Augmentation of Atrazine biodegradation by two Bacilli immobilized on α-Fe2O3 magnetic nanoparticles

In this study, a novel immobilizing carrier with α-Fe2O3 magnetic nanoparticles was developed and used for immobilization of atrazine-degrading bacterial isolates of Bacillus spp. Since the free cells of microorganisms generally not succeed to degrade pollutants; thus, extra treatments are alluring to make strides biodegradation. Scanning electron microscope (SEM) images appeared that after immobilization the bacterial cells were totally retained and entirely distributed on the surface of α-Fe2O3 magnetic nanoparticles. The performance of α-Fe2O3 immobilized cells in atrazine (ATZ) degradation was compared with the free cells, which was about 90.56% in 20 days. Experimental results exhibited that ATZ could be degraded at a broad range of physicochemical parameters viz. pH (4.0 to 9.0), temperature (20 to 45 °C), ATZ concentration (50 to 300 mg L−1) and agitation speed (50 to 300 rpm), which underlines that α-Fe2O3 immobilized cells could tolerate a higher range of ATZ concentration as compared to free cells. This research demonstrated that α-Fe2O3 could be applied as a potential carrier in cell immobilization and biodegradation of ATZ herbicide with greater efficiency.

best of our knowledge, the α-Fe 2 O 3 immobilizing material with the modifications of structural and morphological characteristics of α-Fe 2 O 3 magnetic nanoparticles after bacterial adhesion has not been reported in the literature. Therefore, the understanding of the morphological and structural characterization of α-Fe 2 O 3 magnetic nanoparticles and its bacterial cell immobilizing properties is of great significance. Hence, in the present study, the two ATZ degrading bacterial isolates of Bacillus spp. i.e. B. badius and B. ensimensis were immobilized and degradation of ATZ by free and α-Fe 2 O 3 immobilized cells in varied environmental conditions were examined, which could pave way to improve biodegradation efficiencies.

Isolation and Characterization of ATZ Degrading Bacterial Strain. Atrazine degrading bacterial
strain ABP6 and ABP8 were isolated from the enrichment culture. Both isolates were gram-positive, aerobic, spore-forming, and rod-shaped. The bacterial isolates ABP6 and ABP8 were tested for 24 different substrates (24 carbohydrates as a carbon source and for two enzymes oxidase and catalase) to study their metabolizing abilities using KB009 part A and B HiCarbohydrateTM Kit. All these biochemical tests were based on the principle of substrate utilization and pH change. ABP6 was positive for oxidase, catalase, and 14 carbohydrates, while ABP8 was positive for oxidase, catalase, and 24 carbohydrates. Table 1 represents the biochemical characteristics of both bacterial isolates.

Determination of Atrazine MIC for Bacterial Growth. The minimum inhibitory concentration(MIC)
was determined on the basis of bacterial growth on mineral salt agar plates and minimal broth supplemented with atrazine at different concentrations (50,100,150,200,250, 300 and 350 mg L −1 ) for both bacterial isolates. Atrazine concentration of 300 mg L −1 was found to exhibit MIC, after that no growth of bacterial cells was observed. Both bacterial isolates exhibited fast growth up to 200 mg L −1 atrazine concentration and also used atrazine as the sole source of carbon and nitrogen. Therefore, this concentration was used in forthcoming experiments on atrazine degradation.
16S rRNA Gene Sequencing of bacterial strains. 16S rRNA partial nucleotide sequence analysis of bacterial isolate was carried out by Chromous Biotech Pvt. Ltd., Bangalore, India. Alignment of the 16S rRNA partial gene sequence of bacterial isolates was performed with sequences present in the GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/blast/). A MEGA version 7.0 software package and neighbor-joining (NJ) method were used for phylogenetic analysis. Submission of the sequence was completed in the GenBank database with accession number MG680921 and MG680922. The bacterial isolates were designated to be B. badius ABP6 and B. encimensis ABP8. The constructed phylogenetic trees for both bacterial isolates are represented in Fig. 1a,b.
Analysis of prepared α-Fe 2 O 3 magnetic nanoparticles. The α-Fe 2 O 3 magnetic nanoparticles were synthesized by Chemical precipitation method 25,26 (Fig. 2) and prepared magnetic (Hematite) nanoparticles were characterized on the basis of XRD, FTIR, UV-Visible Spectra analysis as well as with SEM images.
XRD Analysis. The XRD pattern of α-Fe 2 O 3 magnetic nanoparticles is presented in Fig. 3a. In the present study, the crystalline size of prepared nanoparticles was found to be approximately 7 nm using scherrer's formula: Here λ is the x-ray wavelength (Cu-Kα, λ = 1.54 Ǻ), k is the machine constant (0.9), β is the full width at half maximum (FWHM in radian) of the peak and θ is the peak angle.

B. encimensis ABP8
Lactose The most intense XRD peak was obtained at 2θ = 32.89°. All the peaks corresponding to the planes (0 1 2), (1 0 4), (1 1 0), (1 1 3) and other planes are found to be matching with the reported information in JCPDS card no.-36-0664 confirming the alpha phase of synthesized iron oxide nanoparticles. Few low intense peaks present in the XRD curve shows the presence of other iron oxide phases in the samples (Fig. 3a). This XRD pattern for α-Fe 2 O 3 magnetic nanoparticles was also compared with other studies 25,26 and confirmed as α-Fe 2 O 3 magnetic nanoparticles peaks in XRD spectrum.
UV-Visible analysis. The absorption spectra in the UV-Vis range of hematite (α-Fe 2 O 3 ) synthesized through chemical precipitation method shows that the absorption curve exhibit an intense absorption in the range of 400-800 nm wavelength. In Fig. 3b, the spectrum of UV-Vis spectroscopy for prepared nanoparticles characterization is shown. This result is consistent with observations of other studies and confirmed as hematite (α-Fe 2 O 3 ) nanoparticles absorption spectra in UV-Visible range 25-29 . Fourier Transform Infra-Red (FT-IR) spectroscopy. The formation of α-Fe 2 O 3 magnetic nanoparticles (hematite) was further confirmed by FT-IR spectroscopy. The FT-IR spectrum of hematite is shown in Fig. 3c. The strong absorption peaks at 535 and 525 cm −1 can be recognized to the Fe-O band vibrations for α-Fe 2 O 3 nanoparticles 25,26,30,31 . The very broad absorption band centered at 3743 cm −1 and reaching a peak at 1646 cm −1 is assigned to the stretching and bending vibrations of the hydroxyl groups and/or water molecules 32 , respectively. In addition, there is a peak at 1515 cm −1 that is assigned to the deformation of CH 3 .  Fig. 4a, where the shape of initial α-Fe 2 O 3 magnetic nanoparticles was spherical, irregular columnar, which provides a large area for free bacteria to get in touch with the α-Fe 2 O 3 surface. Figure 4b represents the scanning image of free bacterial cells (B. badius and B. encimensis consortium). Figure 4c represents α-Fe 2 O 3 nanoparticles and bacterial cells after immobilization, where all the bacterial cells were fully immobilized with nanoparticles and formed a bacterial-α-Fe 2 O 3 immobilized composite. α-Fe 2 O 3 nanoparticles served as a support material for immobilization of bacteria, because of its size, shape, surface functional groups and natural porous structure, which provides large surface area and high loading volume for bacterial cell adsorption.
Effects of reaction conditions on ATZ degradation. The reaction conditions that might influence the ATZ-degrading activity of the microbial composite were explored for four experiments: pH, temperature, atrazine concentration and agitation speed with different treatments (Treatment-1: α-Fe 2 O 3 without bacterial cells, Treatment-2: free bacterial cells, Treatment-3: α-Fe 2 O 3 immobilized bacterial cells). Figure 5, shows the optimal pH, temperature, atrazine concentration and agitation speed for degradation in all treatments were 7.0, 30 °C, 200 mg L −1 and 150 rpm respectively. Figure 5a revealed that the capability of α-Fe 2 O 3 without bacterial cells and free bacterial cells on the degradation of atrazine decreased significantly when the pH shifted toward the alkaline or acidic conditions. In contrast, the degradation of atrazine by α-Fe 2 O 3 immobilized cells changed slowly and maintained at a high level in the whole pH range of 4-9 (>50%) after 10 days of incubation period. Figure 5b depicted the effect of temperature variations on atrazine degradation. At 30 °C temperature, both free and immobilized bacterial cells displayed increasing atrazine degradation. With the further increase in temperature (>30 °C), the atrazine-degradation declined. Nevertheless, Fig. 5b shows that for all temperature ranges the α-Fe 2 O 3 immobilized bacterial cells represents a better ability to tolerate at different temperature as compared with treatment 1 and 2. When the temperature reached 45 °C the degradation of atrazine using α-Fe 2 O 3 immobilized cells (treatment 3) decreased slightly to 35.29 ± 1.9%, but it dropped significantly to 21.62 ± 0.98% and 26.73 ± 1.08% with treatment 1 and 2. The change of percent degradation rate with the increment of ATZ initial concentrations is presented in Fig. 5c, which clearly depicted that all treatments possessed greater degradation ability at low concentration of ATZ i.e. 50 mg L −1 and after that decreased as the concentration increased up to 300 mg L −1 . The effect of different agitation speeds (50 to 300 rpm) on atrazine degradation is represented in The viability of bacterial cells before and after immobilization. The stability of immobilized as well as free bacterial cells was checked with viability count (OD600 and CFU mL −1 ), and the required data is represented in Table 2, which showed growth and viability of bacterial cells at different incubation period before and after immobilization, to quantify the immobilized cells on magnetic nanoparticles, as well as free bacterial cells.
Results exhibited that immobilized bacterial cells have the better capability as compare to free cells and their growth has been increased as the incubation period increased. At earlier incubation period (0 days) the growth and viability for both the bacterial cells were found to be same, but as the incubation period increased from 0 to 15 days, the growth and viability were also increased and after 15 days of incubation, the growth of both free and immobilized bacterial cells declined gradually. Fig. 6 and Table 3  Whilst, with other treatments (1-5) the ATZ degradation rate was lowest as compared to immobilized cells in different incubation periods.

Discussion
Generally, herbicides applied in culturable lands are included in sorption, desorption, transport, volatilization, and alteration processes 33,34 . Based on the understanding of these physicochemical or biochemical behaviors, superior procedures could be got for compelling utilization of dynamic compounds as well as remediation of herbicide-contaminated locales. Biodegradation includes the utilization of living microorganisms or their enzymes to detoxify pollutants, which has been by and large considered as a successful and cost-effective method for the removal of contaminants 35 . It was emphasized that different microbes had the capacity of efficiently degrading ATZ including Chelatobacter heintzii 36 , Enterobacter (E. cloacae), Bacillus (B. cereus and B. anthracis), Pseudomonas (P. aeruginosa, P. balearica, P. indica and P. otitidis), Ochrobactrum (O. intermedium) and Providencia (P. vermicola) 37 and P. fluorescence + P. putida 38 . In Present study, two exceedingly successful strain of Bacillus spp. (Bacillus badius ABP6 and Bacillus encimensis ABP8) used for ATZ degradation were able to utilized ATZ as carbon and nitrogen source. While comparing to the treatments with higher introductory concentrations, the degradation at lower concentrations was more noteworthy and proficient; this empowered the strain to operate in circumstances with low residues. Prior studies have demonstrated that generally low levels of herbicide residues would influence and cause a chain of impacts on the biological system 39,40 .
The obtained results indicated that the bacterial cells immobilized on α-Fe 2 O 3 magnetic nanoparticles have greater ability to tolerate under high acidic and alkaline conditions as compared to free bacterial cells. The bacterial cell diversity, their enzyme activity as well as nutrient solubility get affected by the pH of culture medium 41,42 . The immobilization of cells has a better capacity to stabilize inner pH value of microbes and which might have led to best external conditions and favorable environment for microorganisms as compared to the free bacterial In the optimization of temperature, results showed that both free and immobilized bacterial cells have reduced capability to degrade atrazine at a higher temperature. This is due to the fact that higher temperature has a negative effect on the activity of the bacteria and   would cause a decline in oxygen solubility, hence, hindered its biodegradation capabilities 16 . After checking the stability of free and immobilized bacterial cells, it was clear that immobilized cells have better growth, viability and higher activity as compared to free cells, which ultimately leads to increase the biodegradability of contaminants by immobilized cells. The surface charge, species/strain, growth phase, growth rate, surface structure and extracellular polymeric substances of bacterial cells could also affect the immobilization process for physicochemical interactions with nanoparticles. In the present experiment, the magnetic nanoparticles were used only for the adsorption of bacterial cells, they do not affect the growth and viability of bacterial cells during immobilization; but they can reduce the bacterial growth when the concentration of nanoparticles reached above the permissible limit 24,[42][43][44][45][46][47] . In feasible application, the degradation implementation was not stable and compelling as tried in research facility since the coordinate utilizes of free cells was affected by numerous variables. Utilizing diverse immobilized microbes may give a conceivable arrangement, which has been demonstrated tediously as a compelling approach for contaminants remediation 13,14 . It was reported that the movement of the immobilized composite is immensely influenced by the properties of support material 16 . Iron oxide nanoparticles (IONs), with their unique magnetic properties, high surface/volume ratio, surface functional groups, size, shape and superior biocompatibility, showed great promise for applications in bioprocesses [47][48][49][50][51][52][53][54] . Therefore, choosing a steady and economical support with great biocompatibility is the key to the immobilized composite and overall results revealed an advantage of α-Fe 2 O 3 immobilized cells on ATZ degradation over free cells. This attribute further signifies the efficient performance of bacterial isolates and α-Fe 2 O 3 magnetic nanoparticles immobilizing support. SEM images represented that bacterial cells completely adhered on the surface of α-Fe 2 O 3 magnetic nanoparticles, and their degrading movement held after immobilization. This statement has been explained in light of the   study made by others 42,47 . Moreover, it is accepted that the immobilization technique should be a generally basic process that does not require a profoundly unadulterated enzyme preparation or a costly support that may not be commercially accessible 15 . The present study demonstrated that the use of combined α-Fe 2 O 3 magnetic nanoparticles in conjunction with indigenous soil microbes could substantially enhance the degradation efficiency of ATZ compared with the individual treatments. In addition, the experimental data suggested that the immobilized α-Fe 2 O 3 magnetic nanoparticles could significantly improve soil microbial populations and enzyme activities, which in turn resulted in improved degradation efficiency of Bacillus spp.

Conclusion
Utilization of dynamic microorganisms is an efficient and economical strategy, while microbial immobilization gives a breakthrough in the restricted application of microbes in in-situ. In the present study, α-Fe 2 O 3 was utilized in herbicide biodegradation as fabulous stacking supports for bacterial immobilization. Compared with freely suspended cells and immobilized cells, the efficiency of ATZ degradation by α-Fe 2 O 3 immobilized cells was improved significantly. On the basis of results obtained, it can be concluded that α-Fe 2 O 3 has much potential application as a carrier in cell immobilization. However, it also requires fine tuning at pilot scale experiments.
ATZ was dissolved in methanol at a stock concentration of 100 mg L −1 and stored at 4 °C prior to use. All reagents used in the synthesis procedure were an analytical grade of Himedia Laboratories, India. Iron (III) chloride hexahydrate (FeCl 3 .6H 2 O) was procured from Sigma-Aldrich, USA, while ammonium hydroxide (NH 4 OH) and ethanol (C 2 H 6 O) from Merck, India.

Soil sample collection and Isolation of ATZ -degrading bacteria. Soil samples at depth of 15 cm
were collected from Norman E. Borlaug, Crop Research Centre, Pantnagar, India, situated at an altitude of 243.84 above mean sea level, 29°N latitude and 79.3°E longitude, after 10 days of herbicide spray in the Maize fields. For the isolation of ATZ-degrading bacteria from contaminated soil, 5 g of Soil samples in Erlenmeyer flasks (250 ml) was added with the minimal broth (50 mL) having 50 mg L −1 ATZ concentration. The enrichment was done by incubating at 30 °C on a rotary incubator (150 rpm). After 7 days, the broth culture (5 mL) from flask was reinoculated into fresh minimal media (50 mL) concentrated with 100 mg L −1 ATZ under the above-mentioned situations. The same procedure was repeated twice up to 200 mg L −1 of ATZ concentration. After that, 0.2 mL of final culture broth was pour plated on agar plates for isolation of a single colony. Each colony, considered as a diverse species, was more than once streaked on agar plates. To obtain a pure culture of best ATZ degradation, 10 times streaking followed screening was done. After the consecutive enrichment culture, bacterial isolates showing prolific growth in ATZ supplemented mineral salt agar medium and designated as ABP6 and ABP8.

Determination of ATZ Minimum Inhibitory Concentration (MIC).
Minimum inhibitory concentration for bacterial isolates ABP6 and ABP8 was determined to check the toxicity and tolerable limit of atrazine on the basis of growth. Bacterial isolates were streaked on mineral salt agar plates supplemented with atrazine as the sole source of carbon and energy at the concentrations of 50, 100, 150, 200, 300, 350 and 400 mg L −1 . After that these agar plates were incubated at 30 °C for 72 h, for the appearance of bacterial colonies.

Identification of ATZ Degrading Bacterial Isolates ABP6 and ABP8. Morphological and
Biochemical tests were done for the identification of most active atrazine degrading bacterial isolates ABP6 and ABP8. These strains were tested for 24 different substrates (24 carbon sources and 2 enzyme activities viz. Catalase and Oxidase) and KB009 part A and B HiCarbohydrateTM Kit were used to check their metabolizing abilities (Table 1). These tests were based on the principle of pH change and substrate utilization.
Synthesis of α-Fe 2 O 3 Nanoparticles. Hematite nanoparticles (α-Fe 2 O 3 ) were synthesized by chemical precipitation method 25,26 . In a typical synthesis procedure, 0.1 M solution was prepared by dissolving iron (III) chloride hexahydrate (FeCl 3 .6H 2 O) in 100 mL of distilled water under vigorous stirring for 45 min at 80 °C. 50 mL aqueous solution of NH 4 OH (2M) was prepared in distilled water. NH 4 OH solution was added dropwise to the solution until the pH reached a value of 11. The solution was stirred at 80 °C for 4 hours to complete the reaction and then centrifuged, washed with distilled water and ethanol. The precipitate was dried in a hot air oven at 80 °C for 6 hours and sintered at 700 °C for 2 hours to get α-Fe 2 O 3 magnetic nanoparticles (Fig. 2).

Growth and Viability of free and immobilized bacterial cells. The viability of bacterial cells with
optimized conditions, before and after immobilization process was checked at different incubation periods (0 to 25 days) by optical density at 600 nm (OD600) using UV-Vis Spectrophotometer (Virion Bio 50) and live cell assessment through colony forming unit (CFU) counting on plates 55 by following formula: No of colonies dilution factor Volumes of the sample taken (mL) and α-Fe 2 O 3 immobilized bacterial cells. Samples from each treatment were withdrawn at intervals of 0, 5, 10, 15 and 20 days and used for the analysis of residual ATZ concentration. Extraction was done with dichloromethane, and the organic layer was dehydrated, dried, and redissolved in methanol. After filtration, the samples were subjected to high-performance liquid chromatography (HPLC) to determine the degradation of ATZ.

Biodegradation of ATZ by free and immobilized bacterial cells.
HPLC analysis of atrazine. The residual ATZ was analyzed on a Dionex ultimate 3000, HPLC equipped with a C18 reversed phase column (250 × 4.6 mm id, 5 μm), and the analytical method is listed in Table 4. Residual ATZ concentration was quantified using a standard curve plotted between detector response (absorbance) and known concentration.
Data analysis. All of the experiments were carried out with three sovereign experiments, and the results were the means of three replicates. The percent degradation of ATZ was analyzed according to the following formula: = − × Percent (%) ATZ degradation Ac As Ac 100 where, As (mg/L) is the residual concentration of sample, and Ac (mg/L) represents the concentration of control sample.
Statistical analysis. The experimental data were processed for calculating the standard error of the means and multi-factorial analysis of variance as available in the SPSS statistical package (Stat Graphics Plus V. 11) and expressed at 0.05 probability level. The significance (p < 0.05) of differences was treated statistically by one and two-way analysis of variance (ANOVA) and evaluated by post hoc comparison of means using the lowest significant differences (LSD).  Table 4. Reaction conditions for HPLC analysis of remaining atrazine residue after biodegradation by bacterial isolates.