Microbial biosynthesis of silver nanoparticles in different culture media

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Microbial biosynthesis of silver nanoparticles in different culture media Ke Luo, Samuel Jung, Kyu-Hwan Park, and Young-Rok Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05092 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Journal of Agricultural and Food Chemistry

Microbial Biosynthesis of Silver Nanoparticles in Different Culture Media

Ke Luo,† Samuel Jung,† Kyu-Hwan Park and Young-Rok Kim*

Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 17104, Korea

*

Corresponding author: [email protected]

Phone: +82-31-201-3830; Fax: +82-31-204-8116

† K.L. and S.J. contributed equally to this work.

1

ACS Paragon Plus Environment


1

ABSTRACT

2

Microbial biosynthesis of metal nanoparticles has been extensively studied for the

3

applications in biomedical sciences and engineering. However, the mechanism for

4

their synthesis through microorganism is not completely understood. In this study,

5

several culture media were investigated for their roles in the microbial biosynthesis of

6

silver nanoparticles (AgNPs). The size and morphology of the synthesized AgNPs

7

were analyzed by UV–Vis spectroscopy, FT-IR, TEM, and DLS. The results

8

demonstrated that nutrient broth (NB) and Mueller-Hinton broth (MHB) among tested

9

media effectively reduced silver ions to form AgNPs with different particle size and

10

shape. Although the involved microorganism enhanced the reduction of silver ions,

11

the size and shape of the particles were shown to mainly depend on the culture media.

12

Our findings suggest that the growth media of bacterial culture play an important role

13

in the synthesis of metallic nanoparticles with regard to their size and shape. We

14

believe our findings would provide useful information for further exploration of

15

microbial biosynthesis of AgNPs and their biomedical applications.

16 17

KEYWORDS: silver nanoparticles, microbial biosynthesis, extracellular, culture

18

media, nutrient broth, Mueller-Hinton broth

19 20

2


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INTRODUCTION

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Metal nanoparticles have attracted considerable attention due to their unique

23

physicochemical characteristics including catalytic activity, optical properties,

24

electronic properties, antimicrobial activity and magnetic properties, etc.1 Amongst

25

them, silver nanoparticles (AgNPs) are used extensively in various applications that

26

includes disinfecting medical devices and home appliances to purifying drinking

27

water.2-4 AgNPs are also well-known antimicrobial agent against various pathogenic

28

microorganisms.5 Furthermore, owing to their unique plasmon-resonance optical

29

scattering properties, AgNPs are currently recognized for their potentials in bio-

30

sensing, medical diagnostics and therapeutics, and biological imaging applications.6-8

31

Due to the environmental issues raised over the past decades, the synthesis of

32

metal nanoparticles by biological process has drawn a great deal of attention.9

33

Numerous biosynthesis methods utilizing bacteria, fungi, plant, and plant extract as

34

reducing agents for the synthesis of AgNPs have been exploited in recent years.5, 10-14

35

Amongst them, the synthesis of AgNPs by living microorganisms is a well-known

36

biomimetic approach, since some microorganisms have been found to synthesize

37

inorganic materials, such as gold nanoparticles,15 magnetite nanoparticles,16 silver

38

nanoparticles,17 and zinc sulfide nanoparticles.18 Bacteria is known to reduce metal

39

ions to metal nanoparticles either intra- or extracellularly. However, the mechanism

40

for intra- and extracellular synthesis of nanoparticles differs depending on the

41

biological agents. In the intracellular synthesis of nanoparticles, metal ions are

42

uptaken by certain microorganisms and bind to either a metal ion reductase or similar

43

proteins, resulting in the reduction of the ions to elemental metals in their zero-valent

44

form, followed by the formation of nanoparticles in cytoplasm.19 In this case, an extra

45

step is required for the purification of the produced nanoparticles if they are to be

46

used for further applications. The electrostatic interaction of the positively charged 3



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metal ions and negatively charged cell wall of the bacteria play important role in ion

48

transportation in the microbial cell. The intracellularly synthesized metallic

49

nanoparticles could eventually be effluxed out of the cell.20 On the other hands, the

50

extracellular synthesis of metal nanoparticles is generally carried out by the reductive

51

components released from the cell. For example, DNA, NADH-dependent reductase

52

and sulfur-contained protein secreted from the cell can induce the reduction of Ag+

53

ion to Ag0 stage leading to the formation of AgNPs.21-23 Since the electronic and

54

optical properties of metal nanoparticles are size- and shape-dependent, the control of

55

size and shape of the particles is one of the most important aspect for their specific

56

applications.24 Moreover, previous studies have reported that antimicrobial nature of

57

AgNPs showed to be depend on their size and shape, where smaller nanoparticles

58

displayed better antimicrobial activity.23 It has been demonstrated that AgNPs with

59

different

60

microorganisms.25 However, the effect of the growth environment, such as culture

61

medium, on the synthesis of AgNPs in a way of affecting their size and shape is not

62

fully understood. In addition, Herein, we report the effect of culture media and several

63

media components on the synthesis of AgNPs in terms of the size and morphology of

64

the particles.

sizes

and

shapes

can

be

synthesized

by

employing

specific

65 66

67

Media, chemicals, and microorganism. Mueller-Hinton broth (MHB) was

68

purchased from Difco (Lawrence, USA). Yeast extract was purchased from Daejung

69

chemical Co. (Siheung, Korea). Peptone A and beef extract were purchased from

70

BIOSESANG (Seongnam, Korea). Silver (I) nitrate was purchased from Daejung

71

chemical Co. (Siheung, Korea). Klebsiella pneumoniae KCTC 2242 and Escherichia

72

coli DH5α were obtained from the Korean Collection for Type Cultures (Daejeon,

MATERIALS AND METHODS

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Korea).

74 75 76

Preparation of culture mediums. To synthesize silver nanoparticles, thirteen

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types of culture mediums were prepared as reducing agent. Abbreviation of different

78

reducing agents was listed in Table 1. MHB, beef extract, peptone A, and yeast extract

79

solutions were prepared by dissolving 21g of MHB, 3g of beef extract, 5g of peptone

80

A, and 5g of yeast extract in 1L distilled water, respectively. Nutrient broth without

81

NaCl (NB) was prepared by dissolving 5g of peptone A and 3g of beef extract in 1L

82

distilled water. NaCl was excluded when preparing Nutrient broth to prevent Cl- effect

83

that could interact with silver ion and produce AgCl sediment. For preparation of

84

culture supernatant (N-KP S, N-EC S, M-KP S, and M-EC S) and inoculum (N-KP I,

85

N-EC I, M-KP I, and M-EC I), MHB and NB media were inoculated with K.

86

pneumoniae or E. coli, and incubated in shaking incubator (HB-201SL, Hanbaek

87

Scientific Co., Korea) at 220 rpm and 37 °C until the inoculum reached an O.D600 of

88

1.0, reflecting the number of K. pneumoniae and E. coli were around 7 × 107 CFU/ml

89

by plate counting. The microbial synthesis of silver nanoparticles was carried out in a

90

reaction containing either E. coli or K. pneumoniae to a final concentration of 3.5 ×

91

106 CFU/ml.

92 93

Biological synthesis of AgNPs. 500 µl of each reducing agent was added into a

94

glass vial containing 10 ml of 1mM AgNO3 and incubated at 25 °C with a gentle

95

rotation (ROTATOR-AG, FINEPCR, KOREA) at 10 rpm. Since light could affect the

96

synthesis of AgNPs,26 the reaction was carried out in dark incubator, in which a

97

fluorescent lamp (FPL27EX-D, Sigma lamp, Incheon, Korea) was equipped to offer

98

controlled irradiation with 10 cm of distance away from the sample during the 5



99

synthesis. To investigate the irradiation effect on the synthesis of AgNPs, we carried

100

out the reaction with or without the visible-light irradiation in MHB and NB for 6

101

hours. To monitor the yield of AgNPs synthesized from the reactions, an aliquot (100

102

µl) of reaction was taken every 0.5 h and its absorbance (O.D430) was measured using

103

UV-vis spectrophotometer (Infinite 200 PRO, TECAN, Switzerland).

104 105

Characterization of synthesized AgNPs. The synthesized AgNPs were washed

106

3 times with water and ethanol (20 %) to remove the medium that could possibly

107

remained on the surface of AgNPs. Absorption spectra of the nanoparticles were

108

analyzed by the UV-vis spectrophotometer (TECAN) at wavelengths ranging from

109

360 nm to 600 nm. One drop of AgNPs suspension was placed onto a carbon-coated

110

copper TEM grid (3.05 mm diameter), and dried at desiccator under vacuum. After

111

the sample was completely dried, the synthesized AgNPs were characterized by using

112

field emission transmission electron microscopy (FE-TEM, JEM-2100F, JEOL, USA).

113

The size distribution of synthesized AgNPs were determined by dynamic light

114

scattering (DLS) (Zetasizer Nano ZS, Malvern, England) under the setting of

115

refractive index (RI) of 1.33 with 25 replications.

116

The FTIR absorption spectra of AgNPs synthesized by NB and MHB media were

117

measured using a Perkin-Elmer Spectrum One System spectrometer (Foster City, CA,

118

USA) with KBr pellets in the range of 500 to 4000 cm−1.

119 120 121

122

Synthesis of silver nanoparticles in different culture media. As the

123

synthesis of AgNPs could be influenced by light irradiation from external

124

environment,27, 28 the reaction solution consisting of silver ions and various culture

RESULTS AND DISCUSSION

6


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media was placed in a dark incubator with a fluorescent lamp providing a controlled

126

irradiation toward the sample with a distance of 10 cm. Abbreviations of different

127

culture media used for the reduction of silver ions were listed in Table 1. Figure 1A

128

shows the color of reaction after incubating 1 mM AgNO3 solution with various

129

media at 25 °C for 2 days. AgNO3 solution without any culture media was used as a

130

control. We did not observe any notable sediment that is an indicative of silver salt

131

formation during or after the reaction with all test media. The results show that the

132

reaction solution containing PA, BE, NB, and MHB exhibited brown color owing to

133

the excitation of surface plasmon vibrations of the synthesized AgNPs,29, 30 while

134

there was no significant color change in the reaction containing YE compared to that

135

of control. The UV–Vis spectra of AgNPs synthesized using different culture media

136

are shown in Figure 1B. AgNPs synthesized by PA, BE, and NB showed a distinct

137

absorbance peaks at the range of 430 nm to 440 nm, which falls between a typical

138

SPR band of spherical AgNPs (400-450 nm),31 whereas no such a characteristic peak

139

was observed in a reaction with YE (Figure 1B). In particular, AgNPs synthesized by

140

MHB showed a remarkable red-shift of absorption peak (480 nm), implying that they

141

would have a different size or shape from those synthesized by other media. The

142

absorption intensity demonstrated that NB and MHB yielded a larger amount of

143

AgNPs, suggesting that catalytic activity in the reduction of silver ions was dependent

144

on the composition of culture media. Since NB is composed of BE and PA, its

145

reducing power would mainly be derived from the synergistic effect of BE and PA on

146

reduction activity. On the other hand, we assume that the casein hydrolysate and

147

starch derivatives in MHB acted as a main reducing agent for the synthesis of AgNPs.

148

The deprotonated form of the hydroxyl and carboxylic groups in these compounds is

149

believed to facilitate the complexation of Ag ions.32 Subsequently, the Ag ions were

150

reduced to form AgNPs through the oxidation of these groups into carbonyl form, 7



151

which also modulated the crystal growth of AgNPs. The shape and size of the

152

synthesized AgNPs were visualized through TEM (Figure 1C). The TEM results

153

showed that silver ‘seeds’ with diameter of about 5 nm appeared along with large

154

spherical AgNPs of an average particle size of 20 nm when the silver ions were

155

reduced by PA and BE media. We assume that the presence of large number of silver

156

seeds is possibly due to the insufficient reduction of AgNPs in PA and BE media,

157

limiting the transition from the nucleation stage to the growth stage during the

158

synthesis process, which was also evidenced by the decreased absorption of AgNPs in

159

a reaction with PA and BE media (Figure 1B). However, for the reaction with NB

160

media consisting of a mixture of PA and BE, we observed highly monodisperse

161

spherical AgNPs with a diameter of about 20 nm. On the other hand, MHB media led

162

to the formation of large AgNPs with a shape of planar triangle and discal

163

nanostructure. It has been also reported that the silver discal nanoplates showed an

164

absorption maximum at around 480 nm,33 which supports the fact that the red shifted

165

absorption of MHB-AgNPs was associated with the significant difference in

166

morphology from those synthesized by other media. Thus, our findings suggested that

167

the size and shape of AgNPs were shown to be depending on the media compositions,

168

such as protein and carbohydrate, which not only affect the nucleation of Ag0 but also

169

influence the growth and formation of AgNPs.34

170 171

Characteristics of AgNPs synthesized by NB and MHB medium. As MHB

172

and NB exhibited higher catalytic activity in the reduction of silver ions among the

173

components of culture media, these two culture media were selected as a model to

174

investigate the effect of media on the synthesis of AgNPs with respect to the size and

175

shape. Considering the irradiation effect that can enhance the reduction of silver

176

ions,35 AgNPs synthesis using MHB (MHB-AgNPs) and NB (NB-AgNPs) were 8


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performed under two different conditions, with or without light exposure (Figure 2A).

178

In both cases, MHB induced a greater increase in absorbance than that of NB. When

179

the reactions were carried out with light, the absorbance (at 480 nm) of the reaction

180

containing MHB increased rapidly within 3 h, followed by a gradual saturation over

181

the rest of reaction time, whereas the reaction with NB media showed a slower

182

increase in the value over the course of 6 h reaction. On the other hand, the reactions

183

without light showed different results. As expected, the absorbance (at 480 nm) of the

184

reaction with MHB was higher than that with NB (at 430 nm), but the overall values

185

were significantly lower than those with light. In addition, the absorption values of the

186

reaction with MHB reached a saturation level after 0.5 h, suggesting that light

187

synergistically participates in the reduction of silver ions together with other reducing

188

components in culture media. In addition, DLS analysis was carried out to investigate

189

the size distribution of the synthesized AgNPs (Figure 2B). According to DLS data,

190

the average hydrodynamic diameter of the AgNPs synthesized by MHB was 78.8 nm,

191

which was approximately four-fold bigger than those synthesized by NB (21.1 nm). In

192

addition, the AgNPs synthesized by NB and MHB medium were subjected to FTIR

193

analysis to investigate the composition of the surface of AgNPs, which could play an

194

important role in determining the sizes and the colloidal stability of the nanoparticles

195

(Figure 2C). FTIR spectrum showed several absorption peaks at 3340, 1645, 1589,

196

1541, 1384, and 1076 cm−1 in the spectrum scanned from 500 to 4000 cm−1. The IR

197

bands observed at 3340, 1075, and 1589 cm-1 in AgNPs synthesized by MHB

198

represent the characteristics of the O-H, C-O-C, and C=C stretching, respectively,36

199

and the band at 1384 cm−1 is derived from the NO3- remained in the solution.37 It

200

should be noted that the absorption peaks of NB-based AgNPs at 1541 cm-1 and 1645

201

cm-1 were originated from the bending vibrations of the amide I and amide II bands of

202

the proteins, respectively, suggesting that the proteins adsorbed on the surface of 9



203

synthesized AgNPs as a capping agent could enhance the colloidal stability of the

204

particles.38 From the results, the protein adsorbed on the surface of the nanoparticles

205

could inhibit the further coprecipitation of Ag on the surface of AgNPs, resulting in

206

the formation of small and highly monodisperse AgNPs in contrast to those

207

synthesized by MHB. In other words, the morphology of synthesized AgNPs could be

208

influenced by the composition of amino acids in NB and MHB. NB media is

209

comprised of water soluble PA and BE, which are derived from partially hydrolyzed

210

proteins and beef infusion, respectively. On the other hand, the primarily component

211

of MHB media is casein hydrolysate whose amino acid composition and sequence are

212

much different from those from NB media. From the results, we propose that the

213

composition of culture media not only play an important role in the synthesis of

214

AgNPs, but also affect the particles size through the interaction between the surface of

215

AgNPs and the media components.

216 217

Microbial biosynthesis of AgNPs. To investigate the effect of bacterial

218

metabolites on the synthesis of AgNPs, NB and MHB media were inoculated with K.

219

pneumoniae and E. coli, and their whole bacterial culture and the culture supernatant,

220

which represents the cellular and extracellular matrices, respectively, was examined.

221

In this study, we tested Klebsiella pneumoniae since this microorganism has

222

intensively been investigated for the cell-associated biosynthesis of AgNPs.17, 26, 39, 40

223

Escherichia coli is also widely used model microorganism in biological study, and we

224

used it as a control microorganism. Reduction of silver ions was not observed in the

225

reaction without NB or MHB media (Figure 3A), indicating that the intrinsic reducing

226

power of the microorganisms at test concentration were not strong enough to reduce

227

silver ions to form AgNPs. On the other hand, regardless of the types of media, the

228

intensity of absorption band was increased by the introduced microorganisms (Figure 10


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3B and 3C), which clearly indicates that the bacterial components or metabolites, such

230

as NADH-dependent reductase and nitrate reductase, could facilitate the synthesis of

231

AgNPs along with media components.13,

232

culture of K. pneumoniae showed higher absorbance compared to that of E. coli,

233

which agrees well with the previous observation.41 This might be due to the higher

234

metabolic activity of K. pneumoniae, because we observed a rapid growth of K.

235

pneumoniae in both culture media comparing to E. coli (see S1 in Supporting

236

Information). Moreover, the absorption amplitude of MHB-AgNPs was greater than

237

that of NB-AgNPs (Figure 3c), regardless of the presence of bacteria or bacterial

238

metabolites, indicating that culture media play a major role for the yield of AgNPs. In

239

addition, we observed a slight blue-shift in the absorption peak of AgNPs synthesized

240

by MHB with bacteria from 480 nm to 475 nm comparing to that without bacteria. On

241

the other hand, no shift of absorption peak was observed for the AgNPs synthesized

242

from NB containing bacterial culture or culture supernatant. TEM and DLS analysis

243

revealed that the presence of bacteria or types of bacteria had no effect on the size and

244

shape of the synthesized AgNPs (Figure 4A and 4B). We speculate that the resulting

245

blue-shift observed in the reaction with MHB and bacteria might be due to the

246

interaction of proteins released from the introduced microorganism with

247

biosynthesized AgNPs.42 Moreover, we found that the diameter of AgNPs synthesized

248

from NB media were significantly smaller than those synthesized from MHB media.

249

The size differences of AgNPs synthesized from NB media with K. pneumoniae and E.

250

coli were insignificant (Figure 4A and 4B). TEM results also showed that the shapes

251

of AgNPs obtained from NB-based group were independent of the type of

252

microorganism. Crystalline nature of the AgNPs synthesized was further examined by

253

the SAED pattern with bright circular spots corresponding to (1 1 1), (2 0 0), (2 2 0),

254

(3 1 1), and (2 2 2) Bragg reflection planes.43 SAED pattern of MHB-AgNPs with

22

Note that AgNPs synthesized with a

11



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bright circular rings corresponding to (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2)

256

planes indicated that the nanoparticles are highly crystalline. In contrast, NB-AgNPs

257

showed weak diffraction spots probably due to the interaction with proteins during the

258

synthesis process, which could affect superstructure of the nanoparticles.44 These

259

findings demonstrate that the use of different culture media predetermined the

260

morphology, size and crystal nature of biosynthesized AgNPs (Figure 4A and 4B).

261

In conclusion, we present the effect of different culture media on the

262

microorganism-mediated synthesis of AgNPs. The results suggest that the components

263

of culture media can affect the size and shape of synthesized AgNPs, independent of

264

whether the synthesis was carried out with microorganism or not. That is, culture

265

media plays a predominant role in determining the physical characteristics of

266

biosynthesized AgNPs. We hope our findings would provide a meaningful

267

information for further elucidating the exact mechanism of microorganism-mediated

268

synthesis of AgNPs.

269 270

271

This work was supported by the Korea Research Foundation Grant (NRF-

272

2016R1D1A1B03934878) and the Cooperative Research Program for Agricultural

273

Science

274

Administration, Republic of Korea.

ACKNOWLEDGEMENTS

&

Technology

Development

(PJ01199303),

Rural

Development

275 276

277

Growth curve of E. coli and K. pneumoniae in NB and MHB media (Figure S1).

SUPPORTING INFORMATION DESCRIPTION

278 279

Notes

280

The authors declare no competing financial interest. 12


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Figure captions Figure 1 Synthesis of AgNPs in different culture media. (A) digital photograph and (B) UV/Vis absorption spectra of reaction solution containing various reducing agents without (left) and with silver ions (right). 1 mM AgNO3 solution without any reducing agent was used as a control. (C) TEM image of the synthesized AgNPs in different culture media. The type of culture media are indicated on top left coner of each image.

Figure 2. (A) O.D values of the reaction after AgNPs synthesis with NB and MHB media without (top) and with light irradiation (down). (B) Size distribution of AgNPs synthesized by NB (blue) and MHB (black), respectively. The synthesis was carried out with light irradiation. (C) FT-IR spectrum of AgNPs synthesized by NB (blue) and MHB media (black).

Figure 3. Digital photograph and corresponding UV/Vis absorption spectra of the reaction after AgNPs synthesis by using culture supernatant and whole bacterial culture in HEPES (A), NB (B) and MHB media (C).

Figure 4. TEM, SAED patterns and DLS analysis of AgNPs synthesized from bacterial cultures in NB (A) and MHB (B) media. TEM images show AgNPs synthesized by using culture supernatant and whole bacterial culture in NB (A) inoculated with K. pneumoniae and E. coli (N-KP C; N-EC C; NKP W; N-EC W), and using that of MHB (B) media (M-KP C; M-EC C; MKP W; M-EC W). The corresponding SAEC patterns and DLS data are shown below. Histograms show the size distribution of the synthesized AgNPs through the condition described above. Solid line and dash line 18


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represent the culture supernatant and whole bacterial culture, respectively.

19



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Table Table 1. The composition of reducing agents used for the synthesis of AgNPs. Reducing agents Abbreviation Medium

Bacteria

Fraction

AgNO3

None

Whole solution

Cont

Peptone A

None

Whole solution

PA

Beef extract

None

Whole solution

BE

Yeast extract

None

Whole solution

YE

Nutrient broth

None

Whole solution

NB

Nutrient broth

K. pneumoniae 2242

Culture supernatant

N-KP C

Nutrient broth

K. pneumoniae 2242

Whole bacterial culture

N-KP W

Nutrient broth

E. coli DH5α

Culture supernatant

N-EC C

Nutrient broth

E. coli DH5α


N-EC W

Muller-Hintone broth

None

Whole solution

MHB


K. pneumoniae 2242

Culture supernatant

M-KP C


K. pneumoniae 2242


M-KP W


E. coli DH5α

Culture supernatant

M-EC C


E. coli DH5α


M-EC W

20


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Figure Graphics Figure 1

21



Figure 2

22


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Figure 3

23



Figure 4

24


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TABLE OF CONTENTS GRAPHICS

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Microbial biosynthesis of silver nanoparticles in different culture media

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