- Email: [email protected]
Silver nanoparticles tolerant bacteria from sewage environment
Journal of Environmental Sciences 2011, 23(2) 346–352
Silver nanoparticles tolerant bacteria from sewage environment Sudheer Khan, Amitava Mukherjee, Natarajan Chandrasekaran ∗ Nanobio-Medicine Laboratory, School of Bio-Sciences and Technology, VIT University, Vellore 632014, India. E-mail: [email protected] Received 29 January 2010; revised 21 June 2010; accepted 02 July 2010
Abstract Silver nanoparticle (SNP) is a threat to soil, water and human health. Protection of environment from silver nanoparticles is a major concern. A sewage isolate, Bacillus pumilus treated with SNPs showed similar growth kinetics to that without nanoparticles. A reduction in the amount of exopolysaccharides was observed after SNPs – B. pumilus culture supernatant interaction. The Fourier transform infrared spectroscopy (FT-IR) peaks for the exopolysaccharides extracted from the bacterial culture supernatant and the interacted SNPs were almost similar. The exopolysaccharide capping of the SNPs was confirmed by UV-Visible, FT-IR and X-ray diffraction analysis. The study of bacterial exopolysaccharides capped SNPs with E. coli, S. aureus and M. luteus showed less toxicity compared to uncoated SNPs. Our studies suggested that the capping of nanoparticles by bacterially produced exopolysaccharides serve as the probable mechanism of tolerance. Key words: Bacillus pumilus; silver nanoparticles; exopolysaccharides; encapsulation; bacterial tolerance; toxicity reduction DOI: 10.1016/S1001-0742(10)60412-3 Citation: Khan S, Mukherjee A, Chandrasekaran N, 2011. Silver nanoparticles tolerant bacteria from sewage environment. Journal of Environmental Sciences, 23(2): 346–352
Introduction With the emergence and increase of multi drug resistant organisms due to the increased usage of multiple antibiotics and other antimicrobial compounds, and the continuing emphasis on health-care, many researchers have tried to develop new, effective antimicrobial reagents. This is a landmark in the field of nanotechnology. Among all the metal nanoparticles, silver nanoparticles (SNPs) have high inhibitory and bactericidal effects (Cho et al., 2005). The researchers believed that the use of Ag-based antiseptics may be linked to broad-spectrum activity and far lower propensity than antibiotics to induce microbial resistance (Jones et al., 2006). Kim et al. (2007) reported that the antimicrobial mechanism of SNPs is due to the formation of free radicals and subsequent free radicalinduced membrane damage. SNPs are largely incorporated in textiles, cotton and surgical masks (Schaller et al., 2004; Li et al., 2006; Duran et al., 2007). A major controversy is that whether SNPs are toxic to bacteria or bacteria develops resistant mechanism against SNPs. If the former is true, there might be a devastating effect to the ecosystem which will lead to a global destabilization. There are reports which states that around 270 tonnes of SNPs enter into sewage treatment plants per year (Blaser et al., 2008). If SNPs reach the environment * Corresponding author. E-mail: [email protected]
it may kill all the beneficial microbes thereby affecting wastewater treatment process (Blaser et al., 2008). Antibacterial activity of SNPs largely has been studied with human pathogenic bacteria, mainly E. coli and S. aureus (Sondi and Salopek-Sondi, 2004; Pal et al., 2007; Shrivastava et al., 2007; Kim et al., 2007; Ruparelia et al., 2008). Nanosilver may compromise to control harmful bacteria. Besides that it may affect the beneficial bacteria present in the soil and sewage treatment plants. Most of these antimicrobial studies were carried out with American Type Culture Collection (ATCC) cultures, and not on environmental strains (Pal et al., 2007; Kim et al., 2007; Shrivastava et al., 2007; Ruparelia et al., 2008). The present study was to investigate the physiological role of bacterial exopolysaccharides in SNPs tolerance and also the possible benefits of this organism in SNPs toxicity reduction.
1 Materials and methods 1.1 Materials All chemicals and media were obtained from Himedia Laboratories Ltd., Mumbai, India. All the experiments were carried out in duplicates or triplicates unless otherwise specified. Nanoparticle concentrations used in our experiments were fixed arbitrarily. The manufactured SNPs were obtained from Sigma Aldrich, USA. Nanopar-
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Silver nanoparticles tolerant bacteria from sewage environment
ticles were dispersed using an ultrasonic processor with a frequency of 132 KHz (Crest, USA). 1.2 Characterization of silver nanoparticles Characterization studies of SNPs were done by using UV-Visible spectroscopy (Shimadzu UV-1700, Japan) and high resolution transmission electron microscope (TEM, FEI, Tecnai G-20, USA) after the dispersion in LB medium. The samples were prepared by placing a drop of homogeneous suspension on a copper grid with a lacey carbon film and allowing it to dry in air. Mean particle size was analyzed from the digitized images with image tool software. The morphological features of the procured SNPs were characterized by scanning electrone microscopy (FEI Sirion, Eindhoven, Netherlands). The surface area was measured using a Smart Sorb 93 Single point BET surface area analyzer (Smart Instruments Co. Pvt. Ltd., Mumbai, India). The received particles were also subjected to X-ray diffraction analysis using a JEOL-JDX 8030 difractometer (Japan). The target was Cu Kα (λ = 1.54 Å. The generator was operated as 45 kV with a 30 mA current. The scanning range (2θ) was selected from 10◦ to 100◦ . 1.3 Isolation and identification organism Bacillus pumilus isolated from sewage environment was used in the present study. The sample collected from the sewage environment (1 mL) was grown on LB broth supplemented with 100 mg/L of SNPs and incubated at 30°C for 24 hr with agitation (200 r/min). The SNPs tolerant bacterial strain was isolated from the above culture and identified by 16S rRNA analysis. The 16S rRNA gene sequencing analysis was done by using the primers 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ). Sequencing reaction was carried out using Shrimpex Biotech (DNA 7500 Series II, DE54700305) with the support of Synergy Scientific Services, Chennai, India. Nearly complete 16S rRNA nucleotides were aligned and bacterial identities were deduced by BLAST search to ascertain their closest relatives. 1.4 Disks diffusion test Bacterial sensitivity to SNPs was done by using disc diffusion method (Bauer et al., 1966). The dispersed SNPs were added in to sterile discs of uniform size (5 mm diameter) containing maximum of 25, 50 and 100 μg of SNPs respectively. The bacterial suspension containing 100 μL of 103 –104 CFU/mL was applied uniformly on the surface of the plate containing nutrient agar, Muller Hinton Agar and LB agar. The SNPs impregnated discs were placed on to the plate and incubated at 30°C for 24 hr. Six replicates were kept for each concentration of nanoparticles. After which the average diameter of the inhibition zone surrounding the disc was measured. The bacteria E. coli (ATCC 13534), E. coli (ATCC 25922), S. aureus (ATCC 25923) and M. luteus (clinical isolate) were used as positive control.
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1.5 Agar-well diffusion method The dispersed SNPs were added into the well carries maximum of 10, 25, 50, 100 and 200 μg of nanoparticles respectively. Well diffusion study for silver nitrate, whose concentrations were 10, 25, 100, 250 and 500 mg/L was also carried out. The bacteria such as E. coli, S. aureus and M. luteus were used as positive control. 1.6 Dilution plate count method In dilution plate count method the different concentrations of SNPs were applied uniformly on the surface of LB agar plates and examined the colony forming units at each concentration. A 100 μL sample of bacterial suspension (with a concentration 100 to 150 CFU of isolate) was plated on a nutrient agar plate. The plates were supplemented with SNPs of 10, 25, 50, 100, and 200 μg concentrations. The plates were incubated at 30°C for 24 hr. The numbers of resultant colonies were counted after 24 hr of incubation. The plates without SNPs incubated under the same conditions were used as control. 1.7 Silver ion concentration measurement SNPs dispersed in LB broth at 100 and 200 mg/L were centrifuged at 15,000 ×g for 30 min. Clear supernatant was carefully collected and filtered through a 0.1 μm sterilized filter. The ion concentrations were measured by an atomic absorption spectroscopy (AAS, Varian SpectrAA 240, Australia) after acidification by 1% nitric acid. 1.8 Growth kinetics study Batch culture studies were done to find out the effect of nanoparticles to the bacterial growth kinetics. The concentration of SNPs used in the study was varied between 10–200 mg/L. Sterile Erlenmeyer side armed flasks (250 mL), each containing 50 mL LB broth were sonicated for 30 min after adding the nanoparticles to prevent aggregation of the nanoparticles. Subsequently, the flasks were inoculated with freshly prepared bacterial culture carrying approximately 102 –103 CFU/mL and then incubated in an orbital shaker at 200 r/min at room temperature (28–30°C. Bacterial growth was measured as increase in absorbance at 600 nm determined using a colorimeter (Elico CL 157, India). A positive control (flask containing nanoparticles and nutrient media, devoid of inoculum) and a negative control (flask containing inoculum and nutrient media, devoid of nanoparticles) were included in this experiment. Growth kinetic profile of the bacteria such as E. coli, S. aureus and M. luteus in the growth medium supplemented with 100 mg/L SNPs were recorded as positive control (details given in Section 1.13). The growth studies of all the bacteria were performed with corresponding amount of silver ions which were released during the time of dispersion of SNPs in the medium. 1.9 Extraction, quantification, and purification of exopolysaccharides The exopolysaccharides was extracted from the culture supernatant of B. pumilus (Kumar et al., 2003) and
Journal of Environmental Sciences 2011, 23(2) 346–352 / Sudheer Khan et al.
quantified (Mitchel et al., 1956). It was purified using the method described by Kumar et al., (2003). At the same time another part of the B. pumilus culture supernatant (10 mL) was collected and interacted with SNPs of 100 mg/L for 4 hr in a rotary shaker at 200 r/min, to maintain a proper interaction with nanoparticles. After the interaction, it was centrifuged and separated out the pellets. The supernatant was collected and quantified the exopolysaccharides left in the culture supernatant after interaction with SNPs. The exopolysaccharides extraction was also done with positive control strains (E. coli (ATCC 13534, ATCC 25922), S. aureus (ATCC 25923) and M. luteus (clinical isolate). 1.10 Fourier transform infrared spectroscopy (FT-IR) analysis A loopful culture of B. pumilus was inoculated in 50 mL of LB broth and incubated in shaker at room temperature for 24 hr. The culture was centrifuged at 10,000 ×g for 10 min and the supernatant was collected. The supernatant was divided into two equal portions. One portion of supernatant used to extract the exopolysaccharides directly (without interaction with SNPs) and performed FT-IR analysis. The second portion was subjected to interaction with SNPs. After 4 hr of interaction, it was centrifuged at 10,000 ×g for 10 min and collected the SNPs pellet. The collected pellet was lyophilized and subjected to the FT-IR analysis (Perkin-Elmer, USA. One instrument in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets). Then the FT-IR peak value of the lyophilized SNPs after interaction with B. pumilus culture supernatant was compared with the FT-IR peak value of the exopolysaccharides extracted from bacterial culture supernatant. FT-IR analysis also performed with the exopolysaccharides extracted from positive control strains. 1.11 UV-Visible spectrophotometer analysis The purified exopolysaccharides was re-suspended in de-ionized water and interacted with different concentrations of SNPs (10–100 mg/L) in rotary shaker at 200 r/min for 4 hr. The absorbance was then measured using UVvisible spectrophotometer (Shimadzu UV-1700, Japan) at 200–700 nm.
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(ATCC 25922, ATCC 13534), S. aureus (ATCC 25923) and M. luteus (clinical isolate) were investigated by culturing the organisms on LB broth (102 –103 CFU/mL was inoculated) supplemented with coated and uncoated SNPs at a concentration of 100 mg/L.
2 Results and discussion 2.1 Characterization of SNPs The dispersed solution of SNPs in the LB medium showed λmax at 425 nm as line a shown in Fig. 1, which is a typical absorption maximum of spherical SNPs due to their surface plasmon (Shahverdi et al., 2007; Pal et al., 2007; Deng et al., 2008). The absorption spectrum of the diluted SNPs solution was identical to the absorption spectrum of the original solution of SNPs (line b, Fig. 1). This confirms the stability of the SNPs in the growth medium. TEM and SEM images of SNPs confirmed that the metal particles were in the nano range and that they were approximately spherical in shape. The surface area of manufactured SNPs was determined to be 0.26 m2 /g. Figure 2 shows the TEM image of manufactured SNPs with polydisperse nanoparticles of the size between 10–40 nm in diameter. Figure 3 shows the SEM image of SNPs with particle size below 40 nm. The XRD pattern of manufactured SNPs was recorded (Fig. 4). The XRD analysis has given 6 peaks for SNPs with a major peak at 38.115. 1.6 1.4 1.2 Absorbance
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a
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450 500 550 600 Wavelength (nm) Fig. 1 UV-Visible absorption spectra of dispersed silver nanoparticles (SNPs) in LB broth. Line a: before dilution; line b: after 10 times dilution.
1.12 X-ray diffraction (XRD) analysis The over night culture of B. pumilus was prepared and centrifuged. The supernatant was collected and discarded the pellets. The exopolysaccharides was extracted from the supernatant and was interacted with SNPs for 4 hr. After the interaction, the SNPs were separated out by centrifugation at 10,000 ×g for 10 min. The settled SNPs were lyophilized and analyzed by XRD (PANalytical XPert Pro, Eindhoven, the Netherlands). 1.13 Toxicity test of exopolysaccharides (produced by B. pumilus) coated SNPs The effect of exopolysaccharides coated SNPs on E. coli
Fig. 2 TEM image of manufactured SNPs after dispersion in the medium.
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2.2 Strain identification The SNPs was supplemented in the LB medium for the selective isolation of SNPs tolerant bacteria. The 16S rRNA analysis showed that the bacterial isolate was B. pumilus. The 16S rRNA sequence was submitted in Genbank (Accession number GQ401238) and the organism was named as B. pumilus VITSCA02. 2.3 Antibacterial tests The antibacterial activity of SNPs with B. pumilus isolated from sewage was studied using disc diffusion and agar well diffusion test. The diameter of inhibition zone (DIZ) reflects the magnitude of susceptibility of the microorganism. The organisms susceptible to disinfectants should exhibit DIZ, whereas resistant organisms would not exhibit any zone. B. pumilus did not give any inhibition zone in disc diffusion test as well as agar well diffusion method (data not shown). In disc diffusion test E. coli 13534, E. coli 25922 and M. luteus showed a small inhibition at 50 μg/disc itself. But S. aureus 25923 gave an inhibition zone at 100 μg/disc. In agar well diffusion, the
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zone of inhibition was observed in all control organisms at 100 and 200 μg (data not shown). SNPs exhibited no difference in anti bacterial activity in all the tested media. According to the studies by Ruparelia et al. (2008) the disc impregnated with SNPs exhibited large inhibition zone for E. coli, S. aureus and B. subtilis. In similar studies by Maliszewska and Sadowski (2009), spherical SNPs of size ranging from 10–100 nm diameters showed excellent antibacterial activity with a clear zone of inhibition against S. aureus, B. cereus, E. coli and P. aeruginosa in agar well diffusion method. In our studies with E. coli, S. aureus and M. luteus, a clear zone of inhibition was observed in both disc diffusion test as well as agar well diffusion method. B. pumilus showed sensitivity in all tested concentrations of silver nitrate. Least sensitivity was observed at 10 mg/L. The observed bacterial count (mean ± standard error) in the agar plates were 95.0 ± 0.966, 95.2 ± 0.833, 96.0 ± 0.931, 94.7 ± 1.145, 94.8 ± 1.138, and 94.8 ± 0.792 for control, 10, 25, 50, 100, and 200 μg of SNPs respectively (figure not shown). When SNPs are present on the surface of the nutrient agar plates, they could completely inhibit the bacterial growth compared to liquid broth. But the growth inhibition was not observed in any of the plates in dilution plate count method. According to Shrivastava et al. (2007) growth inhibition was concentration dependent, i.e., when the concentration of SNPs was increased from 5 to 25 mg/L, an increased reduction was observed in the growth of E. coli and S. typhi. Figure 5 shows the bacterial growth profile for different concentrations of SNPs. The growth pattern of B. pumilus with all the studied SNPs concentrations was similar to that of control (without SNPs) indicating that SNPs would not affect the growth of B. pumilus. Most of the studies reported the bacterial growth inhibition by SNPs at some optimal concentrations (Sondi and Salopek-Sondi, 2004; Morones et al., 2005; Shrivastava et al., 2007; Ruparelia et al., 2008). Shrivastava et al. (2007) reported that multi-drug resistant strain of S. typhus was sensitive to SNPs. Over use of nanoparticles in consumer products and washing of these into sewage system, might have induced resistance to
Fig. 3 SEM image of manufactured SNPs after dispersion in the medium.
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Absorbance at 600 nm
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(degree) Fig. 4 XRD pattern of SNPs after dispersion in the medium.
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Time (hr) Fig. 5 Representative batch growth profile of B. pumilus in LB broth dosed with different SNPs concentrations and control broth (without SNPs) at 28–30°C.
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Table 1 Summary of the FT-IR spectra of exopolysaccharides produced by B. pumilus
environmental strains. The positive control strains showed sensitivity to 100 mg/L SNPs.
2.5 Role of exopolysaccharides in bacterial tolerance The exopolysaccharides quantification showed that (63.4 ± 0.647) mg/L of exopolysaccharides was present in the culture supernatant of B. pumilus. In order to determine the role of exopolysaccharides, the B. pumilus culture supernatant was interacted with SNPs. After the interaction, the SNPs were separated out and the exopolysaccharides left in the culture supernatant was quantified (51.9 ± 0.600 mg/L). There was a reduction in the amount of exopolysaccharides in the B. pumilus culture supernatant after interaction with SNPs. This result suggests that some amount of exopolysaccharides might be coated with SNPs during interaction and were separated along with the SNPs while centrifugation. Figure 6a shows the FT-IR spectra (400–4000 cm−1 ) of exopolysaccharides extracted directly from the culture supernatant of B. pumilus. The exopolysaccharides produced by B. pumilus have an –OH stretch, an N–H stretch, a C–H stretch, β-diketone (enols), COO stretch, C–N stretch, P–O stretch, CH deformation and C–S stretch (Table 1). After the interaction of SNPs with B. pumilus culture supernatant, the SNPs were subjected to lyophilization. The lyophilized SNPs were subjected to FT-IR analysis to find out coating of SNPs with exopolysaccharides, secreted by B. pumilus (Fig. 6b). The peaks values of the lyophilized interacted SNPs were almost matching to the peaks value of the exopolysaccharides extracted from the B. pumilus. FT-IR spectra suggested that compound, which is coated to the SNPs and the exopolysaccharide which is extracted from the bacterial culture supernatant, was almost similar. The exopolysaccharides production was not found in the case of M. luteus. FT-IR spectra of exopolysaccharides secreted by E. coli (ATCC 13534, ATCC 25922), S. aureus
Functional groups
3670–3230 3500–3300 2980–2850 1650–1540 1400–1310 1340–1240 1050–990 900–650 700–550
OH with intermolecular hydrogen bonding N–H stretch C–H stretch β-diketone enols COO stretch C–N stretch P–O Stretch CH deformation C–S stretch
Transmittance (%)
During dispersion a substantial amount of silver ions was quickly released from SNPs into the growth medium. The 100 mg/L of SNPs released (1.46 ± 0.003) mg/L silver ions and 200 mg/L of SNPs released (2.92 ± 0.010) mg/L silver ions into the LB medium. To evaluate the toxic effect of silver ions released during the dispersion of SNPs, the LB medium was centrifuged at 15,000 ×g for 30 min after dispersion, thus SNPs settled down and the silver ions remained in the medium. Growth kinetic studies of B. pumilus did not show any growth reduction in the above medium. And growth kinetic studies with positive control strains showed a negligible growth reduction. The release of silver ions from SNPs may vary depending on the time and medium used for dispersion. Morones et al. (2005) reported that the bactericidal mechanism of SNPs and silver ions are distinctly different. For treatment with silver nitrate, a low molecular weight central region was formed within the cells as a defense mechanism, whereas for treatment with nanoparticles, no such phenomenon was observed, although the nanoparticles were found to penetrate through the cell wall.
Wave numbers (cm−1 )
Transmittance (%)
2.4 Action of released silver ions
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Fig. 6 Fourier transform infrared (FT-IR) spectra of exopolysaccharides extracted from B. pumilus culture supernatant (a), and the spectra of lyophilized SNPs after interaction with B. pumilus culture supernatant (b).
were recorded (Figure not shown). Sulfides (inorganic or thiol) are the most important group that can forms a strong complex with silver, with binding constants of 1012 to 1015 for Ag-S complex stoichiometry (Kramer et al., 2009). Other complexes are much weaker, with amino complexes of about 103 to 105 and hydroxyl, carboxyl, carbonato, and phosphato groups of only 100 to 102 (Kramer et al., 2009). Hydroxyl, carboxyl, phosphato, amino and sulfide groups were found in the FT-IR spectra of exopolysaccharides. The FT-IR spectra of the control bacteria were different from that of the exopolysaccharides spectra of B. pumilus. Noble metal SNPs exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR) dependent on shape, size and size distribution of the nanoparticles. When SNPs were interacted with exopolysaccharides of B. pumilus, UV-Visible spectrophotometric analysis showed that there was a peak shift (blue shift) for SNPs. It has given a peak at 394 nm. On the other hand we got a peak for SNPs at 425 nm before interaction with exopolysaccharides. The peak shift might be due to the repelling action of exopolysaccharides capped SNPs.
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Silver nanoparticles tolerant bacteria from sewage environment
Control
Coated SNPs
Absorbance at 600 nm
E. coli 25922
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2.0 1.8 M. luteus (Clinical isolate) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 4 6 8 10 12 0 2 4 6 Time (hr) Time (hr) Fig. 7 Toxicity studies of exopolysacharides coated and uncoated SNPs with bacteria.
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Absorbance at 600 nm
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The XRD analysis of lyophilized SNPs after interaction with exopolysaccharides did not give the characteristic peak for silver. The XRD study corroborated with adsorption of exopolysaccharides on nanoparticles surface, the coverage was so strong and no characteristic band of silver could be revealed. Ravindran et al. (2010) reported that XRD analysis did not give the characteristic peak for silver which was interacted with BSA, due to the coating of SNPs with BSA.
ing to the nanoparticles and there would be no interaction with the bacterial cell wall. Due to the covering, it reduces the toxicity of nanoparticles and it was confirmed by the experiments conducted with S. aureus, E. coli and M. luteus. The present study adds to a better knowledge of the involvement of bacteria exopolysaccharides in nanoparticle tolerance.
2.6 Reduction of toxicity by exopolysaccharides coating
Authors thank VIT University Chancellor for providing us with funding to carry out our research.
The antimicrobial effects of bacterial exopolysaccharides coated and uncoated SNPs against various microorganisms were evaluated. A reduction in the growth rate of positive control bacteria was observed when treated with uncoated SNPs when comparing to the control growth profile (without nanoparticles) (Fig. 7). There was not much reduction in the growth rate of these bacteria observed, when the culture medium supplemented with exopolysaccharides coated SNPs. It indicates the reduction in the toxicity of exopolysaccharides coated SNPs. The exopolysaccharides coated SNPs showed a less growth inhibition which were almost similar to control growth (without nanoparticles).
3 Conclusions The antimicrobial property of SNPs against the isolate B. pumilus have been investigated by disc diffusion test, agar well diffusion method, dilution plate count method and growth kinetic study. There was no growth reduction observed in any of the cases. Our studies showed that bacterial tolerance to nanoparticles might be due to the secretion of exopolysaccharides. This would give a cover-
Acknowledgments
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Silver nanoparticles tolerant bacteria from sewage environment Sudheer Khan, Amitava Mukherjee, Natarajan Chandrasekaran ∗ Nanobio-Medicine Laboratory, School of Bio-Sciences and Technology, VIT University, Vellore 632014, India. E-mail: [email protected] Received 29 January 2010; revised 21 June 2010; accepted 02 July 2010
Abstract Silver nanoparticle (SNP) is a threat to soil, water and human health. Protection of environment from silver nanoparticles is a major concern. A sewage isolate, Bacillus pumilus treated with SNPs showed similar growth kinetics to that without nanoparticles. A reduction in the amount of exopolysaccharides was observed after SNPs – B. pumilus culture supernatant interaction. The Fourier transform infrared spectroscopy (FT-IR) peaks for the exopolysaccharides extracted from the bacterial culture supernatant and the interacted SNPs were almost similar. The exopolysaccharide capping of the SNPs was confirmed by UV-Visible, FT-IR and X-ray diffraction analysis. The study of bacterial exopolysaccharides capped SNPs with E. coli, S. aureus and M. luteus showed less toxicity compared to uncoated SNPs. Our studies suggested that the capping of nanoparticles by bacterially produced exopolysaccharides serve as the probable mechanism of tolerance. Key words: Bacillus pumilus; silver nanoparticles; exopolysaccharides; encapsulation; bacterial tolerance; toxicity reduction DOI: 10.1016/S1001-0742(10)60412-3 Citation: Khan S, Mukherjee A, Chandrasekaran N, 2011. Silver nanoparticles tolerant bacteria from sewage environment. Journal of Environmental Sciences, 23(2): 346–352
Introduction With the emergence and increase of multi drug resistant organisms due to the increased usage of multiple antibiotics and other antimicrobial compounds, and the continuing emphasis on health-care, many researchers have tried to develop new, effective antimicrobial reagents. This is a landmark in the field of nanotechnology. Among all the metal nanoparticles, silver nanoparticles (SNPs) have high inhibitory and bactericidal effects (Cho et al., 2005). The researchers believed that the use of Ag-based antiseptics may be linked to broad-spectrum activity and far lower propensity than antibiotics to induce microbial resistance (Jones et al., 2006). Kim et al. (2007) reported that the antimicrobial mechanism of SNPs is due to the formation of free radicals and subsequent free radicalinduced membrane damage. SNPs are largely incorporated in textiles, cotton and surgical masks (Schaller et al., 2004; Li et al., 2006; Duran et al., 2007). A major controversy is that whether SNPs are toxic to bacteria or bacteria develops resistant mechanism against SNPs. If the former is true, there might be a devastating effect to the ecosystem which will lead to a global destabilization. There are reports which states that around 270 tonnes of SNPs enter into sewage treatment plants per year (Blaser et al., 2008). If SNPs reach the environment * Corresponding author. E-mail: [email protected]
it may kill all the beneficial microbes thereby affecting wastewater treatment process (Blaser et al., 2008). Antibacterial activity of SNPs largely has been studied with human pathogenic bacteria, mainly E. coli and S. aureus (Sondi and Salopek-Sondi, 2004; Pal et al., 2007; Shrivastava et al., 2007; Kim et al., 2007; Ruparelia et al., 2008). Nanosilver may compromise to control harmful bacteria. Besides that it may affect the beneficial bacteria present in the soil and sewage treatment plants. Most of these antimicrobial studies were carried out with American Type Culture Collection (ATCC) cultures, and not on environmental strains (Pal et al., 2007; Kim et al., 2007; Shrivastava et al., 2007; Ruparelia et al., 2008). The present study was to investigate the physiological role of bacterial exopolysaccharides in SNPs tolerance and also the possible benefits of this organism in SNPs toxicity reduction.
1 Materials and methods 1.1 Materials All chemicals and media were obtained from Himedia Laboratories Ltd., Mumbai, India. All the experiments were carried out in duplicates or triplicates unless otherwise specified. Nanoparticle concentrations used in our experiments were fixed arbitrarily. The manufactured SNPs were obtained from Sigma Aldrich, USA. Nanopar-
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Silver nanoparticles tolerant bacteria from sewage environment
ticles were dispersed using an ultrasonic processor with a frequency of 132 KHz (Crest, USA). 1.2 Characterization of silver nanoparticles Characterization studies of SNPs were done by using UV-Visible spectroscopy (Shimadzu UV-1700, Japan) and high resolution transmission electron microscope (TEM, FEI, Tecnai G-20, USA) after the dispersion in LB medium. The samples were prepared by placing a drop of homogeneous suspension on a copper grid with a lacey carbon film and allowing it to dry in air. Mean particle size was analyzed from the digitized images with image tool software. The morphological features of the procured SNPs were characterized by scanning electrone microscopy (FEI Sirion, Eindhoven, Netherlands). The surface area was measured using a Smart Sorb 93 Single point BET surface area analyzer (Smart Instruments Co. Pvt. Ltd., Mumbai, India). The received particles were also subjected to X-ray diffraction analysis using a JEOL-JDX 8030 difractometer (Japan). The target was Cu Kα (λ = 1.54 Å. The generator was operated as 45 kV with a 30 mA current. The scanning range (2θ) was selected from 10◦ to 100◦ . 1.3 Isolation and identification organism Bacillus pumilus isolated from sewage environment was used in the present study. The sample collected from the sewage environment (1 mL) was grown on LB broth supplemented with 100 mg/L of SNPs and incubated at 30°C for 24 hr with agitation (200 r/min). The SNPs tolerant bacterial strain was isolated from the above culture and identified by 16S rRNA analysis. The 16S rRNA gene sequencing analysis was done by using the primers 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ). Sequencing reaction was carried out using Shrimpex Biotech (DNA 7500 Series II, DE54700305) with the support of Synergy Scientific Services, Chennai, India. Nearly complete 16S rRNA nucleotides were aligned and bacterial identities were deduced by BLAST search to ascertain their closest relatives. 1.4 Disks diffusion test Bacterial sensitivity to SNPs was done by using disc diffusion method (Bauer et al., 1966). The dispersed SNPs were added in to sterile discs of uniform size (5 mm diameter) containing maximum of 25, 50 and 100 μg of SNPs respectively. The bacterial suspension containing 100 μL of 103 –104 CFU/mL was applied uniformly on the surface of the plate containing nutrient agar, Muller Hinton Agar and LB agar. The SNPs impregnated discs were placed on to the plate and incubated at 30°C for 24 hr. Six replicates were kept for each concentration of nanoparticles. After which the average diameter of the inhibition zone surrounding the disc was measured. The bacteria E. coli (ATCC 13534), E. coli (ATCC 25922), S. aureus (ATCC 25923) and M. luteus (clinical isolate) were used as positive control.
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1.5 Agar-well diffusion method The dispersed SNPs were added into the well carries maximum of 10, 25, 50, 100 and 200 μg of nanoparticles respectively. Well diffusion study for silver nitrate, whose concentrations were 10, 25, 100, 250 and 500 mg/L was also carried out. The bacteria such as E. coli, S. aureus and M. luteus were used as positive control. 1.6 Dilution plate count method In dilution plate count method the different concentrations of SNPs were applied uniformly on the surface of LB agar plates and examined the colony forming units at each concentration. A 100 μL sample of bacterial suspension (with a concentration 100 to 150 CFU of isolate) was plated on a nutrient agar plate. The plates were supplemented with SNPs of 10, 25, 50, 100, and 200 μg concentrations. The plates were incubated at 30°C for 24 hr. The numbers of resultant colonies were counted after 24 hr of incubation. The plates without SNPs incubated under the same conditions were used as control. 1.7 Silver ion concentration measurement SNPs dispersed in LB broth at 100 and 200 mg/L were centrifuged at 15,000 ×g for 30 min. Clear supernatant was carefully collected and filtered through a 0.1 μm sterilized filter. The ion concentrations were measured by an atomic absorption spectroscopy (AAS, Varian SpectrAA 240, Australia) after acidification by 1% nitric acid. 1.8 Growth kinetics study Batch culture studies were done to find out the effect of nanoparticles to the bacterial growth kinetics. The concentration of SNPs used in the study was varied between 10–200 mg/L. Sterile Erlenmeyer side armed flasks (250 mL), each containing 50 mL LB broth were sonicated for 30 min after adding the nanoparticles to prevent aggregation of the nanoparticles. Subsequently, the flasks were inoculated with freshly prepared bacterial culture carrying approximately 102 –103 CFU/mL and then incubated in an orbital shaker at 200 r/min at room temperature (28–30°C. Bacterial growth was measured as increase in absorbance at 600 nm determined using a colorimeter (Elico CL 157, India). A positive control (flask containing nanoparticles and nutrient media, devoid of inoculum) and a negative control (flask containing inoculum and nutrient media, devoid of nanoparticles) were included in this experiment. Growth kinetic profile of the bacteria such as E. coli, S. aureus and M. luteus in the growth medium supplemented with 100 mg/L SNPs were recorded as positive control (details given in Section 1.13). The growth studies of all the bacteria were performed with corresponding amount of silver ions which were released during the time of dispersion of SNPs in the medium. 1.9 Extraction, quantification, and purification of exopolysaccharides The exopolysaccharides was extracted from the culture supernatant of B. pumilus (Kumar et al., 2003) and
Journal of Environmental Sciences 2011, 23(2) 346–352 / Sudheer Khan et al.
quantified (Mitchel et al., 1956). It was purified using the method described by Kumar et al., (2003). At the same time another part of the B. pumilus culture supernatant (10 mL) was collected and interacted with SNPs of 100 mg/L for 4 hr in a rotary shaker at 200 r/min, to maintain a proper interaction with nanoparticles. After the interaction, it was centrifuged and separated out the pellets. The supernatant was collected and quantified the exopolysaccharides left in the culture supernatant after interaction with SNPs. The exopolysaccharides extraction was also done with positive control strains (E. coli (ATCC 13534, ATCC 25922), S. aureus (ATCC 25923) and M. luteus (clinical isolate). 1.10 Fourier transform infrared spectroscopy (FT-IR) analysis A loopful culture of B. pumilus was inoculated in 50 mL of LB broth and incubated in shaker at room temperature for 24 hr. The culture was centrifuged at 10,000 ×g for 10 min and the supernatant was collected. The supernatant was divided into two equal portions. One portion of supernatant used to extract the exopolysaccharides directly (without interaction with SNPs) and performed FT-IR analysis. The second portion was subjected to interaction with SNPs. After 4 hr of interaction, it was centrifuged at 10,000 ×g for 10 min and collected the SNPs pellet. The collected pellet was lyophilized and subjected to the FT-IR analysis (Perkin-Elmer, USA. One instrument in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets). Then the FT-IR peak value of the lyophilized SNPs after interaction with B. pumilus culture supernatant was compared with the FT-IR peak value of the exopolysaccharides extracted from bacterial culture supernatant. FT-IR analysis also performed with the exopolysaccharides extracted from positive control strains. 1.11 UV-Visible spectrophotometer analysis The purified exopolysaccharides was re-suspended in de-ionized water and interacted with different concentrations of SNPs (10–100 mg/L) in rotary shaker at 200 r/min for 4 hr. The absorbance was then measured using UVvisible spectrophotometer (Shimadzu UV-1700, Japan) at 200–700 nm.
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(ATCC 25922, ATCC 13534), S. aureus (ATCC 25923) and M. luteus (clinical isolate) were investigated by culturing the organisms on LB broth (102 –103 CFU/mL was inoculated) supplemented with coated and uncoated SNPs at a concentration of 100 mg/L.
2 Results and discussion 2.1 Characterization of SNPs The dispersed solution of SNPs in the LB medium showed λmax at 425 nm as line a shown in Fig. 1, which is a typical absorption maximum of spherical SNPs due to their surface plasmon (Shahverdi et al., 2007; Pal et al., 2007; Deng et al., 2008). The absorption spectrum of the diluted SNPs solution was identical to the absorption spectrum of the original solution of SNPs (line b, Fig. 1). This confirms the stability of the SNPs in the growth medium. TEM and SEM images of SNPs confirmed that the metal particles were in the nano range and that they were approximately spherical in shape. The surface area of manufactured SNPs was determined to be 0.26 m2 /g. Figure 2 shows the TEM image of manufactured SNPs with polydisperse nanoparticles of the size between 10–40 nm in diameter. Figure 3 shows the SEM image of SNPs with particle size below 40 nm. The XRD pattern of manufactured SNPs was recorded (Fig. 4). The XRD analysis has given 6 peaks for SNPs with a major peak at 38.115. 1.6 1.4 1.2 Absorbance
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1.0
a
0.8 0.6
b
0.4 0.2 0 300
350
400
450 500 550 600 Wavelength (nm) Fig. 1 UV-Visible absorption spectra of dispersed silver nanoparticles (SNPs) in LB broth. Line a: before dilution; line b: after 10 times dilution.
1.12 X-ray diffraction (XRD) analysis The over night culture of B. pumilus was prepared and centrifuged. The supernatant was collected and discarded the pellets. The exopolysaccharides was extracted from the supernatant and was interacted with SNPs for 4 hr. After the interaction, the SNPs were separated out by centrifugation at 10,000 ×g for 10 min. The settled SNPs were lyophilized and analyzed by XRD (PANalytical XPert Pro, Eindhoven, the Netherlands). 1.13 Toxicity test of exopolysaccharides (produced by B. pumilus) coated SNPs The effect of exopolysaccharides coated SNPs on E. coli
Fig. 2 TEM image of manufactured SNPs after dispersion in the medium.
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Silver nanoparticles tolerant bacteria from sewage environment
2.2 Strain identification The SNPs was supplemented in the LB medium for the selective isolation of SNPs tolerant bacteria. The 16S rRNA analysis showed that the bacterial isolate was B. pumilus. The 16S rRNA sequence was submitted in Genbank (Accession number GQ401238) and the organism was named as B. pumilus VITSCA02. 2.3 Antibacterial tests The antibacterial activity of SNPs with B. pumilus isolated from sewage was studied using disc diffusion and agar well diffusion test. The diameter of inhibition zone (DIZ) reflects the magnitude of susceptibility of the microorganism. The organisms susceptible to disinfectants should exhibit DIZ, whereas resistant organisms would not exhibit any zone. B. pumilus did not give any inhibition zone in disc diffusion test as well as agar well diffusion method (data not shown). In disc diffusion test E. coli 13534, E. coli 25922 and M. luteus showed a small inhibition at 50 μg/disc itself. But S. aureus 25923 gave an inhibition zone at 100 μg/disc. In agar well diffusion, the
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zone of inhibition was observed in all control organisms at 100 and 200 μg (data not shown). SNPs exhibited no difference in anti bacterial activity in all the tested media. According to the studies by Ruparelia et al. (2008) the disc impregnated with SNPs exhibited large inhibition zone for E. coli, S. aureus and B. subtilis. In similar studies by Maliszewska and Sadowski (2009), spherical SNPs of size ranging from 10–100 nm diameters showed excellent antibacterial activity with a clear zone of inhibition against S. aureus, B. cereus, E. coli and P. aeruginosa in agar well diffusion method. In our studies with E. coli, S. aureus and M. luteus, a clear zone of inhibition was observed in both disc diffusion test as well as agar well diffusion method. B. pumilus showed sensitivity in all tested concentrations of silver nitrate. Least sensitivity was observed at 10 mg/L. The observed bacterial count (mean ± standard error) in the agar plates were 95.0 ± 0.966, 95.2 ± 0.833, 96.0 ± 0.931, 94.7 ± 1.145, 94.8 ± 1.138, and 94.8 ± 0.792 for control, 10, 25, 50, 100, and 200 μg of SNPs respectively (figure not shown). When SNPs are present on the surface of the nutrient agar plates, they could completely inhibit the bacterial growth compared to liquid broth. But the growth inhibition was not observed in any of the plates in dilution plate count method. According to Shrivastava et al. (2007) growth inhibition was concentration dependent, i.e., when the concentration of SNPs was increased from 5 to 25 mg/L, an increased reduction was observed in the growth of E. coli and S. typhi. Figure 5 shows the bacterial growth profile for different concentrations of SNPs. The growth pattern of B. pumilus with all the studied SNPs concentrations was similar to that of control (without SNPs) indicating that SNPs would not affect the growth of B. pumilus. Most of the studies reported the bacterial growth inhibition by SNPs at some optimal concentrations (Sondi and Salopek-Sondi, 2004; Morones et al., 2005; Shrivastava et al., 2007; Ruparelia et al., 2008). Shrivastava et al. (2007) reported that multi-drug resistant strain of S. typhus was sensitive to SNPs. Over use of nanoparticles in consumer products and washing of these into sewage system, might have induced resistance to
Fig. 3 SEM image of manufactured SNPs after dispersion in the medium.
1.6
Absorbance at 600 nm
1.4 1.2 1.0 0.8
Control 10 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L
0.6 0.4 0.2 0
(degree) Fig. 4 XRD pattern of SNPs after dispersion in the medium.
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28
Time (hr) Fig. 5 Representative batch growth profile of B. pumilus in LB broth dosed with different SNPs concentrations and control broth (without SNPs) at 28–30°C.
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Table 1 Summary of the FT-IR spectra of exopolysaccharides produced by B. pumilus
environmental strains. The positive control strains showed sensitivity to 100 mg/L SNPs.
2.5 Role of exopolysaccharides in bacterial tolerance The exopolysaccharides quantification showed that (63.4 ± 0.647) mg/L of exopolysaccharides was present in the culture supernatant of B. pumilus. In order to determine the role of exopolysaccharides, the B. pumilus culture supernatant was interacted with SNPs. After the interaction, the SNPs were separated out and the exopolysaccharides left in the culture supernatant was quantified (51.9 ± 0.600 mg/L). There was a reduction in the amount of exopolysaccharides in the B. pumilus culture supernatant after interaction with SNPs. This result suggests that some amount of exopolysaccharides might be coated with SNPs during interaction and were separated along with the SNPs while centrifugation. Figure 6a shows the FT-IR spectra (400–4000 cm−1 ) of exopolysaccharides extracted directly from the culture supernatant of B. pumilus. The exopolysaccharides produced by B. pumilus have an –OH stretch, an N–H stretch, a C–H stretch, β-diketone (enols), COO stretch, C–N stretch, P–O stretch, CH deformation and C–S stretch (Table 1). After the interaction of SNPs with B. pumilus culture supernatant, the SNPs were subjected to lyophilization. The lyophilized SNPs were subjected to FT-IR analysis to find out coating of SNPs with exopolysaccharides, secreted by B. pumilus (Fig. 6b). The peaks values of the lyophilized interacted SNPs were almost matching to the peaks value of the exopolysaccharides extracted from the B. pumilus. FT-IR spectra suggested that compound, which is coated to the SNPs and the exopolysaccharide which is extracted from the bacterial culture supernatant, was almost similar. The exopolysaccharides production was not found in the case of M. luteus. FT-IR spectra of exopolysaccharides secreted by E. coli (ATCC 13534, ATCC 25922), S. aureus
Functional groups
3670–3230 3500–3300 2980–2850 1650–1540 1400–1310 1340–1240 1050–990 900–650 700–550
OH with intermolecular hydrogen bonding N–H stretch C–H stretch β-diketone enols COO stretch C–N stretch P–O Stretch CH deformation C–S stretch
Transmittance (%)
During dispersion a substantial amount of silver ions was quickly released from SNPs into the growth medium. The 100 mg/L of SNPs released (1.46 ± 0.003) mg/L silver ions and 200 mg/L of SNPs released (2.92 ± 0.010) mg/L silver ions into the LB medium. To evaluate the toxic effect of silver ions released during the dispersion of SNPs, the LB medium was centrifuged at 15,000 ×g for 30 min after dispersion, thus SNPs settled down and the silver ions remained in the medium. Growth kinetic studies of B. pumilus did not show any growth reduction in the above medium. And growth kinetic studies with positive control strains showed a negligible growth reduction. The release of silver ions from SNPs may vary depending on the time and medium used for dispersion. Morones et al. (2005) reported that the bactericidal mechanism of SNPs and silver ions are distinctly different. For treatment with silver nitrate, a low molecular weight central region was formed within the cells as a defense mechanism, whereas for treatment with nanoparticles, no such phenomenon was observed, although the nanoparticles were found to penetrate through the cell wall.
Wave numbers (cm−1 )
Transmittance (%)
2.4 Action of released silver ions
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Fig. 6 Fourier transform infrared (FT-IR) spectra of exopolysaccharides extracted from B. pumilus culture supernatant (a), and the spectra of lyophilized SNPs after interaction with B. pumilus culture supernatant (b).
were recorded (Figure not shown). Sulfides (inorganic or thiol) are the most important group that can forms a strong complex with silver, with binding constants of 1012 to 1015 for Ag-S complex stoichiometry (Kramer et al., 2009). Other complexes are much weaker, with amino complexes of about 103 to 105 and hydroxyl, carboxyl, carbonato, and phosphato groups of only 100 to 102 (Kramer et al., 2009). Hydroxyl, carboxyl, phosphato, amino and sulfide groups were found in the FT-IR spectra of exopolysaccharides. The FT-IR spectra of the control bacteria were different from that of the exopolysaccharides spectra of B. pumilus. Noble metal SNPs exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR) dependent on shape, size and size distribution of the nanoparticles. When SNPs were interacted with exopolysaccharides of B. pumilus, UV-Visible spectrophotometric analysis showed that there was a peak shift (blue shift) for SNPs. It has given a peak at 394 nm. On the other hand we got a peak for SNPs at 425 nm before interaction with exopolysaccharides. The peak shift might be due to the repelling action of exopolysaccharides capped SNPs.
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Silver nanoparticles tolerant bacteria from sewage environment
Control
Coated SNPs
Absorbance at 600 nm
E. coli 25922
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 0 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0
Uncoated SNPs
1.8
2
4
6 Time (hr)
8
10
1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 0
12
E. coli 13534
2
4
6 Time (hr)
2.0 1.8 M. luteus (Clinical isolate) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 4 6 8 10 12 0 2 4 6 Time (hr) Time (hr) Fig. 7 Toxicity studies of exopolysacharides coated and uncoated SNPs with bacteria.
8
10
12
8
10
12
S. aureus 25923
Absorbance at 600 nm
Absorbance at 600 nm
Absorbance at 600 nm
1.8 1.6
351
0
2
The XRD analysis of lyophilized SNPs after interaction with exopolysaccharides did not give the characteristic peak for silver. The XRD study corroborated with adsorption of exopolysaccharides on nanoparticles surface, the coverage was so strong and no characteristic band of silver could be revealed. Ravindran et al. (2010) reported that XRD analysis did not give the characteristic peak for silver which was interacted with BSA, due to the coating of SNPs with BSA.
ing to the nanoparticles and there would be no interaction with the bacterial cell wall. Due to the covering, it reduces the toxicity of nanoparticles and it was confirmed by the experiments conducted with S. aureus, E. coli and M. luteus. The present study adds to a better knowledge of the involvement of bacteria exopolysaccharides in nanoparticle tolerance.
2.6 Reduction of toxicity by exopolysaccharides coating
Authors thank VIT University Chancellor for providing us with funding to carry out our research.
The antimicrobial effects of bacterial exopolysaccharides coated and uncoated SNPs against various microorganisms were evaluated. A reduction in the growth rate of positive control bacteria was observed when treated with uncoated SNPs when comparing to the control growth profile (without nanoparticles) (Fig. 7). There was not much reduction in the growth rate of these bacteria observed, when the culture medium supplemented with exopolysaccharides coated SNPs. It indicates the reduction in the toxicity of exopolysaccharides coated SNPs. The exopolysaccharides coated SNPs showed a less growth inhibition which were almost similar to control growth (without nanoparticles).
3 Conclusions The antimicrobial property of SNPs against the isolate B. pumilus have been investigated by disc diffusion test, agar well diffusion method, dilution plate count method and growth kinetic study. There was no growth reduction observed in any of the cases. Our studies showed that bacterial tolerance to nanoparticles might be due to the secretion of exopolysaccharides. This would give a cover-
Acknowledgments
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