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Biosynthesis of Cr(III) nanoparticles from electroplating wastewater using chromiumresistant Bacillus subtilis and its cytotoxicity and antibacterial activity A Kanakalakshmi, V Janaki, K Shanthi & S Kamala-Kannan To cite this article: A Kanakalakshmi, V Janaki, K Shanthi & S Kamala-Kannan (2016): Biosynthesis of Cr(III) nanoparticles from electroplating wastewater using chromium-resistant Bacillus subtilis and its cytotoxicity and antibacterial activity, Artificial Cells, Nanomedicine, and Biotechnology To link to this article: http://dx.doi.org/10.1080/21691401.2016.1228660
Published online: 09 Sep 2016.
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Date: 09 September 2016, At: 07:30
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016 http://dx.doi.org/10.1080/21691401.2016.1228660
ORIGINAL ARTICLE
Biosynthesis of Cr(III) nanoparticles from electroplating wastewater using chromium-resistant Bacillus subtilis and its cytotoxicity and antibacterial activity A Kanakalakshmia, V Janakib, K Shanthia and S Kamala-Kannanc a Department of Environmental Science, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India; bDepartment of Chemistry, Sri Sarada College for Women, Salem, Tamil Nadu, India; cDivision of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, South Korea
ABSTRACT
ARTICLE HISTORY
The aim of this study was to synthesize and characterize Cr(III) nanoparticles using wastewater from electroplating industries and chromium-resistant Bacillus subtilis. Formation of Cr(III) nanoparticles was confirmed by UV–visible (UV–Vis) spectroscopy at 300 nm. The size of the nanoparticles varied from 4 to 50 nm and energy dispersive spectroscopy profile shows strong Cr peak approximately at 4.45 and 5.2 keV. The nanoparticles inhibited the growth of pathogenic bacteria Staphylococcus aureus and Escherichia coli. The cytotoxic effect of the synthesized Cr(III) nanoparticle was studied using HEK 293 cells, and the cell viability was found to decrease with increasing concentration of Cr(III) nanoparticles.
Received 22 June 2016 Revised 15 August 2016 Accepted 22 August 2016 Published online 8 September 2016
Introduction Chromium (Cr), a naturally occurring heavy metal, is widely used in electroplating, pigmenting, wood preserving, leather tanning, steel manufacturing, textile dyeing, and paper and pulp industries (Sukumar et al. 2014). Effluents from these industries carry large quantities of Cr; for example, wastewater from electroplating industries contains 10–100 mg/l of Cr (Xi et al. 1996). Although it exists in different oxidation states, trivalent [Cr(III)] and hexavalent [Cr(VI)] are the most common forms of Cr in industrial effluents (Park et al. 2006). Cr(III) is an essential element needed for normal metabolic activity. It reduces blood glucose and cholesterol levels and also helps transport amino acids into heart and liver, but high concentration of Cr(III) induces allergic reactions in the organisms (Wang and Cefalu 2010). Cr(VI), on the other hand, is highly toxic than Cr(III) and causes liver damage, pulmonary congestion, epigastric pain, skin ulcers, and mutation and cancer in humans (Baruthio 1992). It is also highly mobile than Cr(III) (Rajeswari et al. 2016). Because of high toxicity, World Health Organization recommends maximum permissible limit of Cr(VI) in drinking water to be 0.05 mg/l, with no relaxation on the permissible limit (WHO 1993). Numerous physical, chemical, and biological methods such as precipitation, adsorption, electrodialysis, ion-exchange resins, and membrane techniques are used to remove Cr from electroplating industrial wastewaters (Benvenuti et al. 2014, Janaki et al. 2014, Park et al. 2006, Sukumar et al. 2014). Biological methods are considered the most promising as they not only recover metal from wastewater, but also
KEYWORDS
Bacillus subtilis; biosynthesis; chromium; electroplating wastewater; nanoparticles
decrease the toxicity of metals by reduction or oxidation processes. Many species of bacteria, fungi, and algae have been reported to have Cr adsorption and reduction potential (Dhal et al. 2013a, Hackbarth et al. 2016), but bacteria are considered the most efficient due to their rapid growth rate, ability to grow in different environmental conditions, and easy handling. Bacillus, Exiguobacterium, Aeromonas, Staphylococcus, Pseudomonas, and Halomonas sp., isolated from the soil effectively adsorb and reduce Cr(VI) from aqueous solution (Dhal et al. 2013b). Dhal et al. (2013a) reported that under optimized conditions, indigenous Bacillus sp. reduce 98% of Cr(VI) present in chromite mine soil. The chromate reductase enzyme present in the bacterial system is mainly involved in the reduction of Cr(VI) (Thatoi et al. 2014). Microbes have been known to produce metallic nanoparticles during detoxification or reduction of heavy metals. For instance, Bacillus cereus XMCr-6 produce Cr(III) nanoparticles when incubated in phosphate buffer containing Cr(VI) (Dong et al. 2013). Marine Enterococcus sp., synthesize CdS nanoparticles when the cells are incubated with 1 mM CdSO4 (Rajeshkumar et al. 2014). Rhodopseudomonas capsulata produces different sizes of gold nanoparticles through extracellular reduction of chloroauric acid solution (He et al. 2007). Bacillus sp. SKK11 produce PbS crystals when cultured in LB medium (1=4 strength) supplemented with Pb(NO3)2 (Govarthanan et al. 2015). Bacillus sp. produces Ag nanoparticles by intracellular reduction of AgNO3 in aqueous solution (Janardhanan et al. 2013). Serratia sp. synthesizes Cu nanoparticles when the cells are incubated in 5 mM CuSO4 solution (Hasan et al. 2007). Thus, nanoparticles/nanocrystals are the
CONTACT K Shanthi [email protected]; S Kamala-Kannan [email protected] Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, 570752, South Korea ß 2016 Informa UK Limited, trading as Taylor & Francis Group
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by-product of bacterial metal resistance, and this property of bacteria could be utilized for the production of metallic nanoparticles. However, the use of metal contaminated industrial wastewater for the microbial synthesis of metallic nanoparticles has not been reported. Metallic nanoparticles have promising applications in biomedical sciences, such as biolabeling, drug delivery, catalysis, nanotherapeutics, and nanodiagnosis (Nasrabadi et al. 2016). Furthermore, they have been widely used as antimicrobial agents to control the dissemination of pathogenic microorganisms. Numerous studies have reported the antibacterial and antifungal activity of metallic nanoparticles; for example, Cr2O3 nanoparticles inhibit the growth of Escherichia coli (Ramesh et al. 2012) and Pseudomonas aeruginosa (Rakesh et al. 2013), Ag nanoparticles significantly inhibit growth of Colletotrichum coccodes, Monilinia sp., Pyricularia sp., B. cereus., P. aeruginosa, and Bacillus anthracis (Lee et al. 2013; Singh et al. 2015), Au nanoparticles inhibit the growth of Staphylococcus aureus, E. coli, and P. aeruginosa (Bindhu and Umadevi 2014, Singh et al. 2014), and CuO nanoparticles inhibit the growth of phytopathogens Ralstonia solanacearum and Xanthomonas axonopodis (Praburaman et al. 2015). Hence, the objectives of this study were to (i) synthesize Cr(III) nanoparticles using wastewater from electroplating industries under optimized conditions, (ii) characterize the synthesized Cr(III) nanoparticles, and (iii) evaluate the antibacterial and cytotoxicity of the synthesized Cr(III) nanoparticles.
Materials and methods Electroplating wastewater Wastewater from electroplating industries located near Saravanampatti village, Coimbatore, Tamil Nadu, India were collected and equally diluted (v/v) with sterile double distilled water and stored at 4 C until required. The pH and heavy metal concentration in the effluent (before and after nanoparticle synthesis) were analyzed according to American Public Health Association (APHA) (APHA 2005).
Biomass preparation Bacillus subtilis was isolated from soil samples collected from domestic waste disposal land sites at Coimbatore, Tamil Nadu, India. Isolation and identification of the isolate were concisely reported in Sukumar et al. (2014). Mid logphase bacterial culture (5 ml) was inoculated in 2 l LuriaBertani medium, and the flasks were incubated in a rotary shaker (Orbital Rotary Shaker, Orbitech, India) (180 rpm) for 24 h at 30 C. The biomass from the flasks were collected by centrifugation (Sigma, 3–16PK, Germany) at 6000g for 10 min and washed several times with phosphate buffer (pH 7) to remove the medium components from the cells. The washed biomass was used for nanoparticle synthesis.
0.1 M HCl. Biomass (1% w/v) were inoculated into the effluents, and the flasks were incubated in a rotary shaker (200 rpm) for 24 h at 30 C (Annamalai et al. 2014a, 2014b). After incubation, the biomass was separated from the medium via centrifugation at 10,000g for 10 min, homogenized twice with 30 s intervals using an ultrasonic homogenizer (JY92-II, Scientz, Ningbo, China) at 40 W for 15 s, centrifuged at 12,000g for 30 min at 4 C and filtered through 0.45 l Millipore filter. The filtrate was scanned in a UV–visible (UV–Vis) spectrophotometer (Spectroquant Pharo300, Merck, Darmstadt, Germany) in the wavelength range of 200–800 nm at 1 nm resolution. The filtrate was freeze-dried under vacuum at 80 C for 24 h and used for the further studies. The nanoparticles were digested with HNO3 at 80 C, and the samples were centrifuged at 6000g for 5 min. Then, 1 ml of the supernatant was filtered through a 0.2 lm membrane and analyzed for total metal concentration using inductively coupled plasma optical emission spectroscopy (ICP-OES) (iCAP 6000, Thermo Fisher Scientific, Waltham, MA) after appropriate dilution. The experiments were carried out in triplicate, and only the average values were considered. Morphology, particle size, and elemental composition of the synthesized nanoparticles were analyzed using transmission electron microscope equipped with energy dispersive spectroscopy (EDS) (TEM, Philips CM20). Fourier transform infrared spectroscopy analysis was carried out using KBr pellet method. The spectra were recorded in the range of 400–4000 cm1 with a Bruker model IFS-55 FTIR spectrometer coupled to a Bruker IR microscope fitted with an IBM compatible computer running OPUS, Version 2.2 software (Opus Software Solutions, Princeton, NJ). Thermogravimetric analysis (TGA) of the synthesized nanoparticles was performed using TGA 4000 thermogravimetric analyzer (PerkinElmer, Waltham, MA) over the temperature range of 35 800 C at a heating rate of 10 C/min under nitrogen atmosphere. Differential scanning calorimetric (DSC) analysis of the nanoparticles was carried out using a Shimadzu DSC-50 system (Shimadzu, Kyoto, Japan) at a heating rate of 10 C/min.
Evaluation of antibacterial activity The clinical pathogens E. coli and S. aureus were collected from the Department of Microbiology, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India. The antibacterial activity of the Cr nanoparticles was determined by well-diffusion method (Bauer et al. 1966). In brief, Muller–Hinton agar (MHA) was prepared and 100 ll of the log-phase cultures were swabbed using a sterile cotton swab. Wells were made with gel puncture, and 20, 30, and 40 ll of 1 mM Cr(III) nanoparticles were loaded onto the wells, and incubated at 37 C for 24 h and observed for bacterial growth. The antibacterial activity was evaluated based on the zone of inhibition around the wells. Experiments were repeated twice, and mean values were considered. Autoclaved water was used as control for the experiment.
Biosynthesis and characterization of nanoparticles Electroplating industrial effluents were supplemented with yeast extract (0.3%), peptone (0.5%) and NaCl (0.25%), and the initial pH was adjusted to 7.0 using 0.1 M NaOH or
Cell culture Human embryonic kidney cell line (HEK 293) was procured from National Centre for Cell Science (NCCS), Pune, India.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
The cells were cultured in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum in humidified (100%) atmosphere with 5% CO2 at 37 C. The medium was replaced every 2 d, and cells between the third and fifth passages were used for cell viability test.
Cytotoxicity test The cytotoxicity of the synthesized Cr(III) nanoparticles was determined according to Mosmann (1983). This assay measures the conversion of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) to dark blue formazan precipitate by succinate dehydrogenase present in the intact mitochondria of living cells. HEK 293 cells (100 ll) were seeded into 96-well plates at a density of 1 104 cells/well in two replicates and incubated for 12 h at 37 C. Later, the cells were exposed to different concentrations of Cr(III) nanoparticles (1 mM) (0.1, 10, 25, and 50 ll) and incubated for 48 h under the same condition. To assess cell viability, 15 ll of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated for 4 h at 37 C. The medium with MTT was flicked off from the wells, and the formazan crystals were solubilized in 100 ll of dimethyl sulfoxide. The optical density was measured at an absorbance of 570 nm using microplate reader, and the percentage of cell viability was calculated using the following formula;
3
Formation of Cr(III) nanoparticles were initially confirmed by UV–Vis spectral analysis. The absorbance band showed a peak wavelength of 300 nm (Figure 1), which is preferentially expected for Cr(III) nanoparticles. The absorbance was due to excitation of surface plasmon vibrations within the synthesized Cr(III) nanoparticles. The result is consistent with previous study reporting UV absorbance of Cr(III) nanoparticles at 300 nm (Chandra and Kumar 2013). ICP-OES analysis showed the ratio of mass of Cr in the nanoparticles to be 80.83 ± 1.04, whereas the concentration of Cu, Fe, Zn, Ni, and Cd was below detection limit. Representative electron micrograph of the biosynthesized Cr(III) nanoparticle is depicted in Figure 2. Most of the Cr(III) particles were circular in shape and evenly distributed without any aggregation. The size of the particles varied from 4 to 50 nm, with a mean size of about 20 nm. The results are in accordance with previous study reporting spherical shape of Cr(III) nanoparticles produced by B. cereus (Dong et al. 2013). To further validate the presence of Cr(III) nanoparticles, the samples were subjected to EDS, and the results are depicted in Figure 3. The EDS profile shows strong Cr peak approximately at 4.45 and 5.2 keV, which is typical for Cr crystallites due to surface plasmon resonance. In addition, peaks corresponding to sodium, oxygen, phosphorus, and carbon were noticed, and these could have come from the bacteria.
% Cell viability ¼ ðAtest =Acontrol Þ 100 1.8
where Atest is the mean absorbance of treated cells, and Acontrol is the mean absorbance of a negative control.
1.6 1.4
pH and heavy metal content of the electroplating effluent before and after Cr(III) nanoparticle synthesis are presented in Table 1. Electroplating effluent was equally diluted with sterile distilled water to decrease the initial Cr concentration to 50 mg/ml, an optimum concentration for the isolate B. subtilis (Annamalai et al. 2014a, 2014b). The dilution decreased the concentration of other metals (Cu, Zn, Ni, Fe, and Cd) in the effluent, and thus contamination of the biofabricated Cr(III) nanoparticles. The isolate, B. subtilis, adsorb and reduce Cr(VI) from aqueous solution (Annamalai et al. 2014a, 2014b). The isolate also efficiently synthesized Cr(III) nanoparticles from potassium dichromate solution. Thus, the same isolate of B. subtilis was used to synthesize Cr(III) nanoparticles from the electroplating effluent.
Absorbance
1.2
Result and discussion
1.0 0.8 0.6 0.4 0.2 0.0 200
300
400 500 600 Wavelength (nm)
700
800
Figure 1. UV–Vis absorbance spectra of biofabricated Cr(III) nanoparticle. The peak was observed at 300 nm.
Table 1. Heavy metals in diluted electroplating effluent before and after Cr(III) nanoparticle synthesis.
Parameters pH Cr Cu Fe Zn Ni Cd
Before Cr(III) nanoparticle synthesis
After Cr(III) nanoparticle synthesis
5.1 55.26 mg/l 0.56 mg/l 2.78 mg/l 0.12 mg/l 0.25 mg/l 0.04 mg/l
6.6 11.05 mg/l 0.25 mg/l 0.95 mg/l BDL 0.1 mg/l BDL
Figure 2. Transmission electron micrograph of biofabricated Cr(III) nanoparticle. The size of the nanoparticles varied from 4 to 50 nm.
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Figure 3. Representative energy dispersive spectra of biofabricated Cr(III) nanoparticles. Strong Cr peak was observed approximately at 4.45 and 5.2 keV.
110
100
100 80
40
830
Mass (%)
Transmittance (%)
90 60
20 0
4000
80 70 60 50
3302 3469 3000
2360
40
1735 1388 1086 1235 2000
30 200
1000 -1
Wavenumber (cm )
FTIR spectra of the biofabricated Cr(III) nanoparticle is shown in Figure 4. The peak at 3469 cm1 could be ascribed to phosphorus compounds, whereas the band at 3302 cm1 is assigned to O H stretching vibrations of adsorbed water molecules (Jayaseelan et al. 2012). The characteristic absorption peak at 2360 cm1 may have originated from the primary amines and sulfur compounds present in the bacterial proteins (Jayaseelan et al. 2012). The band at 1735 cm1 shows evidence for ester group (Sanghi and Verma 2009). The absorption peak at 1388 cm1 is characteristic for O H plane bending (Masood and Malik 2011). The peaks at 1235 and 1086 cm1 correspond to secondary amide and methoxyl groups (Chen et al. 2009). The adsorption at 830 cm1 may correspond to stretching vibrations of S ¼ O, which indicates the presence of sulfonate group (Aravindhan et al. 2004). The stretching vibrations in carboxyl, hydroxyl, methoxyl, ester, sulfur, and amide groups indicate the involvement of several macromolecules in Cr(VI) reduction and subsequent formation of Cr(III) nanoparticles. TGA curve of the biofabricated Cr(III) nanoparticles is depicted in Figure 5. A minor decrease in the mass was observed between 90–100 and 240–380 C, and it could be due to the loss of water molecules and degradation of organic materials present in the nanoparticles (Pei et al. 2009). However, a gradual weight loss was observed from
800
Figure 5. Thermal gravimetric curve of biofabricated Cr(III) nanoparticles. A gradual weight loss was observed from 400 to 800 C.
22 Heat Flow Endo Up (mW)
Figure 4. FTIR spectra of Cr(III) nanoparticles synthesized from electroplating wastewater using Bacillus subtilis.
400 600 Temperature (°C)
20 18 16 14 12 0
100
200 300 Temperature (°C)
400
500
Figure 6. Differential scanning calorimetric curve of the biofabricated Cr(III) nanoparticles. Two exothermic peaks were observed at 90 and 380 C.
400 C, and the residual mass at the end of analysis (800 C) was 35.03%. The results are in accordance with the previous studies reporting greater loss in iron-chromium oxide nanoparticles mass (83% wt) at 750 C (Iacob et al. 2015). DSC curve of the biofabricated Cr(III) nanoparticles is shown in Figure 6. The exothermic peak at 90 C could be assigned to the evaporation of water molecules present in the
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
Table 2. Antibacterial activity of the biofabricated Cr(III) nanoparticles. Inhibition zone (mm) Concentration of Cr(III) nanoparticles (ll) 20 30 40
E. coli
S. aureus
17.66 ± 1.1 20.5 ± 0.8 31.5 ± 2.1
12.2 ± 2.8 18.3 ± 1.4 25.1 ± 1.8
Table 3. Viability of HEK 293 cells after treatment with biofabricated Cr(III) nanoparticles. Concentration of Cr(III) nanoparticles (ll) 1 10 25 50
Viability of HEK 293 cells (%) 98.1 ± 0.4 97.2 ± 2.6 88.9 ± 1.5 83.5 ± 1.6
5
Conclusion This study concludes that it is feasible to produce Cr(III) nanoparticles from electroplating industry wastewaters through biological methods – a way for solving environmental problem related to Cr contamination. Simplicity and greener approach make the process ideal for the removal of contaminants. Furthermore, the properties of the synthesized nanoparticles are found to be comparable with nanoparticles produced by conventional methods, making the process more attractive.
Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Reference nanoparticles. However, the sharp exothermic peak at 380 C could be due to the oxidation of organic functional groups present in the sample (Pei et al. 2009). The TGA and DSC results reveal good thermal stability of the biofabricated Cr(III) nanoparticles. Biofabricated Cr(III) nanoparticles effectively inhibited the growth of E. coli and S. aureus on MHA after 12 h incubation at 37 C (Table 2). The zone of inhibition is directly proportional to the concentration of nanoparticles, and maximum zone of inhibition (31.5 ± 2.1 mm for E. coli and 25.1 ± 1.8 mm for S. aureus) was observed in the wells loaded with 40 ll of 1 mM Cr(III) nanoparticles. However, no growth inhibition was observed in the control wells. Smaller size and larger surface area of the biofabricated Cr(III) nanoparticles enable efficient interaction with the bacterial cell walls and make them more penetrating than macro-sized structures (Azam et al. 2012, Govarthanan et al. 2016, Konwarh et al 2011). Moreover, the spherical shape of the synthesized Cr(III) nanoparticle has a critical role in antibacterial activity (Konwarh et al. 2011). The zone of inhibition was found to be higher in Gram-negative bacteria compared to Gram-positive bacteria, which could be due to the thick and chemically complex peptidoglycan layer present in the cell wall of Gram-positive bacteria. The results are in agreement with previous studies that reported Gram-negative bacteria to be highly sensitive to metallic nanoparticles compared to Gram-Positive bacteria (Azam et al. 2012, Konwarh et al. 2011). Metallic nanoparticles have been shown to induce reactive oxygen species followed by oxidative damage to biomolecules and, thus, cell death (Schrand et al. 2010). Cytotoxicity of the biofabricated Cr(III) nanoparticles was evaluated using MTT assay, and the results are shown in Table 3. Cr(III) nanoparticles induced dose-dependent cytotoxicity at concentrations ranging from 0.1 to 50 ll (1 mM). At 50-ll concentration, the viability of HEK 293 cells was decreased to 83.5 ± 1.66% compared to the initial level, and longer exposure resulted in increased toxicity to the cells (data not shown). The results are consistent with previous studies reporting cytotoxic potential of metallic nanoparticles (Praburaman et al. 2015).
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Biosynthesis of Cr(III) nanoparticles from electroplating wastewater using chromiumresistant Bacillus subtilis and its cytotoxicity and antibacterial activity A Kanakalakshmi, V Janaki, K Shanthi & S Kamala-Kannan To cite this article: A Kanakalakshmi, V Janaki, K Shanthi & S Kamala-Kannan (2016): Biosynthesis of Cr(III) nanoparticles from electroplating wastewater using chromium-resistant Bacillus subtilis and its cytotoxicity and antibacterial activity, Artificial Cells, Nanomedicine, and Biotechnology To link to this article: http://dx.doi.org/10.1080/21691401.2016.1228660
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Date: 09 September 2016, At: 07:30
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016 http://dx.doi.org/10.1080/21691401.2016.1228660
ORIGINAL ARTICLE
Biosynthesis of Cr(III) nanoparticles from electroplating wastewater using chromium-resistant Bacillus subtilis and its cytotoxicity and antibacterial activity A Kanakalakshmia, V Janakib, K Shanthia and S Kamala-Kannanc a Department of Environmental Science, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India; bDepartment of Chemistry, Sri Sarada College for Women, Salem, Tamil Nadu, India; cDivision of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, South Korea
ABSTRACT
ARTICLE HISTORY
The aim of this study was to synthesize and characterize Cr(III) nanoparticles using wastewater from electroplating industries and chromium-resistant Bacillus subtilis. Formation of Cr(III) nanoparticles was confirmed by UV–visible (UV–Vis) spectroscopy at 300 nm. The size of the nanoparticles varied from 4 to 50 nm and energy dispersive spectroscopy profile shows strong Cr peak approximately at 4.45 and 5.2 keV. The nanoparticles inhibited the growth of pathogenic bacteria Staphylococcus aureus and Escherichia coli. The cytotoxic effect of the synthesized Cr(III) nanoparticle was studied using HEK 293 cells, and the cell viability was found to decrease with increasing concentration of Cr(III) nanoparticles.
Received 22 June 2016 Revised 15 August 2016 Accepted 22 August 2016 Published online 8 September 2016
Introduction Chromium (Cr), a naturally occurring heavy metal, is widely used in electroplating, pigmenting, wood preserving, leather tanning, steel manufacturing, textile dyeing, and paper and pulp industries (Sukumar et al. 2014). Effluents from these industries carry large quantities of Cr; for example, wastewater from electroplating industries contains 10–100 mg/l of Cr (Xi et al. 1996). Although it exists in different oxidation states, trivalent [Cr(III)] and hexavalent [Cr(VI)] are the most common forms of Cr in industrial effluents (Park et al. 2006). Cr(III) is an essential element needed for normal metabolic activity. It reduces blood glucose and cholesterol levels and also helps transport amino acids into heart and liver, but high concentration of Cr(III) induces allergic reactions in the organisms (Wang and Cefalu 2010). Cr(VI), on the other hand, is highly toxic than Cr(III) and causes liver damage, pulmonary congestion, epigastric pain, skin ulcers, and mutation and cancer in humans (Baruthio 1992). It is also highly mobile than Cr(III) (Rajeswari et al. 2016). Because of high toxicity, World Health Organization recommends maximum permissible limit of Cr(VI) in drinking water to be 0.05 mg/l, with no relaxation on the permissible limit (WHO 1993). Numerous physical, chemical, and biological methods such as precipitation, adsorption, electrodialysis, ion-exchange resins, and membrane techniques are used to remove Cr from electroplating industrial wastewaters (Benvenuti et al. 2014, Janaki et al. 2014, Park et al. 2006, Sukumar et al. 2014). Biological methods are considered the most promising as they not only recover metal from wastewater, but also
KEYWORDS
Bacillus subtilis; biosynthesis; chromium; electroplating wastewater; nanoparticles
decrease the toxicity of metals by reduction or oxidation processes. Many species of bacteria, fungi, and algae have been reported to have Cr adsorption and reduction potential (Dhal et al. 2013a, Hackbarth et al. 2016), but bacteria are considered the most efficient due to their rapid growth rate, ability to grow in different environmental conditions, and easy handling. Bacillus, Exiguobacterium, Aeromonas, Staphylococcus, Pseudomonas, and Halomonas sp., isolated from the soil effectively adsorb and reduce Cr(VI) from aqueous solution (Dhal et al. 2013b). Dhal et al. (2013a) reported that under optimized conditions, indigenous Bacillus sp. reduce 98% of Cr(VI) present in chromite mine soil. The chromate reductase enzyme present in the bacterial system is mainly involved in the reduction of Cr(VI) (Thatoi et al. 2014). Microbes have been known to produce metallic nanoparticles during detoxification or reduction of heavy metals. For instance, Bacillus cereus XMCr-6 produce Cr(III) nanoparticles when incubated in phosphate buffer containing Cr(VI) (Dong et al. 2013). Marine Enterococcus sp., synthesize CdS nanoparticles when the cells are incubated with 1 mM CdSO4 (Rajeshkumar et al. 2014). Rhodopseudomonas capsulata produces different sizes of gold nanoparticles through extracellular reduction of chloroauric acid solution (He et al. 2007). Bacillus sp. SKK11 produce PbS crystals when cultured in LB medium (1=4 strength) supplemented with Pb(NO3)2 (Govarthanan et al. 2015). Bacillus sp. produces Ag nanoparticles by intracellular reduction of AgNO3 in aqueous solution (Janardhanan et al. 2013). Serratia sp. synthesizes Cu nanoparticles when the cells are incubated in 5 mM CuSO4 solution (Hasan et al. 2007). Thus, nanoparticles/nanocrystals are the
CONTACT K Shanthi [email protected]; S Kamala-Kannan [email protected] Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, 570752, South Korea ß 2016 Informa UK Limited, trading as Taylor & Francis Group
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by-product of bacterial metal resistance, and this property of bacteria could be utilized for the production of metallic nanoparticles. However, the use of metal contaminated industrial wastewater for the microbial synthesis of metallic nanoparticles has not been reported. Metallic nanoparticles have promising applications in biomedical sciences, such as biolabeling, drug delivery, catalysis, nanotherapeutics, and nanodiagnosis (Nasrabadi et al. 2016). Furthermore, they have been widely used as antimicrobial agents to control the dissemination of pathogenic microorganisms. Numerous studies have reported the antibacterial and antifungal activity of metallic nanoparticles; for example, Cr2O3 nanoparticles inhibit the growth of Escherichia coli (Ramesh et al. 2012) and Pseudomonas aeruginosa (Rakesh et al. 2013), Ag nanoparticles significantly inhibit growth of Colletotrichum coccodes, Monilinia sp., Pyricularia sp., B. cereus., P. aeruginosa, and Bacillus anthracis (Lee et al. 2013; Singh et al. 2015), Au nanoparticles inhibit the growth of Staphylococcus aureus, E. coli, and P. aeruginosa (Bindhu and Umadevi 2014, Singh et al. 2014), and CuO nanoparticles inhibit the growth of phytopathogens Ralstonia solanacearum and Xanthomonas axonopodis (Praburaman et al. 2015). Hence, the objectives of this study were to (i) synthesize Cr(III) nanoparticles using wastewater from electroplating industries under optimized conditions, (ii) characterize the synthesized Cr(III) nanoparticles, and (iii) evaluate the antibacterial and cytotoxicity of the synthesized Cr(III) nanoparticles.
Materials and methods Electroplating wastewater Wastewater from electroplating industries located near Saravanampatti village, Coimbatore, Tamil Nadu, India were collected and equally diluted (v/v) with sterile double distilled water and stored at 4 C until required. The pH and heavy metal concentration in the effluent (before and after nanoparticle synthesis) were analyzed according to American Public Health Association (APHA) (APHA 2005).
Biomass preparation Bacillus subtilis was isolated from soil samples collected from domestic waste disposal land sites at Coimbatore, Tamil Nadu, India. Isolation and identification of the isolate were concisely reported in Sukumar et al. (2014). Mid logphase bacterial culture (5 ml) was inoculated in 2 l LuriaBertani medium, and the flasks were incubated in a rotary shaker (Orbital Rotary Shaker, Orbitech, India) (180 rpm) for 24 h at 30 C. The biomass from the flasks were collected by centrifugation (Sigma, 3–16PK, Germany) at 6000g for 10 min and washed several times with phosphate buffer (pH 7) to remove the medium components from the cells. The washed biomass was used for nanoparticle synthesis.
0.1 M HCl. Biomass (1% w/v) were inoculated into the effluents, and the flasks were incubated in a rotary shaker (200 rpm) for 24 h at 30 C (Annamalai et al. 2014a, 2014b). After incubation, the biomass was separated from the medium via centrifugation at 10,000g for 10 min, homogenized twice with 30 s intervals using an ultrasonic homogenizer (JY92-II, Scientz, Ningbo, China) at 40 W for 15 s, centrifuged at 12,000g for 30 min at 4 C and filtered through 0.45 l Millipore filter. The filtrate was scanned in a UV–visible (UV–Vis) spectrophotometer (Spectroquant Pharo300, Merck, Darmstadt, Germany) in the wavelength range of 200–800 nm at 1 nm resolution. The filtrate was freeze-dried under vacuum at 80 C for 24 h and used for the further studies. The nanoparticles were digested with HNO3 at 80 C, and the samples were centrifuged at 6000g for 5 min. Then, 1 ml of the supernatant was filtered through a 0.2 lm membrane and analyzed for total metal concentration using inductively coupled plasma optical emission spectroscopy (ICP-OES) (iCAP 6000, Thermo Fisher Scientific, Waltham, MA) after appropriate dilution. The experiments were carried out in triplicate, and only the average values were considered. Morphology, particle size, and elemental composition of the synthesized nanoparticles were analyzed using transmission electron microscope equipped with energy dispersive spectroscopy (EDS) (TEM, Philips CM20). Fourier transform infrared spectroscopy analysis was carried out using KBr pellet method. The spectra were recorded in the range of 400–4000 cm1 with a Bruker model IFS-55 FTIR spectrometer coupled to a Bruker IR microscope fitted with an IBM compatible computer running OPUS, Version 2.2 software (Opus Software Solutions, Princeton, NJ). Thermogravimetric analysis (TGA) of the synthesized nanoparticles was performed using TGA 4000 thermogravimetric analyzer (PerkinElmer, Waltham, MA) over the temperature range of 35 800 C at a heating rate of 10 C/min under nitrogen atmosphere. Differential scanning calorimetric (DSC) analysis of the nanoparticles was carried out using a Shimadzu DSC-50 system (Shimadzu, Kyoto, Japan) at a heating rate of 10 C/min.
Evaluation of antibacterial activity The clinical pathogens E. coli and S. aureus were collected from the Department of Microbiology, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India. The antibacterial activity of the Cr nanoparticles was determined by well-diffusion method (Bauer et al. 1966). In brief, Muller–Hinton agar (MHA) was prepared and 100 ll of the log-phase cultures were swabbed using a sterile cotton swab. Wells were made with gel puncture, and 20, 30, and 40 ll of 1 mM Cr(III) nanoparticles were loaded onto the wells, and incubated at 37 C for 24 h and observed for bacterial growth. The antibacterial activity was evaluated based on the zone of inhibition around the wells. Experiments were repeated twice, and mean values were considered. Autoclaved water was used as control for the experiment.
Biosynthesis and characterization of nanoparticles Electroplating industrial effluents were supplemented with yeast extract (0.3%), peptone (0.5%) and NaCl (0.25%), and the initial pH was adjusted to 7.0 using 0.1 M NaOH or
Cell culture Human embryonic kidney cell line (HEK 293) was procured from National Centre for Cell Science (NCCS), Pune, India.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
The cells were cultured in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum in humidified (100%) atmosphere with 5% CO2 at 37 C. The medium was replaced every 2 d, and cells between the third and fifth passages were used for cell viability test.
Cytotoxicity test The cytotoxicity of the synthesized Cr(III) nanoparticles was determined according to Mosmann (1983). This assay measures the conversion of 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) to dark blue formazan precipitate by succinate dehydrogenase present in the intact mitochondria of living cells. HEK 293 cells (100 ll) were seeded into 96-well plates at a density of 1 104 cells/well in two replicates and incubated for 12 h at 37 C. Later, the cells were exposed to different concentrations of Cr(III) nanoparticles (1 mM) (0.1, 10, 25, and 50 ll) and incubated for 48 h under the same condition. To assess cell viability, 15 ll of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated for 4 h at 37 C. The medium with MTT was flicked off from the wells, and the formazan crystals were solubilized in 100 ll of dimethyl sulfoxide. The optical density was measured at an absorbance of 570 nm using microplate reader, and the percentage of cell viability was calculated using the following formula;
3
Formation of Cr(III) nanoparticles were initially confirmed by UV–Vis spectral analysis. The absorbance band showed a peak wavelength of 300 nm (Figure 1), which is preferentially expected for Cr(III) nanoparticles. The absorbance was due to excitation of surface plasmon vibrations within the synthesized Cr(III) nanoparticles. The result is consistent with previous study reporting UV absorbance of Cr(III) nanoparticles at 300 nm (Chandra and Kumar 2013). ICP-OES analysis showed the ratio of mass of Cr in the nanoparticles to be 80.83 ± 1.04, whereas the concentration of Cu, Fe, Zn, Ni, and Cd was below detection limit. Representative electron micrograph of the biosynthesized Cr(III) nanoparticle is depicted in Figure 2. Most of the Cr(III) particles were circular in shape and evenly distributed without any aggregation. The size of the particles varied from 4 to 50 nm, with a mean size of about 20 nm. The results are in accordance with previous study reporting spherical shape of Cr(III) nanoparticles produced by B. cereus (Dong et al. 2013). To further validate the presence of Cr(III) nanoparticles, the samples were subjected to EDS, and the results are depicted in Figure 3. The EDS profile shows strong Cr peak approximately at 4.45 and 5.2 keV, which is typical for Cr crystallites due to surface plasmon resonance. In addition, peaks corresponding to sodium, oxygen, phosphorus, and carbon were noticed, and these could have come from the bacteria.
% Cell viability ¼ ðAtest =Acontrol Þ 100 1.8
where Atest is the mean absorbance of treated cells, and Acontrol is the mean absorbance of a negative control.
1.6 1.4
pH and heavy metal content of the electroplating effluent before and after Cr(III) nanoparticle synthesis are presented in Table 1. Electroplating effluent was equally diluted with sterile distilled water to decrease the initial Cr concentration to 50 mg/ml, an optimum concentration for the isolate B. subtilis (Annamalai et al. 2014a, 2014b). The dilution decreased the concentration of other metals (Cu, Zn, Ni, Fe, and Cd) in the effluent, and thus contamination of the biofabricated Cr(III) nanoparticles. The isolate, B. subtilis, adsorb and reduce Cr(VI) from aqueous solution (Annamalai et al. 2014a, 2014b). The isolate also efficiently synthesized Cr(III) nanoparticles from potassium dichromate solution. Thus, the same isolate of B. subtilis was used to synthesize Cr(III) nanoparticles from the electroplating effluent.
Absorbance
1.2
Result and discussion
1.0 0.8 0.6 0.4 0.2 0.0 200
300
400 500 600 Wavelength (nm)
700
800
Figure 1. UV–Vis absorbance spectra of biofabricated Cr(III) nanoparticle. The peak was observed at 300 nm.
Table 1. Heavy metals in diluted electroplating effluent before and after Cr(III) nanoparticle synthesis.
Parameters pH Cr Cu Fe Zn Ni Cd
Before Cr(III) nanoparticle synthesis
After Cr(III) nanoparticle synthesis
5.1 55.26 mg/l 0.56 mg/l 2.78 mg/l 0.12 mg/l 0.25 mg/l 0.04 mg/l
6.6 11.05 mg/l 0.25 mg/l 0.95 mg/l BDL 0.1 mg/l BDL
Figure 2. Transmission electron micrograph of biofabricated Cr(III) nanoparticle. The size of the nanoparticles varied from 4 to 50 nm.
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Figure 3. Representative energy dispersive spectra of biofabricated Cr(III) nanoparticles. Strong Cr peak was observed approximately at 4.45 and 5.2 keV.
110
100
100 80
40
830
Mass (%)
Transmittance (%)
90 60
20 0
4000
80 70 60 50
3302 3469 3000
2360
40
1735 1388 1086 1235 2000
30 200
1000 -1
Wavenumber (cm )
FTIR spectra of the biofabricated Cr(III) nanoparticle is shown in Figure 4. The peak at 3469 cm1 could be ascribed to phosphorus compounds, whereas the band at 3302 cm1 is assigned to O H stretching vibrations of adsorbed water molecules (Jayaseelan et al. 2012). The characteristic absorption peak at 2360 cm1 may have originated from the primary amines and sulfur compounds present in the bacterial proteins (Jayaseelan et al. 2012). The band at 1735 cm1 shows evidence for ester group (Sanghi and Verma 2009). The absorption peak at 1388 cm1 is characteristic for O H plane bending (Masood and Malik 2011). The peaks at 1235 and 1086 cm1 correspond to secondary amide and methoxyl groups (Chen et al. 2009). The adsorption at 830 cm1 may correspond to stretching vibrations of S ¼ O, which indicates the presence of sulfonate group (Aravindhan et al. 2004). The stretching vibrations in carboxyl, hydroxyl, methoxyl, ester, sulfur, and amide groups indicate the involvement of several macromolecules in Cr(VI) reduction and subsequent formation of Cr(III) nanoparticles. TGA curve of the biofabricated Cr(III) nanoparticles is depicted in Figure 5. A minor decrease in the mass was observed between 90–100 and 240–380 C, and it could be due to the loss of water molecules and degradation of organic materials present in the nanoparticles (Pei et al. 2009). However, a gradual weight loss was observed from
800
Figure 5. Thermal gravimetric curve of biofabricated Cr(III) nanoparticles. A gradual weight loss was observed from 400 to 800 C.
22 Heat Flow Endo Up (mW)
Figure 4. FTIR spectra of Cr(III) nanoparticles synthesized from electroplating wastewater using Bacillus subtilis.
400 600 Temperature (°C)
20 18 16 14 12 0
100
200 300 Temperature (°C)
400
500
Figure 6. Differential scanning calorimetric curve of the biofabricated Cr(III) nanoparticles. Two exothermic peaks were observed at 90 and 380 C.
400 C, and the residual mass at the end of analysis (800 C) was 35.03%. The results are in accordance with the previous studies reporting greater loss in iron-chromium oxide nanoparticles mass (83% wt) at 750 C (Iacob et al. 2015). DSC curve of the biofabricated Cr(III) nanoparticles is shown in Figure 6. The exothermic peak at 90 C could be assigned to the evaporation of water molecules present in the
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
Table 2. Antibacterial activity of the biofabricated Cr(III) nanoparticles. Inhibition zone (mm) Concentration of Cr(III) nanoparticles (ll) 20 30 40
E. coli
S. aureus
17.66 ± 1.1 20.5 ± 0.8 31.5 ± 2.1
12.2 ± 2.8 18.3 ± 1.4 25.1 ± 1.8
Table 3. Viability of HEK 293 cells after treatment with biofabricated Cr(III) nanoparticles. Concentration of Cr(III) nanoparticles (ll) 1 10 25 50
Viability of HEK 293 cells (%) 98.1 ± 0.4 97.2 ± 2.6 88.9 ± 1.5 83.5 ± 1.6
5
Conclusion This study concludes that it is feasible to produce Cr(III) nanoparticles from electroplating industry wastewaters through biological methods – a way for solving environmental problem related to Cr contamination. Simplicity and greener approach make the process ideal for the removal of contaminants. Furthermore, the properties of the synthesized nanoparticles are found to be comparable with nanoparticles produced by conventional methods, making the process more attractive.
Disclosure statement The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Reference nanoparticles. However, the sharp exothermic peak at 380 C could be due to the oxidation of organic functional groups present in the sample (Pei et al. 2009). The TGA and DSC results reveal good thermal stability of the biofabricated Cr(III) nanoparticles. Biofabricated Cr(III) nanoparticles effectively inhibited the growth of E. coli and S. aureus on MHA after 12 h incubation at 37 C (Table 2). The zone of inhibition is directly proportional to the concentration of nanoparticles, and maximum zone of inhibition (31.5 ± 2.1 mm for E. coli and 25.1 ± 1.8 mm for S. aureus) was observed in the wells loaded with 40 ll of 1 mM Cr(III) nanoparticles. However, no growth inhibition was observed in the control wells. Smaller size and larger surface area of the biofabricated Cr(III) nanoparticles enable efficient interaction with the bacterial cell walls and make them more penetrating than macro-sized structures (Azam et al. 2012, Govarthanan et al. 2016, Konwarh et al 2011). Moreover, the spherical shape of the synthesized Cr(III) nanoparticle has a critical role in antibacterial activity (Konwarh et al. 2011). The zone of inhibition was found to be higher in Gram-negative bacteria compared to Gram-positive bacteria, which could be due to the thick and chemically complex peptidoglycan layer present in the cell wall of Gram-positive bacteria. The results are in agreement with previous studies that reported Gram-negative bacteria to be highly sensitive to metallic nanoparticles compared to Gram-Positive bacteria (Azam et al. 2012, Konwarh et al. 2011). Metallic nanoparticles have been shown to induce reactive oxygen species followed by oxidative damage to biomolecules and, thus, cell death (Schrand et al. 2010). Cytotoxicity of the biofabricated Cr(III) nanoparticles was evaluated using MTT assay, and the results are shown in Table 3. Cr(III) nanoparticles induced dose-dependent cytotoxicity at concentrations ranging from 0.1 to 50 ll (1 mM). At 50-ll concentration, the viability of HEK 293 cells was decreased to 83.5 ± 1.66% compared to the initial level, and longer exposure resulted in increased toxicity to the cells (data not shown). The results are consistent with previous studies reporting cytotoxic potential of metallic nanoparticles (Praburaman et al. 2015).
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