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Efficient Bacterial Inactivation by Transition Metal Catalyzed Auto-oxidation of Sulfite Long Chen, Min Tang, Chuan Chen, Mingguang Chen, Kai Luo, Jing Xu, Danna Zhou, and Feng Wu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03705 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Environmental Science & Technology

1

Efficient Bacterial Inactivation by Transition Metal

2

Catalyzed Auto-oxidation of Sulfite

3 4

Long Chen†, ‡, Min Tang‡, Chuan Chen†, Mingguang Chen†, Kai Luo§, Jing Xu‡, Danna

5

Zhou*∥, Feng Wu*‡, ⊥

6

†

7

Riverside, Riverside, California, 92521, USA

8

‡

9

School of Resources and Environmental Science, Wuhan University, Wuhan, 430079, P.

Department of Chemical and Environmental Engineering, University of California,

Hubei Key Lab of Biomass Resource Chemistry and Environmental Biotechnology,

10

R. China

11

§

12

MN 55455, USA

13

∥

14

430074, P. R. China

15

⊥

16

Institute of Eco-Environmental Science & Technology, Guangzhou, 510650, P. R. China

Masonic Cancer Center, University of Minnesota, 2231 6th Street SE, Minneapolis,

Faculty of Material Science and Chemistry, China University of Geosciences, Wuhan,

Guangdong Key Laboratory of Agro-Environmental Integrated Control, Guangdong

17 18 19

ACS Paragon Plus Environment


20

Abstract

21

Disinfection is an indispensable process in wastewater treatment plants. New bacterial

22

inactivation technologies are of increasing interest and persistent demand. A category

23

of simple and efficient bactericidal systems have been established in this study, i.e., the

24

combination of divalent transition metal (Mn(II), Co(II), Fe(II), or Cu(II)) and sulfite. In

25

these systems, metal catalyzed auto-oxidation of sulfite was manifested to generate

26

reactive intermediary SO4•- that played the major role in Escherichia coli inactivation at

27

pH 5-8.5. Increasing concentrations of metal ion or sulfite, and lower pH, led to higher

28

bacterial deaths. Bacterial inactivation by Me(II)/Sulfite systems was demonstrated to be

29

a surface-bound oxidative damage process through destructing vital cellular

30

components, such as NADH and proteins. Additionally, the developed Me(II)/Sulfite

31

systems also potently killed other microbial pathogens, i.e. Pseudomonas aeruginosa,

32

Bacillus subtilis, and Cu(II)-/antibiotic-resistant E. coli. The efficacy of Me(II)/Sulfite in

33

treating real water samples was further tested with two sewages from a wastewater

34

treatment plant and a natural lake water body, and Cu(II)/Sulfite and Co(II)/Sulfite

35

rapidly inactivated viable bacteria regardless of bacteria species and cell density,

36

therefore holding great promises for wastewater disinfection.

37 38 39 40 41


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42

Introduction

43

Waterborne pathogenic microbes are of great concern to public health. Two methods

44

are conventionally employed for bacteria removal, i.e. membrane filtration and bacterial

45

disinfection. Membrane filtration, taking advantage of the small pore size of synthetic

46

sheets, however, has the drawbacks of easy fouling, incomplete removal of tiny

47

organisms such as virus, and heavy demand of high pressure.1,2 Hence, a secondary

48

disinfection process is necessary for post-membrane treatment. Up to now, an

49

expanding range of disinfection processes or their combinations have been widely

50

applied. Traditionally used chlorine and ozone are powerful oxidants disinfecting

51

bacterial virulence, but the large-scale production, storage, and transportation are

52

challenging owing to their acute toxicities.3,4 Moreover, certain fraction of chlorination

53

byproducts are carcinogenic and their presences in potable water may secondarily

54

impair human health.5 Alternatively, advanced oxidation processes (AOPs) generating

55

various oxidative radicals have been considered for bacterial inactivation purposes.

56

Hydroxyl radical, HO•, derived from Fenton reaction has been verified to be potent in

57

killing bacteria via degrading vital cellular components.6,7 Another emerging strong

58

radical, sulfate radical (SO4•-), has gained a lot of attractions as well due to its selective

59

degradation towards electron-rich recalcitrant contaminants.8,9 Recently, it has been

60

reported that SO4•- is especially effective at bacterial/viral inactivation.10-13

61

Previous SO4•- generating processes heavily relied on peroxymonosulfate (PMS) or

62

persulfate (PS) through a one-step activation,8,9,14-16 while another intricate sulfite-

63

associated radical chain reaction capable of deriving SO4•- has been largely ignored.

64

Sulfite auto-oxidation catalyzed by transition metal ions with the presence of molecular



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65

oxygen is a key part of the sulfur cycle that mediates the transformation of S(IV) to

66

S(VI).17-19 Briefly, at first SO3•- is generated after the instant decomposition of transient

67

complex between high-valence metal ion and sulfite via internal electron transfer, but it

68

rapidly

69

disproportionally oxidizes sulfite producing SO4•- and sulfate anion (Table S1). The most

70

intriguing property of the sulfite oxidation process is that, plethoral SO4•- is effectively

71

generated. As a result, without exogenous competing substrates, SO4•- exclusively

72

oxidizes parent sulfite, leading to the kinetically favored auto-oxidation of sulfite; on the

73

other hand, the pool of total SO4•- is supposed to be re-assigned with the simultaneous

74

presences of sulfite and other heterologous substrates. We have thus utilized this

75

attractive

76

decontamination purpose.20-23 However, up to date, the application of sulfite-based

77

AOPs for waterborne bacteria disinfection still remains unexplored, and there were

78

lacking

79

decontamination/disinfection.

80

In this study, we tested four divalent transition metals, i.e. Mn(II), Co(II), Fe(II), and

81

Cu(II), catalyzed sulfite oxidation (denoted as Me(II)/Sulfite reactions) and therefore the

82

proliferation of SO4•- bactericide. The main aims of this work were to comparatively

83

investigate the potential of Me(II)/Sulfite systems in killing various microbial pathogens

84

and to reveal the associated bactericidal mechanisms. Our work has vital environmental

85

implications as both components of metal elements and sulfite are abundantly present

86

in various environmental media, and thus the associated cost is economically effective.

87

Materials and Methods

reacts

with

feature,

dissolved

and

oxygen

managed

comparisons

on

to

metal

to

drive

produce

SO4•-

catalyzed


SO5•-.

Followingly,

generation

sulfite

for

SO5•-

wastewater

oxidation

for

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88

Materials

89

Bacteria culture LB (Luria-Bertani) medium, disposable γ-ray sterilized 1-mL deep 96-

90

well plates for bactericidal assays, and PBS buffer (pH 7.2) were purchased from

91

Thermo Fisher Scientific Inc. (Shanghai, PRC). Antibiotics (kanamycin, tetracyclin,

92

ampicillin, and chloramphenicol) were bought from Aladdin Industrial Corporation

93

(Shanghai, PRC). Other chemicals including sodium sulfite (Na2SO3), manganese

94

chloride (MnCl2), cobalt chloride (CoCl2), ferrous sulfate (FeSO4), copper sulfate

95

(CuSO4), tert-butanol (TBA), thiourea, and 2,2’-dipyridyl were obtained from Sinopharm

96

Chemical Reagent Co. Ltd. (Shanghai, PRC). Bovine Serum Albumin (BSA) was from

97

Shanghai Boao Biochemical Technology (Shanghai, China). All chemicals were of

98

reagent grade and used without further purification. Throughout the assays, Milli-Q

99

water was used (18 MΩ•cm, Millipore Co., USA). All chemicals used to assess cellular

100

NADH

content

(ethanol,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

101

bromide, phenazine ethosulfate, EDTA, and Bicine buffer) were bought from Sigma

102

(Shanghai, China). HClO4 or NaOH was used to adjust the pH of PBS.

103

Methods

104

Cell culturing and bactericidal assay quantification. Single colony of Escherichia

105

coli BL21(DE3) strain was cultured in 5 mL liquid LB medium at 37 °C and 250 rpm for

106

overnight incubation. 1% (50 µL) of saturated bacterial culture was then added into

107

fresh 5 mL LB medium, followed by constant shaking at 37 °C and 250 rpm for another

108

2 h until the cells reached exponential phase (OD600 ~ 0.4-0.6). Cells were harvested

109

by centrifugation at 3000 g for 3 min. Pelleted cells were then resuspended and washed

110

with PBS buffer of desired pHs for three times to remove residual nutrients. The freshly



111

obtained cells were immediately used without storage. Bactericidal assays were initiated

112

by addition of metal ions and/or sodium sulfite at indicated concentrations. 200 µL liquid

113

samples were taken at pre-determined intervals, 10-fold serially diluted with PBS (pH

114

7.2), and eventually 5 µL liquid of each sample was dropped on LB-agar plates and

115

incubated at 37 °C for 12 h. Bacterial survival rate was quantified by counting the CFU

116

(Colony Forming Unit) on the plates. Each assay was repeated for at least three times.

117

Bactericidal Assays of Me(II)/Sulfite in Sewages and Natural Lake Water. Sewages

118

of biological treatment tank and secondary sedimentation tank from Erlangmiao

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wastewater treatment plant (Wuhan, China) were sampled. The sampling sites were set

120

at the well-mixed regions that were representative of the whole tank. For each tank, we

121

chose at least two sampling points. Natural lake water of a nearby lake was also

122

sampled to test the bactericidal effect of Me(II)/Sulfite (Scheme S1). Samples were

123

stored in sterilized brown bottles to avoid light irradiation, and bactericidal assays were

124

performed within the same day of sampling. PCA (plate count agar) was used to

125

quantify the survival rate of total bacteria, while Endo agar was used to monitor the

126

survival rate of coliforms. For the bactericidal assays of added E. coli, sewages or

127

natural lake water were centrifuged at 1,000 g for 3 min followed by filtration with 0.45

128

µm membranes, in order to remove original bacteria. Then exponential phase E. coli

129

cultured in LB media was washed with desired water bodies (i.e. sewages or natural

130

lake water), and then spiked into the same volume of corresponding sewages or natural

131

lake water for Me(II)/Sulfite bactericidal assays (0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM

132

Fe(II), 0.1 mM Cu(II), 2 mM sulfite).


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133

Radical-quenching

134

Me(II)/Sulfite reactions (0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM Cu(II), 2

135

mM sulfite, pH 6), either of 400 mM TBA (HO• scavenger), 400 mM thiourea (SO4•-/HO•

136

scavenger), or 2 mM dipyridyl (metal ions chelator) was added into the solution at the

137

beginning of the reactions. The individual effect of TBA, thiourea, or dipyridyl was also

138

investigated in the same condition without the presence of metal ions or sulfite.

139

Samples were taken and analyzed after 2 h.

140

Scanning Electron Microscopy analysis. 5 mL exponential phase bacteria after

141

treatment for 2 h were collected and resuspended into an Eppendorf vial containing 1

142

mL PBS buffer (pH 7.2). 2.5% glutaraldehyde was used to fix the bacterial cells at room

143

temperature for 1.5 h. Samples were then collected and exposed for 10 min to a series

144

of ethanol concentration solutions (30, 50, 70, 80, 95, or 100% ethanol/PBS) for

145

dehydration. The 100% ethanol treatment was repeated for twice, and the solutions

146

were then further dehydrated with anhydrous sodium sulfate. 10 µL of bacterial solution

147

in 100% ethanol was dropped on glass substrate, and then a uniform layer of 10 nm Au

148

was coated on the bacterial samples with an E-beam evaporator (QPrep400, Mantis,

149

England). Samples were observed under 10-5 Torr vacuum in SEM (JSM-6510, JEOL,

150

Japan).

151

Protein Degradation Assay with Me(II)/Sulfite. The oxidative degradation capability

152

towards polypeptide (0.1 mg/mL BSA) of Me(II)/Sulfite (0.8 mM Mn(II), 0.4 mM Co(II),

153

0.4 mM Fe(II), 0.1 mM Cu(II), and 2 mM sulfite) was investigated in PBS buffer (pH 6).

154

Reactions were carried out in a 96-well plate with constant shaking at 37 °C. After 2 h,

155

samples were boiled at 98 °C for 5 min, and 40 µL boiled samples were then loaded on

Assay

of

Me(II)/Sulfite.

During bactericidal assays with



156

10% SDS-PAGE for resolution. Protein gels were run at 200 V for 60 min with a Bio-rad

157

electrophoresis cell.

158

NADH Measurement. Conventional cofactor recycling assay was used to determine

159

intracellular NADH concentrations.24 5mL cells after treatment were at first collected via

160

centrifugation at 8000 g for 2 min, then 300 µL of 0.2 M NaOH was added to the cell

161

pellets, followed by incubation at 55 °C for 10 min. The resulting extracts were then

162

neutralized with 300 µL of 0.1 M HCl. Samples were then centrifuged at 12000 g for 5

163

min, and supernatants were collected for further analysis. NADH recycling assay was

164

performed with 50 µL neutralized supernatant, 50 µL of yeast ADH with 25 units activity,

165

and 0.9 mL reagents mixture (11% ethanol, 4.4 mM EDTA, 0.47 mM 3-(4,5-dimethyl-2-

166

thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and 3.7 mM phenazine ethosulfate in

167

100 mM Bicine buffer, pH 8). The absorbance changes at 570 nm after 2 min reactions

168

were recorded. The same amount of cells without any treatment was used as a control.

169

Results and Discussion

170

Inactivation of Escherichia coli with Me(II)/Sulfite systems

171

Divalent metal ions, i.e. Mn(II), Co(II), Fe(II), Cu(II), and sulfite combinatorically killed

172

exponential phase E. coli cells at pH 6. In control experiments with single component of

173

metal ion or sulfite, negligible cell deaths were observed (Figure 1a). The bactericidal

174

effect of Me(II)/Sulfite systems exhibited a time- and metal species-dependent manner.

175

Me(II)/Sulfite systems, except Cu(II)/Sulfite system, resulted in less than 0.5 log

176

inactivation at 1 h while more than 1.2 log inactivation at 3 h. Although lower

177

concentration of cupric ion (0.1 mM) was used, Cu(II)/Sulfite exhibited higher degree of


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bactericidal effect than the other three metal ions combined with sulfite. Cu(II)/Sulfite

179

inactivated 1.3 log of E. coli at 1 h, and more than 2.2 log of cells at 3 h.

180

Auto-oxidation of (bi)sulfite catalyzed by metal ions has been widely recognized through

181

radical chain reactions, involving several intermediary short-lived oxysulfur radicals, i.e.

182

SO3•-, SO5•-, and SO4•-.17-19 During the radical chain reactions, SO5•- and SO4•- were

183

secondarily derived from initially generated SO3•- (Table S1). As expected, evident

184

signals corresponding to DMPO/SO3•- adduct (aHβ = 16.0 G, aN = 14.7 G)25,26 were

185

visualized in the ESR spectra of Me(II)/Sulfite systems during the first 1 min reaction

186

(Figure 1b). Moreover, consistent with above observation, Cu(II)/Sulfite produced the

187

most pronounced signals indicating the highest abundance of oxidative radicals (Figure

188

1b). Among the three oxysulfur radicals, SO4•- possesses a standard reduction potential

189

of 2.5-3.1 V (NHE),27 comparable to that of HO• (1.8-2.7 V, NHE).28,29 SO4•- owned

190

preeminent oxidative hydrogen abstraction capability, and Fe(II)/Sulfite system has

191

been tentatively used for wastewater decontamination.20-22 Hence the most possible

192

effective bactericidal species was SO4•-.

193

Effect of metal ions or sulfite concentrations

194

E. coli survival rates were determined after inactivation by Me(II)/Sulfite systems for 2 h

195

with varying concentrations of metal ions or sulfite at pH 7. Figure 2 showed significant

196

enhancements of bactericidal effect with increasing concentrations of sulfite (0-8 mM) or

197

metal ions (for Mn(II), Co(II) and Fe(II): 0.05-0.8 mM; for Cu(II): 0.0125-0.2 mM).

198

Cu(II)/Sulfite system mediated the most evident E. coli inactivation. For instance,

199

addition of 8 mM sulfite and 0.1 mM Cu(II) resulted in 31-fold enhancement of bacterial

200

death compared with the mere presence of Cu(II). Further, 2 mM sulfite combined with



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0.2 mM Cu(II) killed bacteria 40-fold more efficient than that combined with 0.0125 mM

202

Cu(II). The sole presence of either sulfite or metal ions at all tested concentrations did

203

not kill bacteria more than 0.6 log of cells (Figure S1). Cu(II) was obviously more toxic

204

than the other three metal ions, as 0.2 mM Cu(II) inactivated over 0.6 log of cells, higher

205

than the inactivation efficiencies by other metal ions at 0.8 mM (Figure S1). The

206

molecular mechanism of Cu(II) toxicity involved enhancement of cell membrane

207

permeability and cellular protein denaturation.30,31 In contrast to previously observed

208

inhibitive growth in nutritious media,32 the presence of 0.05-0.8 mM Co(II) surprisingly

209

led to around 2-fold growth of E. coli in PBS buffer without any carbon source (Figure

210

S1), indicating its pivotal role of participating in the synthesis of metalloprotein with poor

211

nutrients.33

212

Effect of pH

213

pH mainly affected the metal ion species distribution, and thus possibly affected the

214

initiation of catalyzed oxidation of sulfite.21 Aside from the bactericidal assays, we also

215

used a triphenylmethane dye (Coomassie Brilliant Blue) to probe the amount of

216

generated oxidative radicals by Me(II)/Sulfite at varying pHs (Figure S2). Through all

217

the assays, near-neutral pHs (5-8.5) were chosen as other pHs adversely inactivated

218

cells by destabilizing cell membrane and denaturing inherent functional enzymes34,35

219

and therefore may cause false positive observations with Me(II)/Sulfite systems.

220

Mn(II)/Sulfite and Fe(II)/Sulfite systems were potent at lower pH, in terms of both dye

221

removal and bacterial inactivation (Figure 3a, Figure S2). At pH 5, Mn(II)/Sulfite

222

inactivated over 3.2 log of cells, but its potency rapidly decayed with increasing pH; at

223

pHs above 8, negligible bacterial inactivation was observed. Fe(II)/Sulfite inactivated 0.9


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log of cells at pH 8.5, while its bactericidal efficiency modestly increased to 1.9 log at pH

225

5.

226

Consistent with our previous results,23 Co(II)/Sulfite was more capable in dye removal at

227

higher pHs (Figure S2), whereas its bactericidal effect exhibited a reverted pattern

228

(Figure 3). At pH 8.5, Co(II)/Sulfite inactivated 0.5 log of cells, but at pH 5 inactivated

229

1.8 log of cells, resulting in a 22-fold enhancement. In sharp contrast, dye removal at pH

230

5 was significantly lower than that at pH 8.5. The mechanism for this contradictory

231

observation was not clear, and one plausible explanation was that higher pHs increased

232

negative charges of bacterial surface and thus efficiently repulsing sulfite anion away

233

from surface-bound catalytically active metal species.

234

Cu(II)/Sulfite at lower pH values tended to mediate higher bacterial deaths, while a

235

notable exception was that Cu(II)/Sulfite at pH 8.5 inactivated 9.6-fold cells of that at pH

236

8 (Figure 3a), resulting in a bell-shaped bacterial inactivation pattern against pH.

237

Different from other metals, Cu(II) was a severely toxic metal, in particular at higher pHs

238

(Figure 3b), because 1) formed Cu(II)-OH complex readily adsorbed on bacterial

239

surface,36,37 and 2) moreover Cu(II)-OH complex was highly toxic and effective at

240

disrupting membrane structure than the other Cu(II) species.38,39 Therefore, at lower

241

pHs the bactericidal effects mainly came from Cu(II)/Sulfite as evidenced by efficient

242

dye removals (Figure S1), while the toxicity of Cu(II)-OH complex played the major role

243

at higher pHs.

244

Morphological and Biochemical Characterizations of E. coli



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Morphological changes of E. coli in response to Me(II)/Sulfite treatments were examined

246

by SEM (Figure 4a). Bacterial samples were coated with a uniform layer of 10 nm

247

atomic Au to improve the resolution of SEM by increasing the conductivity of the

248

samples and thus reducing the accumulation of charges upon irradiation of electron

249

beams. As a result, intensive darkness within the cytoplasmic region was observed for

250

treated cells indicating severe cellular components disruption, but untreated cells

251

exhibited normal homogeneity. In addition, the Mn(II)/Sulfite and Fe(II)/Sulfite

252

treatments altered a fraction of E. coli with rod shape into fragmented irregular shape.

253

The contents of cellular reduced nicotinamide adenine dinucleotide, NADH, an

254

important biochemical indicator of the reducing power in cytoplasmic environment, was

255

quantified through a cofactor cycling method.24 NADH was especially important for

256

respiration-associated electron transport chain (ETC) and synthesis of certain

257

metabolites.40,41 Remarkable decreases of NADH contents of cells treated by

258

Me(II)/Sulfite were observed, wherein Cu(II)/Sulfite treatment particularly eliminated

259

93.7% of total cellular NADH (Figure 4b).

260

Furthermore, inspired by strong potencies of Me(II)/Sulfite systems in depleting NADH,

261

we attempted to investigate their abilities in degrading proteins. A model protein, bovine

262

serum albumin (BSA), was chosen as our target for polypeptide degradation assays. 0.1

263

mg/mL BSA was spiked into PBS buffer (pH = 6) together with metal ion and sulfite, and

264

40 µL samples were subjected to protein gel electrophoresis. The most evident

265

decrease in band intensity was observed with Cu(II)/Sulfite, whereas the other

266

Me(II)/Sulfite systems did not show detectable weakenings (Figure 4c). Densitometry

267

analysis assisted by ImageJ software showed that around 56% of BSA was degraded


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by Cu(II)/Sulfite. Although the other three Me(II)/Sulfite systems (i.e. Mn(II)/Sulfite,

269

Co(II)/Sulfite, and Fe(II)/Sulfite) did not exhibit polypeptide degradation capabilities,

270

however, it was possible for them to inactivate cells through deactivating functional

271

enzymes.25,42 During the course of running SDS-PAGE, denatured linearized BSA

272

polypeptide was resolved based on its molecular weight. The denaturation of BSA due

273

to Me(II)/Sulfite prior to SDS-PAGE thus cannot be told. A recent example supportive of

274

protein denaturation by SO4•- was disease-associated prion protein inactivation.11

275

Roles of Radicals in Bactericidal Effect of Me(II)/Sulfite Systems

276

Me(II)/Sulfite reactions rapidly depleted dissolved oxygen (Figure S3a) accompanied by

277

proton generation (Table S1). However, oxygen deprivation did not cause detectable

278

bacterial death without Me(II)/Sulfite treatments (Figure S4), and generated proton did

279

not acidify the buffered solution (Figure S3b) and was thus not responsible for bacterial

280

inactivation (Figure 3). Moreover, three oxysulfur radicals, i.e. SO3•-, SO4•-, and SO5•-,

281

were directly produced during sulfite oxidation catalyzed by metal ions (Table S1). SO3•-

282

is fairly inert towards organic chemicals such as alcohols, but is extremely prone to

283

oxidation by oxygen into SO5•- with an estimated second-order rate constant of 109 M-1

284

s-1.43 It was thus concluded that SO3•- did not likely cause bacterial death. In addition,

285

because SO5•- was scarcely generated and less oxidative than SO4•-,43,44 the possibility

286

of its bactericidal action was also excluded. Hence, SO4•- could be the most possible

287

bactericide responsible for cell inactivation during Me(II)/Sulfite treatments.

288

Besides SO4•-, another plausible possibility is that formed hydroxyl radical (HO•), due to

289

the electron-scavenging of H2O by SO4•- (eq 1),8,9 inactivated bacterial cells. We

290

therefore used a scavenging method to distinguish the roles of SO4•- and HO• in



Page 14 of 38

291

Me(II)/Sulfite systems at pH 6.8,9 Tert-butanol (TBA) quenched HO• with a 1000-fold

292

selectivity higher than SO4•-, while thiourea non-selectively quenched both radicals. For

293

all Me(II)/Sulfite systems, addition of SO4•-/HO•-scavenging thiourea greatly suppressed

294

bacterial inactivation, more evidently for Mn(II)/Sulfite and Cu(II)/Sulfite, while the

295

addition of HO•-scavenging TBA exhibited less potent inhibition (Figure 5a). It was

296

therefore concluded that SO4•- played the major role in inactivating cells, in agreement

297

with previous report.21 We further used a strong metal ion chelator, 2,2’-dipyridyl, to fully

298

stall the catalyzed sulfite oxidation. As expected, since there was no intermediary

299

oxidative species generated, negligible bacterial deaths were observed (Figure 5a). It

300

should be noted that, conventionally used ethanol (SO4•-/HO• scavenger) and EDTA

301

(metal ions chelator) were avoided because they were reported to be detrimental to

302

bacteria.45,46 But TBA, thiourea, and dipyridyl at used concentrations did not affect cell

303

survival rates (Figure S4).

304

SO4•- + H2O SO42- + HO• + H+

(k2 = (103-104) s-1)

(1)

305 306

Possible Mechanisms of Bactericidal Effect of Me(II)/Sulfite Systems

307

We reasoned that, generated reactive species, such as oxidative short-lived SO4•-, had

308

to act on cellular components for bacterial inactivation purpose. Accordingly, the

309

catalytically active metal ion species had to be bound onto cell surface. We hence

310

ultrasonically cracked the cells treated by either metal ions alone or Me(II)/Sulfite

311

together, centrifuged and collected the supernatants, and subsequently quantified

312

cellular metal ions with AAS (Figure 5b). As expected, significant cellular uptakes were


Page 15 of 38


313

found for all four metal ions. Among them, cells treated by 0.1 mM Cu(II) mediated a

314

most evident 134-fold increasement in cellular Cu(II) content by comparison with

315

untreated cells, presumably because Cu(II) readily complexed with cellular polypeptide

316

through highly affinitive Cu(II)-thiol group interactions.47,48 Additionally, we also found

317

that cellular metal ion uptakes by Me(II)/Sulfite treatments were generally less effective

318

than the treatments of metal ions alone (Figure 5b), likely because the presence of

319

sulfite complexed with metal ions and thus reducing the chances of metal ions adsorbed

320

onto bacterial surface. The observed less Cu(II) adsorption in the presence of sulfite

321

also supportively explained the fact that bacterial inactivation efficiencies of

322

Cu(II)/Sulfite were even lower than that of the single role of Cu(II) at pH 8 and 8.5

323

(Figure 3). We concluded that the toxicity of Cu(II)/Sulfite at higher pHs was mainly

324

from Cu(II), and therefore less surface-bound Cu(II) due to sulfite complexation led to

325

attenuated bacterial deaths. Hence, we believed the bacterial inactivations by

326

Me(II)/Sulfite systems were a surface-bound in situ oxidative damage process.

327

Cu(II)/Sulfite was an especially attractive system as it owned the most potent

328

bactericidal effects. During the initiation of radical chain reactions, Cu(I) was generated

329

via reduction of Cu(II) by sulfite (Table S1), and the presence of Cu(I) was

330

experimentally verified (Figure S5). More importantly, Cu(I) has been reported to be an

331

effective bactericide.49 We thus assessed the bactericidal role of Cu(I) in an anaerobic

332

chamber purged with nitrogen. The exclusion of oxygen did not allow the propagation of

333

radical chain reactions for Cu(II)/Sulfite system (Table S1), and the effect of Cu(I) could

334

be specifically investigated. We surprisingly found that Cu(I) formed by Cu(II)/Sulfite

335

with the absence of oxygen absolutely inactivated all the cells (~ 8 log), while the



Page 16 of 38

336

presence of oxygen led to 1.8 log of bacterial inactivation (Figure 5c). Since oxygen

337

played inverse roles in the generation of Cu(I) (Figure S5) and SO4•- (Table S1), the net

338

effect of oxygen on Cu(II)/Sulfite bactericidal performance was still vague, and therefore

339

the role of oxygen was investigated in more details (Figure S6). Oxygen ratio of 0, 0.01,

340

0.05, 0.2, and 0.4 in oxygen/nitrogen mixing gas at fixed flow rate of 400 mL/min were

341

constantly purged into 200 mL of Cu(II)/Sulfite reaction solution. When oxygen ratio

342

slightly increased from 0 to 0.01, Cu(II)/Sulfite killing efficiency dramatically decreased

343

from 3.7-log to 0.8-log after reaction for 1 h, indicating the strong bactericidal potency of

344

Cu(I). The increase of oxygen ratio 0.01 up to 0.4 led to the improvement of

345

Cu(II)/Sulfite bactericidal efficiency from 0.8-log to 2.1-log. While the increase of oxygen

346

ratio reduced Cu(I) amount, it improved the SO4•- production, and therefore can further

347

enhance bactericidal effect of Cu(II)/Sulfite. As expected, without molecular oxygen, no

348

reactive species were generated for Mn(II)/Sulfite, Co(II)/Sulfite, or Fe(II)/Sulfite, and

349

hence bacterial deaths were greatly suppressed (Figure 5c). Conclusively, based on

350

the obtained results, triple components majorly contributed to the bactericidal effect of

351

Cu(II)/Sulfite, i.e. 1) oxidative SO4•-, 2) toxic Cu(II)-OH complex at higher pHs, and 3)

352

monovalent Cu(I) (Scheme 1). The triply synergistic actions may also explain the fact

353

that Cu(II)/Sulfite system was the most efficient system among the Me(II)/Sulfite

354

systems investigated in this work.

355

Disinfection of Sewages and Natural Lake Water with Me(II)/Sulfite Systems

356

Application of Me(II)/Sulfite systems in inactivation of other bacteria genres, e.g. various

357

antibiotic-resistant E. coli strains (Figure S7), Cu(II)-resistant E. coli (Figure S8),

358

Pseudomonas aeruginosa and Bacillus subtilis (Figure S9) were proved to be valid


Page 17 of 38


359

(Text S1). Despite all this, our aim was to apply the established Me(II)/Sulfite systems

360

into real wastewater treatment plants. Sewage samples were taken at two

361

representative sites from Erlangmiao wastewater treatment plant, i.e. biological

362

treatment tank and secondary sedimentation tank. The samples had typical sewage

363

characters, such as near-neutral pH, and high contents of ammonium and phosphate

364

ions (Table S2). Bactericidal assays were performed immediately after sampling, in

365

order to avoid possible bacterial inactivation during storage. Moreover, to mimic real

366

condition, sewage samples were directly used without filtration, and reaction solutions

367

were subjected to minimum stirring. Furtherly, we used PCA (plate count agar), a rich

368

and non-selective medium, to recover total viable bacteria. And Endo agar, a selective

369

medium, was used to isolate coliform colonies that formed green metallic sheen on the

370

colorless plate, such as E. coli. Initially, total bacteria CFUs per mL of biological

371

treatment tank and secondary sedimentation tank were 5 × 105 and 5 × 103,

372

respectively. In both tanks, coliforms were around 10% of total bacteria. In biological

373

treatment tank, Cu(II)/Sulfite inactivated 2 log of both total bacteria and coliforms after

374

reaction for 3 h, while the other three Me(II)/Sulfite systems did not show detectable

375

bacterial inactivation (Figure 6a, 6b). In secondary sedimentation tank, both

376

Cu(II)/Sulfite and Co(II)/Sulfite rapidly and absolutely inactivated total bacteria and

377

coliforms within 2 h, whereas Mn(II)/Sulfite and Fe(II)/Sulfite did not mediate significant

378

bactericidal effects (Figure 6d, 6e). Additionally, it has been observed that Cu(II)/Sulfite

379

and Co(II)/Sulfite killed coliforms more efficiently than total bacteria, likely due to the

380

complex bacterial species within total bacteria.



Page 18 of 38

381

The upper limit of bacterial density in aqueous solution is around 108 CFU/mL, even in

382

the case of nutritious media. We therefore attempted to examine the bactericidal

383

efficiencies of Me(II)/Sulfite systems with the presence of maximum bacterial density.

384

Exponential phase E. coli with OD600 around 0.4-0.6, corresponding to 108 CFU/mL,

385

was collected and added into the same volume of biological treatment tank and

386

secondary sedimentation tank sewage samples. Consistent with above results,

387

Cu(II)/Sulfite absolutely inactivated bacteria within 1 h, and Co(II)/Sulfite killed all

388

bacteria at 2 h. The preeminent performance of Co(II)/Sulfite may associate with the

389

toxicity of Co(II) with the presence of sewage carbon source.32

390

Disinfection of lake water is important as it is a possible source of drinking water. Hence,

391

besides sewage samples, we also tested bacterial inactivation efficiencies of

392

Me(II)/Sulfite systems towards natural lake water (Figure S10). Cu(II)/Sulfite could

393

rapidly inactivate total bacteria (4 log) in lake water within 1 h. Co(II)/Sulfite also potently

394

killed bacteria but slower than Cu(II)/Sulfite, and Co(II)/Sulfite inactivated all bacteria

395

after 2 h. Neither Mn(II)/Sulfite nor Fe(II)/Sulfite showed significant bacterial inactivation

396

(Figure S10a). Consistently, Cu(II)/Sulfite and Co(II)/Sulfite inactivated 8 log and 3.2 log

397

of added E. coli (~ 108 CFU/mL) in natural lake water after reactions for 3 h (Figure

398

S10b). Moreover, CT (concentration [mg/L] × time [min]) value of Cu(II)/Sulfite to

399

inactivate 4-log E. coli varied between 1600 and 9600 in terms of different water bodies

400

(Figure 6c, 6f, Figure S10), comparable to the CT values of chlorine (i.e. 1575) and

401

ozone (i.e. 1440).50 Considering the costs/risks associated with production, storage,

402

and/or carcinogenic byproducts of chlorine and ozone, the cost-effectiveness of

403

Cu(II)/Sulfite is especially attractive. In conclusion, Cu(II)/Sulfite exhibited especially


Page 19 of 38


404

potent bactericidal performances in all tested water bodies, and therefore it ought to be

405

considered as a promising disinfection method in real practice.

406

A major finding of this study was that SO4•- generated from sulfite autoxidation catalyzed

407

by divalent metal ion, i.e. Mn(II), Co(II), Fe(II) or Cu(II), was potent in killing expanding

408

varieties of bacteria regardless of their antibiotic resistance. Cu(II)-resistant E. coli

409

persisters were especially sensitive to Cu(II)/Sulfite treatment, highlighting its significant

410

environmental implication as proliferative waterborne bacteria usually develop

411

resistance to aqueous toxic species. Concerns of oxygen-dependence and organic

412

matter interference may be raised towards Me(II)/Sulfite systems in treating real sewage

413

samples with complex components, and we therefore attempted the bactericidal

414

performance of Me(II)/Sulfite systems into sewage disinfection. As a result, Cu(II)/Sulfite

415

and Co(II)/Sulfite remarkably inactivated total bacteria and coliforms without additional

416

aeration, validating the feasibility of these two systems in real practice. Notably,

417

catalysts for sulfite oxidation are not limited to the four herein investigated metal ions,

418

and other heavy metals such as Cr(III/VI) or Ni(II) also own powerful catalysis

419

capabilities,17,51 expanding the application scope of our methods. Hereby, we propose

420

two ways of applying Me(II)/Sulfite into real wastewater disinfection. At first, free metal

421

ions can be added into wastewater together with sulfite. Tandem post-Me(II)/Sulfite

422

treatment units, including coagulation/sedimentation, sludge disposal, and adsorption,

423

can eliminate most of present metal ions. Alternatively, efficient heterogeneous

424

catalysts, such as cobalt ferrite nanoparticles,23 can be used to activate sulfite oxidation

425

in wastewater. Heterogeneous catalysts own the advantages of minimum metal ion

426

leaching, stable performance, and high catalytic activity,23,52 therefore holding more



Page 20 of 38

427

promises. The most important aspect of environmental implications is that catalytically

428

active metal ions and sulfite can be readily obtained from industrial wastewater and

429

combustion flue gas respectively, and eventually multiple purposes could be achieved,

430

i.e. flue gas desulfurization, bacterial inactivation, and organics decontamination if

431

present.

432 433

■ ASSOCIATED CONTENT

434

Supporting Information

435

Methods, reaction equations, water quality characterizations, and additional figures

436

depicting bacteria survival rate under various treatments. This material is available free

437

of charge via the Internet athttp://pubs.acs.org.

438 439

■ AUTHOR INFORMATION

440

Corresponding Authors

441

Danna Zhou, e-mail: [email protected].

442

Feng Wu, e-mail: [email protected].

443

Notes

444

The authors declare no competing financial interest.

445

■ ACKNOWLEDGMENTS


Page 21 of 38


446

This work was supported by the National Natural Science Foundation of China (NSFC

447

No. 21477090 and 41103066), the NSFC-CNRS_PRC Cooperation Project (No.

448

21711530144) and Science and Technology Project of Guangdong Province (No.

449

2014B030301055). Comments from anonymous reviewers are also appreciated.

450 451



Page 22 of 38

452

Reference:

453

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Prog. React. Kinet. 1989, 15, 35−75.

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599

.



Table of Content

600

O2

Me(IIa/IIIb)

SO32-

Me(Ia/IIb)

a. Cu b. Mn, Co, Fe

O2

SO32-

SO3● SO5●-

SO32-

SO4●-

601 602


Page 30 of 38

Page 31 of 38


603 604

Figure 1. E. coli inactivation efficiencies (a) and ESR spectra (b) of Me(II)/Sulfite. (0.8

605

mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM Cu(II), 2 mM sulfite, PBS, pH 6).

606



Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

100 10 1 0

607

0.5

1

2

4

8

Sulfite concentration (mM)


b100 Survival rate (%)

Survival rate (%)

a

Page 32 of 38

10 1

Mn(II)/Co(II)/Fe(II)0.05 0.1 Cu(II) 0.0125 0.025

0.2 0.05

0.4 0.1

0.8 0.2

Metal ion concentration (mM)

608

Figure 2. Effects of sulfite (a) and metal ion (b) concentration on the E. coli inactivation

609

efficiencies of Me(II)/Sulfite. (a) 0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM

610

Cu(II), sulfite concentrations varied between 0 and 8 mM, PBS, pH 7, reaction for 2 h.

611

(b) 2 mM sulfite, Mn(II) (0.05, 0.1, 0.2, 0.4, 0.8 mM), Co(II) (0.05, 0.1, 0.2, 0.4, 0.8 mM),

612

Fe(II) (0.05, 0.1, 0.2, 0.4, 0.8 mM), Cu(II) (0.0125, 0.025, 0.05, 0.1, 0.2 mM). PBS, pH 7,

613

incubation for 2h.

614 615


Page 33 of 38


a

Survival rate (%)

Survival rate (%)

100 10 1 0.1 5

616

b


6

7

8

8.5

Sulfite Mn(II) Co(II) Fe(II) Cu(II)

100

1 0.1

5

6

7

8

8.5

pH

pH

617

Figure 3. Effect of pH on the E. coli inactivation efficiencies of Me(II)/Sulfite (a) and

618

sulfite or Me(II) (b). Magenta curve in panel a represented the trendline of Cu(II)/Sulfite

619

bactericidal performance against pH. 0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1

620

mM Cu(II), 2 mM sulfite, incubation in PBS for 2h.

621



a No treatment

Mn(II)/Sulfite

Fe(II)/Sulfite

Cu(II)/Sulfite

c Residual NADH (%)

b

622

Co(II)/Sulfite

120

kD 95 72 55

80 40

43 0

t en ulfite ulfite ulfite ulfite m t a /S /S /S /S tre n(II) o(II) e(II) u(II) o F C C M N

34

623

Figure 4. Morphological characterization of E. coli with SEM (a), cellular NADH

624

depletion (b), and BSA degradation (c) after treatments with Me(II)/Sulfite. 0.8 mM

625

Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II), 0.1 mM Cu(II), 2 mM sulfite. PBS, pH 6, incubation

626

for 2h.

627


Page 34 of 38

Page 35 of 38


628



Page 36 of 38

629

Figure 5. (a) Me(II)/Sulfite bactericidal performances on E. coli after quenching with

630

TBA, thiourea, or dipyridyl. (b) Cellular metal content comparative quantifications by

631

treatments of Me(II) alone or Me(II)/Sulfite towards E. coli. (c) Effect of oxygen on E. coli

632

inactivation efficiencies of Me(II)/Sulfite, 2h. 0.8 mM Mn(II), 0.4 mM Co(II), 0.4 mM Fe(II),

633

0.1 mM Cu(II), 2 mM sulfite. PBS, pH 6, incubation for 2 h. (n.d., not detectable).

634


Page 37 of 38


O2

Me(IIa/IIIb)

O2

SO3

636

2-

Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite

SO32-

Me(Ia/IIb)

a. Cu b. Mn, Co, Fe

635

SO32-

SO4●-

SO3

●-

Me(II)-OH complex

SO5●SO4●-

SO32-

Cu(I)

Cu(II)/Sulfite

SO4●Cu(II)-OH SO 23

Scheme 1. Bactericidal mechanisms of Me(II)/Sulfite.

637



10 Mn(II)/Sulfite Co(II)/Sulfite Fe(II)/Sulfite Cu(II)/Sulfite

0

1

2


1

3

0

d100

e100

Survival rate (%)

Survival rate (%)


0

638

1

2

2

3

10 1


0.1 0.01 0

Time (h)

Time (h)

1

1

Added E. coli

c 100

3

f


1 0.1 0

Time (h)

1

2

1

2

3

Time (h)

Survival rate (%)

1

Coliforms

b100

Survival rate (%)

Total bacteria

Survival rate (%)

Survival rate (%)

a100

Page 38 of 38

3

Time (h)

100 1 0.01


1E-4 1E-6

0

1

2

3

Time (h)

639

Figure 6. Bactericidal performance of Me(II)/Sulfite on sewage samples. Samples from

640

biological treatment tank (a-c) and secondary sedimentation tank (d-f) in Erlangmiao

641

wastewater treatment plant were tested. Bactericidal efficiencies with total bacteria (a,

642

d), coliforms (b, e), and added E. coli (c, f) were monitored. Mn(II) 0.8mM, Co(II) 0.4mM,

643

Fe(II) 0.4mM, Cu(II) 0.1mM, 2mM sulfite.

644 645 646 647 648


Efficient Bacterial Inactivation by Transition Metal ... - ACS Publications

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