Materials Science and Engineering C 49 (2015) 183–189
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Microbial-assisted synthesis and evaluation the cytotoxic effect of tellurium nanorods Hamid Forootanfar a, Sahar Amirpour-Rostami b, Mandana Jafari b, Amir Forootanfar c, Zahra Yousefizadeh d, Mojtaba Shakibaie b,⁎ a
Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d The Student Research Committee, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran b c
a r t i c l e
i n f o
Article history: Received 18 August 2014 Received in revised form 21 December 2014 Accepted 23 December 2014 Available online 24 December 2014 Keywords: Tellurium nanorods Biosynthesis Pseudomonas pseudoalcaligenes Cytotoxicity Potassium tellurite
a b s t r a c t The present study was designed to isolate bacterial strain capable of tellurium nanorods' (Te NRs) production followed by purification and evaluation of the cytotoxic effect of Te NRs. Among 25 environmental samples collected for screening of Te NR-producer bacterial strains one bacterial colony (isolated from hot spring and identified as Pseudomonas pseudoalcaligenes strain Te) was selected and applied for biosynthesis of Te NRs. Thereafter, an organic–aqueous partitioning system was applied for the purification of the biogenic Te NRs and the purified Te NRs were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX), X-ray diffraction spectroscopy (XRD), UV–visible spectroscopy, and Fourier transform infrared spectroscopy (FTIR) techniques. The cytotoxic effect of biologically synthesized Te NRs and potassium tellurite on four cell lines of MCF-7, HT1080, HepG2 and A549 was then determined using the MTT assay method. The obtained results revealed lower toxicity for the rod-shaped biogenic tellurium nanostructures (~22 nm diameter by 185 nm length) compared to K2TeO3. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Conspicuous physicochemical and biological characteristics of materials at nano-scale compared to those of bulk materials introduced nanostructures as an attractive trend during last decades [1–4]. Metal nanoparticles received more interest due to remarkable photoelectrochemical and catalytic properties [5]. Excellent thermal, optical and electrical properties of tellurium (Te), one of the chalcogen elements, made it as an important metalloid in different industries such as steel and glass making as well as solar panels, sensor production and rechargeable battery manufacturing [6–8]. The fluorescent feature of Tecontaining compounds such as CdTe quantum dots has raised the possibility that such substances may serve as efficient biological markers [6, 9]. Unlike selenium, Te does not seem to be an essential trace element for biological systems [6,7]. Behind the traditional application of Te compounds for the treatment of bacterial infectious diseases such as tuberculosis, leprosy, cystitis and severe eye infections [5] other biological ⁎ Corresponding author. E-mail address: [email protected] (M. Shakibaie).
http://dx.doi.org/10.1016/j.msec.2014.12.078 0928-4931/© 2014 Elsevier B.V. All rights reserved.
activities like the inhibition of cytokine production by T cells, immunomodulatory activities and antisickling have been recently reported for Te-containing compounds [10]. 2− Tellurite (TeO2− 3 ) and tellurate (TeO4 ), the most abundant form of Te in nature [11], could be changed into the insoluble and less toxic elemental Te (Te0) via physicochemical techniques like laser ablation, electrodeposition, pyrolysis, and chemical vapor deposition [12,13]. However, time and cost consuming properties of physicochemical methods applied for the production of elemental nano-tellurium [14] encouraged investigators to screen for biological resources as clean, nontoxic and eco-friendly methods for the synthesis of elemental nano-tellurium. For example, in the study of Zare et al. [5] it was shown that Bacillus sp. BZ, isolated from environmental samples, was able to produce nanorod elemental Te NPs after cultivation in the presence of potassium tellurite. A moderately halophilic bacterium, Salinicoccus sp. QW6, capable of reducing tellurite anion to elemental tellurium was isolated from salty environment samples in the study of Amoozegar et al. [15]. The present study focused on the screening of bacterial strains able to produce Te NRs followed by identification of selected strain. The
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produced nanorods were then purified and characterized. Subsequently, the cytotoxic effects of pure Te NRs were investigated on different cell lines by using an in vitro cytotoxicity assay.
2. Materials and methods 2.1. Chemicals Potassium tellurite (K2TeO3·3H2O) and n-octanol were provided by Merck Chemicals (Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-2H-tetrazolium bromide (MTT) and sodium borohydride (NaBH4) were supplied by Sigma–Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle medium (DMEM) and antibiotics were supplied by Gibco (Life Sciences Inc., USA). All other chemicals and solvents were of analytical grade.
2.4. Measurement of Te4+ ion concentration A modified version of the spectrophotometric method described by Molina et al. [18] was applied for the determination of residual Te4 + ion concentration in bacterial culture media. Briefly, different concentrations of Te4 + ion (1.56–200 μg/mL) were prepared by dissolving the K2TeO3·3H2O in nutrient broth medium and 1 mL of each solution was reduced using freshly prepared solution of sodium borohydride (NaBH4, 4.5 mM) followed by recording the optical densities of all prepared colloids at 500 nm using a UV–visible Double Beam PC Scanning spectrophotometer (UV-1800, Shimadzu CO, USA). The calibration curve was set up by plotting the measured absorbance against the Te4+ ion concentration. In all absorption measurements, 1 mL of nutrient broth medium was subjected to the same steps and was employed as the blank. These procedures were repeated three times, and the mean of the absorbances was used to draw a suitable standard curve. 2.5. Cultivation of the isolate in submerged culture and Te ion reduction
2.2. Screening for Te NRs producing bacteria Water samples were collected from different springs in Kerman (30°15 N, 56°58 E), Iran during the winter of 2012 in order to isolate Te NR-producing bacterial strains. Each sample (5 mL) was then diluted using sterile solution of 0.9% NaCl (20 mL) and 500 μL of each prepared sample was then streaked on nutrient agar (NA) plates containing 100 μg/mL of Te4+ ions (equivalent to 241.4 mg of K2TeO3·3H2O per liter). The plates were then incubated at 30 °C until black colonies (reduction of Te4 + to Te NRs) were observed. Positive colonies were subcultured in NA medium with and without Te4 + ions to confirm that the black color was not an organic bacterial pigment (for example, melanin). The minimal inhibitory concentration (MIC) of Te4+ on selected isolate was achieved using agar dilution method [5]. The selected bacterial colony was then maintained at 4 °C on NA plates supplemented with Te4+ ions. The isolate was also preserved at −80 °C in glycerolsupplemented nutrient broth medium.
2.3. Identification of the isolate Morphological and biochemical characteristics of the selected isolate were studied according to Bergey's Manual of Determinative Bacteriology [16]. Furthermore, 16S rDNA gene sequence analysis was performed as follow: After the cultivation of the isolate in nutrient broth medium for 16 h, 100 μL of bacterial culture was harvested and the produced biomass was separated using centrifugation (5000 rpm, 5 min) followed by washing the obtained bacterial cells using sterile deionized water for three times. The harvested cells were subsequently resuspended in sterile water (50 μL) and boiled at 94 °C for 10 min. After removing the cell debris by centrifugation (14,000 rpm, 10 min), the obtained supernatant was used as a DNA template for PCR amplification. The primer pair of 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) as the forward primer and 1525R (5 -AAGGAGGTGATCCAGCC-3 ) as the reverse primer [17] was used to amplify the 16S rDNA gene. After the preparation of the PCR mixture (Tris, 50 mM pH 8.3; dNTP mixture 250 μM of each; forward primer 400 nM; reverse primer 400 nM; MgCl2 2.5 mM; and Taq DNA polymerase 0.5 U) in 200 μL microtubes, the template DNA (40–60 ng) was added and amplification was performed in a Primus 96 advanced thermal cycler (PEQLAB, Erlangen, Germany) programmed as follows: (a) initial denaturation at 94 °C for 3 min; (b) 30 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 45 s and synthesis at 72 °C for 90 s; and (c) the final extension at 72 °C for 90 s. The amplified DNA fragment was purified from agarose gel (1%) and sent for automated sequencing using the mentioned primers (Bioneer Corporation, South Korea). The basic local alignment search tool (BLAST) was then applied for comparing the sequence of amplified DNA with the sequences in GenBank.
In order to investigate on the time course of Te reduction the selected bacterial strain was grown in NB medium and 1 mL of fresh inoculum (OD600 0.1) was transferred to 500-mL Erlenmeyer flask containing 100 mL of NB medium (pH 7) including sub-MIC concentration of Te4+ ions (100 μg/mL and 200 μg/mL). The flasks were then aerobically cultivated at 30 °C and 150 rpm for 96 h. Negative controls were designed by introducing of Te ions to un-inoculated culture media. In a similar trial the selected isolate was cultured in the absence of Te ions. Samples were taken periodically and subjected for determining of Te4+ concentration and OD600. Before Te analysis, the produced biomass and Te NRs were separated from the culture media by centrifugation (11,000 g for 30 min). For the spectrophotometric determination of the remaining Te4+ ions, 1 mL of the supernatant from the inoculated media containing Te4+ ions, and 1 mL of the supernatant from the inoculated media without Te4+ ions were separately used as the test and blank controls, respectively. To examine the ability of the cell-free supernatant for Te ion reduction, 100 mL of sterile NB was inoculated by fresh inoculum of bacterial isolate (OD600 0.1) and incubated for 24 h followed by removing the produced cells by centrifugation at (4000 g, 10 min). Subsequently, the obtained supernatant was aseptically passed through 0.22 μm filter and collected in a sterile 500-mL Erlenmeyer flask. Thereafter, the Te4+ ions were added to the obtained supernatant to reach final concentration of 100 μg/mL and 200 μg/mL. The flasks were then incubated (30 °C, 150 rpm), and checked periodically for Te NR formation. All mentioned experiments were done in triplicate. 2.6. Production, purification and characterization of Te NRs For the preparation of Te NRs the sterile nutrient broth (NB) medium containing Te ions (100 μg/mL) was inoculated with 1 mL of the fresh inoculums (OD600, 0.1) of the selected bacterial strain and incubated at 30 °C and 150 rpm for 80 h followed by harvesting the bacterial cells using centrifugation (4000 ×g for 10 min). After washing the obtained pellets with sterile solution of NaCl (0.9%) for three times, the bacterial cells were disrupted by grinding the frozen cells in liquid nitrogen using a mortar and pestle. The resulting slurry was then ultrasonicated at 100 W for 5 min and washed three times by sequential centrifugation (14,000 ×g, 5 min) with a 1.5 M Tris–HCl buffer (pH 8.3) containing SDS (1%) and deionized water, respectively. Subsequently, Te NRs were extracted and purified by organic–aqueous partitioning system (n-octanol–water), as previously described [1,5]. Micrographs of the prepared biogenic Te NRs were obtained using a TEM apparatus (Zeiss 902A) operated at accelerating voltage of 80 kV. In order to investigate the surface and elemental composition of biologically synthesized Te NR samples were analyzed using an SEM (Philips XL30, 16 kV) apparatus equipped with an EDX (energy dispersive X-ray) microanalyzer. The particle size distribution pattern of Te NRs was achieved using a
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Zetasizer MS2000 (Malvern Instruments, UK). The crystalline structure of the as synthesized Te NRs was checked by the X-ray diffractometer (Philips PW1710) with CuKα radiation (λ = 1.5405 A°) at room temperature in the 2θ range of 5°–90° with a scanning rate of 0.02°/S. A Shimadzu UV–visible Double Beam PC Scanning spectrophotometer (UV-1800, Shimadzu CO, USA) was applied to record the UV–visible spectrum of the purified Te NRs. The surface chemistry of oven-dried Te NRs (50 °C, 48 h) was also investigated by an infrared spectrophotometer (Shimadzu IR-470, Japan) at a resolution of 4 cm−1 in KBr disks. 2.7. Cell culture and cytotoxicity assay MCF-7, HT1080, HepG2 and A549 cell lines were obtained from the National Cell Bank of Iran, Pasteur Institute of Iran (Tehran, Iran). The cells were maintained in DMEM medium supplemented with FBS (10%, v/v) and antibiotics [penicillin (100 units/mL) and streptomycin (100 μg/mL)] at 37 °C in a CO2 incubator (5% CO2 and 95% relative humidity). In order to evaluate the cytotoxic effect of the biologically synthesized Te NRs and K2TeO3, the cell lines were harvested in the exponential phase of growth, seeded separately into 96-well tissue culture plates (20,000 per well) and allowed to adhere for 24 h. Thereafter, Te NRs and K2TeO3 were added to the desired wells to reach different concentrations. After 24 h of incubation, 20 μL of DMEM medium containing MTT (5 mg/mL) was added to each well and incubated for 4 h. Consequently, the medium was replaced with 100 μl of DMSO, and optical densities were determined at 570 nm. MTT assay was performed in three replicates for each experiment. 2.8. Statistical analysis Each value was expressed as mean ± SD. SPSS software 15 for Windows (SPSS Inc., Chicago) was used for statistical analysis. Differences between the groups were determined using one-way
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analysis of variance (ANOVA) and p-values less than 0.05 were considered significant. 3. Results and discussion 3.1. Isolation and identification of the microorganism Out of 25 samples collected from various springs in Kerman, only one sample collected from Sirch hot springs contained a bacterium with the ability to convert Te4 + ions into Te0. As presented in Fig. 1a and d the cultivation of the bacterial isolate in the absence of Te4 + ions shows no color change indicating the absence of any probable pigment production. However, in culture media containing K2TeO3 (Fig. 1b and c) the reduction of Te4+ ions to elemental Te was evident from the color change of cultivation medium to black due to generation of Te0 during the exponential growth phase. In morphological studies, the isolated strain showed up as a motile, rod-shaped, and Gram-negative bacterium with yellowish white colonies on the nutrient agar medium. The results of biochemical characteristics are summarized in Table 1. Both morphological and biochemical properties of the selected isolate, candidate it as Pseudomonas strain. The alignment of the amplified 16S rDNA gene sequence of the selected isolate against the present sequences of GenBank using BLAST tool represented 99% identity of the obtained gene to Pseudomonas pseudoalcaligenes. The 1403 bp sequence was then submitted to GenBank under accession number of KF055346. The ability of microbial strains for the reduction of chalcogen oxyanions to elemental form introduced this mechanism as a safe, cost-effective, clean, non-toxic, and environmental friendly alternative for time and cost consuming physicochemical techniques applied for the production of metalloids such as Te0 and Se0 nanoparticles [19]. In the study of Zare et al. [5] a tellurium-transforming bacterial strain Bacillus sp. BZ was isolated from the Caspian Sea in northern of Iran. Out of 49 strains of moderately halophilic bacteria isolated from the salty environments of Iran, a Gram-positive coccus designated as
Fig. 1. Nutrient agar plates of P. pseudoalcaligenes strain Te in the a) absence and b) presence of Te ions. Culture flasks of P. pseudoalcaligenes strain Te cultivated c) with and d) without Te4+ ions, at 30 °C for 24 h.
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H. Forootanfar et al. / Materials Science and Engineering C 49 (2015) 183–189 Table 1 Some biochemical characteristics of P. pseudoalcaligenes strain Te. Characteristics
Results
Catalase production Oxidase activity Voges–Proskauer test pH in Voges–Proskauer broth b6 Methyl red test Acid production from D-Glucose Maltose D-Galactose Sucrose Hydrolysis of Casein Gelatin Starch Utilization of citrate Nitrate reduced to nitrite Formation of Indole Dihydroxyacetone Growth in NaCl 2% 5% 7% 10% Growth at 4 °C 10 °C 25 °C 35 °C 45 °C
− − − + − + − − − − − + + +
Fig. 3. Reduction patterns of Te ions in uninoculated and inoculated culture media by P. pseudoalcaligenes strain Te.
− − + + + − − + + + +
Salinicoccus sp. strain QW6 showed high ability for reduction of tellurium ions [15].
3.2. Te4+ ion reduction to Te NRs The spectrophotometric measurement of the Te4 + ions showed good linearity between the absorbance obtained at 500 nm and serial concentrations of K2TeO3·3H2O (0.07–5 mM). The determination of the MIC value showed that P. pseudoalcaligenes strain Te didn't grow at K2TeO3·3H2O concentrations above 5 mM (Te+4, 640 μg/mL). Therefore, sub-MIC concentrations were used for Te4+ ion reduction experiments. In the study of Amoozegar et al. [15] who investigated on the ability of moderately halophilic bacterial strains for the reduction of potassium tellurite, it was reported that Salinicoccus sp. strain QW6 (isolated from a textile factory effluent) was able to grow in the presence of 12 mM of K2TeO3. Furthermore, resistance to ~ 10 mM of K2TeO3 has been described in some isolates from marine hydrothermal vents [20].
Fig. 2. Growth curve of P. pseudoalcaligenes strain Te in the absence and presence of Te+4 ions.
Fig. 4. a) Transmission electron micrograph and b) SEM image of Te NRs synthesized by P. pseudoalcaligenes strain Te.
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Zanaroli et al. [21] showed that P. pseudoalcaligenes KF707 can grow in the presence of the K2TeO3 at concentration b 0.6 mM. Fig. 2 shows the effect of the sub-inhibitory concentrations of Te4+ ions (100 μg/mL and 200 μg/mL) on the growth of P. pseudoalcaligenes strain Te. Increasing the absorbance of inoculated Te4+-supplemented culture media (100 μg/mL) compared to that of inoculated plain culture broth (without Te4+) contributed to Te NR accumulation in the cell biomass (Fig. 2). For Te4+-supplemented culture media (200 μg/mL) the decreasing of absorbance was observed compared with uninoculated media. Thus, it seems that the closer to the minimum inhibitory concentration of Te4+ ions, the lower the biosynthesis of Te NRs. Same profile of increase in optical densities of Bacillus sp. BZ culture under different concentrations of Te4 + ions (0 mg/L, 50 mg/L, and 100 mg/L) was reported by Zare et al. [5] during 48 h incubation at 30 °C. Chien et al. [22] who isolated Pseudomonas sp. strain TeU (resistant to high concentration of TeO2− 3 , 2000 μM) determined same pattern of
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growth curve during 50 h cultivation in the presence of K2TeO3 (1000 μM). The time course of Te4+ ion reduction by P. pseudoalcaligenes strain Te (Fig. 3) revealed that the reduction process took place with different profiles in the presence of different concentrations of Te4+ ions. The tellurium ions were completely reduced in the nutrient broth culture supplemented with 100 μg/mL K2TeO3; so, Te4 + was not detectable in culture medium after 80 h (Fig. 3). In contrast, in the culture broth containing 200 μg/mL of Te4 +, total reduction of Te4 + ions was not observed after 80 h (Fig. 3). No reduction of Te4 + was detected in the uninoculated nutrient broth (Fig. 3) and reduction wasn't observed for the 24-h-culture supernatant of P. pseudoalcaligenes strain Te at the mentioned incubation time (data not shown). Baesman et al. [11] who isolated two haloalkaliphilic bacterial strains (Sulfurospirillum barnesii and Bacillus selenitireducens) from Mono lake determined the biological removal of 5.5 mM of Te4+ ions
Fig. 5. a) Size distribution patterns, b) UV–visible spectrum, c) energy dispersive X-ray spectrum, d) Fourier transform infrared (FTIR) spectrum, and e) XRD pattern of biologically synthesized Te NRs purified by the liquid–liquid two-phase system.
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after 30 days of incubation. After 50 h treatment of nutrient broth culture supplemented with 50 mg/L and 100 mg/L of K2TeO3 using Bacillus sp. BZ the Te ion concentration reduced to 1 mg/L and 23 mg/L, respectively [5]. The cultivation of Pseudomonas sp. strains TeU in Luria Bertani medium containing 1000 μM of tellurite for 50 h decreased the Te4+ ions to 27 μM [22]. 3.3. Te NR purification and characterization Nanorods were separated from the cell debris during extraction by n-octyl alcohol. Cell debris materials got dissolved in the n-octyl alcohol/water system or remained at the interface of the aqueous and alcoholic phases in the first run of the solvent extraction. According to Fig. 4a, which shows the TEM image, tellurium nanostructures have a rod shape (~ 22 nm diameter by 185 nm length). Such observation was also confirmed by the obtained SEM micrograph (Fig. 4b). Fig. 5a is the corresponding size distribution patterns of Te NRs measured by laser light scattering method after partitioning by the liquid–liquid two-phase system. For the purified Te NRs, one modal peak was clearly revealed in the range of 58–220 nm, and nanorods with a size of 92 nm was the most frequent ones (Fig. 5a). The three phase partitioning system (using organic solvent such as n-octanol) has been successfully applied for the purification of biologically synthesized Te NPs and Se NPs [1,5]. The result of the present study revealed the potential application of this extraction method for the purification of biogenic Te NRs synthesized by P. pseudoalcaligenes
strain Te. In general, the reduction of tellurite compounds assisted by microbial strains led to the formation of nano-rod structures [19]. For example, Baesman et al. [11] reported the formation of rod-shape Te NPs (~10-nm diameter by 200-nm length) by Bacillus selenitireducens. The obtained TEM micrographs of the biologically synthesized Te NPs in the study of Zare et al. [5] represented nano-rod structures with 180 nm in length by less than 20 nm in width. Mohanty et al. [23] reported about the ability of Shewanella oneidensis MR-1 for the reduction of tellurite and production of nanorod Te NPs with the average size of 10–20 nm. Te NRs extracted from P. pseudoalcaligenes strain Te (the present study) exhibited a rod shape (~ 22 nm diameter by 185 nm length). However, P. pseudoalcaligenes KF707 produced Te NPs of spherical shape with the average particle size of 30 nm [24]. The UV–visible spectrum of the purified Te NRs is shown in Fig. 5b. Same spectrum with maximum absorbance at 210 nm was determined by Zare et al. [5]. They ascribed it to the formation of biogenic Te NPs. EDX microanalysis of the purified Te NRs exhibited Te absorption peak at 3.72 keV (Fig. 5c). Elemental composition analysis showed the presence of strong signals from the Te atoms and the Cu peaks were from the TEM copper grid (Fig. 5c). The FTIR spectrum of the biogenic Te NRs produced by P. pseudoalcaligenes strain Te (Fig. 5d) did not show any typical and strong absorption band which confirmed the absence of functional groups on the surface of the purified Te NRs. Generally, the formation of nanoparticles assisted by microorganisms led to the insertion of some unknown compounds on the surface of nanoparticles which is evident from their FTIR spectra [25]. The obtained results of our previous study [1] revealed that the Se NPs produced by Bacillus sp. MSh-1 represented different functional group such as hydroxyl and carbonyl groups on their surface. Same results was reported for other metalloid elements such as Te NPs synthesized by Bacillus sp. BZ [5]. The related XRD pattern of biologically synthesized Te NRs was illustrated in Fig. 5e that all diffraction peaks can be indexed as hexagonal phases of tellurium (space group: P3121 (no. 152)), (JCPDS 36–1452). Similar XRD pattern was reported by Panahi-Kalamuei et al. [3] who applied a simple chemical reduction method for the synthesis of tellurium nanostructures. In addition, the one-step synthesized Te tubular structures (using hydrothermal method) in the study of Qin et al. [26] exhibited the same XRD profile. 3.4. Cytotoxicity assay
Fig. 6. Effect of a) biogenic Te NRs and b) K2TeO3 on viability of A549, HEPG2, HT1080, and MCF-7 cell lines determined by MTT assay. Each value is represented as mean ± SD of three independent experiments.
MTT assay was applied in order to investigate the cytotoxic effect of the biogenic Te NRs and K2TeO3. The obtained results showed a direct dose–response result for the Te NRs (Fig. 6a). For A549, HEPG2, HT1080, and MCF-7 cell lines treated with Te NRs, the concentration necessary for causing 50% cell death (IC50) was found to be 352.2 ± 17.5 ng/mL, 471.9 ± 23.1 ng/mL, 93.7 ± 2.7 ng/mL, and 634.8 ± 24.8 ng/mL, respectively (Fig. 6a). However, in the case of K2TeO3 same effect (i.e. 50% cell death) was determined at the concentration of 12 ± 0.6 ng/mL, 34.1 ± 1.7 ng/mL, 32.4 ± 1.6 ng/mL, and 93.7 ± 1.3 ng/mL for A549, HEPG2, HT1080, and MCF-7 cell lines, respectively (Fig. 6b). For all cell lines the biogenic Te NRs represented lower cytotoxicity compared to that of K2TeO3 (p b 0.05). The obtained results of the present study was in agreement with the results of Mohanty et al. [23] who didn't observe toxic effect of biogenic Te NPs (produced by bacterial strain of Shewanella oneidensis MR-1) in the presence of human bronchial epithelial cells (BEAS-2B) and murine macrophages (RAW264.7). Decreasing the toxicity of other chalcogen oxyanions such as sodium selenite following microbial reduction was previously reported by both in vivo and in vitro studies [27]. The obtained results of a recently published investigation [28] on the cytotoxicity of the chemically synthesized nanosized and microsized tellurium powders on HeLa cells showed that the toxicity of the synthetic nanosized tellurium powders (90–270 nm diameter by 185 nm length) was significantly greater than the microsized one (diameter of 6–9 μm) indicating that the toxicity of Te NPs depends not only on the applied production
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methods (even biological or chemical techniques) but also on the related size of applied Te NPs. 4. Conclusions To sum up, Te NR-producing bacterial strain was isolated from environmental samples and identified as P. pseudoalcaligenes strain Te based on 16S rDNA sequencing analysis. The obtained results of the cytotoxicity studies revealed that biogenic Te NRs represented lower toxicity on four applied cell line compared to that of Te4+ ions. Acknowledgment This work was financially supported by a grant from the Kerman University of Medical Sciences (Kerman, Iran). The authors should also acknowledge the Iranian Nanotechnology Initiative Council for its admirable participation in this study. References [1] M. Shakibaie, M.R. Khorramizadeh, M.A. Faramarzi, O. Sabzevari, A.R. Shahverdi, Biotechnol. Appl. Biochem. 56 (2010) 7–15. [2] M.A. Faramarzi, A. Sadighi, Adv. Colloid Interf. Sci. 189–190 (2013) 1–20. [3] M. Panahi-Kalamuei, F. Mohandes, M. Mousavi-Kamazani, M. Salavati-Niasari, Z. Fereshteh, M. Fathi, Mater. Sci. Semicond. Process. 27 (2014) 1028–1035. [4] H. Zhu, H. Zhang, J.K. Liang, G. Rao, J. Li, G. Liu, Z. Du, H. Fan, J. Luo, J. Phys. Chem. C 115 (2011) 6375–6380. [5] B. Zare, M.A. Faramarzi, Z. Sepehrizadeh, M. Shakibaie, S. Rezaie, A.R. Shahverdi, Mater. Res. Bull. 47 (2012) 3719–3725. [6] L.A. Ba, M. Doring, V. Jamier, C. Jacob, Org. Biomol. Chem. 8 (2010) 4203–4216. [7] D.-H. Kim, R.A. Kanaly, H.-G. Hur, Bioresour. Technol. 125 (2012) 127–131. [8] M. Panahi-Kalamuei, M. Mousavi-Kamazani, M. Salavati-Niasari, Mater. Lett. 136 (2014) 218–221. [9] P. Li, S. Liu, S. Yan, X. Fan, Y. He, Colloids Surf. A 392 (2011) 7–15.
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[10] B. Sredni, R. Geffen-Aricha, W. Duan, M. Albeck, F. Shalit, H.M. Lander, N. Kinor, O. Sagi, A. Albeck, S. Yosef, M. Brodsky, D. Sredni-Kenigsbuch, T. Sonino, D.L. Longo, M.P. Mattson, G. Yadid, FASEB J. 21 (2007) 1870–1883. [11] S.M. Baesman, T.D. Bullen, J. Dewald, D. Zhang, S. Curran, S.F. Islam, T.J. Beveridge, R.S. Oremland, Appl. Environ. Microbiol. 73 (2007) 2135–2143. [12] S.C. Cho, Y.C. Hong, H.S. Uhm, TeO2 nanoparticles synthesized by evaporation of tellurium in atmospheric microwave-plasma torch-flame, Chem. Phys. Lett. 429 (2006) 214–218. [13] J. Pola, D. Pokorna, J. Bohacek, Z. Bastl, A. Ouchi, J. Anal. Appl. Pyrol. 71 (2004) 739–746. [14] K.B. Narayanan, N. Sakthivel, Adv. Colloid Interf. Sci. 156 (2010) 1–13. [15] M.A. Amoozegar, M. Ashengroph, F. Malekzadeh, M.R. Razavi, S. Naddaf, M. Kabiri, Microbiol. Res. 163 (2008) 456–465. [16] J.G. Holt, N.R. Krieg, P.H.A. Sneath, J.T. Staley, S.T. Williams, Bergey's Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins, Baltimore, 1994. [17] M. Hasan-Beikdashti, H. Forootanfar, M.S. Safiarian, A. Ameri, M.H. Ghahremani, M.R. Khoshayand, M.A. Faramarzi, J. Taiwan Inst. Chem. Eng. 43 (2012) 670–677. [18] R.C. Molina, R. Burra, J.M. Pe'rez-Donoso, A.O. Elı'as, C. Mun˜oz, R.A. Montes, T.G. Chasteen, C.C. Va' squez, Appl. Environ. Microbiol. 76 (2010) 4901–4904. [19] R.J. Turner, R. Borghese, D. Zannoni, Biotechnol. Adv. 30 (2012) 954–963. [20] C. Rathgeber, N. Yurkova, E. Stackebrandt, J.T. Beatty, V. Yurkov, Appl. Environ. Microbiol. 68 (2002) 4613–4622. [21] G. Zanaroli, S. Fedi, M. Carnevali, F. Fava, D. Zannoni, Res. Microbiol. 153 (2002) 353–360. [22] C.-C. Chien, M.-H. Jiang, M.-R. Tsai, C.-C. Chien, Environ. Toxicol. Chem. 30 (2011) 2202–2207. [23] A. Mohanty, M.H. Kathawala, J. Zhang, W.N. Chen, J.S.C. Loo, S. Kjelleberg, L. Yang, B. Cao, Biotechnol. Bioeng. 111 (2014) 858–865. [24] G. Di Tomaso, S. Fedi, M. Carnevali, M. Manegatti, C. Taddei, D. Zannoni, Microbiology 148 (2002) 1699–1708. [25] M. Shakibaie, H. Forootanfar, K. Mollazadeh-Moghaddam, Z. Bagherzadeh, N. Nafissi-Varcheh, A.R. Shahverdi, M.A. Faramarzi, Biotechnol. Appl. Biochem. 57 (2010) 71–75. [26] A.-M. Qin, Y.-P. Fang, C.-Y. Su, Inorg. Chem. Commun. 7 (2004) 1014–1016. [27] H. Forootanfar, M. Adeli-Sardou, M. Nikkhoo, M. Mehrabani, B. Amir-Heidari, A.R. Shahverdi, M. Shakibaie, J. Trace Elem. Med. Biol. 28 (2014) 75–79. [28] H. Wen, J. Zhong, B. Shen, T. Gan, C. Fu, Z. Zhu, L. Rui, Front. Biol. 8 (2013) 444–450.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Microbial-assisted synthesis and evaluation the cytotoxic effect of tellurium nanorods Hamid Forootanfar a, Sahar Amirpour-Rostami b, Mandana Jafari b, Amir Forootanfar c, Zahra Yousefizadeh d, Mojtaba Shakibaie b,⁎ a
Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d The Student Research Committee, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran b c
a r t i c l e
i n f o
Article history: Received 18 August 2014 Received in revised form 21 December 2014 Accepted 23 December 2014 Available online 24 December 2014 Keywords: Tellurium nanorods Biosynthesis Pseudomonas pseudoalcaligenes Cytotoxicity Potassium tellurite
a b s t r a c t The present study was designed to isolate bacterial strain capable of tellurium nanorods' (Te NRs) production followed by purification and evaluation of the cytotoxic effect of Te NRs. Among 25 environmental samples collected for screening of Te NR-producer bacterial strains one bacterial colony (isolated from hot spring and identified as Pseudomonas pseudoalcaligenes strain Te) was selected and applied for biosynthesis of Te NRs. Thereafter, an organic–aqueous partitioning system was applied for the purification of the biogenic Te NRs and the purified Te NRs were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX), X-ray diffraction spectroscopy (XRD), UV–visible spectroscopy, and Fourier transform infrared spectroscopy (FTIR) techniques. The cytotoxic effect of biologically synthesized Te NRs and potassium tellurite on four cell lines of MCF-7, HT1080, HepG2 and A549 was then determined using the MTT assay method. The obtained results revealed lower toxicity for the rod-shaped biogenic tellurium nanostructures (~22 nm diameter by 185 nm length) compared to K2TeO3. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Conspicuous physicochemical and biological characteristics of materials at nano-scale compared to those of bulk materials introduced nanostructures as an attractive trend during last decades [1–4]. Metal nanoparticles received more interest due to remarkable photoelectrochemical and catalytic properties [5]. Excellent thermal, optical and electrical properties of tellurium (Te), one of the chalcogen elements, made it as an important metalloid in different industries such as steel and glass making as well as solar panels, sensor production and rechargeable battery manufacturing [6–8]. The fluorescent feature of Tecontaining compounds such as CdTe quantum dots has raised the possibility that such substances may serve as efficient biological markers [6, 9]. Unlike selenium, Te does not seem to be an essential trace element for biological systems [6,7]. Behind the traditional application of Te compounds for the treatment of bacterial infectious diseases such as tuberculosis, leprosy, cystitis and severe eye infections [5] other biological ⁎ Corresponding author. E-mail address: [email protected] (M. Shakibaie).
http://dx.doi.org/10.1016/j.msec.2014.12.078 0928-4931/© 2014 Elsevier B.V. All rights reserved.
activities like the inhibition of cytokine production by T cells, immunomodulatory activities and antisickling have been recently reported for Te-containing compounds [10]. 2− Tellurite (TeO2− 3 ) and tellurate (TeO4 ), the most abundant form of Te in nature [11], could be changed into the insoluble and less toxic elemental Te (Te0) via physicochemical techniques like laser ablation, electrodeposition, pyrolysis, and chemical vapor deposition [12,13]. However, time and cost consuming properties of physicochemical methods applied for the production of elemental nano-tellurium [14] encouraged investigators to screen for biological resources as clean, nontoxic and eco-friendly methods for the synthesis of elemental nano-tellurium. For example, in the study of Zare et al. [5] it was shown that Bacillus sp. BZ, isolated from environmental samples, was able to produce nanorod elemental Te NPs after cultivation in the presence of potassium tellurite. A moderately halophilic bacterium, Salinicoccus sp. QW6, capable of reducing tellurite anion to elemental tellurium was isolated from salty environment samples in the study of Amoozegar et al. [15]. The present study focused on the screening of bacterial strains able to produce Te NRs followed by identification of selected strain. The
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produced nanorods were then purified and characterized. Subsequently, the cytotoxic effects of pure Te NRs were investigated on different cell lines by using an in vitro cytotoxicity assay.
2. Materials and methods 2.1. Chemicals Potassium tellurite (K2TeO3·3H2O) and n-octanol were provided by Merck Chemicals (Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-2H-tetrazolium bromide (MTT) and sodium borohydride (NaBH4) were supplied by Sigma–Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle medium (DMEM) and antibiotics were supplied by Gibco (Life Sciences Inc., USA). All other chemicals and solvents were of analytical grade.
2.4. Measurement of Te4+ ion concentration A modified version of the spectrophotometric method described by Molina et al. [18] was applied for the determination of residual Te4 + ion concentration in bacterial culture media. Briefly, different concentrations of Te4 + ion (1.56–200 μg/mL) were prepared by dissolving the K2TeO3·3H2O in nutrient broth medium and 1 mL of each solution was reduced using freshly prepared solution of sodium borohydride (NaBH4, 4.5 mM) followed by recording the optical densities of all prepared colloids at 500 nm using a UV–visible Double Beam PC Scanning spectrophotometer (UV-1800, Shimadzu CO, USA). The calibration curve was set up by plotting the measured absorbance against the Te4+ ion concentration. In all absorption measurements, 1 mL of nutrient broth medium was subjected to the same steps and was employed as the blank. These procedures were repeated three times, and the mean of the absorbances was used to draw a suitable standard curve. 2.5. Cultivation of the isolate in submerged culture and Te ion reduction
2.2. Screening for Te NRs producing bacteria Water samples were collected from different springs in Kerman (30°15 N, 56°58 E), Iran during the winter of 2012 in order to isolate Te NR-producing bacterial strains. Each sample (5 mL) was then diluted using sterile solution of 0.9% NaCl (20 mL) and 500 μL of each prepared sample was then streaked on nutrient agar (NA) plates containing 100 μg/mL of Te4+ ions (equivalent to 241.4 mg of K2TeO3·3H2O per liter). The plates were then incubated at 30 °C until black colonies (reduction of Te4 + to Te NRs) were observed. Positive colonies were subcultured in NA medium with and without Te4 + ions to confirm that the black color was not an organic bacterial pigment (for example, melanin). The minimal inhibitory concentration (MIC) of Te4+ on selected isolate was achieved using agar dilution method [5]. The selected bacterial colony was then maintained at 4 °C on NA plates supplemented with Te4+ ions. The isolate was also preserved at −80 °C in glycerolsupplemented nutrient broth medium.
2.3. Identification of the isolate Morphological and biochemical characteristics of the selected isolate were studied according to Bergey's Manual of Determinative Bacteriology [16]. Furthermore, 16S rDNA gene sequence analysis was performed as follow: After the cultivation of the isolate in nutrient broth medium for 16 h, 100 μL of bacterial culture was harvested and the produced biomass was separated using centrifugation (5000 rpm, 5 min) followed by washing the obtained bacterial cells using sterile deionized water for three times. The harvested cells were subsequently resuspended in sterile water (50 μL) and boiled at 94 °C for 10 min. After removing the cell debris by centrifugation (14,000 rpm, 10 min), the obtained supernatant was used as a DNA template for PCR amplification. The primer pair of 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) as the forward primer and 1525R (5 -AAGGAGGTGATCCAGCC-3 ) as the reverse primer [17] was used to amplify the 16S rDNA gene. After the preparation of the PCR mixture (Tris, 50 mM pH 8.3; dNTP mixture 250 μM of each; forward primer 400 nM; reverse primer 400 nM; MgCl2 2.5 mM; and Taq DNA polymerase 0.5 U) in 200 μL microtubes, the template DNA (40–60 ng) was added and amplification was performed in a Primus 96 advanced thermal cycler (PEQLAB, Erlangen, Germany) programmed as follows: (a) initial denaturation at 94 °C for 3 min; (b) 30 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 45 s and synthesis at 72 °C for 90 s; and (c) the final extension at 72 °C for 90 s. The amplified DNA fragment was purified from agarose gel (1%) and sent for automated sequencing using the mentioned primers (Bioneer Corporation, South Korea). The basic local alignment search tool (BLAST) was then applied for comparing the sequence of amplified DNA with the sequences in GenBank.
In order to investigate on the time course of Te reduction the selected bacterial strain was grown in NB medium and 1 mL of fresh inoculum (OD600 0.1) was transferred to 500-mL Erlenmeyer flask containing 100 mL of NB medium (pH 7) including sub-MIC concentration of Te4+ ions (100 μg/mL and 200 μg/mL). The flasks were then aerobically cultivated at 30 °C and 150 rpm for 96 h. Negative controls were designed by introducing of Te ions to un-inoculated culture media. In a similar trial the selected isolate was cultured in the absence of Te ions. Samples were taken periodically and subjected for determining of Te4+ concentration and OD600. Before Te analysis, the produced biomass and Te NRs were separated from the culture media by centrifugation (11,000 g for 30 min). For the spectrophotometric determination of the remaining Te4+ ions, 1 mL of the supernatant from the inoculated media containing Te4+ ions, and 1 mL of the supernatant from the inoculated media without Te4+ ions were separately used as the test and blank controls, respectively. To examine the ability of the cell-free supernatant for Te ion reduction, 100 mL of sterile NB was inoculated by fresh inoculum of bacterial isolate (OD600 0.1) and incubated for 24 h followed by removing the produced cells by centrifugation at (4000 g, 10 min). Subsequently, the obtained supernatant was aseptically passed through 0.22 μm filter and collected in a sterile 500-mL Erlenmeyer flask. Thereafter, the Te4+ ions were added to the obtained supernatant to reach final concentration of 100 μg/mL and 200 μg/mL. The flasks were then incubated (30 °C, 150 rpm), and checked periodically for Te NR formation. All mentioned experiments were done in triplicate. 2.6. Production, purification and characterization of Te NRs For the preparation of Te NRs the sterile nutrient broth (NB) medium containing Te ions (100 μg/mL) was inoculated with 1 mL of the fresh inoculums (OD600, 0.1) of the selected bacterial strain and incubated at 30 °C and 150 rpm for 80 h followed by harvesting the bacterial cells using centrifugation (4000 ×g for 10 min). After washing the obtained pellets with sterile solution of NaCl (0.9%) for three times, the bacterial cells were disrupted by grinding the frozen cells in liquid nitrogen using a mortar and pestle. The resulting slurry was then ultrasonicated at 100 W for 5 min and washed three times by sequential centrifugation (14,000 ×g, 5 min) with a 1.5 M Tris–HCl buffer (pH 8.3) containing SDS (1%) and deionized water, respectively. Subsequently, Te NRs were extracted and purified by organic–aqueous partitioning system (n-octanol–water), as previously described [1,5]. Micrographs of the prepared biogenic Te NRs were obtained using a TEM apparatus (Zeiss 902A) operated at accelerating voltage of 80 kV. In order to investigate the surface and elemental composition of biologically synthesized Te NR samples were analyzed using an SEM (Philips XL30, 16 kV) apparatus equipped with an EDX (energy dispersive X-ray) microanalyzer. The particle size distribution pattern of Te NRs was achieved using a
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Zetasizer MS2000 (Malvern Instruments, UK). The crystalline structure of the as synthesized Te NRs was checked by the X-ray diffractometer (Philips PW1710) with CuKα radiation (λ = 1.5405 A°) at room temperature in the 2θ range of 5°–90° with a scanning rate of 0.02°/S. A Shimadzu UV–visible Double Beam PC Scanning spectrophotometer (UV-1800, Shimadzu CO, USA) was applied to record the UV–visible spectrum of the purified Te NRs. The surface chemistry of oven-dried Te NRs (50 °C, 48 h) was also investigated by an infrared spectrophotometer (Shimadzu IR-470, Japan) at a resolution of 4 cm−1 in KBr disks. 2.7. Cell culture and cytotoxicity assay MCF-7, HT1080, HepG2 and A549 cell lines were obtained from the National Cell Bank of Iran, Pasteur Institute of Iran (Tehran, Iran). The cells were maintained in DMEM medium supplemented with FBS (10%, v/v) and antibiotics [penicillin (100 units/mL) and streptomycin (100 μg/mL)] at 37 °C in a CO2 incubator (5% CO2 and 95% relative humidity). In order to evaluate the cytotoxic effect of the biologically synthesized Te NRs and K2TeO3, the cell lines were harvested in the exponential phase of growth, seeded separately into 96-well tissue culture plates (20,000 per well) and allowed to adhere for 24 h. Thereafter, Te NRs and K2TeO3 were added to the desired wells to reach different concentrations. After 24 h of incubation, 20 μL of DMEM medium containing MTT (5 mg/mL) was added to each well and incubated for 4 h. Consequently, the medium was replaced with 100 μl of DMSO, and optical densities were determined at 570 nm. MTT assay was performed in three replicates for each experiment. 2.8. Statistical analysis Each value was expressed as mean ± SD. SPSS software 15 for Windows (SPSS Inc., Chicago) was used for statistical analysis. Differences between the groups were determined using one-way
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analysis of variance (ANOVA) and p-values less than 0.05 were considered significant. 3. Results and discussion 3.1. Isolation and identification of the microorganism Out of 25 samples collected from various springs in Kerman, only one sample collected from Sirch hot springs contained a bacterium with the ability to convert Te4 + ions into Te0. As presented in Fig. 1a and d the cultivation of the bacterial isolate in the absence of Te4 + ions shows no color change indicating the absence of any probable pigment production. However, in culture media containing K2TeO3 (Fig. 1b and c) the reduction of Te4+ ions to elemental Te was evident from the color change of cultivation medium to black due to generation of Te0 during the exponential growth phase. In morphological studies, the isolated strain showed up as a motile, rod-shaped, and Gram-negative bacterium with yellowish white colonies on the nutrient agar medium. The results of biochemical characteristics are summarized in Table 1. Both morphological and biochemical properties of the selected isolate, candidate it as Pseudomonas strain. The alignment of the amplified 16S rDNA gene sequence of the selected isolate against the present sequences of GenBank using BLAST tool represented 99% identity of the obtained gene to Pseudomonas pseudoalcaligenes. The 1403 bp sequence was then submitted to GenBank under accession number of KF055346. The ability of microbial strains for the reduction of chalcogen oxyanions to elemental form introduced this mechanism as a safe, cost-effective, clean, non-toxic, and environmental friendly alternative for time and cost consuming physicochemical techniques applied for the production of metalloids such as Te0 and Se0 nanoparticles [19]. In the study of Zare et al. [5] a tellurium-transforming bacterial strain Bacillus sp. BZ was isolated from the Caspian Sea in northern of Iran. Out of 49 strains of moderately halophilic bacteria isolated from the salty environments of Iran, a Gram-positive coccus designated as
Fig. 1. Nutrient agar plates of P. pseudoalcaligenes strain Te in the a) absence and b) presence of Te ions. Culture flasks of P. pseudoalcaligenes strain Te cultivated c) with and d) without Te4+ ions, at 30 °C for 24 h.
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Results
Catalase production Oxidase activity Voges–Proskauer test pH in Voges–Proskauer broth b6 Methyl red test Acid production from D-Glucose Maltose D-Galactose Sucrose Hydrolysis of Casein Gelatin Starch Utilization of citrate Nitrate reduced to nitrite Formation of Indole Dihydroxyacetone Growth in NaCl 2% 5% 7% 10% Growth at 4 °C 10 °C 25 °C 35 °C 45 °C
− − − + − + − − − − − + + +
Fig. 3. Reduction patterns of Te ions in uninoculated and inoculated culture media by P. pseudoalcaligenes strain Te.
− − + + + − − + + + +
Salinicoccus sp. strain QW6 showed high ability for reduction of tellurium ions [15].
3.2. Te4+ ion reduction to Te NRs The spectrophotometric measurement of the Te4 + ions showed good linearity between the absorbance obtained at 500 nm and serial concentrations of K2TeO3·3H2O (0.07–5 mM). The determination of the MIC value showed that P. pseudoalcaligenes strain Te didn't grow at K2TeO3·3H2O concentrations above 5 mM (Te+4, 640 μg/mL). Therefore, sub-MIC concentrations were used for Te4+ ion reduction experiments. In the study of Amoozegar et al. [15] who investigated on the ability of moderately halophilic bacterial strains for the reduction of potassium tellurite, it was reported that Salinicoccus sp. strain QW6 (isolated from a textile factory effluent) was able to grow in the presence of 12 mM of K2TeO3. Furthermore, resistance to ~ 10 mM of K2TeO3 has been described in some isolates from marine hydrothermal vents [20].
Fig. 2. Growth curve of P. pseudoalcaligenes strain Te in the absence and presence of Te+4 ions.
Fig. 4. a) Transmission electron micrograph and b) SEM image of Te NRs synthesized by P. pseudoalcaligenes strain Te.
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Zanaroli et al. [21] showed that P. pseudoalcaligenes KF707 can grow in the presence of the K2TeO3 at concentration b 0.6 mM. Fig. 2 shows the effect of the sub-inhibitory concentrations of Te4+ ions (100 μg/mL and 200 μg/mL) on the growth of P. pseudoalcaligenes strain Te. Increasing the absorbance of inoculated Te4+-supplemented culture media (100 μg/mL) compared to that of inoculated plain culture broth (without Te4+) contributed to Te NR accumulation in the cell biomass (Fig. 2). For Te4+-supplemented culture media (200 μg/mL) the decreasing of absorbance was observed compared with uninoculated media. Thus, it seems that the closer to the minimum inhibitory concentration of Te4+ ions, the lower the biosynthesis of Te NRs. Same profile of increase in optical densities of Bacillus sp. BZ culture under different concentrations of Te4 + ions (0 mg/L, 50 mg/L, and 100 mg/L) was reported by Zare et al. [5] during 48 h incubation at 30 °C. Chien et al. [22] who isolated Pseudomonas sp. strain TeU (resistant to high concentration of TeO2− 3 , 2000 μM) determined same pattern of
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growth curve during 50 h cultivation in the presence of K2TeO3 (1000 μM). The time course of Te4+ ion reduction by P. pseudoalcaligenes strain Te (Fig. 3) revealed that the reduction process took place with different profiles in the presence of different concentrations of Te4+ ions. The tellurium ions were completely reduced in the nutrient broth culture supplemented with 100 μg/mL K2TeO3; so, Te4 + was not detectable in culture medium after 80 h (Fig. 3). In contrast, in the culture broth containing 200 μg/mL of Te4 +, total reduction of Te4 + ions was not observed after 80 h (Fig. 3). No reduction of Te4 + was detected in the uninoculated nutrient broth (Fig. 3) and reduction wasn't observed for the 24-h-culture supernatant of P. pseudoalcaligenes strain Te at the mentioned incubation time (data not shown). Baesman et al. [11] who isolated two haloalkaliphilic bacterial strains (Sulfurospirillum barnesii and Bacillus selenitireducens) from Mono lake determined the biological removal of 5.5 mM of Te4+ ions
Fig. 5. a) Size distribution patterns, b) UV–visible spectrum, c) energy dispersive X-ray spectrum, d) Fourier transform infrared (FTIR) spectrum, and e) XRD pattern of biologically synthesized Te NRs purified by the liquid–liquid two-phase system.
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after 30 days of incubation. After 50 h treatment of nutrient broth culture supplemented with 50 mg/L and 100 mg/L of K2TeO3 using Bacillus sp. BZ the Te ion concentration reduced to 1 mg/L and 23 mg/L, respectively [5]. The cultivation of Pseudomonas sp. strains TeU in Luria Bertani medium containing 1000 μM of tellurite for 50 h decreased the Te4+ ions to 27 μM [22]. 3.3. Te NR purification and characterization Nanorods were separated from the cell debris during extraction by n-octyl alcohol. Cell debris materials got dissolved in the n-octyl alcohol/water system or remained at the interface of the aqueous and alcoholic phases in the first run of the solvent extraction. According to Fig. 4a, which shows the TEM image, tellurium nanostructures have a rod shape (~ 22 nm diameter by 185 nm length). Such observation was also confirmed by the obtained SEM micrograph (Fig. 4b). Fig. 5a is the corresponding size distribution patterns of Te NRs measured by laser light scattering method after partitioning by the liquid–liquid two-phase system. For the purified Te NRs, one modal peak was clearly revealed in the range of 58–220 nm, and nanorods with a size of 92 nm was the most frequent ones (Fig. 5a). The three phase partitioning system (using organic solvent such as n-octanol) has been successfully applied for the purification of biologically synthesized Te NPs and Se NPs [1,5]. The result of the present study revealed the potential application of this extraction method for the purification of biogenic Te NRs synthesized by P. pseudoalcaligenes
strain Te. In general, the reduction of tellurite compounds assisted by microbial strains led to the formation of nano-rod structures [19]. For example, Baesman et al. [11] reported the formation of rod-shape Te NPs (~10-nm diameter by 200-nm length) by Bacillus selenitireducens. The obtained TEM micrographs of the biologically synthesized Te NPs in the study of Zare et al. [5] represented nano-rod structures with 180 nm in length by less than 20 nm in width. Mohanty et al. [23] reported about the ability of Shewanella oneidensis MR-1 for the reduction of tellurite and production of nanorod Te NPs with the average size of 10–20 nm. Te NRs extracted from P. pseudoalcaligenes strain Te (the present study) exhibited a rod shape (~ 22 nm diameter by 185 nm length). However, P. pseudoalcaligenes KF707 produced Te NPs of spherical shape with the average particle size of 30 nm [24]. The UV–visible spectrum of the purified Te NRs is shown in Fig. 5b. Same spectrum with maximum absorbance at 210 nm was determined by Zare et al. [5]. They ascribed it to the formation of biogenic Te NPs. EDX microanalysis of the purified Te NRs exhibited Te absorption peak at 3.72 keV (Fig. 5c). Elemental composition analysis showed the presence of strong signals from the Te atoms and the Cu peaks were from the TEM copper grid (Fig. 5c). The FTIR spectrum of the biogenic Te NRs produced by P. pseudoalcaligenes strain Te (Fig. 5d) did not show any typical and strong absorption band which confirmed the absence of functional groups on the surface of the purified Te NRs. Generally, the formation of nanoparticles assisted by microorganisms led to the insertion of some unknown compounds on the surface of nanoparticles which is evident from their FTIR spectra [25]. The obtained results of our previous study [1] revealed that the Se NPs produced by Bacillus sp. MSh-1 represented different functional group such as hydroxyl and carbonyl groups on their surface. Same results was reported for other metalloid elements such as Te NPs synthesized by Bacillus sp. BZ [5]. The related XRD pattern of biologically synthesized Te NRs was illustrated in Fig. 5e that all diffraction peaks can be indexed as hexagonal phases of tellurium (space group: P3121 (no. 152)), (JCPDS 36–1452). Similar XRD pattern was reported by Panahi-Kalamuei et al. [3] who applied a simple chemical reduction method for the synthesis of tellurium nanostructures. In addition, the one-step synthesized Te tubular structures (using hydrothermal method) in the study of Qin et al. [26] exhibited the same XRD profile. 3.4. Cytotoxicity assay
Fig. 6. Effect of a) biogenic Te NRs and b) K2TeO3 on viability of A549, HEPG2, HT1080, and MCF-7 cell lines determined by MTT assay. Each value is represented as mean ± SD of three independent experiments.
MTT assay was applied in order to investigate the cytotoxic effect of the biogenic Te NRs and K2TeO3. The obtained results showed a direct dose–response result for the Te NRs (Fig. 6a). For A549, HEPG2, HT1080, and MCF-7 cell lines treated with Te NRs, the concentration necessary for causing 50% cell death (IC50) was found to be 352.2 ± 17.5 ng/mL, 471.9 ± 23.1 ng/mL, 93.7 ± 2.7 ng/mL, and 634.8 ± 24.8 ng/mL, respectively (Fig. 6a). However, in the case of K2TeO3 same effect (i.e. 50% cell death) was determined at the concentration of 12 ± 0.6 ng/mL, 34.1 ± 1.7 ng/mL, 32.4 ± 1.6 ng/mL, and 93.7 ± 1.3 ng/mL for A549, HEPG2, HT1080, and MCF-7 cell lines, respectively (Fig. 6b). For all cell lines the biogenic Te NRs represented lower cytotoxicity compared to that of K2TeO3 (p b 0.05). The obtained results of the present study was in agreement with the results of Mohanty et al. [23] who didn't observe toxic effect of biogenic Te NPs (produced by bacterial strain of Shewanella oneidensis MR-1) in the presence of human bronchial epithelial cells (BEAS-2B) and murine macrophages (RAW264.7). Decreasing the toxicity of other chalcogen oxyanions such as sodium selenite following microbial reduction was previously reported by both in vivo and in vitro studies [27]. The obtained results of a recently published investigation [28] on the cytotoxicity of the chemically synthesized nanosized and microsized tellurium powders on HeLa cells showed that the toxicity of the synthetic nanosized tellurium powders (90–270 nm diameter by 185 nm length) was significantly greater than the microsized one (diameter of 6–9 μm) indicating that the toxicity of Te NPs depends not only on the applied production
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methods (even biological or chemical techniques) but also on the related size of applied Te NPs. 4. Conclusions To sum up, Te NR-producing bacterial strain was isolated from environmental samples and identified as P. pseudoalcaligenes strain Te based on 16S rDNA sequencing analysis. The obtained results of the cytotoxicity studies revealed that biogenic Te NRs represented lower toxicity on four applied cell line compared to that of Te4+ ions. Acknowledgment This work was financially supported by a grant from the Kerman University of Medical Sciences (Kerman, Iran). The authors should also acknowledge the Iranian Nanotechnology Initiative Council for its admirable participation in this study. References [1] M. Shakibaie, M.R. Khorramizadeh, M.A. Faramarzi, O. Sabzevari, A.R. Shahverdi, Biotechnol. Appl. Biochem. 56 (2010) 7–15. [2] M.A. Faramarzi, A. Sadighi, Adv. Colloid Interf. Sci. 189–190 (2013) 1–20. [3] M. Panahi-Kalamuei, F. Mohandes, M. Mousavi-Kamazani, M. Salavati-Niasari, Z. Fereshteh, M. Fathi, Mater. Sci. Semicond. Process. 27 (2014) 1028–1035. [4] H. Zhu, H. Zhang, J.K. Liang, G. Rao, J. Li, G. Liu, Z. Du, H. Fan, J. Luo, J. Phys. Chem. C 115 (2011) 6375–6380. [5] B. Zare, M.A. Faramarzi, Z. Sepehrizadeh, M. Shakibaie, S. Rezaie, A.R. Shahverdi, Mater. Res. Bull. 47 (2012) 3719–3725. [6] L.A. Ba, M. Doring, V. Jamier, C. Jacob, Org. Biomol. Chem. 8 (2010) 4203–4216. [7] D.-H. Kim, R.A. Kanaly, H.-G. Hur, Bioresour. Technol. 125 (2012) 127–131. [8] M. Panahi-Kalamuei, M. Mousavi-Kamazani, M. Salavati-Niasari, Mater. Lett. 136 (2014) 218–221. [9] P. Li, S. Liu, S. Yan, X. Fan, Y. He, Colloids Surf. A 392 (2011) 7–15.
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