerate vary ing concentrations (500 mg•L 1 , 1000 mg•L 1 and 1500 mg•L 1 ) of atrazine in MSM as sole carbon source (Ah mad et al. 2022).

Molecular identification
Pure bacterial culture grown on Luria broth at 37 °C was used to extract genomic DNA and centrifuged for 2 min at 5000 rpm.The pellets were homogenized in 100 µL warmed Livak grind buffer (1.6 mL, 5M NaCl, 5.48 g su crose, 1.57 g Tris, 10.16 mL 0.5M EDTA, 2.5 mL 20% SDS) and incubated at 65 °C for 30 min.About 14 µL of 8 M Kacetate was added and centrifuged for 20 min at 4 °C.The pellet was rinsed in 100 µL icecold 70% ethanol dried and suspended in 100 µL dH 2 O incubated for 10 min at 65 °C and the concentration of DNA was determined us ing a Nanodrop spectrophotometer (2000/2000c, Thermo Fisher Scientific, USA) (Brahmbhatt 2012).
The PCR reaction was carried out using KAPATaq DNA polymerase.Total volume of 25 µL and com prise of 2 µL genomic DNA, 2.5 µL of 10 TaqA Buffer, 0.4 M (0.85 µL) of each of forward primer Bact1442 F (AGAGTTGATCCTGGCTCAG) and reverse primer Bact1492R (GGTTACCTTGTTACGACTT) 1.25 mM (1.5 µL) of MgCl 2 , 0.25 mM (0.2 µL) of dNTPs mixes and 0.2 µL of Taq DNA polymerase, in ddH 2 O (Sawangjit 2016).PCR amplification products were observed using agarose gel electrophoresis.A 14well comb was placed, and the gel was allowed to set for 30 min.After the gel so lidified, the comb was removed and the gel tray was placed in the buffer tank.The gel was submerged to a depth of 2 to 5 mm by pouring 0.5× TBE into the tank (Li et al. 2020).
Purified PCR products were further amplified in one direction with 16S primer using Big DyeTM Terminator Ready Reaction Mix (ABI) and sequencing of the ampli fied product was done with an ABI 3130 Genetic Analyzer version (Applied Biosystem) (Singh and Singh 2016).The 16S rRNA gene sequence of the isolate was analyzed with nucleotide BLAST search in GenBank.Phylogenetic re lationship was analyzed with other closely related in Gen Bank (Swissa et al. 2014).The sequences were obtained in FASTA format, checked for Multiple Sequence Align ment (MSA) using TCoffee tool from EBI (EMBL), and a tree was created (Brahmbhatt 2012).

Characterization of atrazine-degrading bacteria
Various environmental factors affect the growth of mi croorganisms.A triplicate experimental run was con ducted using one factor at a time (OFAT) to study the effect of growth parameters.

Effect of initial pH on atrazine degradation
The pH of the MSM (50 mL) supplemented with atrazine (100 mg•L 1 ) was varied in a range between 5.5-8.5, bac terial culture (100 µL) was then inoculated and incubated at 37 °C for 120 h.Bacterial aliquot (1 mL) was taken every 24 h to measure at OD 600 (Shehu et al. 2021).

Effect of temperature on atrazine degradation
The incubation temperature of the inoculated MSM (50 mL) supplemented with atrazine (100 mg•L 1 ) was var ied from 25-50 °C, and incubated for 120 h.Bacterial aliquot (1 mL) was taken every 24 h to measure the OD 600 (Muhammad et al. 2021).

Effect of substrate concentration on atrazine degradation
Into MSM (50 mL) supplemented with various concentra tions of atrazine (100-1000 mg•L 1 ) was inoculated with 100 µL bacterial culture and incubated at 37 °C for 120 h.Bacterial aliquot (1 mL) was taken every 24 h to measure the OD 600 .

Effect of incubation time on atrazine degrada tion
A culture media containing 50 mL of MSM supplemented with atrazine (100 mg•L 1 ) was inoculated with 100 µL of bacterial culture and incubated at 37 °C.Bacterial aliquot (1 mL) was taken every 24 h to measure the OD 600 for 120 h.

Effect of inoculum size on atrazine degradation
Various inoculum sizes ranging between 100-1000 µL were inoculated into 50 mL each of MSM supplemented with atrazine (100 mg•L 1 ) and incubated for 120 h at 37 °C.An aliquot (1 mL) was taken every 24 h to measure the OD 600 .

Effect of heavy metals on atrazine degradation
Heavy metals solution (2 ppm) was added into each MSM (50 mL) supplemented with atrazine (100 mg•L 1 ) and in oculated with 100 µL of bacterial culture, then incubated at 37 °C for 120 h.An aliquot (1 mL) was used at each 24 h to measure the OD 600 .

Biodegradation study
An experiment with optimum conditions was set in 250 mL Erlenmeyer flasks with 100 mL of MSM, for 120 h.Microbial growth was assessed spectrophotometrically by measuring OD 600 .Final atrazine residue at 120 h were ex tracted with 12 mL acetonitrile, and centrifuged at 9980 ×g for 6 min; extracts were concentrated to dryness by a ro tary evaporator at 25 °C.The dried residue was redissolved in 0.5 mL acetonitrile/water (20:80, v/v), and the final so lutions were filtered through a 0.22 μm filter membrane before GCMS analysis on an Agilent 19091S433UI (HP 5MS Ultra Inert).The mobile phase consisted of acetoni trile and water (v/v, 80:20).Following injection of 2 μL of the sample onto the column, the analytes were eluted using an isocratic mode with a flow rate of 0.3 mL•min 1 .The GC temperature program was 50 °C for 1 min, ramped to 50 °C at 10 °C min 1, ramped to 180 °C at 5 °C min 1, and finally ramped to 230 °C at 10 °C min 1 and held for 5 min.The injector temperature was held at 300 °C for 5 min.Splitless injection was used.Helium was used as the carrier gas at a 1 mL•min 1 flow rate.The transfer line between the GC and mass spectrometer was held at 325 °C, and the ion trap manifold was set to 330 °C.A Varian 8200 Auto Sampler was used to perform sample injection and solvent flushing of the needle.Atrazine standard was 100 μg•L 1 in 100% acetonitrile.After completion of the analysis, the GCMS postrun analysis was checked on the desktop.The data required for the report preparation was opened and integration was done.The mass spectrum of the atrazine standard was compared with that of the test sample to determine the metabolite present.The degradation percentage was calculated according to the formula: Note: ACS= Area under the curve of the standard and ACT= Area under the curve of the test sample.

Isolation, screening and identification atrazinedegrading bacteria
A total of six isolates (A1, A2, B1, B2, C1 and C2) were isolated and screened for atrazine degradation, of which isolates A1, A2, B2, C1 and C2 tolerated only concentra tions between 500 mg•L 1 and 1000 mg•L 1 of atrazine.However, only isolate B1 tolerated up to 1500 mg•L 1 atrazine; hence, it was selected for the characterization work (Table 1).The 16S rRNA gene sequences of the bacterium were compared with related sequences from the Gen Bank database using Blast Server at NCBI, and results show that the DNA sequences obtained were closely re lated to the partial sequences of several Bacillus sp. with almost 99% similarity.Molecular phylogenetic stud ies using the neighborjoining method linked the iden The first pure bacterial culture to be isolated with po tential for mineralization and utilization of atrazine as a sole carbon and nitrogen source for growth was Pseu domonas sp., which fully degrades it, making it a model of interest for analyzing the genetic and enzymatic basis of atrazine mineralization (Fernandes et al. 2020).To date, a number of Bacillus with atrazine degrading capability have been isolated from soils around the world.Interest ingly, this work was the first to report the isolation of B. safensis from Nigerian soil that could tolerate up to 1500 mg•L 1 atrazine and tolerated a number of toxic heavy met als, which is quite a novel feat.

Effect of initial pH on atrazine degradation
The effect of pH on the growth of atrazinedegrading B. safensis in MSM is presented in Figure 2. The growth of this bacterium was shown to be optimum at pH 7.5 fol lowing 48 h incubation at 37 °C.A significant decrease in growth was observed at a pH higher than 7.5 (p < 0.05).
The optimum pH of 7.5 for atrazine degradation ob served in this study (Figure 2), was in agreement with find ings from literature reports (Abigail et 2018) reported a pH of 9.0.Research has in dicated that an increase in soil pH may lead to an increase in soil microbial biomass and enzymatic process, it was also observed that atrazine degradation was higher at a pH FIGURE 2 Effect of pH on the growth of strain BUK_BCH_BTE6 in MSM containing 100 mg•L -1 atrazine, incubated at 37 °C for 48 h.Each value represents mean ± SD of three replicates.range of 6.5-7.5 (Omotayo et al. 2016).Perhaps, pH is among the factors influencing the rate of some chemical and biological processes (Shamsedini et al. 2015).The changes in pH values affect both the structure and activity of protein, this also affects the activity of cellular enzymes.Drastic change in pH also damages the cell and membrane proteins, which in turn debilitate permeability barrier of cell (Cho et al. 2016).

Effect of temperature on atrazine degradation
The temperature effect on the growth of atrazine degrading Bacillus safensis in MSM is presented in Figure 3.The result shows that a temperature between 35 and 40 °C was optimum for growth following 48 h incubation.A significant decline in growth (p < 0.05) was observed at temperatures below 30 °C higher than 40 °C.
The optimum temperature between 35 and 40 °C for the growth of atrazine degrading isolate reported in this study (Figure 3 2013) reported an opti mum temperature of 28 °C and 29.3 °C, respectively for atrazine biodegradation.The variation in optimum tem perature could be due to the differences in the species of isolates, geographical location from which the isolate was isolated, targeted concentration of atrazine, as well as other conditions prevailed.This study shows that growth and degradation abilities of the bacteria were both inhib ited at temperatures higher than 40 °C or lower than 35 °C, indicating that the isolate is a mesophile (growth in moderate temperature) and that temperature plays an ac tive role in bacterial metabolism and atrazine degradation.All life activities leading to growth are catalyzed by the en zymes found in the cell, the activity of which is affected by temperature, which also influences growth (Charpe et al. 2019).Thus, the effect of accelerated degradation in trop FIGURE 3 Effect of temperature on growth of strain BUK_BCH_BTE6 in MSM containing 100 mg•L -1 atrazine, incubated for 48 h.Each value represents mean ± SD of three replicates.
ical zones might be stronger than in temperate regions (Omotayo et al. 2016).

Effect of atrazine (carbon source) concentration on its degradation
The effect of atrazine (substrate) concentration on the growth of B. safensis in MSM is presented in Figure 4.A concentration of 400 mg•L 1 atrazine was seen to produce optimum growth for this bacterium following incubation at 37 °C for 48 h.A significant decrease in growth (p < 0.05) was observed at concentrations higher than 400 mg•L 1 , though the difference was between 600 mg•L 1 and 1000 mg•L 1 .The linear increase in growth observed at atrazine con centrations between 50-400 mg•L 1 (Figure 4), shows that lower concentrations of this herbicide did not produce tox icity to this bacterial isolate.However, the decline in OD 600 as concentration increased beyond 400 mg•L 1 , re veals that concentrations above the optimum are toxic to the bacterial cell, slowing down the growth of the isolate.The optimum concentration found in this study was sim ilar to that reported by Abigail et al. (2013), but deviated from 50 mg•L 1 reported (Shiri et al. 2016; Zhao et al. 2017, 2018; Zhang et al. 2019).Similarly, an optimum concentration of 100 mg•L 1 atrazine was reported (Zhang et al. 2009; Kolekar et al. 2014; Ye et al. 2016; Ariole and Abubakar 2015; Li et al. 2019).Other studies (Wang et al. 2014; Zhu et al. 2019)  inhibit the metabolism of microorganisms (Khatoon and Rai 2020).This result shows that this isolate is a good atrazine degrader that could be used for remediation in a broader range of atrazine concentrations.

Effect of inoculum size on atrazine degradation
The effect of inoculum size on the growth of atrazine degrading B. safensis in MSM is presented in Figure 5.The growth of the bacterium was found to be optimum when the inoculum was 600 µL following 48 h incuba tion at 37 °C.A significant decrease in growth (p < 0.05) was observed at inoculum sizes below 400 µL and higher than 600 µL.
The optimum inoculum size from this study was found to be 600 µL (Figure 5).The insignificant effect ob served after increasing the inoculum from 600-800 µL could probably be due to the attainment of equilibrium of microbial load.Similar studies on atrazine degrada tion reported different inoculum sizes; Zhang et al. (2009) and Abigail et al. (2013) reported 3%, Debasmita and Ra jasimman (2013) reported 5%, Zhao et al. (2017) reported 100 µL and 10% was reported by Zhao et al. (2018).Ac cording to Zhang et al. (2009), a lower inoculum size pre vents sufficient contact of the strain with the atrazine in the medium, resulting in a lower degradation rate, while a higher inoculum size results in carbon source competi tion at the initial stage of the strain growth causing malnu trition in strains and a significant influence on biodegra dation rate.A high inoculum size results in an increase in atrazinedegrading capability and may play a simula tive role in the degradation of atrazine by the isolate and increase the pollutant removal capacity.Hence, different inoculum size levels result in different degradation rates (Zhao et al. 2017).

Effect of incubation time on atrazine degrada tion
The effect of incubation time on the growth of atrazine degrading B. safensis in MSM is presented in Figure 6.The growth of this isolate reveals a linear pattern within the first 24 h, reaching optimum after 48 h of incubation at 37 °C.A significant decrease in growth was observed at incubation times above 48 h (p < 0.05).
In this study, the optimum growth was observed af ter 48 h of incubation (Figure 6), which could be due to the lag phase of the isolate.Atrazine degradation was in efficient at low incubation time, but increases with higher incubation period, indicating that time might positively af fect atrazine degradation.Similar findings were reported (Abigail et al. 2013; Zhao et al. 2018; Li et al. 2019; Zhang et al. 2019; Li et al. 2020), and deviated from 72 h reported (Wang et al. 2014; Andleeb et al. 2016; Omotayo et al. 2016).Moreover, Ye et al. (2016) 2013) reported 24 h, while Ariole and Abubakar (2015) and Mansee, Ayman and Bakry, Nabila and Doaa (2017) reported an optimum incubation time of 20 days for atrazine degradation.It is well established that to ensure an optimal reproducible physiological state of bacterial cells during growth incubation time needs to be strictly respected (Chikere and Ugueri 2014).

Effect of heavy metals on atrazine degradation
Assessing the effect of heavy metals on the growth of atrazinedegrading B. safensis in MSM showed that the metals affect the growth of this bacterium differently.At 2 ppm concentration, Fe, Zn and Cu were found to pro mote the growth of this bacterium as indicated by their in creased OD 600 relative to control.Whereas Cd, Ar, Cr, Ni, Hg and Pb were found to inhibit the growth of this isolate relative to control.The order of inhibition was Pb>Hg>Ni>Cr>Ar>Cd following incubation at 37 °C for 72 h (Figure 7).Statistical analysis also showed signifi cant differences (p < 0.05) between control, Fe, Hg, Zn, Cd, Cr, Cu, Pb, Ar and Ni.
The effect of various heavy metals on the growth of this isolate (Figure 7) showed that the growth and max imum tolerance level of the isolate is between 2-4 ppm.At concentrations higher than 4 ppm, the growth and metabolic activities of the isolate were inhibited.This result is in agreement with that of Alvarez et al. (2017) and Zhang et al. (2019).The need to assess the effect of various heavy metals on the growth of atrazinedegrading isolate arises from the fact that these metals often oc cur as cocontaminants with other herbicides in the pol luted environment.Heavy metals like Cd, Ni, Cr, Pb, As, Hg are discharged by industries, which in trace amounts can cause toxicity to flora and fauna (Meenambigai et al. 2016).While other heavy metals are essential elements to all known life forms as they have a number of functions in biological systems.The toxicity depends on factors like the absorbed dose, the route of exposure, and the period of exposure.A number of homeostatic mechanisms are possessed by the cells to deal with the level of heavy met als and decrease the toxicity produced by excessive level   Indonesian Journal of Biotechnology 28(3), 2023, 127-136 (Alvarez et al. 2017).

Atrazine biodegradation study
The gas chromatography mass spectroscopy (GCMS) analysis of the atrazine standard is presented in Figure 8. Atrazine, with a molecular weight of 215.0 was identified at a retention time (Rt) of 18.029.

Conclusions
A bacterium with potential to tolerate up to 1500 mg•L 1 of atrazine herbicide (as sole carbon source) was isolated from agricultural soil in Kano, Northwestern Nigeria and identified as Bacillus safensis strain BUK_BCH_BTE6 based on 16S rRNA sequencing and molecular phyloge netic analysis.The isolate tolerated 2 ppm heavy metals and degraded 88.85% of 400 mg•L 1 atrazine to cyanuric acid at an optimum pH of 7.5, temperature 35 °C, and in oculum size of 600 µL after 120 h.With the characteristics of this isolate, this finding has provided basic information about indigenous isolates that could be exploited for biore mediation of atrazinepolluted sites.This isolate is a high atrazine tolerant and efficient atrazine degrader that could be employed for bioremediation of atrazinepolluted sites.

FIGURE 1
FIGURE 1 Cladogram indicating genetic relationship between B. safensis strain BUK_BCH_BTE6 and 16S rRNA sequences of related microorganisms.

FIGURE 4
FIGURE 4Effect of atrazine (substrate) concentration on growth of strain BUK_BCH_BTE6 in MSM incubated at 37 °C.Each value represents mean ± SD of three replicates.

FIGURE 5
FIGURE 5Effect of inoculum sizes on growth of strain BUK_BCH_BTE6 in MSM containing 100 mg•L -1 atrazine, incubated at 37 °C for 48 h.Each value represents mean ± SD of three replicates.

FIGURE 6 FIGURE 7
FIGURE 6Effect of incubation time on growth of strain BUK_BCH_BTE6 in MSM containing 100 mg•L -1 atrazine, incubated at 37 °C.Each value represents mean ± SD of three replicates.

FIGURE 8
FIGURE 8 GC-MS mass spectrum, peak with MW of 215.0, Rt at 18.029 min indicates atrazine standard.

FIGURE 9
FIGURE 9 (a) GC-MS mass spectrum of 400 mg•L -1 residual atrazine, peak with MW of 147.1 indicates desethyldeisopropylatrazine.The culture (600 µL) of strain BUK_BCH_BTE6 was inoculated and incubated at 35 °C for 120 h.(b) GC-MS mass spectrum of 400 mg•L -1 residual atrazine, peak with MW of 173.1 indicates deisopropylatrazine.The culture (600 µL) of strain BUK_BCH_BTE6 was inoculated and incubated at 35 °C for 120 h.(c) GC-MS mass spectrum of 400 mg•L -1 residual atrazine, peak with MW of 155.2 indicates N-ethylammelide.The culture (600 µL) of strain BUK_BCH_BTE6 was inoculated and incubated for 120 h at 35 °C.(d) GC-MS mass spectrum of 400 mg•L -1 residual atrazine, peak with MW 129.0 indicates cyanuric acid.The culture (600 µL) of strain BUK_BCH_BTE6 was inoculated and incubated for 120 h at 35 °C.
% Degradation = (ACS-ACT)/(ACS) x 100 ACS= Area under the curve of the standard ACT= Area under the curve of the test sample