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Effect of culture medium on the extracellular synthesis of silver nanoparticles using Klebsiella pneumoniae, Escherichia coli and Pseudomonas jessinii
Author’s Accepted Manuscript Effect of culture medium on the extracellular synthesis of silver nanoparticles using Klebsiella pneumoniae, Escherichia coli and Pseudomonas jessinii Alexandra Müller, Diana Behsnilian, Elke Walz, Volker Gräf, Lola Hogekamp, Ralf Greiner www.elsevier.com/locate/bab
PII: DOI: Reference:
S1878-8181(16)30035-4 http://dx.doi.org/10.1016/j.bcab.2016.02.012 BCAB368
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 2 November 2015 Revised date: 23 January 2016 Accepted date: 29 February 2016 Cite this article as: Alexandra Müller, Diana Behsnilian, Elke Walz, Volker Gräf, Lola Hogekamp and Ralf Greiner, Effect of culture medium on the extracellular synthesis of silver nanoparticles using Klebsiella pneumoniae, Escherichia coli a n d Pseudomonas jessinii, Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2016.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of culture medium on the extracellular synthesis of silver nanoparticles using Klebsiella pneumoniae, Escherichia coli and Pseudomonas jessinii
Alexandra Müller*, Diana Behsnilian, Elke Walz, Volker Gräf, Lola Hogekamp, Ralf Greiner
Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Department of Food Technology and Bioprocess Engineering, Haid-und-Neu-Str. 9, 76131 Karlsruhe (Germany)
*Corresponding author: Alexandra Müller, Federal Research Institute of Nutrition and Food, Department of Food Technology and Bioprocess Engineering, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany. Tel:
+49 721 6625 363, Fax: +49 721 6625 303
Email: [email protected]
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Abstract Because green biosynthesis using microorganisms is advertised as a promising, ecofriendly and biocompatible approach for the production of silver nanoparticles (AgNPs), many studies were focused on the extracellular production of nanoparticles enabling an easy downstream processing. However, the influence of the culture media on extracellular AgNP synthesis was rarely investigated. In this study, the effect of various culture media and their components on the extracellular AgNP synthesis of Klebsiella pneumoniae UVHC5, Escherichia coli ATCC 8739 and Pseudomonas jessinii UVKS19) was investigated. UV-Vis spectroscopy, DLS, SEM and EDX spectroscopy were used to verify the particles formed. Culture media, bacterial supernatants and the individual medium components were added to AgNO3 solutions. After 7 days of incubation the liquids contained particles of different shapes, sizes and composition. The formation of specific particle species mainly depended on the composition of the culture media (especially the Cl- content), in which the bacteria were inoculated. Samples incubated with bacterial supernatant of STI, NB, LB or MH showed AgCl particles of cubic or star-/flowerlike shapes coated with AgNPs. In contrast, silver nanoclusters were observed in the inoculants of bacterial supernatants grown in MRS or AgNPNB. However, small effects of bacterial metabolites on the formation of the particles could be observed and indicate an influence of the media alteration by bacterial growth on the particle formation. When the individual medium components were added to AgNO3, different absorbance spectra were obtained. Therefore, we conclude that the formation of Ag/AgCl nanoparticles results from the interaction of all medium components.
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Keywords: Culture media, Escherichia coli, extracellular synthesis, Klebsiella pneumoniae, Pseudomonas jessinii, silver nanoparticles
1 Introduction Due to the antimicrobial activity of silver, silver nanoparticles (AgNPs) gained importance in various industrial sectors. While established in the medical and textile industry, promising applications of these nanoparticles emerge in the food sector such as their inclusion in packaging materials (Guo 2013; Iravani et al. 2014; Rai et al. 2013). In order to circumvent the use of toxic substances or expensive physical methods for the production process of AgNPs, green biosynthesis using microorganisms is advertised as a promising, eco-friendly and biocompatible approach (Anthony et al. 2013; Gopinath and Velusamy 2013; Jeevan et al. 2012; Kannan and Subbalaxmi 2011; Narayanan and Sakthivel 2010; Rai et al. 2013). Therefore, the number of publications reporting the synthesis of AgNPs by the use of bacteria continuously increases (Anthony et al. 2013; Deepak et al. 2011; Mohanpuria et al. 2008; Narayanan and Sakthivel 2010; Rai et al. 2013). In this context, the AgNP synthesis is attributed to the bacterial formation of insoluble non-toxic nanoclusters by reducing or precipitating the silver ions in order to minimize the toxicity (Ganesh Babu and Gunasekaran 2013; Klaus-Joerger et al. 2001; Mandal et al. 2006; Narayanan and Sakthivel 2010; Shenashen et al. 2013). These particles can be produced either intra or extracellular (Klaus-Joerger et al. 2001; Narayanan and Sakthivel 2010). However, the underlying mechanisms of AgNP synthesis are still under investigation and various studies suggest
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the involvement of cell wall, the transmembrane proton gradient and the nitrate reductase in the formation of the nanoparticles as well as proteins and other biomolecules as stabilizers and capping agents (Deepak et al. 2011; Kumar et al. 2007; Parikh et al. 2008; Prakash et al. 2011; Priyadarshini et al. 2013; Rai et al. 2013; Sharma et al. 2009; Shenashen et al. 2013; Vaidyanathan et al. 2010). Concerning the biosynthesis, many studies were focused on extracellular methods, because of their easy downstream processing and high scale-up potential (Das et al. 2014; Gopinath and Velusamy 2013; Gurunathan et al. 2009; Priyadarshini et al. 2013). A popular and frequently used method is the addition of bacterial supernatant to AgNO3. However, the influence of the culture media on this approach of AgNP synthesis was rarely investigated. In this study, the effect of culture media on the extracellular AgNP synthesis was investigated using the supernatant of Klebsiella (K.) pneumoniae UVHC5, Escherichia (E.) coli ATCC 8739 and Pseudomonas (P.) jessinii UVKS19 grown in 6 different broths: Standard I nutrient -, de Man, Rogosa and Sharpe -, nutrient -, AgNP nutrient -, lysogeny – and Mueller Hinton broth. The mixtures (1 mM AgNO3 + 1 % v/v supernatants/corresponding uninoculated culture media (control) or the solution of individual components) were incubated at ambient conditions for 7 days. UV-Visspectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) analysis were performed in order to verify the particles formed.
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2 Materials and Methods 2.1 Culture media and bacteria For the experiments six different culture media with the following compositions (in g L1
) were used. Standard I nutrient broth (STI, Merck, Darmstadt, Germany): 15 peptone,
3 yeast extract, 6 NaCl, 1 Glucose, pH 7.5; de Man, Rogosa and Sharpe broth (MRS, Roth, Karlsruhe, Germany): 10 peptone, 4 yeast extract, 8 beef extract, 20 glucose, 2 dipotassium phosphate, 5 sodium acetate, 2 ammonium citrate, 0.05 magnesium sulfate, 1 Tween 80, pH 6.2; nutrient broth (NB, individual components were purchased from Merck, Darmstadt, Germany): 5 peptone, 3 meat extract, pH 7.0; AgNP nutrient broth (AgNPNB, individual components were purchased from Merck, Darmstadt, Germany): 10 peptone, 3 yeast extract, 2 glucose, pH 7.0; lysogeny broth (LB, individual components were purchased from Merck, Darmstadt, Germany): 10 peptone, 5 yeast extract, 10 NaCl, pH 7; Mueller Hinton broth (MH, Merck, Darmstadt, Germany): 17.5 casein hydrolysate, 2 meat infusion, 1.5 starch, pH 7.4. For the production of STI-agar, 15 g L-1 agar-agar (Roth, Karlsruhe, Germany) was added to STI broth. In order to investigate the effect of the individual components on the AgNP synthesis, the chemicals were solved in deionized water in the given quantity. All media were prepared in the given composition and autoclaved at 121 °C for 20 min. STI-agar was then poured in 94x16 mm petri dishes. The chloride concentration of the culture media was determined by titration with silver nitrate according to the Mohr method (Nielsen 2010). The following three bacteria were used in this study. K. pneumoniae UVHC5 was isolated from tiger nut (Cyperus esculentus). This ubiquitous bacterium naturally occurs in the soil, but forms also a normal part of human microbiota in the mouth and
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intestines. P. jessinii UVKS19 was gained by isolation from carrot juice. As residents of the upper layers of the soil, pseudomonades are frequently found food contaminants. Escherichia coli ATCC 8739 isolated from feces is a standard organism for several quality controls and food testing. Bacteria were stored in cryo vials and sub-cultured twice in STI broth before used in experiments.
2.2 Synthesis of AgNPs Bacterial overnight cultures (K. pneumoniae UVHC5, E. coli ATCC 8739, P. jessinii UVKS19) were inoculated (1 % v/v) in the six different culture media and grown at 30 °C for 18 h. In order to enumerate the viable counts 1 ml of the overnight cultures was taken, ten-fold serially diluted in Quarter-strength Ringer’s solution (Merck, Darmstadt, Germany) and plated out on STI-agar. After incubation for 18 h at 30 °C, colonies were enumerated. The remaining overnight culture was centrifuged at 3220 g for 15 min and the supernatant was sterile-filtered through a 0.2 µm filter (CHROMAFIL®PET-20/25, Macherey-Nagel GmbH & Co. KG, Düren, Germany). In order to synthesize AgNPs, 200 µl of the supernatants as well as 200 µl of the corresponding uninoculated culture media (control) or the solution of individual components (except dipotassium phosphate, ammonium citrate and magnesium sulfate as present in MRS) were added to 20 ml of 1 mM AgNO3 aqueous solutions and incubated at ambient conditions (room temperature = 22 ± 2 °C and normal daylight). The formation of AgNPs was daily characterized by UV-Vis analyses as well as DLS. All experiments were done in triplicate.
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2.3 Characterization of AgNPs 2.3.1 UV-Vis spectrophotometry Because of their unique optical properties noble metals as Ag-dispersion can be characterized by UV-VIS spectroscopy using the surface plasmon resonance effect. Therefore, the UV-Vis spectra of the incubated AgNO3 solutions were recorded between 300 – 700 nm in 2 nm intervals using a UV/Vis-Spectrometer (UV.2, UNICAM, Kassel, Germany) in order to receive information about the presence of AgNPs in the reaction mixtures.
2.3.2 DLS The formation of particles was verified by particle size measurement using dynamic light scattering (DLS) performed on a Zetasizer Nano ZS (Malvern Instruments Ltd, UK) with temperature control (25 °C). The correlation functions were analyzed using the General Purpose Model algorithm of the instrument software (version 7.02); viscosity and refractive index (RI) of the dispersion media were assumed as viscosity and RI of water (0.8872 cP; 1.330). To avoid erroneous data because of the different refractivities of silver and silver chloride particles, in this study only the refractivity independent harmonic intensity-weighted arithmetic average particle diameter (ZAverage) and polydispersity index (PDI) has been used for characterization. The PDI is a dimensionless value, which gives an indication of the width of the particle size distributions. With PDI values of about 0.2 a moderate polydisperse size distribution can be assumed. Each sample was measured in triplicate.
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2.3.3 SEM and EDX The verification of the particles species (Ag or Ag/AgCl nanoparticles, respectively) was determined by SEM and EDX analyses, which were conducted on day 7 after addition of media or supernatants to AgNO3. Furthermore, analyses were also conducted directly after addition of STI or NB to AgNO3. Morphological analysis of the particles contained in all samples was carried out using a FEG-SEM Quanta 250 (FEI, Czech Republic) equipped with Everhart-Thornley (ETD) and gaseous analytical backscattered electrons (GAD) detectors. Energy-dispersive Xray spectroscopy was used for elemental analysis of the particles (Apollo X SDD, EDAX, USA). Analytical conditions were: acceleration voltage 10 kV; working distance 10 mm (optimal distance for EDX analysis). Sample aliquots (10 µL) were deposited onto silicium substrates which were mounted onto aluminum stubs with double coated carbon conductive tabs; samples were then left to dry in a desiccator.
2.3.4 Statistical analysis The values of the absorbance at 420 nm as well as Z-Averages of AgNO3 incubated with culture media or supernatant of culture media inoculated with bacteria were checked for significant differences using a t-test (Sigma Plot for Windows 12.3, Systat Software GmbH, Erkrath, Germany). The confidence level was set at 95 % (p < 0.05).
3 Results 3.1 Culture media mediated synthesis of AgNPs
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Figure 1 a shows an image of 1 mM AgNO3 solutions immediately after adding the culture media (1 % v/v) and figure 1 d shows the corresponding absorbance spectra. The turbidity determined indicates the immediate formation of AgCl in AgNO3 incubated with STI, LB and MH and correlates well with the Cl- concentration of the media, in which values of 6.16 ± 0.02 g L-1 (LB), 4.05 ± 0.03 g L-1 (MH), 3.93 ± 0.01 g L-1 (STI), 0.35 ± 0.02 g L-1 (MRS), 0.22 ± 0.01 g L-1 (NB) and 0.12 ± 0.01 g L-1 (AgNPNB) were measured. DLS analysis confirmed the formation of particles (table 1) whereby mean particle diameters (Z-Average) of about 100 nm were determined in the incubated mixtures containing STI, LB and MH. In the samples incubated with MRS, NB and AgNPNB culture medium, the concentration of particles directly after the addition of media was too low for DLS analysis. The detection limit of the DLS technique depends on the amount of scattered light caused by the particles. If the difference in scattering between the dispersant and the sample is too low, the quality of the raw data is too poor for particles sizes analysis. The very fast formation of AgCl particles after addition of medium, which contains a high Cl- concentration (ST I, LB, MH), to AgNO3 solution was confirmed using SEM/EDX analysis (figure 1b, c). Here, the REM image and the EDX analysis of the AgCl particles formed immediately after adding STI to AgNO3 is also representative for the particles as present after the addition of LB and MH to silver nitrate. The EDX spectrum (figure 1c) of the cubic particles visible in figure 1b shows strong characteristic signals for Ag as well as for Cl, indicating that they consist of AgCl. The high Si peak and the small C peak are caused by the substrate on which the sample was deposited. Traces of Na resulting from the culture media were also found by EDX analysis. During 7 days of incubation at ambient conditions the absorbance at 420 nm of AgNO3 incubated with culture media significantly (p < 0.05) increased, mainly rising in the first
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hours of incubation and indicates a culture media mediated formation of AgNPs (figure 1e). The highest increase of the absorbance at 420 nm (A420nm 0.334 ± 0.008 cm-1 up to 2.335 ± 0.146 cm-1) was observed in AgNO3 incubated with STI, followed by incubated mixtures containing LB and MH. Here, the values at 420 nm rose from 0.637 ± 0.117 cm-1 and 0.736 ± 0.051 cm-1 up to 1.781 ± 0.126 cm-1 and 1.519 ± 0.106 cm-1, respectively. In contrast, the absorbance at 420 nm of the sample containing AgNPNB only slightly increased from 0.002 ± 0.001 cm-1 to 0.108 ± 0.015 cm-1 during 7 days of incubation. Whereas, similar absorbance at 420 nm was determined for the samples incubated with MRS and NB, rising from 0.030 ± 0.011 cm-1 and 0.006 ± 0.001 cm-1 up to 0.625 ± 0.172 cm-1 and 0.384 ± 0.156 cm-1, respectively, at day 7 (figure 1e).
3.2 Effect of culture media on the extracellular synthesis of AgNPs After 7 days of incubation at ambient conditions the incubated mixtures of AgNO3 with culture media or supernatant of K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19 grown in various media contained different concentrations of particles with different sizes and shapes resulting in different signals of surface plasmon resonance (figure 2 and 3, table 1). As shown in figure 2, the absorbance spectra of the incubated mixtures mainly depended on the media, in which the bacteria were inoculated. Therefore, the shapes of the absorbance spectra were similar for each medium tested, independent of whether they were previously inoculated or not with bacteria. However, small effects of bacterial metabolites on the formation of the particles could be observed. A significant (p < 0.05) reduced absorbance at 420 nm was determined when AgNO3 was incubated with the supernatant of E. coli ATCC 8739 grown in STI compared to the incubation using uninoculated STI (figure 2a). In contrast, the bacterial growth in MRS did not significantly affect the particle formation,
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although the supernatant of K. pneumoniae UVHC5 tended to result in a higher (whereas E. coli ATCC 8739 a lower) absorbance at 420 nm (figure 2b). However, different absorbance spectra were determined for samples incubated using the supernatant of K. pneumoniae UVHC5 and P. jessinii UVKS19 grown in NB compared to the control (incubation with uninoculated NB) (figure 2c), whereby significant higher absorbances at 420 nm were observed when using the supernatant of K. pneumoniae UVHC5 and P. jessinii UVKS19. In addition, despite the general low absorbance of the samples containing AgNPNB medium (figure 2d), the bacterial metabolites significantly increased the signal of surface plasmon resonance of the incubated mixture and therefore, promoted particle formation. These results could also be visually confirmed, as shown in figure 2g. Here, the samples containing AgNPNB or supernatants of E. coli ATCC 8739, P. jessinii UVKS19 and K. pneumoniae UVHC5 got darker in ascending order. In contrast, no significant difference was observed using LB culture medium (figure 2e) and only P. jessinii UVKS19 significantly affected the absorbance spectra of the samples incubated with MH medium (figure 2f). Although, significant effects of bacterial growth on the formation of the particles were detected, no correlation between absorbance spectra and bacterial counts could be observed (data of the bacterial counts not shown). The results of DLS indicated the presence of particles after 7 days of incubation (table 1). Here Z-Averages of about 100 nm were measured in samples containing both uninoculated culture media and bacterial supernatant with exception of the incubated mixture of MRS and AgNPNB. In this context, Z-Averages of about 50 nm were observed and suggest the presence of AgNPs or Ag nanoclusters. In several cases, a significant effect (p < 0.05) of bacterial growth in the culture media on the particle size was determined regarding the particles formation. E.g. the incubated
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mixtures containing the supernatant of K. pneumoniae UVHC5 and E. coli ATCC 8739 in STI and MRS showed significant differences in particle size after 7 days of incubation compared to the addition of uninoculated culture media, as well as the bacterial growth in MH resulted in significant different particle size determined (table 1). The overall appearance of UV-Vis and DLS measurements was confirmed by SEM and EDX analysis after 7 days of incubation (figure 3 and 4). Correlating to the Cl - content of the media, samples incubated with bacterial supernatant grown in STI, LB and MH show sharp-edged AgCl particles of cubic or star-/flowerlike shapes coated with silver nanoparticles. Whereas, the basic structure of the Ag/AgCl particles presented in the samples containing NB was not sharp-edged but rounder (figure 3). In contrast, silver nanoclusters were observed in the incubated mixtures of bacterial supernatants grown in MRS and AgNPNB (figure 3). Figure 4a shows a SEM image of Ag/AgCl particles (particles magnified in figure 4b) as observed in samples incubated 7 days with bacterial supernatants containing STI, NB, LB or MH. In this connection, EDX measurements were performed on a homogenous spot near the center of the star shaped particle near the left edge of image 4a (spectrum in 4c), as well as on one of the small spherical particles attached to the surface of the large structure (figure 4d). The spectrum in figure 4c shows Ag and Cl peaks of similar magnitude, indicating that the particle is composed of AgCl at this location. In contrast, the Cl peak of the spectrum shown in figure 4d is negligible compared to the Ag peak, indicating that the spherical nanoparticle on the surface of the star shaped particle consists of silver. Here, the Cl signal is due to the volume below the targeted AgNP.
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3.3 The role of individual culture medium components in the extracellular synthesis of AgNPs The absorbance spectra of 1 mM AgNO3 incubated (7 days, ambient conditions) with individual culture media components having different curve shapes are shown in figure 5. Here, the highest signal of surface plasmon resonance (Amax approximately 0.6 cm-1) was observed in the samples containing 8 g L-1 meat extract or 5 g L-1 yeast extract, as the components and concentrations are present in MRS and LB, respectively. Lower absorbance maxima were determined with decreasing concentrations of meat and yeast extract as well as in samples incubated with 17.5 g L-1 peptone and 17.5 g L-1 NaCl. All other concentrations of medium components tested resulted in no or only a small signal and therefore, absorbance spectra were not included in figure 5. However, the incubation of 1 mM AgNO3 with culture media and medium components resulted in different absorbance spectra. In addition, the sum of the absorbance maxima of the incubated mixtures containing media components differed from the maximum of the mixtures using the entire culture media. E.g. an absorbance maximum of 2.33 ± 0.15 cm-1 was measured for 1 mM AgNO3 incubated with STI (figure 2a), whereas the sum of Amax containing solely the medium components amounted about 0.37 cm-1 (figure 5). In general, a higher absorbance maximum was calculated for the medium components compared to the surface plasmon resonance signal of the corresponding culture medium, except for MRS and AgNPNB samples. Here, the single absorbance maxima of the components (approximately 0.95 and 0.37 cm-1) exceeded the absorbance of the culture media (0.64 ± 0.18 and 0.11 ± 0.02 cm-1).
4 Discussion
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In this study, the effect of various culture media and their media components on the extracellular AgNP synthesis of K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19 was investigated using UV-Vis-spectroscopy, DLS, SEM and EDX analysis in order to characterize the particles formed. The spontaneous formation of large particles of about 100 nm diameter after addition of media containing high Cl- concentrations to AgNO3 solution in contrast to the addition of media with low Cl- concentrations is an indication of the formation of AgCl particles rather than silver nanoparticles or reaction products caused by other medium components. In addition, the formation of the AgCl particles, which were determined by UV-Vis and DLS was verified by SEM/EDX analysis. The results of particle size measurements of the present study are in good accordance with those of the study of Song et al. (Song et al. 2013), where the edge length of AgCl nanocubes produced using AgNO3, Poly-vinyl alcohol (PVA) and NaCl was between 57-170 nm depending on process temperature. In addition, Loza et al. determined the precipitation of AgCl when adding AgNO3 to biological media such as phosphate-buffered saline solution (PBS) or LB medium due to the presence of Cl- in the media. The authors conclude that the size of AgCl particles formed depends on the presence of biomolecules, which coat the particles and prevent them from further growth (Loza et al. 2014). In the study of Mokhtari et al., a white precipitate was observed in the reaction vessels after the addition of culture supernatant of Klebsiella pneumonia grown in MH media (Mokhtari et al. 2009). Here again, subsequent XRD analysis confirmed the presence of AgCl particles, which were ascribed to the presence of Cl- ions in the culture media. Our results show that during 7 days of incubation at ambient conditions the absorbance at 420 nm of AgNO3 incubated with culture media significantly increased and indicates a culture media mediated formation of AgNPs. After 7 days of incubation, the mixture
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of AgNO3 with culture media or supernatant contained particles of different concentrations, size and shapes resulting in different signals of surface plasmon resonance, DLS and SEM/EDX analysis. In this context, the formation of specific particle species mainly depended on the composition of culture media, in which the bacteria were inoculated. Samples incubated with bacterial supernatant grown in STI, LB or MH show sharp-edged AgCl particles of cubic or star-/flowerlike shapes coated with silver nanoparticles. Whereas, the basic structure of the Ag/AgCl particles of NB samples were rounder. In contrast, silver nanoclusters were observed in the incubated mixtures of bacterial supernatants grown in MRS or AgNPNB. However, some small effects of bacterial metabolites on the formation of the particles could be observed by UV-Vis and DLS. These results indicate that both the original composition of the media and the modified composition of the media, which results as a consequence of bacterial growth, play an important role on the particle formation. The kinetic of maximum absorbance using the supernatant of E. coli and Morganella spp. grown in NB, LB and LB without NaCl was investigated by several authors (Gurunathan et al. 2009; Natarajan et al. 2010; Parikh et al. 2008). In all those studies the highest increase in the absorbance was observed in the first hours of incubation and matches very well with our results concerning the addition of uninoculated culture media. Numerous studies report a surface plasmon resonance signal of AgNP particles after incubation of AgNO3 with the supernatant of various bacteria grown in several culture media (such as NB, LB and MH) at ambient conditions (Das et al. 2014; Ganesh Babu and Gunasekaran 2013; Gopinath and Velusamy 2013; Jeevan et al. 2012; Kalishwaralal et al. 2008; Kannan and Subbalaxmi 2011; Minaeian et al. 2008; Natarajan et al. 2010; Priyadarshini et al. 2013). Unfortunately, most of these studies did not check the absorbance spectra of the incubated mixture applying the uninoculated corresponding culture media. However,
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Shivaji et al. tested both supernatant of various bacteria and the corresponding culture media in 1 mM AgNO3 and determined an absorbance maximum of about 1.6 cm-1 for modified NB (10 g L-1 beef extract, 10 g L-1 peptone, 5 g L-1 NaCl, pH 7) and a higher absorbance of approximately 2.3 cm-1 for the supernatant of Bacillus indicus after 4 h of incubation at ambient conditions (Shivaji et al. 2011). Similar results were obtained in the present study, where the absorbance of the incubated mixture containing STI rose to 1.24 ± 0.07 cm-1 after 3 h (figure 1b). In addition, according to our findings Natarajan et al. observed a higher absorbance of AgNO3 incubated with the supernatant of E. coli grown in LB when compared to the supernatant of NB (1 % v/v) (Natarajan et al. 2010). Therefore, both the cited and the present studies indicate an influence of the media composition and its alteration by bacterial growth on the particle formation. When the individual components of culture media were solely used, the absorbance spectra showed different trends. In addition, the sum of the absorbance maxima of the incubated mixtures containing media components differed to the maximum of the mixtures using the entire culture media. Therefore, the formation of Ag/AgCl nanoparticles appears to be due to the interaction of all medium components. Shivaij et al. tested peptone, beef extract and NaCl for their ability to form AgNPs. They observed that the individual components were less efficient in producing AgNPs compared to the supernatant of Arthrobacter kerguelensis and Pseudomonas antarctica grown in NB. In addition, a decreasing stability of the produced AgNPs was determined using bacterial supernatant, uninoculated nutrient broth and individual medium components of NB for the synthesis. Therefore, Shivaij et al. assumed that AgNP stabilizing factors were present in the bacterial supernatant. The results of the various studies indicate that the particles formation results from the interaction of all components present in the incubated mixture.
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In addition, since no Ag signal for surface plasmon resonance was observed in preliminary studies after incubation in the absence of light for 7 days (data not shown), we conclude that the reaction was based on photochemical processes. Other studies also reported that the AgNP synthesis only took place when samples were exposed to light (Minaeian et al. 2008; Mokhtari et al. 2009; Nam et al. 2008; Wei et al. 2012). Therefore, medium components and/or secreted bacterial products supported the photochemical AgNP synthesis by reaction between AgNO3 and bacterial supernatant. In order to ensure that AgNPs are produced by the microorganisms used or caused by microbial metabolites, the effects of media components and light should be excluded. Since cell extracts, and therefore cell contents can also contribute to the particle formation (Wei et al. 2012), AgNP synthesis can occur after the release of organics by dead cells as a result of osmotic pressure or silver intoxication (Morsy 2015). In view of the various parameters influencing the method of extracellular AgNP synthesis, the interpretation of the results obtained and the conclusions drawn should be carefully considered.
Acknowledgements The authors would like to thank their teams and colleagues for their kind support and scientific contribution, especially M. Knörr, S. Brümmer and F. Mohr.
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Das VL, Thomas R, Varghese RT, Soniya EV, Mathew J, Radhakrishnan EK (2014) Extracellular synthesis of silver nanoparticles by the Bacillus strain CS 11 isolated from industrialized area 3 Biotech 4:121-126 Deepak V, Kalishwaralal K, Pandian SRK, Gurunathan S (2011) An insight into the bacterial biogenesis of silver nanoparticles, industrial production and scale-up. In: Rai M, Durán N (eds) Metal nanoparticles in microbiology. Springer-Verlag, pp 17-36 Ganesh Babu MM, Gunasekaran P (2013) Extracellular synthesis of crystalline silvernanoparticles and its characterization Materials Letters 90:162–164 Gopinath V, Velusamy P (2013) Extracellular biosynthesis of silver nanoparticles using Bacillus sp. GP-23 and evaluation of their antifungal activity towards Fusarium oxysporum Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106:170–174 Guo KW (2013) Green technology for nanoparticles in biomedical applications. In: Rai M, Posten C (eds) Green biosynthesis of nanoparticles - Mechanisms and applications. CAB International, pp 1-12 Gurunathan S et al. (2009) Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli Colloids and Surfaces B: Biointerfaces 74:328-335 Iravani S, Korbekandi H, Mirmohammadi SV, Zolfaghari B (2014) Synthesis of silver nanoparticles: chemical, physical and biological methods Research in Pharmaceutical Sciences 9:385-406 Jeevan P, Ramya K, Rena AE (2012) Extracellular biosynthesis of silver nanoparticles by culture supernant of Pseudomonas aeruginosa Indian Journal of Biotechnology 11:72-76 Kalishwaralal K, Deepak V, Ramkumarpandian S, Nellaiah H, Sangiliyandi G (2008) Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheniformis Materials Letters 62:4411-4413 Kannan N, Subbalaxmi S (2011) Green synthesis of silver nanoparticles using Bacillus subtills IA751 and its antimicrobial activity Research Journal of Nanoscience and Nanotechnology 1:87-94 Klaus-Joerger T, Joerger R, Olsson E, Granqvist C (2001) Bacteria as workers in the living factory: Metal-accumulating bacteria and their potential for materials science Trends in Biotechnology 19:15-20 Kumar AS, Abyaneh MK, Gosavi SW, Kulkarni SK, Pasricha R, Ahmad A, Khan MI (2007) Nitrate reductase-mediated synthesis of silver nanoparticles from AgNO3 Biotechnology Letters 29:439-445 Loza K, Sengstock C, Chernousova S, Köller M, Epple M (2014) The predominant species of ionic silver in biological media is colloidally dispersed nanoparticulate silver chloride Royal Society of Chemistry 4:35290–35297 Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Mukherjee P (2006) The use of microorganisms for the formation of metal nanoparticles and their application Applied Microbiology and Biotechnology 69:485-492 Minaeian S, Shahverdi AR, Nohi AS, Shahverdi HR (2008) Extracellular biosynthesis of silver nanoparticles by some bacteria J Sci I A U (JSIAU) 17:1-4 Mohanpuria P, Rana NK, Yadav SK (2008) Biosynthesis of nanoparticles: technological concepts and future applications Journal of Nanoparticle Research 10:507–517 Mokhtari N et al. (2009) Biological synthesis of very small silver nanoparticles by culture supernatant of Klebsiella pneumonia: The effects of visible-light
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irradiation and the liquid mixing process Materials Research Bulletin 44:1415– 1421 Morsy FM (2015) Toward revealing the controversy of bacterial biosynthesis versus bactericidal properties of silver nanoparticles (AgNPs): bacteria and other microorganisms do not per se viably synthesize AgNPs Archives of Microbiology 197:645–655 Nam KT, Lee YJ, Krauland EM, Kottmann ST, Belcher AM (2008) Peptide-mediated reduction of silver ions on engineered biological scaffolds ACSNano 2:14801486 Narayanan KB, Sakthivel N (2010) Biological synthesis of metal nanoparticles by microbes Advances in Colloid and Interface Science 156:1-13 Natarajan K, Selvaraj S, Murty VR (2010) Microbial production of silver nanoparticles Digest Journal of Nanomaterials and Biostructures 5:135-140 Nielsen SS (2010) Sodium determination using ion selective electrodes, Mohr titration, and test strips. In: Nielson SS (ed) Food Analysis Laboratory Manual. 2nd edn. Springer Science & Buisiness Media, N.Y., USA, New York, Parikh RY, Singh S, Prasad BLV, Patole MS, Sastry M, Shouche YS (2008) Extracellular synthesis of crystalline silver nanoparticles and molecular evidence of silver resistance from Morganella sp.: Towards understanding biochemical synthesis mechanism ChemBioChem 9:1415 – 1422 doi:DOI: 10.1002/cbic.200700592 Prakash A, Sharma S, Ahmad N, Ghosh A, Sinha P (2011) Synthesis of AgNPs by Bacillus cereus bacteria and their antimicrobial potential Journal of Biomaterials and Nanobiotechnology 2:156-162 Priyadarshini S, Gopinath V, Priyadharsshini NM, MubarakAli D, Velusamy P (2013) Synthesis of anisotropic silver nanoparticles using novel strain, Bacillus flexus and its biomedical application Colloids and Surfaces B: Biointerfaces 102:232237 Rai M, Ingle AP, Gupta IR, Birla SS, Yadav AP, Abd-Elsalam KA (2013) Potential role of biological systems in formation of nanoparticles: Mechanism of synthesis and biomedical applications Current Nanoscience 9:576-587 Sharma VK, Yngard RA, Lin Y (2009) Silver nanoparticles: Green synthesis and their antimicrobial activities Advances in Colloid and Interface Science 145:83–96 Shenashen MA, El-Safty SA, Elshehy EA (2013) Synthesis, morphological control, and properties of silver nanoparticles in potential applications Particle & Particle System Characterization 96:13611-13614 Shivaji S, Madhu S, Singh S (2011) Extracellular synthesis of antibacterial silver nanoparticles using psychrophilic bacteria Process Biochemistry 46:1800–1807 Song J, Roh J, Lee I, Jang J (2013) Low temperature aqueous phase synthesis of silver/silver chloride plasmonic nanoparticles as visible light photocatalysts Dalton Transactions 42:13897–13904 Vaidyanathan R, Gopalram S, Kalishwaralal K, Deepak V, Pandian SRK, Gurunathan S (2010) Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity Colloids and Surfaces B: Biointerfaces 75:335–341 Wei X et al. (2012) Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgNO3 Bioresource Technology 103:273-278
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Table Table 1 Z-Average and PDI (n = 9) of 1mM AgNO3 incubated with uninoculated culture media or supernatant of various culture media inoculated with K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19. * significant difference to the incubated mixture containing uninoculated culture media (control)
Figures Figure 1 a) Image of 1 mM AgNO3 solutions immediately after the addition of culture media b) REM image of AgCl particles formed immediately after adding STI to AgNO 3 c) EDX-analysis of the particles shown in b d) Absorbance spectra of 1 mM AgNO3 incubated with various culture media directly after the addition and e) evolution of absorbance at 420 nm during the incubation for 7 days at ambient conditions. Figure 2 a) - f) Absorbance spectra and g) image of 1mM AgNO3 incubated (7 days) with uninoculated culture media (1 % v/v) or supernatant of various culture media (1 % v/v) inoculated with K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19, respectively. Figure 3 SEM images of 1 mM AgNO3 incubated with bacterial supernatant (1 % v/v) after 7 days of incubation at ambient conditions. Figure 4 a) SEM image of Ag/AgCl particles as observed in samples incubated 7 days with bacterial supernatants containing STI, NB, LB or MH; b) magnification of the cubes and flower; c) EDX spectrum of the particle core indicating that it is composed of
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AgCl; d) EDX spectrum of a bright dot located on the surface of the particle core, indicating that it is composed of Ag, the small contribution of chloride corresponds to the surrounding core. Figure 5 Absorbance spectra of 1 mM AgNO3 incubated with individual culture media components for 7 days; ME = meat extract, PE = peptone, YE = yeast extract.
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Figure 1
Figure 2
Figure 3_1
Figure 3_2
Figure 4
Figure 5
7d AgNO3 + MH / supernatant
7d AgNO3 + LB / supernatant
7d AgNO3 + AgNPNB / supernatant
7d AgNO3 + NB / supernatant
7d AgNO3 + MRS / supernatant
7d AgNO3 + STI / supernatant
0h AgNO3 + medium
Table 1
STI MRS NB AgNPNB LB
Z-Average mean ± SD 103.87 ± 15.23 n.a. n.a. n.a. 117.62 ± 1.44
PDI mean ± SD 0.23 ± 0.01 n.a. n.a. n.a. 0.26 ± 0.01
MH STI K. pneumoniae E. coli
118.56 ± 6.25 100.68 ± 3.20 119.90 ± 2.85* 103.21 ± 2.57
0.20 ± 0.01 0.22 ± 0.02 0.27 ± 0.06 0.19 ± 0.01
P. jessinii MRS K. pneumoniae E. coli
92.36 ± 1.50* 56.29 ± 8.29 45.70 ± 4.66* 59.05 ± 13.07
0.20 ± 0.01 0.24 ± 0.07 0.25 ± 0.04 0.31 ± 0.09
P. jessinii NB K. pneumoniae E. coli
42.70 ± 13.05* n.a. 111.18 ± 2.17 n.a.
0.22 ± 0.04 n.a. 0.16 ± 0.01 n.a.
P. jessinii AgNPNB K. pneumoniae E. coli
123.34 ± 85.76 53.23 ± 12.52 57.73 ± 9.76 n.a.
0.26 ± 0.10 0.28 ± 0.08 0.24 ± 0.07 n.a.
P. jessinii LB K. pneumoniae E. coli
n.a. 107.21 ± 4.81 105.51 ± 3.14 109.00 ± 2.96
n.a. 0.21 ± 0.02 0.21 ± 0.01 0.21 ± 0.01
P. jessinii MH K. pneumoniae E. coli
103.88 ± 3.95 110.17 ± 2.09 112.83 ± 1.39* 115.71 ± 3.36*
0.22 ± 0.01 0.21 ± 0.01 0.21 ± 0.01 0.23 ± 0.02
P. jessinii
96.63 ± 0.86*
0.25 ± 0.01
graphical abstract
PII: DOI: Reference:
S1878-8181(16)30035-4 http://dx.doi.org/10.1016/j.bcab.2016.02.012 BCAB368
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 2 November 2015 Revised date: 23 January 2016 Accepted date: 29 February 2016 Cite this article as: Alexandra Müller, Diana Behsnilian, Elke Walz, Volker Gräf, Lola Hogekamp and Ralf Greiner, Effect of culture medium on the extracellular synthesis of silver nanoparticles using Klebsiella pneumoniae, Escherichia coli a n d Pseudomonas jessinii, Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2016.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of culture medium on the extracellular synthesis of silver nanoparticles using Klebsiella pneumoniae, Escherichia coli and Pseudomonas jessinii
Alexandra Müller*, Diana Behsnilian, Elke Walz, Volker Gräf, Lola Hogekamp, Ralf Greiner
Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Department of Food Technology and Bioprocess Engineering, Haid-und-Neu-Str. 9, 76131 Karlsruhe (Germany)
*Corresponding author: Alexandra Müller, Federal Research Institute of Nutrition and Food, Department of Food Technology and Bioprocess Engineering, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany. Tel:
+49 721 6625 363, Fax: +49 721 6625 303
Email: [email protected]
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Abstract Because green biosynthesis using microorganisms is advertised as a promising, ecofriendly and biocompatible approach for the production of silver nanoparticles (AgNPs), many studies were focused on the extracellular production of nanoparticles enabling an easy downstream processing. However, the influence of the culture media on extracellular AgNP synthesis was rarely investigated. In this study, the effect of various culture media and their components on the extracellular AgNP synthesis of Klebsiella pneumoniae UVHC5, Escherichia coli ATCC 8739 and Pseudomonas jessinii UVKS19) was investigated. UV-Vis spectroscopy, DLS, SEM and EDX spectroscopy were used to verify the particles formed. Culture media, bacterial supernatants and the individual medium components were added to AgNO3 solutions. After 7 days of incubation the liquids contained particles of different shapes, sizes and composition. The formation of specific particle species mainly depended on the composition of the culture media (especially the Cl- content), in which the bacteria were inoculated. Samples incubated with bacterial supernatant of STI, NB, LB or MH showed AgCl particles of cubic or star-/flowerlike shapes coated with AgNPs. In contrast, silver nanoclusters were observed in the inoculants of bacterial supernatants grown in MRS or AgNPNB. However, small effects of bacterial metabolites on the formation of the particles could be observed and indicate an influence of the media alteration by bacterial growth on the particle formation. When the individual medium components were added to AgNO3, different absorbance spectra were obtained. Therefore, we conclude that the formation of Ag/AgCl nanoparticles results from the interaction of all medium components.
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Keywords: Culture media, Escherichia coli, extracellular synthesis, Klebsiella pneumoniae, Pseudomonas jessinii, silver nanoparticles
1 Introduction Due to the antimicrobial activity of silver, silver nanoparticles (AgNPs) gained importance in various industrial sectors. While established in the medical and textile industry, promising applications of these nanoparticles emerge in the food sector such as their inclusion in packaging materials (Guo 2013; Iravani et al. 2014; Rai et al. 2013). In order to circumvent the use of toxic substances or expensive physical methods for the production process of AgNPs, green biosynthesis using microorganisms is advertised as a promising, eco-friendly and biocompatible approach (Anthony et al. 2013; Gopinath and Velusamy 2013; Jeevan et al. 2012; Kannan and Subbalaxmi 2011; Narayanan and Sakthivel 2010; Rai et al. 2013). Therefore, the number of publications reporting the synthesis of AgNPs by the use of bacteria continuously increases (Anthony et al. 2013; Deepak et al. 2011; Mohanpuria et al. 2008; Narayanan and Sakthivel 2010; Rai et al. 2013). In this context, the AgNP synthesis is attributed to the bacterial formation of insoluble non-toxic nanoclusters by reducing or precipitating the silver ions in order to minimize the toxicity (Ganesh Babu and Gunasekaran 2013; Klaus-Joerger et al. 2001; Mandal et al. 2006; Narayanan and Sakthivel 2010; Shenashen et al. 2013). These particles can be produced either intra or extracellular (Klaus-Joerger et al. 2001; Narayanan and Sakthivel 2010). However, the underlying mechanisms of AgNP synthesis are still under investigation and various studies suggest
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the involvement of cell wall, the transmembrane proton gradient and the nitrate reductase in the formation of the nanoparticles as well as proteins and other biomolecules as stabilizers and capping agents (Deepak et al. 2011; Kumar et al. 2007; Parikh et al. 2008; Prakash et al. 2011; Priyadarshini et al. 2013; Rai et al. 2013; Sharma et al. 2009; Shenashen et al. 2013; Vaidyanathan et al. 2010). Concerning the biosynthesis, many studies were focused on extracellular methods, because of their easy downstream processing and high scale-up potential (Das et al. 2014; Gopinath and Velusamy 2013; Gurunathan et al. 2009; Priyadarshini et al. 2013). A popular and frequently used method is the addition of bacterial supernatant to AgNO3. However, the influence of the culture media on this approach of AgNP synthesis was rarely investigated. In this study, the effect of culture media on the extracellular AgNP synthesis was investigated using the supernatant of Klebsiella (K.) pneumoniae UVHC5, Escherichia (E.) coli ATCC 8739 and Pseudomonas (P.) jessinii UVKS19 grown in 6 different broths: Standard I nutrient -, de Man, Rogosa and Sharpe -, nutrient -, AgNP nutrient -, lysogeny – and Mueller Hinton broth. The mixtures (1 mM AgNO3 + 1 % v/v supernatants/corresponding uninoculated culture media (control) or the solution of individual components) were incubated at ambient conditions for 7 days. UV-Visspectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) analysis were performed in order to verify the particles formed.
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2 Materials and Methods 2.1 Culture media and bacteria For the experiments six different culture media with the following compositions (in g L1
) were used. Standard I nutrient broth (STI, Merck, Darmstadt, Germany): 15 peptone,
3 yeast extract, 6 NaCl, 1 Glucose, pH 7.5; de Man, Rogosa and Sharpe broth (MRS, Roth, Karlsruhe, Germany): 10 peptone, 4 yeast extract, 8 beef extract, 20 glucose, 2 dipotassium phosphate, 5 sodium acetate, 2 ammonium citrate, 0.05 magnesium sulfate, 1 Tween 80, pH 6.2; nutrient broth (NB, individual components were purchased from Merck, Darmstadt, Germany): 5 peptone, 3 meat extract, pH 7.0; AgNP nutrient broth (AgNPNB, individual components were purchased from Merck, Darmstadt, Germany): 10 peptone, 3 yeast extract, 2 glucose, pH 7.0; lysogeny broth (LB, individual components were purchased from Merck, Darmstadt, Germany): 10 peptone, 5 yeast extract, 10 NaCl, pH 7; Mueller Hinton broth (MH, Merck, Darmstadt, Germany): 17.5 casein hydrolysate, 2 meat infusion, 1.5 starch, pH 7.4. For the production of STI-agar, 15 g L-1 agar-agar (Roth, Karlsruhe, Germany) was added to STI broth. In order to investigate the effect of the individual components on the AgNP synthesis, the chemicals were solved in deionized water in the given quantity. All media were prepared in the given composition and autoclaved at 121 °C for 20 min. STI-agar was then poured in 94x16 mm petri dishes. The chloride concentration of the culture media was determined by titration with silver nitrate according to the Mohr method (Nielsen 2010). The following three bacteria were used in this study. K. pneumoniae UVHC5 was isolated from tiger nut (Cyperus esculentus). This ubiquitous bacterium naturally occurs in the soil, but forms also a normal part of human microbiota in the mouth and
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intestines. P. jessinii UVKS19 was gained by isolation from carrot juice. As residents of the upper layers of the soil, pseudomonades are frequently found food contaminants. Escherichia coli ATCC 8739 isolated from feces is a standard organism for several quality controls and food testing. Bacteria were stored in cryo vials and sub-cultured twice in STI broth before used in experiments.
2.2 Synthesis of AgNPs Bacterial overnight cultures (K. pneumoniae UVHC5, E. coli ATCC 8739, P. jessinii UVKS19) were inoculated (1 % v/v) in the six different culture media and grown at 30 °C for 18 h. In order to enumerate the viable counts 1 ml of the overnight cultures was taken, ten-fold serially diluted in Quarter-strength Ringer’s solution (Merck, Darmstadt, Germany) and plated out on STI-agar. After incubation for 18 h at 30 °C, colonies were enumerated. The remaining overnight culture was centrifuged at 3220 g for 15 min and the supernatant was sterile-filtered through a 0.2 µm filter (CHROMAFIL®PET-20/25, Macherey-Nagel GmbH & Co. KG, Düren, Germany). In order to synthesize AgNPs, 200 µl of the supernatants as well as 200 µl of the corresponding uninoculated culture media (control) or the solution of individual components (except dipotassium phosphate, ammonium citrate and magnesium sulfate as present in MRS) were added to 20 ml of 1 mM AgNO3 aqueous solutions and incubated at ambient conditions (room temperature = 22 ± 2 °C and normal daylight). The formation of AgNPs was daily characterized by UV-Vis analyses as well as DLS. All experiments were done in triplicate.
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2.3 Characterization of AgNPs 2.3.1 UV-Vis spectrophotometry Because of their unique optical properties noble metals as Ag-dispersion can be characterized by UV-VIS spectroscopy using the surface plasmon resonance effect. Therefore, the UV-Vis spectra of the incubated AgNO3 solutions were recorded between 300 – 700 nm in 2 nm intervals using a UV/Vis-Spectrometer (UV.2, UNICAM, Kassel, Germany) in order to receive information about the presence of AgNPs in the reaction mixtures.
2.3.2 DLS The formation of particles was verified by particle size measurement using dynamic light scattering (DLS) performed on a Zetasizer Nano ZS (Malvern Instruments Ltd, UK) with temperature control (25 °C). The correlation functions were analyzed using the General Purpose Model algorithm of the instrument software (version 7.02); viscosity and refractive index (RI) of the dispersion media were assumed as viscosity and RI of water (0.8872 cP; 1.330). To avoid erroneous data because of the different refractivities of silver and silver chloride particles, in this study only the refractivity independent harmonic intensity-weighted arithmetic average particle diameter (ZAverage) and polydispersity index (PDI) has been used for characterization. The PDI is a dimensionless value, which gives an indication of the width of the particle size distributions. With PDI values of about 0.2 a moderate polydisperse size distribution can be assumed. Each sample was measured in triplicate.
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2.3.3 SEM and EDX The verification of the particles species (Ag or Ag/AgCl nanoparticles, respectively) was determined by SEM and EDX analyses, which were conducted on day 7 after addition of media or supernatants to AgNO3. Furthermore, analyses were also conducted directly after addition of STI or NB to AgNO3. Morphological analysis of the particles contained in all samples was carried out using a FEG-SEM Quanta 250 (FEI, Czech Republic) equipped with Everhart-Thornley (ETD) and gaseous analytical backscattered electrons (GAD) detectors. Energy-dispersive Xray spectroscopy was used for elemental analysis of the particles (Apollo X SDD, EDAX, USA). Analytical conditions were: acceleration voltage 10 kV; working distance 10 mm (optimal distance for EDX analysis). Sample aliquots (10 µL) were deposited onto silicium substrates which were mounted onto aluminum stubs with double coated carbon conductive tabs; samples were then left to dry in a desiccator.
2.3.4 Statistical analysis The values of the absorbance at 420 nm as well as Z-Averages of AgNO3 incubated with culture media or supernatant of culture media inoculated with bacteria were checked for significant differences using a t-test (Sigma Plot for Windows 12.3, Systat Software GmbH, Erkrath, Germany). The confidence level was set at 95 % (p < 0.05).
3 Results 3.1 Culture media mediated synthesis of AgNPs
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Figure 1 a shows an image of 1 mM AgNO3 solutions immediately after adding the culture media (1 % v/v) and figure 1 d shows the corresponding absorbance spectra. The turbidity determined indicates the immediate formation of AgCl in AgNO3 incubated with STI, LB and MH and correlates well with the Cl- concentration of the media, in which values of 6.16 ± 0.02 g L-1 (LB), 4.05 ± 0.03 g L-1 (MH), 3.93 ± 0.01 g L-1 (STI), 0.35 ± 0.02 g L-1 (MRS), 0.22 ± 0.01 g L-1 (NB) and 0.12 ± 0.01 g L-1 (AgNPNB) were measured. DLS analysis confirmed the formation of particles (table 1) whereby mean particle diameters (Z-Average) of about 100 nm were determined in the incubated mixtures containing STI, LB and MH. In the samples incubated with MRS, NB and AgNPNB culture medium, the concentration of particles directly after the addition of media was too low for DLS analysis. The detection limit of the DLS technique depends on the amount of scattered light caused by the particles. If the difference in scattering between the dispersant and the sample is too low, the quality of the raw data is too poor for particles sizes analysis. The very fast formation of AgCl particles after addition of medium, which contains a high Cl- concentration (ST I, LB, MH), to AgNO3 solution was confirmed using SEM/EDX analysis (figure 1b, c). Here, the REM image and the EDX analysis of the AgCl particles formed immediately after adding STI to AgNO3 is also representative for the particles as present after the addition of LB and MH to silver nitrate. The EDX spectrum (figure 1c) of the cubic particles visible in figure 1b shows strong characteristic signals for Ag as well as for Cl, indicating that they consist of AgCl. The high Si peak and the small C peak are caused by the substrate on which the sample was deposited. Traces of Na resulting from the culture media were also found by EDX analysis. During 7 days of incubation at ambient conditions the absorbance at 420 nm of AgNO3 incubated with culture media significantly (p < 0.05) increased, mainly rising in the first
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hours of incubation and indicates a culture media mediated formation of AgNPs (figure 1e). The highest increase of the absorbance at 420 nm (A420nm 0.334 ± 0.008 cm-1 up to 2.335 ± 0.146 cm-1) was observed in AgNO3 incubated with STI, followed by incubated mixtures containing LB and MH. Here, the values at 420 nm rose from 0.637 ± 0.117 cm-1 and 0.736 ± 0.051 cm-1 up to 1.781 ± 0.126 cm-1 and 1.519 ± 0.106 cm-1, respectively. In contrast, the absorbance at 420 nm of the sample containing AgNPNB only slightly increased from 0.002 ± 0.001 cm-1 to 0.108 ± 0.015 cm-1 during 7 days of incubation. Whereas, similar absorbance at 420 nm was determined for the samples incubated with MRS and NB, rising from 0.030 ± 0.011 cm-1 and 0.006 ± 0.001 cm-1 up to 0.625 ± 0.172 cm-1 and 0.384 ± 0.156 cm-1, respectively, at day 7 (figure 1e).
3.2 Effect of culture media on the extracellular synthesis of AgNPs After 7 days of incubation at ambient conditions the incubated mixtures of AgNO3 with culture media or supernatant of K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19 grown in various media contained different concentrations of particles with different sizes and shapes resulting in different signals of surface plasmon resonance (figure 2 and 3, table 1). As shown in figure 2, the absorbance spectra of the incubated mixtures mainly depended on the media, in which the bacteria were inoculated. Therefore, the shapes of the absorbance spectra were similar for each medium tested, independent of whether they were previously inoculated or not with bacteria. However, small effects of bacterial metabolites on the formation of the particles could be observed. A significant (p < 0.05) reduced absorbance at 420 nm was determined when AgNO3 was incubated with the supernatant of E. coli ATCC 8739 grown in STI compared to the incubation using uninoculated STI (figure 2a). In contrast, the bacterial growth in MRS did not significantly affect the particle formation,
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although the supernatant of K. pneumoniae UVHC5 tended to result in a higher (whereas E. coli ATCC 8739 a lower) absorbance at 420 nm (figure 2b). However, different absorbance spectra were determined for samples incubated using the supernatant of K. pneumoniae UVHC5 and P. jessinii UVKS19 grown in NB compared to the control (incubation with uninoculated NB) (figure 2c), whereby significant higher absorbances at 420 nm were observed when using the supernatant of K. pneumoniae UVHC5 and P. jessinii UVKS19. In addition, despite the general low absorbance of the samples containing AgNPNB medium (figure 2d), the bacterial metabolites significantly increased the signal of surface plasmon resonance of the incubated mixture and therefore, promoted particle formation. These results could also be visually confirmed, as shown in figure 2g. Here, the samples containing AgNPNB or supernatants of E. coli ATCC 8739, P. jessinii UVKS19 and K. pneumoniae UVHC5 got darker in ascending order. In contrast, no significant difference was observed using LB culture medium (figure 2e) and only P. jessinii UVKS19 significantly affected the absorbance spectra of the samples incubated with MH medium (figure 2f). Although, significant effects of bacterial growth on the formation of the particles were detected, no correlation between absorbance spectra and bacterial counts could be observed (data of the bacterial counts not shown). The results of DLS indicated the presence of particles after 7 days of incubation (table 1). Here Z-Averages of about 100 nm were measured in samples containing both uninoculated culture media and bacterial supernatant with exception of the incubated mixture of MRS and AgNPNB. In this context, Z-Averages of about 50 nm were observed and suggest the presence of AgNPs or Ag nanoclusters. In several cases, a significant effect (p < 0.05) of bacterial growth in the culture media on the particle size was determined regarding the particles formation. E.g. the incubated
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mixtures containing the supernatant of K. pneumoniae UVHC5 and E. coli ATCC 8739 in STI and MRS showed significant differences in particle size after 7 days of incubation compared to the addition of uninoculated culture media, as well as the bacterial growth in MH resulted in significant different particle size determined (table 1). The overall appearance of UV-Vis and DLS measurements was confirmed by SEM and EDX analysis after 7 days of incubation (figure 3 and 4). Correlating to the Cl - content of the media, samples incubated with bacterial supernatant grown in STI, LB and MH show sharp-edged AgCl particles of cubic or star-/flowerlike shapes coated with silver nanoparticles. Whereas, the basic structure of the Ag/AgCl particles presented in the samples containing NB was not sharp-edged but rounder (figure 3). In contrast, silver nanoclusters were observed in the incubated mixtures of bacterial supernatants grown in MRS and AgNPNB (figure 3). Figure 4a shows a SEM image of Ag/AgCl particles (particles magnified in figure 4b) as observed in samples incubated 7 days with bacterial supernatants containing STI, NB, LB or MH. In this connection, EDX measurements were performed on a homogenous spot near the center of the star shaped particle near the left edge of image 4a (spectrum in 4c), as well as on one of the small spherical particles attached to the surface of the large structure (figure 4d). The spectrum in figure 4c shows Ag and Cl peaks of similar magnitude, indicating that the particle is composed of AgCl at this location. In contrast, the Cl peak of the spectrum shown in figure 4d is negligible compared to the Ag peak, indicating that the spherical nanoparticle on the surface of the star shaped particle consists of silver. Here, the Cl signal is due to the volume below the targeted AgNP.
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3.3 The role of individual culture medium components in the extracellular synthesis of AgNPs The absorbance spectra of 1 mM AgNO3 incubated (7 days, ambient conditions) with individual culture media components having different curve shapes are shown in figure 5. Here, the highest signal of surface plasmon resonance (Amax approximately 0.6 cm-1) was observed in the samples containing 8 g L-1 meat extract or 5 g L-1 yeast extract, as the components and concentrations are present in MRS and LB, respectively. Lower absorbance maxima were determined with decreasing concentrations of meat and yeast extract as well as in samples incubated with 17.5 g L-1 peptone and 17.5 g L-1 NaCl. All other concentrations of medium components tested resulted in no or only a small signal and therefore, absorbance spectra were not included in figure 5. However, the incubation of 1 mM AgNO3 with culture media and medium components resulted in different absorbance spectra. In addition, the sum of the absorbance maxima of the incubated mixtures containing media components differed from the maximum of the mixtures using the entire culture media. E.g. an absorbance maximum of 2.33 ± 0.15 cm-1 was measured for 1 mM AgNO3 incubated with STI (figure 2a), whereas the sum of Amax containing solely the medium components amounted about 0.37 cm-1 (figure 5). In general, a higher absorbance maximum was calculated for the medium components compared to the surface plasmon resonance signal of the corresponding culture medium, except for MRS and AgNPNB samples. Here, the single absorbance maxima of the components (approximately 0.95 and 0.37 cm-1) exceeded the absorbance of the culture media (0.64 ± 0.18 and 0.11 ± 0.02 cm-1).
4 Discussion
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In this study, the effect of various culture media and their media components on the extracellular AgNP synthesis of K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19 was investigated using UV-Vis-spectroscopy, DLS, SEM and EDX analysis in order to characterize the particles formed. The spontaneous formation of large particles of about 100 nm diameter after addition of media containing high Cl- concentrations to AgNO3 solution in contrast to the addition of media with low Cl- concentrations is an indication of the formation of AgCl particles rather than silver nanoparticles or reaction products caused by other medium components. In addition, the formation of the AgCl particles, which were determined by UV-Vis and DLS was verified by SEM/EDX analysis. The results of particle size measurements of the present study are in good accordance with those of the study of Song et al. (Song et al. 2013), where the edge length of AgCl nanocubes produced using AgNO3, Poly-vinyl alcohol (PVA) and NaCl was between 57-170 nm depending on process temperature. In addition, Loza et al. determined the precipitation of AgCl when adding AgNO3 to biological media such as phosphate-buffered saline solution (PBS) or LB medium due to the presence of Cl- in the media. The authors conclude that the size of AgCl particles formed depends on the presence of biomolecules, which coat the particles and prevent them from further growth (Loza et al. 2014). In the study of Mokhtari et al., a white precipitate was observed in the reaction vessels after the addition of culture supernatant of Klebsiella pneumonia grown in MH media (Mokhtari et al. 2009). Here again, subsequent XRD analysis confirmed the presence of AgCl particles, which were ascribed to the presence of Cl- ions in the culture media. Our results show that during 7 days of incubation at ambient conditions the absorbance at 420 nm of AgNO3 incubated with culture media significantly increased and indicates a culture media mediated formation of AgNPs. After 7 days of incubation, the mixture
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of AgNO3 with culture media or supernatant contained particles of different concentrations, size and shapes resulting in different signals of surface plasmon resonance, DLS and SEM/EDX analysis. In this context, the formation of specific particle species mainly depended on the composition of culture media, in which the bacteria were inoculated. Samples incubated with bacterial supernatant grown in STI, LB or MH show sharp-edged AgCl particles of cubic or star-/flowerlike shapes coated with silver nanoparticles. Whereas, the basic structure of the Ag/AgCl particles of NB samples were rounder. In contrast, silver nanoclusters were observed in the incubated mixtures of bacterial supernatants grown in MRS or AgNPNB. However, some small effects of bacterial metabolites on the formation of the particles could be observed by UV-Vis and DLS. These results indicate that both the original composition of the media and the modified composition of the media, which results as a consequence of bacterial growth, play an important role on the particle formation. The kinetic of maximum absorbance using the supernatant of E. coli and Morganella spp. grown in NB, LB and LB without NaCl was investigated by several authors (Gurunathan et al. 2009; Natarajan et al. 2010; Parikh et al. 2008). In all those studies the highest increase in the absorbance was observed in the first hours of incubation and matches very well with our results concerning the addition of uninoculated culture media. Numerous studies report a surface plasmon resonance signal of AgNP particles after incubation of AgNO3 with the supernatant of various bacteria grown in several culture media (such as NB, LB and MH) at ambient conditions (Das et al. 2014; Ganesh Babu and Gunasekaran 2013; Gopinath and Velusamy 2013; Jeevan et al. 2012; Kalishwaralal et al. 2008; Kannan and Subbalaxmi 2011; Minaeian et al. 2008; Natarajan et al. 2010; Priyadarshini et al. 2013). Unfortunately, most of these studies did not check the absorbance spectra of the incubated mixture applying the uninoculated corresponding culture media. However,
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Shivaji et al. tested both supernatant of various bacteria and the corresponding culture media in 1 mM AgNO3 and determined an absorbance maximum of about 1.6 cm-1 for modified NB (10 g L-1 beef extract, 10 g L-1 peptone, 5 g L-1 NaCl, pH 7) and a higher absorbance of approximately 2.3 cm-1 for the supernatant of Bacillus indicus after 4 h of incubation at ambient conditions (Shivaji et al. 2011). Similar results were obtained in the present study, where the absorbance of the incubated mixture containing STI rose to 1.24 ± 0.07 cm-1 after 3 h (figure 1b). In addition, according to our findings Natarajan et al. observed a higher absorbance of AgNO3 incubated with the supernatant of E. coli grown in LB when compared to the supernatant of NB (1 % v/v) (Natarajan et al. 2010). Therefore, both the cited and the present studies indicate an influence of the media composition and its alteration by bacterial growth on the particle formation. When the individual components of culture media were solely used, the absorbance spectra showed different trends. In addition, the sum of the absorbance maxima of the incubated mixtures containing media components differed to the maximum of the mixtures using the entire culture media. Therefore, the formation of Ag/AgCl nanoparticles appears to be due to the interaction of all medium components. Shivaij et al. tested peptone, beef extract and NaCl for their ability to form AgNPs. They observed that the individual components were less efficient in producing AgNPs compared to the supernatant of Arthrobacter kerguelensis and Pseudomonas antarctica grown in NB. In addition, a decreasing stability of the produced AgNPs was determined using bacterial supernatant, uninoculated nutrient broth and individual medium components of NB for the synthesis. Therefore, Shivaij et al. assumed that AgNP stabilizing factors were present in the bacterial supernatant. The results of the various studies indicate that the particles formation results from the interaction of all components present in the incubated mixture.
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In addition, since no Ag signal for surface plasmon resonance was observed in preliminary studies after incubation in the absence of light for 7 days (data not shown), we conclude that the reaction was based on photochemical processes. Other studies also reported that the AgNP synthesis only took place when samples were exposed to light (Minaeian et al. 2008; Mokhtari et al. 2009; Nam et al. 2008; Wei et al. 2012). Therefore, medium components and/or secreted bacterial products supported the photochemical AgNP synthesis by reaction between AgNO3 and bacterial supernatant. In order to ensure that AgNPs are produced by the microorganisms used or caused by microbial metabolites, the effects of media components and light should be excluded. Since cell extracts, and therefore cell contents can also contribute to the particle formation (Wei et al. 2012), AgNP synthesis can occur after the release of organics by dead cells as a result of osmotic pressure or silver intoxication (Morsy 2015). In view of the various parameters influencing the method of extracellular AgNP synthesis, the interpretation of the results obtained and the conclusions drawn should be carefully considered.
Acknowledgements The authors would like to thank their teams and colleagues for their kind support and scientific contribution, especially M. Knörr, S. Brümmer and F. Mohr.
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Das VL, Thomas R, Varghese RT, Soniya EV, Mathew J, Radhakrishnan EK (2014) Extracellular synthesis of silver nanoparticles by the Bacillus strain CS 11 isolated from industrialized area 3 Biotech 4:121-126 Deepak V, Kalishwaralal K, Pandian SRK, Gurunathan S (2011) An insight into the bacterial biogenesis of silver nanoparticles, industrial production and scale-up. In: Rai M, Durán N (eds) Metal nanoparticles in microbiology. Springer-Verlag, pp 17-36 Ganesh Babu MM, Gunasekaran P (2013) Extracellular synthesis of crystalline silvernanoparticles and its characterization Materials Letters 90:162–164 Gopinath V, Velusamy P (2013) Extracellular biosynthesis of silver nanoparticles using Bacillus sp. GP-23 and evaluation of their antifungal activity towards Fusarium oxysporum Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 106:170–174 Guo KW (2013) Green technology for nanoparticles in biomedical applications. In: Rai M, Posten C (eds) Green biosynthesis of nanoparticles - Mechanisms and applications. CAB International, pp 1-12 Gurunathan S et al. (2009) Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli Colloids and Surfaces B: Biointerfaces 74:328-335 Iravani S, Korbekandi H, Mirmohammadi SV, Zolfaghari B (2014) Synthesis of silver nanoparticles: chemical, physical and biological methods Research in Pharmaceutical Sciences 9:385-406 Jeevan P, Ramya K, Rena AE (2012) Extracellular biosynthesis of silver nanoparticles by culture supernant of Pseudomonas aeruginosa Indian Journal of Biotechnology 11:72-76 Kalishwaralal K, Deepak V, Ramkumarpandian S, Nellaiah H, Sangiliyandi G (2008) Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheniformis Materials Letters 62:4411-4413 Kannan N, Subbalaxmi S (2011) Green synthesis of silver nanoparticles using Bacillus subtills IA751 and its antimicrobial activity Research Journal of Nanoscience and Nanotechnology 1:87-94 Klaus-Joerger T, Joerger R, Olsson E, Granqvist C (2001) Bacteria as workers in the living factory: Metal-accumulating bacteria and their potential for materials science Trends in Biotechnology 19:15-20 Kumar AS, Abyaneh MK, Gosavi SW, Kulkarni SK, Pasricha R, Ahmad A, Khan MI (2007) Nitrate reductase-mediated synthesis of silver nanoparticles from AgNO3 Biotechnology Letters 29:439-445 Loza K, Sengstock C, Chernousova S, Köller M, Epple M (2014) The predominant species of ionic silver in biological media is colloidally dispersed nanoparticulate silver chloride Royal Society of Chemistry 4:35290–35297 Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Mukherjee P (2006) The use of microorganisms for the formation of metal nanoparticles and their application Applied Microbiology and Biotechnology 69:485-492 Minaeian S, Shahverdi AR, Nohi AS, Shahverdi HR (2008) Extracellular biosynthesis of silver nanoparticles by some bacteria J Sci I A U (JSIAU) 17:1-4 Mohanpuria P, Rana NK, Yadav SK (2008) Biosynthesis of nanoparticles: technological concepts and future applications Journal of Nanoparticle Research 10:507–517 Mokhtari N et al. (2009) Biological synthesis of very small silver nanoparticles by culture supernatant of Klebsiella pneumonia: The effects of visible-light
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irradiation and the liquid mixing process Materials Research Bulletin 44:1415– 1421 Morsy FM (2015) Toward revealing the controversy of bacterial biosynthesis versus bactericidal properties of silver nanoparticles (AgNPs): bacteria and other microorganisms do not per se viably synthesize AgNPs Archives of Microbiology 197:645–655 Nam KT, Lee YJ, Krauland EM, Kottmann ST, Belcher AM (2008) Peptide-mediated reduction of silver ions on engineered biological scaffolds ACSNano 2:14801486 Narayanan KB, Sakthivel N (2010) Biological synthesis of metal nanoparticles by microbes Advances in Colloid and Interface Science 156:1-13 Natarajan K, Selvaraj S, Murty VR (2010) Microbial production of silver nanoparticles Digest Journal of Nanomaterials and Biostructures 5:135-140 Nielsen SS (2010) Sodium determination using ion selective electrodes, Mohr titration, and test strips. In: Nielson SS (ed) Food Analysis Laboratory Manual. 2nd edn. Springer Science & Buisiness Media, N.Y., USA, New York, Parikh RY, Singh S, Prasad BLV, Patole MS, Sastry M, Shouche YS (2008) Extracellular synthesis of crystalline silver nanoparticles and molecular evidence of silver resistance from Morganella sp.: Towards understanding biochemical synthesis mechanism ChemBioChem 9:1415 – 1422 doi:DOI: 10.1002/cbic.200700592 Prakash A, Sharma S, Ahmad N, Ghosh A, Sinha P (2011) Synthesis of AgNPs by Bacillus cereus bacteria and their antimicrobial potential Journal of Biomaterials and Nanobiotechnology 2:156-162 Priyadarshini S, Gopinath V, Priyadharsshini NM, MubarakAli D, Velusamy P (2013) Synthesis of anisotropic silver nanoparticles using novel strain, Bacillus flexus and its biomedical application Colloids and Surfaces B: Biointerfaces 102:232237 Rai M, Ingle AP, Gupta IR, Birla SS, Yadav AP, Abd-Elsalam KA (2013) Potential role of biological systems in formation of nanoparticles: Mechanism of synthesis and biomedical applications Current Nanoscience 9:576-587 Sharma VK, Yngard RA, Lin Y (2009) Silver nanoparticles: Green synthesis and their antimicrobial activities Advances in Colloid and Interface Science 145:83–96 Shenashen MA, El-Safty SA, Elshehy EA (2013) Synthesis, morphological control, and properties of silver nanoparticles in potential applications Particle & Particle System Characterization 96:13611-13614 Shivaji S, Madhu S, Singh S (2011) Extracellular synthesis of antibacterial silver nanoparticles using psychrophilic bacteria Process Biochemistry 46:1800–1807 Song J, Roh J, Lee I, Jang J (2013) Low temperature aqueous phase synthesis of silver/silver chloride plasmonic nanoparticles as visible light photocatalysts Dalton Transactions 42:13897–13904 Vaidyanathan R, Gopalram S, Kalishwaralal K, Deepak V, Pandian SRK, Gurunathan S (2010) Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity Colloids and Surfaces B: Biointerfaces 75:335–341 Wei X et al. (2012) Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgNO3 Bioresource Technology 103:273-278
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Table Table 1 Z-Average and PDI (n = 9) of 1mM AgNO3 incubated with uninoculated culture media or supernatant of various culture media inoculated with K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19. * significant difference to the incubated mixture containing uninoculated culture media (control)
Figures Figure 1 a) Image of 1 mM AgNO3 solutions immediately after the addition of culture media b) REM image of AgCl particles formed immediately after adding STI to AgNO 3 c) EDX-analysis of the particles shown in b d) Absorbance spectra of 1 mM AgNO3 incubated with various culture media directly after the addition and e) evolution of absorbance at 420 nm during the incubation for 7 days at ambient conditions. Figure 2 a) - f) Absorbance spectra and g) image of 1mM AgNO3 incubated (7 days) with uninoculated culture media (1 % v/v) or supernatant of various culture media (1 % v/v) inoculated with K. pneumoniae UVHC5, E. coli ATCC 8739 and P. jessinii UVKS19, respectively. Figure 3 SEM images of 1 mM AgNO3 incubated with bacterial supernatant (1 % v/v) after 7 days of incubation at ambient conditions. Figure 4 a) SEM image of Ag/AgCl particles as observed in samples incubated 7 days with bacterial supernatants containing STI, NB, LB or MH; b) magnification of the cubes and flower; c) EDX spectrum of the particle core indicating that it is composed of
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AgCl; d) EDX spectrum of a bright dot located on the surface of the particle core, indicating that it is composed of Ag, the small contribution of chloride corresponds to the surrounding core. Figure 5 Absorbance spectra of 1 mM AgNO3 incubated with individual culture media components for 7 days; ME = meat extract, PE = peptone, YE = yeast extract.
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Figure 1
Figure 2
Figure 3_1
Figure 3_2
Figure 4
Figure 5
7d AgNO3 + MH / supernatant
7d AgNO3 + LB / supernatant
7d AgNO3 + AgNPNB / supernatant
7d AgNO3 + NB / supernatant
7d AgNO3 + MRS / supernatant
7d AgNO3 + STI / supernatant
0h AgNO3 + medium
Table 1
STI MRS NB AgNPNB LB
Z-Average mean ± SD 103.87 ± 15.23 n.a. n.a. n.a. 117.62 ± 1.44
PDI mean ± SD 0.23 ± 0.01 n.a. n.a. n.a. 0.26 ± 0.01
MH STI K. pneumoniae E. coli
118.56 ± 6.25 100.68 ± 3.20 119.90 ± 2.85* 103.21 ± 2.57
0.20 ± 0.01 0.22 ± 0.02 0.27 ± 0.06 0.19 ± 0.01
P. jessinii MRS K. pneumoniae E. coli
92.36 ± 1.50* 56.29 ± 8.29 45.70 ± 4.66* 59.05 ± 13.07
0.20 ± 0.01 0.24 ± 0.07 0.25 ± 0.04 0.31 ± 0.09
P. jessinii NB K. pneumoniae E. coli
42.70 ± 13.05* n.a. 111.18 ± 2.17 n.a.
0.22 ± 0.04 n.a. 0.16 ± 0.01 n.a.
P. jessinii AgNPNB K. pneumoniae E. coli
123.34 ± 85.76 53.23 ± 12.52 57.73 ± 9.76 n.a.
0.26 ± 0.10 0.28 ± 0.08 0.24 ± 0.07 n.a.
P. jessinii LB K. pneumoniae E. coli
n.a. 107.21 ± 4.81 105.51 ± 3.14 109.00 ± 2.96
n.a. 0.21 ± 0.02 0.21 ± 0.01 0.21 ± 0.01
P. jessinii MH K. pneumoniae E. coli
103.88 ± 3.95 110.17 ± 2.09 112.83 ± 1.39* 115.71 ± 3.36*
0.22 ± 0.01 0.21 ± 0.01 0.21 ± 0.01 0.23 ± 0.02
P. jessinii
96.63 ± 0.86*
0.25 ± 0.01
graphical abstract