Differential Microbicidal Effects of Bimetallic Iron-Copper

Subscriber access provided by UNIV OF TASMANIA

Remediation and Control Technologies

Differential Microbicidal Effects of Bimetallic Iron-Copper Nanoparticles on Escherichia coli and MS2 Coliphage Hyung-Eun Kim, Hye-Jin Lee, Min Sik Kim, Taewan Kim, Hongshin Lee, Hak-Hyeon Kim, Min Cho, Seok Won Hong, and Changha Lee Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06077 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on February 2, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Environmental Science & Technology

Differential Microbicidal Effects of Bimetallic IronCopper Nanoparticles on Escherichia coli and MS2 Coliphage

Hyung-Eun Kim†,1, Hye-Jin Lee‡,1, Min Sik Kim§, Taewan Kim∥, Hongshin Lee∥, Hak-Hyeon Kim∥, Min Cho⊥, Seok-Won Hong†, Changha Lee§,*

†

Center for Water Resource Cycle Research, KIST School, Korea Institute of Science and Technology (KIST), 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea

‡

Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada

§

School of Chemical and Biological Engineering, Institute of Chemical Process (ICP), Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea

∥School

of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology

(UNIST), 50 UNIST-gil, Ulju-gun, Ulsan 44919, Republic of Korea ⊥ Division

of Biotechnology, Advanced Institute of Environmental and Bioscience, Chonbuk National

University, 79 Gobong-ro, Iksan 54596, Republic of Korea

Submitted to Environmental Science and Technology

1These

authors contributed equally to this work.

*Corresponding author Tel.: +82‒2‒880‒8630, Fax: +82‒2‒888‒7295, E‒mail: [email protected] 1

ACS Paragon Plus Environment


TOC/Abstract art

2


Page 2 of 30

Page 3 of 30

1


ABSTRACT

2

Bimetallic iron-copper nanoparticles (Fe/Cu-NPs) were synthesized by a single-pot

3

surfactant-free method in aqueous solution (the reduction of ferrous ion to zero-valent iron

4

nanoparticles (Fe-NPs) and the subsequent copper-coating by metal ion exchange). The

5

produced Fe/Cu-NPs formed aggregates of spherical nanoparticles (approx. 3070 nm) of Fe-

6

Cu core-shell structures with 11 wt.% copper content. The microbicidal effects of Fe/Cu-NPs

7

were explored on E. coli and MS2 coliphage, surrogates for bacterial and viral pathogens,

8

respectively. Fe/Cu-NPs exhibited synergistically enhanced activity for the inactivation of E.

9

coli and MS2, compared to single metal nanoparticles (i.e., Fe-NPs and Cu-NPs). Various

10

experiments (microbial inactivation tests under different conditions, fluorescence staining

11

assays, experiments using ELISA and qRT-PCR, etc.) suggested that Fe/Cu-NPs inactivate E.

12

coli and MS2 via dual microbicidal mechanisms. Two biocidal copper species (Cu(I) and

13

Cu(III)) can be generated by different redox reactions of Fe/Cu-NPs. It is suggested that E.

14

coli is strongly influenced by the cytotoxicity of Cu(I), while MS2 is inactivated mainly due

15

to the oxidative damages of protein capsid and RNA by Cu(III).

16

3



17

INTRODUCTION

18

As nanotechnology rapidly advances, research to apply nanomaterials in environmental

19

engineering is attracting attention.1 Water disinfection using antimicrobial nanoparticles is

20

one of the promising environmental applications of nanotechnology.2,3 For disinfection and

21

microbial control in water, a variety of metal and nonmetal nanoparticles have been studied.

22

In particular, metal nanoparticles in different forms (zero-valent metals, metal oxides, alloys,

23

and composites) containing silver, zinc, magnesium, iron, and copper have been reported to

24

exhibit antimicrobial properties.49 The disinfection using nanomaterials has been envisioned

25

as a prospective technology that is free from the production of harmful disinfection

26

byproducts (DBPs), and is advantageous for small-scale point-of-use applications.2,3

27

Silver-based nanomaterials, mainly silver nanoparticles (Ag-NPs), have been widely

28

studied for microbial control due to the strong antimicrobial activity of silver.4,1012 The

29

antimicrobial mechanism of Ag-NPs has been explained by the cytotoxicity of released silver

30

ion (which binds to thiol moiety in proteins, causing manifold detrimental effects),10,11 and

31

the disturbance of cell integrity by physicochemical interactions between Ag-NPs and cell

32

components (e.g., affecting the cell membrane permeability).12 Copper is also known as a

33

strong microbicide, and copper-based nanomaterials including copper and copper oxide

34

nanoparticles have been tested for their antimicrobial activity.4,1315 Similar to the case of Ag-

35

NPs, the toxicity of both released copper ion and nanoparticles themselves were suggested to

36

be responsible for the antimicrobial activity of copper nanoparticles (Cu-NPs).

37

Iron nanoparticles, particularly nanoparticulate zero-valent iron (nZVI or Fe-NPs), have

38

been reported to exhibit antimicrobial properties.1619 Investigators found that Fe-NPs

39

inactivate E. coli and MS2 coliphage mainly by inducing oxidative damage to the cells and

40

viral particles.1619 Fe-NPs and ferrous ion released from Fe-NPs generate reactive oxidants, 4


Page 4 of 30

Page 5 of 30


41

such as hydroxyl radical (OH) and ferryl ion (Fe(IV)), via the Fenton (-like) reactions.20 Fe-

42

NPs exhibited superior microbicidal activity to silver nanoparticles under anoxic conditions.16

43

However, the activity of Fe-NPs drastically decreases in the presence of oxygen due to rapid

44

iron corrosion,16 which limits the practical application of Fe-NPs in water disinfection.

45

Meanwhile, a few recent studies have tested iron-based bimetallic nanoparticles for their

46

antimicrobial properties.21,22 They showed that Fe-NPs doped with copper or silver exhibit

47

enhanced antibacterial or antifungal activities.

48

In this study, we report the synergistically-enhanced microbicidal activity of bimetallic

49

iron-copper nanoparticles (Fe/Cu-NPs). Fe/Cu-NPs showed greater inactivation efficacies of

50

E. coli and MS2 compared to Fe-NPs and Cu-NPs under both oxic and anoxic conditions.

51

The bactericidal activity of Fe/Cu-NPs for E. coli has been tested by a previous study,

52

together with Fe-NPs and three other iron-based bimetallic nanoparticles.22 However, the

53

synergistic microbicidal activity of Fe/Cu-NPs has not yet been clearly addressed. Moreover,

54

little is known about the redox reactions underlying the microbicidal actions of Fe/Cu-NPs

55

(in particular, the reactions involving different valencies of copper species), which are

56

believed to be the key factors in interpreting the microbicidal mechanisms of Fe/Cu-NPs. In

57

addition, this study found that when exposed to Fe/Cu-NPs, the E. coli and MS2 coliphage

58

are inactivated in different mechanisms.

59

The objectives of this study were i) to evaluate the enhanced microbicidal activity of

60

Fe/Cu-NPs (compared to Fe-NPs and Cu-NPs) and ii) to elucidate the microbicidal

61

mechanisms in terms of the roles of biocidal agents that are generated by the redox reactions

62

of Fe/Cu-NPs.

63 64

MATERIALS AND METHODS 5



65

Reagents. All chemicals were of reagent grade, and were used as received without further

66

purification (refer to Text S1 of the Supporting Information (SI), for the list of chemicals

67

used in this study). All solutions were prepared using deionized water (>18 M·cm,

68

Millipore, USA).

69 70

Synthesis and Characterization of Nanoparticles. Fe-NPs were synthesized by the

71

aqueous-phase reduction of ferrous ion, in which FeSO4 and NaBH4 were used as an iron

72

source and a reducing agent, respectively. Details about the synthetic procedures are

73

described elsewhere.16 In the same manner, Cu-NPs were prepared by the aqueous-phase

74

reduction of cupric ion (using CuSO4). Bimetallic nanoparticles (Fe/Cu-NPs) were

75

synthesized by the surface modification of Fe-NPs. The solution of CuSO4 (6.3 mM, 50 mL)

76

was slowly added into the suspension of Fe-NPs (prepared from 17.9 mM FeSO4 solution

77

200 mL) while stirring the suspension vigorously. Nanoparticles were collected by

78

centrifugation. Collected nanoparticles were washed with acetone three times, dried and

79

stored in a N2 chamber at room temperature (24 ± 1˚C) before use.

80

The surface morphologies and composition of Fe/Cu-NPs were analyzed by high

81

resolution transmission electron microscopy (HRTEM) equipped with energy dispersive

82

spectrometry (EDS) (JEM-2100F, JEOL, Japan). The phase and crystalline properties of

83

Fe/Cu-NPs were analyzed by high-power X-ray diffractometry (XRD; D/MAX 2500 V/PC,

84

Rigaku, Japan) with Cu Kα radiation. The chemical states of copper and iron were

85

characterized using X-ray photoelectron spectroscopy (XPS; K-alpha, Thermo Fisher, USA).

86

The specific surface area of Fe/Cu-NPs was measured using the Brunauer-Emmett-Teller

87

(BET) method on Gemini V system (Micromeritics, USA).

88 6


Page 6 of 30

Page 7 of 30


89

Culture and Analysis of Microorganisms. E. coli (ATCC8739) stock was cultivated in 30

90

mL of Difco nutrient broth at 37˚C for 18–24 h. The cells were collected by centrifugation at

91

3000 g for 15 min, and washed 3 times with phosphate buffered solution (PBS, 10 mM, pH

92

7.2). The obtained E. coli cells were resuspended in 20 mL PBS and kept in the refrigerator at

93

4˚C. The spread plate method was employed to determine the population of E. coli using

94

nutrient agar.23 The plates were incubated for 18–24 h at 37˚C, and the numbers of colonies

95

were then counted.

96

MS2 (ATCC 15597-B1) was used with E. coli C3000 (ATCC 15597) as a host. The host

97

E. coli was cultivated in tryptone broth containing 10 g/L tryptone, 8 g/L NaCl, 1 g/L yeast

98

extract, 1 g/L glucose, 2 mM CaCl2, and 0.01 g/L thiamine. MS2 was inoculated in the

99

suspension of host E. coli for 18–24 h at 37˚C. Then, the mixture of E. coli and MS2 was

100

centrifuged at 3000 g for 15 min, and the supernatant was filtered with a 0.22 μm PTFE

101

syringe filter. For further purification, the cultured MS2 was filtered by ultrafiltration (20,000

102

MWCO for collecting the filtrate followed by 10,000 MWCO for collecting the retentate).24

103

The population of MS2 was determined by the plaque assay method using media of top and

104

bottom double layer containing 0.5% and 1.5% of agar.23

105 106

Inactivation Experiments. All experiments were performed using 60 mL Pyrex flasks with

107

E. coli or MS2 suspensions (107 CFU or PFU/mL) at room temperature (24 ± 1˚C) and

108

neutral pH (pH = 6.8, unbuffered). The inactivation was initiated by adding the microbicidal

109

agent into the microbial suspension under vigorous mixing. A 1 mL sample aliquot was

110

withdrawn at pre-determined time, immediately mixed with ethylenediaminetetraacetic acid

111

(EDTA), and consecutively diluted with PBS to quench the reaction. Aliquots of samples

112

were assayed on agar plates for the counting of viable cells (or viral particles). 7



113

For inactivation experiments under anoxic conditions, the microbial suspension was

114

deaerated by sparging ultrapure N2 gas. 2 mM of 2,9-Dimethyl-1,10-phenanthroline (DMP)

115

and EDTA were used as Cu(I)- and Cu(II)- chelating reagents, respectively, and 10 mM

116

methanol was employed as a scavenger of reactive oxidants (i.e., OH, cupryl (Cu(III))

117

species, and Fe(IV)) for some experiments. DMP, EDTA, and methanol were added into the

118

microbial suspension before adding the microbial agent.

119 120

Measurement of Intracellular Oxidants. The fluorescent probe compounds, hydroxyphenyl

121

fluorescein (HPF) and hydroethidine (HE), were used to analyze the reactive oxidants (i.e.,

122

HPF for OH or Cu(III), and HE for superoxide radical anion (O2)) generated in the

123

intracellular region.2527 By the specific reactions with the oxidants, HPF and HE are

124

transformed into strong fluorescent compounds. To measure intracellular oxidants, E. coli

125

cells were stained with the probe compounds; E. coli cells were suspended in the probe

126

compound solution (100 µM) while stirring the suspension for 1 h at 100 rpm in the dark.

127

Then, the stained cells were centrifuged and washed three times with PBS, and resuspended

128

in the reactor for the treatment by NPs. The treated cells were sampled, and the fluorescence

129

intensity was measured by microplate reader (Infinite M200, Tecan, Switzerland) at 485 nm

130

excitation and 535 nm emission for HPF and at 535 nm excitation and 590 nm emission for

131

HE. The fluorescence intensity ratio (FIR) relative to the control was presented. More details

132

are described elsewhere.18

133 134

Analysis of Cell (or Viral Particle) Integrity. The LIVE/DEAD® BacLight™ bacterial

135

viability kit (L7012, Molecular Probes, Thermo Fisher, USA) was used to evaluate the cell

136

membrane integrity of E. coli. Live and dead cells were stained with SYTO® 9 and propidium 8


Page 8 of 30

Page 9 of 30


137

iodide, respectively; the excitation/emission maxima are 480 nm/500 nm for SYTO® 9 and

138

490 nm/635 nm for propidium iodide. The stained cells (live and dead cells, respectively)

139

were analyzed by confocal laser scanning microscopy (FV1000, Olympus, Japan). Standard

140

filter sets, FITC and Texas Red® were used for live and dead cells, and the fluorescence

141

signal was quantified using the IMARIS image processing software (Bitplane, Switzerland).

142

To analyze the antigenicity loss and the protein oxidation of MS2, the MS2 bacteriophage

143

BioThreat Alert® (Tetracore, USA) and OxiSelect™ Protein Carbonyl (Cell Biolabs, USA)

144

ELISA Kits were used, respectively. The absorbance at 450 nm was measured by microplate

145

reader for quantification. Detailed procedures for these assays are described elsewhere.18,19

146 147

qRT-PCR Analysis. The quantitative real-time PCR (qRT-PCR) analysis was carried out to

148

quantify the damage of RNA in MS2 treated by NPs. Viral RNA was extracted from the

149

sampled suspensions of MS2 with the QIAamp® Viral RNA mini kit (QIAGEN, Germany),

150

and the qRT-PCR signals were obtained on a CFX96 Real-Time system (Bio-Rad, USA). In

151

this study, two RNA sites, which are known to be important for protein maturation and RNA

152

replication, were selected for the analysis. Details of the qRT-PCR analysis are described in

153

the SI (Text S2 and Table S1).

154 155

RESULTS

156

Morphology and Surface Properties of Fe/Cu-NPs. TEM images show that Fe/Cu-NPs

157

form aggregates of spherical nanoparticles, of which the sizes range from 20 to 70 nm in

158

diameter (Figure 1a). Magnified TEM images with EDS analyses show that Fe/Cu-NPs have

159

core-shell structures, in which iron and copper dominate in the inner core and the outer shell,

160

respectively (Figures 1b and 1c). The oxygen content in the outer layers is higher, indicating 9



161

that zero-valent iron and copper are partially oxidized to their oxide forms (Sites B and E);

162

the estimated thickness of oxide layers ranges from 3.3 to 6.1 nm, and these oxide layers can

163

hinder the electron transfer from the core to the surface. In some cases, smaller particulates of

164

zero-valent copper (< 10 nm) are deposited on the outer layers (Site D). The total contents of

165

iron and copper in Fe/Cu-NPs were measured to be 85wt% and 11wt%, respectively

166

(measured by atomic absorption spectroscopy after dissolving Fe/Cu-NPs in HNO3). The

167

specific surface area of Fe/Cu-NPs was 35.5 m2/g.

168

The XRD spectrum of Fe/Cu-NPs shows a broad peak centered at 2θ = 44.9° which

169

indicates zero-valent iron (α-Fe) (SI Figure S1a).28,29 XPS spectra identified iron and copper

170

in their oxide forms on the Fe/Cu-NPs surfaces (SI Figure S1b). The peaks at 710.9 eV and

171

724.4 eV are assigned Fe 2p3/2 and 2p1/2 of iron oxides, respectively. A shoulder observed at

172

around 706.6 eV is attributed to a Fe 2p3/2 peak of zero-valent iron.29 The peaks at 932.5 and

173

952 eV are Cu 2p3/2 and 2p1/2 of CuO.30

174 175

Inactivation of E. coli and MS2 by Fe-, Cu-, and Fe/Cu-NPs. The inactivation of E. coli

176

and MS2 was examined in the presence of different nanoparticles ([NPs]0 = 50 mg/L) under

177

oxic and anoxic conditions (Figure 2). Under oxic conditions, Fe/Cu-NPs and Cu-NPs

178

resulted in the E. coli inactivation of 4.4 log in 10 min and 3.4 log in 15 min, respectively,

179

whereas Fe-NPs showed negligible bactericidal effect on E. coli (Figure 2a). Under anoxic

180

conditions, the inactivation efficacies of E. coli by Fe/Cu-NPs and Fe-NPs greatly increased

181

(Figure 2b); the enhanced bactericidal activity of Fe-NPs under anoxic conditions is

182

consistent with the previous observations.16 Meanwhile, Cu-NPs showed somewhat less

183

inactivation efficacy of E. coli under anoxic conditions than under oxic conditions.

10


Page 10 of 30

Page 11 of 30


184

Similar to the bactericidal activity, Fe/Cu-NPs also exhibited the greatest virucidal

185

activity toward MS2 under oxic conditions, followed by Cu-NPs and Fe-NPs (Figure 2c).

186

Under anoxic conditions, all NPs did not significantly inactivate MS2 (less than 0.7 log

187

inactivation in 15 min, Figure 2d).

188 189

Effects of a Reactive Oxidant Scavenger, Copper-Chelating Agents, and Hydrogen

190

Peroxide. To investigate the effects of reactive oxidants, and Cu(I) and Cu(II) species on the

191

inactivation of E. coli and MS2 by Fe/Cu-NPs,

192

(Figures 3a and 3b); the average microbial inactivation rates in the absence and presence of

193

reagents are presented. The addition of 10 mM methanol decreased the inactivation rates of E.

194

coli and MS2 by 15% and 54%, respectively. Copper-chelating agents significantly inhibited

195

the inactivation of both E. coli and MS2. The addition of Cu(I)-chelating agent (DMP)

196

decreased the inactivation rates of E. coli and MS2 by 69% and 67%, respectively. The

197

Cu(II)-chelating agent (EDTA) decreased the inactivation rates of E. coli and MS2 by more

198

than 90%.

methanol, EDTA, and DMP were employed

199

Meanwhile, under anoxic conditions, no inhibitory effect of methanol was observed on

200

the E. coli inactivation, whereas DMP and EDTA almost eliminated the bactericidal activity

201

of Fe/Cu-NPs (SI Figure S2).

202

The oxidation of Fe/Cu-NPs under oxic conditions is believed to generate hydrogen

203

peroxide (H2O2) as an intermediate, which subsequently reacts with copper species to

204

produce reactive oxidants, mainly Cu(III) (refer to the Discussion section for details). To test

205

this possibility, the inactivation of E. coli and MS2 by Cu-NPs in combination with H2O2 was

206

performed, and the results were compared with the microbial inactivation using Fe/Cu-NPs

207

and Cu-NPs in the absence of H2O2 (Figures 3c and 3d). The addition of H2O2 inhibited the E. 11



208

coli inactivation by Cu-NPs (Figure 3c), although it increased the intracellular oxidant levels

209

in E. coli cells (the inset of Figure 3c, also refer to SI Figure S3).

210

On the other hand, the addition of H2O2 greatly enhanced the MS2 inactivation by Cu-

211

NPs (Figure 3d); increasing the concentration of H2O2 further increased the inactivation rate

212

of MS2. SI Figure S4 shows the kinetic data for the microbial inactivation by Cu-NPs in the

213

presence of H2O2.

214 215

Viability Test of E. coli by a Staining Assay. A staining assay using the LIVE/DEAD®

216

BacLight™ bacterial viability kit was employed to test the viability of E. coli cells during the

217

treatment by different NPs. Inactivated cells (measured by the spread plate method) can be

218

considered as live if their cell membranes are not disrupted.

219

The live/dead cell ratio decreased with reaction time during the treatment by NPs (Figure

220

4a). Similar to the results of inactivation tests (Figure 2a), Fe/Cu-NPs exhibited the greatest

221

bactericidal activity, followed by Cu-NPs and Fe-NPs. To assess the relationship between the

222

cell membrane damage and culturability, the ratios of cell decay measured by the two

223

methods (i.e., the spread plate method and the LIVE/DEAD staining assay) were calculated

224

using the data of Figures 2a and 4a ( = Log(N/N0)Live/dead / Log(N/N0)Inactivation, Figure 4b).

225

The cells treated by Fe-NPs showed the highest  values, and those treated by Fe/Cu-NPs

226

and Cu-NPs followed.

227 228

Antigenicity, Protein Oxidation, and RNA damage of MS2. The reduction of antigenicity

229

of MS2 was examined during the treatment by NPs (Figure 5a). The antigenicity measured by

230

ELISA (expressed by normalized absorbance) decreased with the reaction time. Fe/Cu-NPs

231

resulted in the greatest reduction of antigenicity. Fe-NPs caused greater reduction of 12


Page 12 of 30

Page 13 of 30


232

antigenicity than Cu-NPs. The oxidation of protein capsid of MS2 (marked by protein

233

carbonylation) was monitored during the treatment by NPs (Figure 5b). The increase in the

234

protein carbonyl groups showed a similar trend to the reduction of antigenicity (i.e., Cu-NPs

235

< Fe-NPs < Fe/Cu-NPs).

236

The qRT-PCR analysis showed that the number of RNA targets in MS2 decreased during

237

the treatment by NPs (Figures 5c and 5d). The RNA damage by NPs increased in the order of

238

Fe-NPs < Cu-NPs < Fe/Cu-NPs.

239 240

DISCUSSION

241

Microbicidal Effects of Fe/Cu-NPs. Different microbicidal agents can be generated by the

242

redox reactions of Fe/Cu-NPs in aqueous suspension. Zero-valent iron and copper release

243

ionic iron and copper species, such as Fe(II), Cu(I), and Cu(II), as they corrode by oxygen

244

(intracellular or extracellular) and cellular components. Reactions 14 describe the corrosion

245

process of zero-valent iron and copper by oxygen.20,31

246

Fe0 + O2 + 2H+ → Fe(II) + H2O2

(1)

247

Fe0 + H2O2 + 2H+ → Fe(II) + 2H2O

(2)

248

Cu0 (or 2Cu0) + O2 + 2H+ → Cu(II) (or 2Cu(I)) + H2O2

(3)

249

Cu0 (or 2Cu0) + H2O2 + 2H+ → Cu(II) (or 2Cu(I)) + 2H2O

(4)

250

Among the ionic iron and copper species, Cu(I) is known as a strong microbicide, and has

251

been suggested to be responsible for the bactericidal actions of different copper-based

252

antimicrobial systems.26,27,32

253

During the oxidation of Fe/Cu-NPs, microbicidal reactive oxidants are also generated via

254

the Fenton (-like) reactions. H2O2 can be produced by the reactions of oxygen with reduced

255

forms of iron and copper. The two-electron reduction of oxygen by Fe0 or Cu0 produces H2O2 13



Page 14 of 30

256

(reactions 1 and 3). A series of one-electron reduction of oxygen by Fe(II) or Cu(I) also

257

produces H2O2 through the intermediate of O2 (reactions 58).33,34

258

Fe(II) + O2 → Fe(III) + O2•−

(5)

259

Fe(II) + O2•− + 2H+ → Fe(III) + H2O2

(6)

260

Cu(I) + O2 → Cu(II) + O2•−

(7)

261

Cu(I) + O2•− + 2H+ → Cu(II) + H2O2

(8)

262

The H2O2 so produced generates reactive oxidants such as Fe(IV), Cu(III), and OH by the

263

Fenton (-like) reactions with Fe(II) and Cu(I) (reactions 9 and 10).

264

Fe(II) + H2O2 → Fe(III) + •OH + OH− or Fe(IV) + H2O

(9)

265

Cu(I) + H2O2 → Cu(II) + •OH + OH− or Cu(III) + H2O

(10)

266

Previous studies suggested that the iron- and copper-based Fenton (-like) reactions

267

preferentially proceed via a two-electron transfer mechanism at neutral pH to produce Fe(IV)

268

and Cu(III), rather than OH.3538

269 270

Meanwhile, zero-valent iron and copper can donate electrons to recycle Fe(III) and Cu(II) to Fe(II) and Cu(I), which are further used to reduce oxygen and H2O2.

271

Under anoxic conditions, no reactive oxidants can be generated in the bulk phase.

272

However, cellular components can lead to the corrosion of Fe0 and Cu0 into their ionic

273

species. Due to the reducing environment, most of ionic iron and copper species will be

274

present as Fe(II) and Cu(I).

275

The redox reactions take place primarily on the surface of Fe/Cu-NPs, and possibly in the

276

diffuse layer in close proximity to the surface; the concentrations of total dissolved iron and

277

copper released from Fe/Cu-NPs (measured after microfiltration) were 1.16 M and < 0.1

278

M, respectively. When Fe/Cu-NPs contact E. coli and MS2, the microbicidal agents

279

generated from Fe/Cu-NPs can be transferred to the cells (or viral particles) through the NP14


Page 15 of 30


280

cell interface. A portion of Fe/Cu-NPs may penetrate into the E. coli cells through the

281

disrupted cell membranes, accelerating the transfer of the biocides into the intracellular

282

region. Cu(I), Cu(III), and Fe(IV) (free or surface-bound) are believed to be the potential

283

biocides that may contribute to the microbial inactivation; however, the minor microbicidal

284

activity of Fe-NPs under oxic conditions (Figures 2a and 2c) indicates that the contribution of

285

Fe(IV) is relatively insignificant. The significant inhibition of microbial inactivation in the

286

presence of DMP and EDTA (Figures 3a and 3b) indicates that Cu(I) and Cu(II) species play

287

key roles in the microbicidal actions of Fe/Cu-NPs; Cu(I) is a biocide as well as an

288

intermediate to produce Cu(III), and Cu(II) is the precursor of Cu(I).

289

The synergistic microbicidal activity of Fe-Cu-NPs is believed to result from the

290

enhanced production of microbicidal copper species (i.e., Cu(I) and Cu(III)) attributed to the

291

reactions of Fe0. As a reducing agent, Fe0 reduces Cu(II) into Cu(I) (reaction 11), and Cu(I) is

292

subsequently used to produce Cu(III) through the oxidation process (reactions 7, 8 and 10).

293

Fe0 + 2Cu(II) → Fe2+ + 2Cu(I)

(11)

294

This explanation is consistent with our previous observation that a reducing agent

295

(hydroxylamine) accelerates the production of Cu(I) and Cu(III) from Cu(II), enhancing the

296

microbicidal activity of Cu(II).27 In addition, Fe0 can produce H2O2 by a direct reaction

297

(reaction 1) and secondary routes via released Fe(II) (reactions 5 and 6); H2O2 is further used

298

to generate Cu(III) (reaction 10).

299

The results obtained in this study collectively show that the inactivation mechanisms of E.

300

coli and MS2 by Fe/Cu-NP are different from each other. These differences are mainly

301

explained by the different roles and contributions of Cu(I) and Cu(III); details will be

302

discussed in the following sections.

303

15



304

Inactivation of E. coli. Cu(I) appears to play a key role in the inactivation of E. coli by

305

Fe/Cu-NPs. This explanation is supported by several experimental evidences. First, the

306

enhanced bactericidal activity of Fe/Cu-NPs under anoxic conditions (Figure 2b) supports

307

that Cu(I) is important, because anoxic conditions increase the stability of Cu(I). Under oxic

308

conditions, Cu(I) is more readily oxidized by different oxygen species (reactions 7, 8, and 10).

309

Second, many observations have not found significant involvement of reactive oxidants,

310

most likely Cu(III), in the E. coli inactivation. The effect of methanol (an oxidant scavenger)

311

on the E. coli inactivation was minor (Figure 3a), indicating that the bactericidal effects of

312

reactive oxidants are not important; the effect of methanol was almost negligible under

313

anoxic conditions where reactive oxidants are not generated (SI Figure S2). The combined

314

use of Cu-NPs and H2O2 (intended to accelerate the generation of Cu(III)) decreased the

315

inactivation rate of E. coli (Figure 3c), indicating that the bactericidal role of Cu(III) is minor.

316

Although the FIR values for intracellular oxidants slightly increased in the presence of H2O2

317

(the inset of Figures 3c and SI Figure S3), this increase (31.850.8 %) is insufficient to cause

318

a substantial inactivation of E. coli; the FIR values increased by more than ten-fold in the

319

Cu(II)/H2O2 system.26

320

Fe/Cu-NPs also exhibited the greatest bactericidal activity in the LIVE/DEAD staining

321

assay of E. coli (Figure 4a), indicating that during the Fe/Cu-NPs treatment, the cell

322

membranes are significantly disrupted. However, the  values ( = Log(N/N0)LIVE/DEAD /

323

Log(N/N0)Inactivation) were the highest when treated by Fe-NPs, followed by Fe/Cu-NPs and

324

then Cu-NPs (Figure 4b). This result implies that the ratios of the membrane-damaged cells

325

to non-culturable cells decreases in the order of Fe-NPs > Fe/Cu-NPs > Cu-NPs. There may

326

be multiple physicochemical effects of NPs to disrupt the cell membranes of E. coli, and no

327

simple explanation can apply for the cell membrane disruption. However, it is assumed that 16


Page 16 of 30

Page 17 of 30


328

the  values are higher when the contribution of reactive oxidants such as Fe(IV) and Cu(III)

329

is high and the contribution of non-oxidizing biocides such as Cu(I) is low. Based on the

330

entire results obtained in this study, Cu(I) is believed to be mainly responsible for the E. coli

331

inactivation, but other effects (e.g., reactive oxidants or physical interactions between NPs

332

and cells) may disrupt cell membranes to facilitate the penetration of Cu(I) into the

333

intracellular region.

334 335

Inactivation of MS2 Coliphage. Previous studies have reported that Cu(III) is a strong MS2

336

virucide.26,27 Cu(III) also appears to play an important role in the inactivation of MS2 by

337

Fe/Cu-NPs. The following observations support this explanation. First, the inactivation rates

338

of MS2 by NPs greatly decreased under anoxic conditions in which reactive oxidants are not

339

generated (Figure 2d). Second, methanol significantly inhibited the MS2 inactivation (Figure

340

3b); the residual virucidal activity in the presence of methanol may be attributed to the

341

incomplete scavenging of reactive oxidants by methanol or other biocidal effects of Fe-Cu-

342

NPs (e.g., Cu(I) or physical actions of NPs). In addition, the combined use of Cu-NPs and

343

H2O2 dramatically enhanced the MS2 inactivation (Figure 3d), indicating that H2O2 is a

344

critical reagent to produce Cu(III) from Cu0. As described earlier, Fe/Cu-NPs accelerates the

345

in situ-generation of H2O2 by different reactions of electron-donating iron species (i.e., Fe0

346

and Fe(II)), leading to the greater production of Cu(III), compared to Cu-NPs.

347

The reduction of antigenicity of MS2 by Fe/Cu-NPs (Figure 5a) is believed to result from

348

the damage of the protein capsid possibly due to the oxidation by Cu(III). This explanation

349

agrees with the increase of protein carbonyl groups by the Fe/Cu-NPs treatment (Figure 5b).

350

However, it is noteworthy that Fe-NPs, which showed minor virucidal activity (Figure 2c),

351

caused substantial protein oxidation (Figures 5a and 5b), indicating that Fe(IV) effectively 17



352

oxidizes proteins. On the other hand, the RNA damage by NPs (Figures 5c and 5d) showed a

353

similar trend to the MS2 inactivation (Figure 2c). The damage of external capsid may

354

facilitate the attack of biocides on the internal RNA. The lack of RNA damage in the Fe-NPs-

355

treated MS2 indicates that Fe(IV) is not effective in destroying RNA. The greater inactivation

356

of MS2 by Fe/Cu-NPs is thought to result from greater damage of both the protein capsid and

357

RNA induced by Cu(III).

358 359

Environmental Implications. Fe/Cu-NPs can be applied in different areas where water

360

disinfection or antimicrobial action is needed. Fe/Cu-NPs were synthesized by a facile single-

361

batch method that can readily be scaled up for mass production by increasing the batch size.

362

Fe/Cu-NPs exhibited great microbicidal activity toward both bacteria and viruses. The

363

inactivation rates of E. coli and MS2 by Fe/Cu-NPs were superior to those of Ag-NPs (SI

364

Table 2), showing its potential as a cost-competitive antimicrobial material. The microbicidal

365

activity of Fe/Cu-NPs did not significantly decrease for at least six months, when stored in a

366

powder form under ambient conditions (data not shown). Fe/Cu-NPs can be applied as both a

367

consumable water disinfectant and an antimicrobial surface-coating material. Depending on

368

their uses, the properties of Fe/Cu-NPs can be further modified (e.g., by controlling the iron

369

and copper content or the degree of surface oxidation); the reactivity and stability of Fe/Cu-

370

NPs are important for the former and latter applications, respectively. Meanwhile, the

371

microbicidal mechanisms of Fe/Cu-NPs suggested in this study (the dual roles of Cu(I) and

372

Cu(III)) provide insight into the design of effective antimicrobial materials and systems using

373

copper species.

374

18


Page 18 of 30

Page 19 of 30


375

Supporting Information Reagents (Text S1), qRT-PCR analysis (Text S2), information on

376

the designs of primers for the two RNA sites used in this study (Table S1), comparison of E.

377

coli inactivation rates by Ag-NPs and Fe/Cu-NPs (Table 2), XRD and XPS spectra of Fe/Cu-

378

NPs (Figure S1), effects of methanol and copper-chelating reagents on the inactivation of E.

379

coli under anoxic condition (Figure S2), intracellular oxidant generation by NPs and Cu-NPs

380

with different concentrations of H2O2 (Figure S3), inactivation kinetics of E. coli and MS2 by

381

NPs and Cu-NPs with different concentrations of H2O2 (Figure S4).

382 383

ACKNOWLEDGMENTS

384

This work was supported by the Korea Ministry of Environment as an “Advanced Industrial

385

Technology Development Project” (2017000140005), and a National Research Foundation of

386

Korea (NRF) Grant (NRF2017R1A2B3006827).

387 388

REFERENCES

389

1. Wiesner, M. R.; Bottero, J.-Y. Environmental nanotechnology: Applications and impacts

390

of nanomaterials; McGraw-Hill: New York, 2007.

391

2. Li, Q.; Mahendra, S.; Lyon, D. Y.; Brunet, L.; Liga, M. V.; Li, D.; Alvarez, P. J. J.

392

Antimicrobial nanomaterials for water disinfection and microbial control: Potential

393

applications and implications. Water Res. 2008, 42 (18), 45914602.

394

3. Hossain, F.; Perales-Perez, O. J.; Hwang, S.; Román, F. Antimicrobial nanomaterials as

395

water disinfectant: Applications, limitations and future perspectives. Sci. Total Environ.

396

2014, 466–467, 10471059.

19



397

4. Yoon, K.-Y.; Byeon, H. J.; Park, J.-H.; Hwang, J. Susceptibility constants of Escherichia

398

coli and Bacillus subtilis to silver and copper nanoparticles. Sci. Total Environ. 2007, 373

399

(2–3), 572–575.

400

5. Feng, Q. L.; Wu, J.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O. A mechanistic study

401

of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J.

402

Biomed. Mater. Res. 2000, 52 (4), 662–668.

403

6. Huang, L.; Li, D.-Q.; Lin, Y.-J.; Wei, M.; Evans, D. G.; Duan, X. Controllable

404

preparation of nano-MgO and investigation of its bactericidal properties. J. Inorg.

405

Biochem. 2005, 99 (5), 986–993.

406 407

7. Yamamoto, O. Influence of particle size on the antibacterial activity of zinc oxide. Int. J. Inorg. Mater. 2001, 3 (7), 643–646.

408

8. Jones, N.; Ray, B.; Ranjit, K. T.; Manna, A. C. Antibacterial activity of ZnO nanoparticle

409

suspensions on a broad spectrum of microorganisms. FEMS Microbiol. Lett. 2008, 279

410

(1), 71–76.

411

9. Shi, L.-E.; Xing, L.; Hou, B.; Ge, H.; Guo, X.; Tang, Z., Inorganic nano mental oxides

412

used as anti-microorganism agents for pathogen control. Curr. Res. Technol. Educ.

413

Topics Appl. Microbiol. Microbial Biotechnol. 2010, 1, 361–368.

414

10. Morones, J. R.; Elechiguerra, J. L.; Camacho, A.; Holt, K.; Kouri, J. B.; Ramirez, J. T.;

415

Yacaman, M. J. The bactericidal effect of silver nanoparticles. Nanotechnology 2005, 16

416

(10), 2346–2353.

417

11. Xiu Z.-M; Zhang Q.-B; Puppala H. L.; Colvin V. L.; Alvarez P. J. J. Negligible particle-

418

specific antibacterial activity of silver nanoparticles. Nano Lett. 2012, 12 (8), 4271–4275.

20


Page 20 of 30

Page 21 of 30


419

12. Sondi, I.; Salopek-Sondi, B. Silver nanoparticles as antimicrobial agent: A case study on

420

E. coli as a model for Gram-negative bacteria. J. Colloid Interf. Sci. 2004, 275 (1), 177–

421

182.

422

13. Ruparelia, J. P.; Chatterjee, A. K.; Duttagupta, S. P.; Mukherji, S. Strain specificity in

423

antimicrobial activity of silver and copper nanoparticles. Acta Biomater. 2008, 4 (3),

424

707–716.

425

14. Rena, G.; Hu, D.; Cheng, E. W. C.; Vargas-Reus, M. A.; Peip, P.; Allaker, R. P.

426

Characterisation of copper oxide nanoparticles for antimicrobial applications. Int. J.

427

Antimicrob. Agents 2009, 33 (6), 587–590.

428

15. Raffi, M.; Mehrwan, S.; Bhatti, T. M.; Akhter, J. I.; Hameed, A.; Yawar, W.; ul Hasan,

429

M. M. Investigations into the antibacterial behavior of copper nanoparticles against

430

Escherichia coli. Ann. Microbiol. 2010, 60 (1), 75–80.

431

16. Lee, C.; Kim, J. Y.; Lee, W. I.; Nelson, K. L.; Yoon, J.; Sedlak, D. L. Bactericidal effect

432

of zero-valent iron nanoparticles on Escherichia coli. Environ. Sci. Technol. 2008, 42

433

(13), 4927–4933.

434

17. Auffan, M.; Achouak, W.; Rose, J.; Roncato, M.-A.; Chanéac, C.; Waite, D. T.; Masion,

435

A.; Woicik, J. C.; Wiesner, M. R.; Bottero, J.-Y., Relation between the redox state of

436

iron-based nanoparticles and their cytotoxicity toward Escherichia coli. Environ. Sci.

437

Technol. 2008, 42 (17), 6730–6735.

438

18. Kim, J. Y.; Park, H.-J.; Lee, C.; Nelson, K. L.; Sedlak, D. L.; Yoon, J. Inactivation of

439

Escherichia coli by nanoparticulate zerovalent iron and ferrous ion. Appl. Environ.

440

Microbiol. 2010, 76 (22), 7668–7670.

21



441

19. Kim, J. Y.; Lee, C.; Love, D. C.; Sedlak, D. L.; Yoon, J.; Nelson, K. L., Inactivation of

442

MS2 coliphage by ferrous ion and zero-valent iron nanoparticles. Environ. Sci. Technol.

443

2011, 45 (16), 6978–6984.

444 445

20. Lee, C. Oxidation of organic contaminants in water by iron-induced oxygen activation: A short review. Environ. Eng. Res. 2015, 20 (3), 205–211.

446

21. Marková, Z.; Šišková, M. K.; Filip, J.; Čuda, J.; Kolář, M.; Šafářová, K.; Medřík, I.;

447

Zbořil, R. Air stable magnetic bimetallic Fe–Ag nanoparticles for advanced antimicrobial

448

treatment and phosphorus removal. Environ. Sci. Technol. 2013, 47 (10), 5285–5293.

449

22. Kim, E.-J.; Le Thanh, T.; Chang, Y.-S., Comparative toxicity of bimetallic Fe

450

nanoparticles toward Escherichia coli: Mechanism and environmental implications.

451

Environ. Sci.: Nano 2014, 1 (3), 233–237.

452 453

23. American Public Health Association (APHA). Standard methods for the examination of water and wastewater; APHA: Washington D. C., 2005.

454

24. Cho, M.; Lee, J.; Mackeyev, Y.; Wilson, L. J.; Alvarez, P. J. J.; Hughes, J. B.; Kim, J.-H.

455

Visible light sensitized inactivation of MS-2 bacteriophage by a cationic amine-

456

Functionalized C60 derivative. Environ. Sci. Technol. 2010, 44 (17), 6685–6691.

457 458

25. Gomes, A.; Fernandes, E.; Lima, J. L. F. C. Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 2005, 65 (2–3), 45–80.

459

26. Nguyen, T. T. M.; Park, H.-J.; Kim, J. Y.; Kim, H.-E.; Lee, H.; Yoon, J.; Lee, C.

460

Microbial inactivation by cupric ion in combination with H2O2: Role of reactive oxidants.

461

Environ. Sci. Technol. 2013, 47 (23), 13661–13667.

462

27. Kim, H.-E.; Nguyen, T. T. M.; Lee, H.; Lee, C. Enhanced inactivation of Escherichia

463

coli and MS2 coliphage by cupric ion in the presence of hydroxylamine: Dual

464

microbicidal effects. Environ. Sci. Technol. 2015, 49 (24), 14416–14423. 22


Page 22 of 30

Page 23 of 30

465 466


28. Sun, Y.-P.; Li, X.-Q.; Cao, J.; Zhang, W.-X.; Wang, H. P. Characterization of zero-valent iron nanoparticles. Adv. Colloid Interface Sci. 2006, 120 (1–3), 47–56.

467

29. Wang, Q.; Kanel, S. R.; Park, H.; Ryu, A.; Choi, H. Controllable synthesis,

468

characterization, and magnetic properties of nanoscale zerovalent iron with specific high

469

Brunauer–Emmett–Teller surface area. J. Nanopart. Res. 2008, 11 (3), 749–755.

470

30. Midander, K.; Cronholm, P.; Karlsson, H. L.; Elihn, K.; Möller, L.; Leygraf, C.;

471

Wallinder, I. O. Surface characteristics, copper release, and toxicity of nano- and

472

micrometer-sized copper and copper(II) oxide particles: A cross-disciplinary study. Small

473

2009, 5 (3), 389–399.

474

31. Zhou, P.; Zhang, J.; Zhang. Y.; Liang, J.; Liu, Y.; Liu, B.; Zhang, W. Activation of

475

hydrogen peroxide during the corrosion of nanoscalezero valent copper in acidic solution.

476

J. Mol. Catal. A: Chem. 2016, 424, 115–120.

477

32. Park, H.-J.; Nguyen, T. T. M.; Yoon, J.; Lee, C. Role of reactive oxygen species in

478

Escherichia coli inactivation by cupric ion. Environ. Sci. Technol. 2012, 46 (20), 11299–

479

11304.

480 481

33. King, D. W.; Lounsbury, H. A.; Millero, F. J. Rates and mechanism of Fe(II) oxidation at nanomolar total iron concentrations. Environ. Sci. Technol. 1995, 29 (3), 818–824.

482

34. Yuan, X.; Pham, A. N.; Xing, G.; Rose, A. L.; Waite, T. D. Effects of pH, chloride, and

483

bicarbonate on Cu(I) oxidation kinetics at circumneutral pH. Environ. Sci. Technol. 2012,

484

46 (3), 1527−1535.

485

35. Johnson, G. R. A.; Nazhat, N. B.; Saadalla-Nazhat, R. A. Reaction of the aquacopper(I)

486

ion with hydrogen peroxide. Evidence for a CuIII(cupryl) intermediate. J. Chem. Soc.,

487

Faraday Trans. 1 1988, 84 (2), 501−510.

23



488

36. Hug, S. J.; Leupin, O. Iron-catalyzed oxidation of arsenic(III) by oxygen and by

489

hydrogen peroxide: pH-dependent formation of oxidants in the Fenton reaction. Environ.

490

Sci. Technol. 2003, 37 (12), 2734−2742.

491

37. Bataineh, H.; Pestovsky, O.; Bakac, A. pH-Induced mechanistic changeover from

492

hydroxyl radicals to iron(IV) in the Fenton reaction. Chem. Sci. 2012, 3, 1594−1599.

493

38.

Lee, H.; Lee, H.-J.; Seo, J.; Kim, H.-E.; Shin, Y. K.; Kim, J.-H.; Lee, C. Activation of

494

oxygen and hydrogen peroxide by copper(II) coupled with hydroxylamine for oxidation

495

of organic contaminants. Environ. Sci. Technol. 2016, 50 (15), 8231−8238.

24


Page 24 of 30

Page 25 of 30


Figure Captions Figure 1. (a)‒(c) HRTEM images and (d) EDS elemental compositions of Fe/Cu-NPs.

Figure 2. Inactivation of E. coli and MS2 by NPs under oxic and anoxic conditions. Insets indicate the inactivation rates ([Fe-NPs] = [Cu-NPs] = [Fe/Cu-NPs] = 0.05 g/L).

Figure 3. Effects of methanol and copper-chelating reagents on the inactivation of (a) E. coli and (b) MS2 coliphage by Fe/Cu-NPs. Inactivation rates of (c) E. coli and (d) MS2 coliphage by NPs and Cu-NPs with different concentrations of H2O2. The inset of (c) indicates the intracellular oxidant generation ([Fe-NPs] = [Cu-NPs] = [Fe/Cu-NPs] = 0.05 g/L, [MeOH] = 10 mM and [DMP] = [EDTA] = 2 mM for (a) and (b), reaction time = 30 min for (c) and (d)).

Figure 4. (a) E. coli cell decay analyzed by the LIVE/DEAD BacLight staining assay, and (b) calculated α values during the treatment of NPs. Insets of (a) indicate the microscopic images ([Fe-NPs] = [Cu-NPs] = [Fe/Cu-NPs] = 0.05 g/L).

Figure 5. Variations in the (a) antigenicity, (b) protein carbonyl, and two viral RNA targets ((c) Site A and (d) Site B, refer to SI Table S2) in MS2 coliphage, during the treatment by NPs ([Fe-NPs] = [Cu-NPs] = [Fe/Cu-NPs] = 0.05 g/L).

25



Figure 1.

26


Page 26 of 30

Page 27 of 30


(a) E. coli, oxic

(b) E. coli, anoxic

-6

4.5 3.6 0.6 0.4 0.2 0.0

Fe-NPs Cu-NPs Fe/Cu-NPs

Ps NPs NPs -N e uF Cu /C Fe

-8 0

5

10

-2 -4 -6


-8

15

0

1

Reaction time (min)

2.7 1.8 0.9 0.0

Ps NPs NPs -N e uF Cu /C Fe

5

10

15

-8 0

4.5 3.6 0.6 0.4 0.2 0.0


Ps Ps Ps -N -N -N u u Fe C /C Fe

5

10

-2 -4 -6

-Log(N/N0)/min

Log(N/N0)

0

-Log(N/N0)/min

Log(N/N0)

-6

3.6

(d) MS2, anoxic

0

-4

3

4.5

Reaction time (min)

(c) MS2, oxic

-2

2

-Log(N/N0)/min

-4

Log(N/N0)

-2

0

-Log(N/N0)/min

Log(N/N0)

0

4.5 3.6 0.2 0.1 0.0

-8

15

0

Reaction time (min)


Ps NPs NPs -N uFe Cu /C Fe

5

10

Reaction time (min)

Figure 2.

27


15

0.2

0.0

EDTA Control MeOH

(c) E. coli

3

4

1

0

0.0

Figure 3.

28

ACS Paragon Plus Environment DMP

Cu-NPs + 100 M H2O2

0.4


0.6

-Log(N/N0)/min 0.8


2

-Log(N/N0)/min

(a) E. coli

Cu-NPs

Fe/Cu-NPs

0.2 Cu-NPs + 100 M H2O2


DMP


0.4 Cu-NPs + 10 M H2O2

Control MeOH


2.0

Cu-NPs

3.0


0.6

Fe/Cu-NPs

4.0

FIR for HPF

-Log(N/N0)/min 0.8

Cu-NPs

Fe/Cu-NPs

-Log(N/N0)/min

Environmental Science & Technology Page 28 of 30

(b) MS2

0.6

0.4

0.2

0.0

EDTA

(d) MS2

3

2

1

0

Page 29 of 30


(a) Log(N/N0)Live/dead

0.0

Fe-NPs

-0.2 -0.4 Cu-NPs Fe/Cu-NPs

-0.6 -0.8 -1.0

0

5

10

15

Log(N/N0)Live/Dead/Log(N/N0)Inactivation

Reaction time (min) 10

(b)

1

0.1 Cu-NPs

Fe-NPs Fe/Cu-NPs 0.01

0

5

10

Reaction time (min) Figure 4.

29


15


Protein carbonyl (nmol/mg)

Absorbance/Absorbance0

(a) 1.0 0.8 0.6 0.4


0.2 0.0

0

5

10

15

1.0

(b)

0.8 0.6 0.4


0.2 0.0 0

Reaction time (min)

5

10

15

Reaction time (min)

(c)

(d) 0.0

Log(RNA/RNA0)

0.0

Log(RNA/RNA0)

Page 30 of 30

-0.5 -1.0 Fe-NPs Cu-NPs Fe/Cu-NPs

-1.5 -2.0 0

2

4

-0.5 -1.0 Fe-NPs Cu-NPs Fe/Cu-NPs

-1.5 -2.0

6

8

10

0

Reaction time (min)

2

4

6

8

Reaction time (min)

Figure 5.

30


10

Differential Microbicidal Effects of Bimetallic Iron-Copper

Recommend Documents