Rapid Hydrolysis of Penicillin Antibiotics Mediated by Adsorbed Zinc

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Environmental Processes

Rapid Hydrolysis of Penicillin Antibiotics Mediated by Adsorbed Zinc on Goethite Surfaces Feng Sheng, Jingyi Ling, Chao Wang, Xin Jin, Xueyuan Gu, Hong Li, Jiating Zhao, Yujun Wang, and Cheng Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02666 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Environmental Science & Technology

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Rapid Hydrolysis of Penicillin Antibiotics Mediated by Adsorbed Zinc on Goethite Surfaces

2 3

Feng Sheng1, Jingyi Ling1, Chao Wang1, Xin Jin1, Xueyuan Gu1, Hong Li2, Jiating Zhao2, Yujun Wang3, Cheng Gu1*

4 5 6

1State

Environment, Nanjing University, Nanjing 210023, P.R. China

7 8

Key Laboratory of Pollution Control and Resource Reuse, School of the

2CAS

Key Laboratory for Biological Effects of Nanomaterials and Nanosafety,

9

HKU-IHEP Joint Laboratory on Metallomics, Institute of High Energy Physics,

10

Chinese Academy of Sciences, Beijing 100049, P.R. China

11 12

3State

Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil

Science, Chinese Academy of Sciences, Nanjing 210008, P. R. China

13 14

*To whom correspondence should be addressed

15

Cheng Gu

16

Professor

17

School of the Environment

18

Nanjing University

19

Nanjing, Jiangsu, 210023

20

P. R. China

21

Phone/Fax: +86-25-89680636

22

E-mail: [email protected] 1

ACS Paragon Plus Environment


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TOC Art

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27

Abstract

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The soil environment is an important sink for penicillin antibiotics released from

29

animal manure and wastewater, but the mineral-catalyzed transformation of

30

penicillins in soil has not been well studied. To simulate this environmental process,

31

we systematically investigated the behavior of penicillin G and amoxicillin, the two

32

most widely-used penicillin antibiotics, in the presence of goethite and metal ions.

33

The results demonstrated that Zn ion significantly promoted the hydrolysis of

34

penicillins in goethite suspensions, as evidenced by the degradation rate nearly three

35

orders of magnitude higher than that of the non-Zn-containing control. Spectroscopic

36

analysis indicated that the specific complexation between penicillins, adsorbed Zn and

37

goethite was responsible for the enhanced degradation. Metastable interactions,

38

involving hydrogen bonds between carbonyl groups in the β-lactam ring and the

39

double/triple hydroxyl groups on goethite surface, and coordination bonding between

40

carboxyl groups and surface irons, were proposed to stabilize the ternary reaction

41

intermediates. Moreover, the surface zinc-hydroxide might act as powerful

42

nucleophile to rapidly rupture the β-lactam ring in penicillins. This study is among the

43

first to identify the synergic roles of Zn ion and goethite in facilitating penicillin

44

degradation and provides insights for β-lactam antibiotics to assess their

45

environmental risk in soil.

46

3



47

Introduction

48

Recently, the widespread antibiotics in aquatic and soil environments have raised

49

a worldwide concern, as the overuse of antibiotics leads to the emergence of resistant

50

bacteria problem threatening human health.1-3 The β-lactam antibiotics are the most

51

frequently used antibiotics, making up over 50% of the total worldwide antibiotic

52

consumption.4,5 Among them, the usage of penicillins accounts for the major

53

consumption of β-lactam antibiotics.6 Previous study indicated that penicillin

54

antibiotics could be degraded through hydrolysis, photolysis and biological reactions

55

in the aqueous environment, where the occurrence of penicillin antibiotics is relatively

56

low ranging from 3 to 200 ng L-1 in wastewater effluent and surface water.7 The

57

metal-assisted degradation of penicillin antibiotics in the presence of Zn2+,8 Cu2+,9

58

Mn2+,10 Fe3+ 4 and Cd2+

59

carbonyl and amino functional groups in penicillins account for the strong tendency to

60

complex with metal ions, which in turn significantly influences the stability of the

61

β-lactam ring.4 For example, coordination between Zn2+ and penicillins could stabilize

62

the tetrahedral intermediate generated from OH- attack on β-lactam ring and promote

63

their degradations.8 Previous studies further indicated that different metal ions

64

exhibited different enhancement for the degradation of penicillin antibiotics due to the

65

different sites of metal-ion coordination.12 Compared to aqueous phase reactions, few

66

studies have investigated the transformation of penicillin antibiotics in soil.

67

Nonetheless, the intense veterinary usage of penicillins in livestock and the

68

subsequent application of antibiotic-containing animal manure on agricultural fields

11

has been widely reported in solution phase. The carboxyl,

4


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as fertilizer have led to the release of considerable amounts of antibiotics into the

70

soil.13,14 According to the previous literatures, microbial-involved hydrolysis reaction

71

in soil environment is deemed as the main degradation pathway for penicillins.15-18 A

72

recent study found that amoxicillin could still be quickly degraded in sterilized soil,14

73

suggesting that abiotic degradation of penicillins might also play an essential role in

74

soil environment. Furthermore, the different physicochemical characteristics of

75

penicillins, such as their solubility, speciation and octanol-water partitioning

76

coefficient (KOW), as well as the properties of the soil, including the pH, water content

77

and microbial activity, could also affect degradation.7,13 The low KOW values of

78

penicillins (0.87–1.83)1 would hinder their association with the organic components

79

of soil, whereas soil minerals, e.g., iron oxides, clays and manganese oxides, may

80

provide matrices to form surface complexes with penicillin functional groups.19,20

81

Goethite is a common mineral, which is ubiquitously distributed in soils and

82

sediments.21 Due to its unique surface structure, goethite is able to strongly adsorb

83

several antibiotics in soil and subsequently catalyze their transformation.22-26 For

84

instance, Zhang and Huang found that fluoroquinolone antimicrobials could be

85

strongly adsorbed onto goethite accompanied by the slow oxidation process.2

86

Furthermore, goethite mineral usually coexists with various heavy metals in natural

87

soil.27 According to the prior studies, goethite particles were considered as the

88

potential sink for many cations (Cu, Mn, Ni and Zn)28 and Zn-bound goethite was also

89

observed in natural loess soil.29,30 The typical concentrations of Zn in agricultural

90

soils were reported to range from 52.17 to 227 mg kg-1.31-33 Even higher 5



91

concentrations of Zn could be detected in industrial polluted soils.27 Therefore, the

92

coexistence of metals and goethite could be ubiquitous in natural soil, which might

93

significantly affect the migration and transformation of organic and inorganic

94

contaminants on goethite surface.34-38 Gräfe et al. investigated the co-sorption of

95

arsenate and zinc ion in soil, and found that the adsorption of arsenate on ferrihydrite

96

increased by ~5 times in the presence of zinc via the formation of surface precipitate

97

[Zn2(AsO4)OH].27 In addition, the study of Gu et al. revealed that copper ion was able

98

to enhance the adsorption of ciprofloxacin on goethite surface by forming

99

goethite-copper-ciprofloxacin ternary complex.25

100

So far, few studies have comprehensively examined the reactions of penicillin

101

antibiotics in metal/goethite systems. Thus, the objectives of this study were to

102

identify the surface-mediated reaction of penicillins on goethite in the presence of

103

common metal ions, and to elucidate the mechanism underlying the rapid hydrolysis

104

of penicillin determined in a Zn/goethite system. The analyses were performed using

105

Fourier transform infrared (FTIR) and Zn K-edge X-ray absorption near edge

106

structure (Zn K-edge XANES) spectrometry. To our knowledge, this study is among

107

the first to demonstrate the synergetic effects of Zn ion and goethite on catalyzing

108

hydrolysis of penicillin antibiotics under mild conditions. As such, these new findings

109

contribute to a better understanding of the environmental persistence and

110

transformation of not only penicillins but also many other β-lactam antibiotics in soil.

6


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111


Materials and Methods

112

Chemicals. Chemicals used in this study were described in the Supporting

113

Information. Goethite was synthesized following the method of Zhao et al26 and the

114

details were also shown in the Supporting Information.

115

Degradation and Adsorption Experiments. Three buffers, MES (10 mM, pH

116

5.5 and 6.5), HEPES (10 mM, pH 7, 7.5 and 8) and CHES (10 mM, pH 9 and 10),

117

were selected due to their previously reported negligible effects on the interactions

118

between contaminants and minerals.9,39 These buffers were prepared by dissolving the

119

required amounts of reagents and then adjusting the solution pH by the addition of 1

120

M HCl or 1 M NaOH. To exclude the effects of organic buffers and glass bottles,

121

control experiments were conducted and the detailed information was listed in

122

Supporting Information. The results (Figure S1-S3) indicated that neither organic

123

buffers nor glass bottles had effects on the degradation process. Before the reaction,

124

the colloid solution (100 mL) containing goethite (0.02 to 0.5 g L-1), Zn ion (1 to 50

125

µM) and different buffer (MES, HEPES or CHES, 10 mM) was thoroughly mixed

126

using magnetic stirring bar at 200 rpm in an amber glass vial (100 mL) for 2 h. Our

127

preliminary results (Figure S4) indicated that the adsorption of Zn, Co, Ni, Cu and Pb

128

on goethite could reach equilibrium within 2 h. The reaction temperature was at 20 ±

129

2 °C, and the variation of pH was controlled within 0.1 units. The initial concentration

130

of antibiotics was 50 µM in the reaction vial. To initiate the reaction, 1 mL antibiotic

131

solution (5 mM) was spiked into the reaction solution (100 mL). At predetermined

132

time interval, 1 mL suspension was sampled and transferred to a tube prefilled with 7



133

0.1 mL EDTA solution (0.2 M). Then the residual antibiotic was extracted by shaking

134

for 5 min, and filtered through a 0.22 µm PTFE membrane. The recovery rates of PG

135

and AMX with EDTA extraction were detected as 103-104% and 99-103%,

136

respectively. The adsorption of penicillins on goethite was proven to be negligible in

137

Zn/goethite system (Figure S5, S6). Similar experiments were conducted for Co, Ni,

138

Cu and Pb ions. A pseudo-first-order kinetics model was used to fit the degradation

139

kinetics and then the rate constant (k, min-1) was calculated.4,10 Calculation details of

140

the pseudo-first order degradation constant were involved in Supporting Information.

141

The degradation of penicillin antibiotics under anoxic condition was also conducted

142

by purging N2 during the whole reaction period. A quenching experiment, in which

143

isopropanol (100 mM) was added as a radical scavenger, was conducted to investigate

144

the potential involvement of hydroxyl radicals in the reaction. All the experiments

145

were conducted in triplicate.

146

We also tested the adsorption of Zn on goethite at different concentrations of Zn

147

and goethite at pH 7 under the same reaction conditions. Before the analysis, the

148

samples were centrifuged at 4,000 g for 20 min and filtered through a 0.22 µm

149

cellulose acetate membrane. The Zn2+ concentration in solution was finally

150

determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES,

151

PQ 9000, Jena, Germany) after the sample was acidified with 6 M HNO3 (10 µL).

152 153 154

Analytical Methods. The details of analytical methods of penicillin antibiotics were described in Supporting Information. Spectroscopic Analysis. IR spectra were obtained by FTIR spectroscopy 8


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(Bruker Tensor 27, Germany), using a system equipped with an attenuated total

156

reflection (ATR) ZnSe crystal flow cell. In this study, H2O was replaced by D2O to

157

eliminate the disturbance due to the strong H-O-H bending absorption at ~1640

158

cm-1.17 To investigate the interaction between AMX and goethite in the presence of

159

Zn, 1 mL of 4 g L-1 goethite suspension in D2O was initially spread on the cell and

160

dried at 40 °C in a vacuum oven overnight to form a uniform goethite coating. The IR

161

spectra were then collected as below. Firstly, 1 mL NaCl solution (10 mM, in D2O)

162

was added into the coated cell and then 20 µL Zn2+ solution (5 mM, in D2O) was

163

applied. After equilibrium of Zn on goethite for 2 h, the background spectrum for

164

each sample was collected. Subsequently, 0.1 mL AMX stock solution (5 mM in

165

D2O) was spiked into the cell and IR spectra were continuously collected every 10

166

min in the range of 1000 to 2000 cm-1 with a resolution of 4 cm-1 and total scan of 64

167

within 30 min. The pD of the solution was adjusted to 7.0 ± 0.1 by addition of 1 M

168

DCl or 1 M NaOD. Similarly, the FTIR spectra of control groups, including AMX

169

solution alone, AMX/Zn, and AMX/goethite systems, were obtained for comparison.

170

The zinc species adsorbed on goethite were characterized by Zn K-edge XANES

171

experiment conducted on the BL14W beamline at Shanghai synchrotron radiation

172

facility. The energy of synchrotron was performed at 3.5 GeV and the current was

173

between 150 and 200 mA. Franklinite (ZnFe2O4), smithsonite (ZnCO3), willenite

174

(Zn2SiO4), Zn(OH)2 and ZnO were served as the reference compounds. The

175

suspension sample, consisting of goethite, NaCl and Zn with the final concentrations

176

of 5 g L-1, 10 mM and 10 mM, was firstly prepared. Then the system pH was adjusted 9



177

to 5.5, 7 and 10 by addition of HCl (1 M) and NaOH (1 M). After the pH was

178

stabilized for 48 h, the suspension was centrifuged at 4000 g for 30 min and the

179

sludge paste was collected to mount in a thin plastic sample holder covered with

180

Kapton tape. The Zn K-edge (9659 eV) was carried out with a Si (111) crystal

181

monochromator, and the Athena program was used for data processing.

182

Results and Discussion

183

Enhanced Degradation of Penicillins on Goethite in the Presence of

184

Different Metals. The degradation of PG in the presence of goethite was negligible

185

(< 4%) at neutral solution (pH = 7) within 48 h (Figure S7). However, when metal

186

ions were present, the degradation rates were significantly enhanced, following the

187

order: Zn > Co > Ni ≈ Cu ≈ Pb (Figure 1a). Especially for Zn, the degradation rate (k

188

= 1.14 h-1) increased ~3 orders of magnitude higher than the control sample with PG

189

and goethite (k = 0.001 h-1). The enhancement of PG degradation by Zn in the

190

presence of goethite was also observed at lower PG concentration (Figure S8). The

191

study by Gu et al. showed the enhanced adsorption of ciprofloxacin in ternary

192

complexation of Cu/goethite/ciprofloxacin.25 However, in our penicillin/Zn/goethite

193

system, the adsorption of penicillins on goethite was negligible (Figure S5, S6).

194

Previous studies also indicated that metals (Cu, Zn, Co, et. al.) could facilitate the

195

degradation of penicillin antibiotics via forming the metal/antibiotics complexes in

196

homogeneous solution, while the rates followed a different pattern as Cu > Zn > Ni ≈

197

Co.27,35 In homogeneous metal-catalyzed degradation, the aqueous metal ions (e.g. 10


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198

Zn2+, Co2+, Fe3+) could coordinate with the functional groups in penicillin molecules

199

to stabilize the tetrahedral intermediate generated from H2O and OH- attack on the

200

beta-lactam ring.10 Moreover, the prominent mechanism for penicillin degradation in

201

the presence of Cu2+ was attributed to Cu-complex oxidation process.9 Therefore, the

202

discrepancy probably reflected the difference in the mechanism of PG degradation in

203

metal/goethite vs. aqueous system. Since Zn exhibited the most significant effect, we

204

would focus on the catalytic degradation of penicillins in the coexistence of Zn ion

205

including adsorbed and aqueous Zn, and goethite suspension (Zn/goethite system) to

206

explore the underlying mechanism in the following study. Degradations of three

207

analogous penicillins (6-APA, AMP and AMX) were also conducted in Zn/goethite

208

system, which had the same basic structure as PG but different substituted groups in

209

the branched chain (Figure 2). As a result, compared to the control groups (Figure

210

S9), the prominent enhancements for degradation of all four penicillins in Zn/goethite

211

system were observed (Figure 1b), and the calculated pseudo-first-order degradation

212

constants of PG, AMP, AMX and 6-APA were 1.14, 0.516, 0.438 and 0.174 h-1,

213

respectively. The different degradation behaviors of these penicillin antibiotics were

214

consistent with their susceptibility to the nucleophilic attack, which was mainly

215

attributed to the inherent strain in β-lactam ring.12 As the structural hydrogen in

216

penicillins was substituted by the electron-donating groups (amino and hydroxyl

217

groups), the torsional rotation of β-lactam ring might decrease, subsequently

218

enhancing the stability.35 The penicillin antibiotics could be rapidly degraded in

219

Zn/goethite system, and the ternary interactions between penicillin antibiotics, 11



220

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goethite and Zn ion might account for this degradation.

221

The degradation of penicillin antibiotics via hydrolysis and oxidation reactions

222

mediated by transition metals or minerals has been widely reported.3,9,19,39 To obtain

223

insights into the mechanism of penicillin degradation in Zn/goethite system, we

224

conducted anoxic and radical quenching experiments with two typical penicillins (PG

225

and AMX) and identified the reaction products arising from their degradation. As

226

shown in Figure S10, neither oxygen nor isopropanol had a significant effect on the

227

degradation, indicating that penicillin degradation did not involve oxygen or hydroxyl

228

radicals. The analysis of degradation products demonstrated that the hydrolysis

229

products of AMX and PG differed in their retention times but had the same molecular

230

weights as 383 (365 + 18) for AMX and 352 (334 + 18) for PG (Figure S11).40

231

According to the mass spectra, the major degradation products were confirmed to be

232

the two amoxicilloic acid isomers for AMX, and two penicilloic acid isomers for PG

233

(Figure S11), which were widely reported as the major hydrolysis products of PG and

234

AMX.4 Whereas, the absence of oxidation products with molecular weights of 381

235

(AMX, 365 + 16) and 350 (PG, 334 + 16) indicated the absence of the oxidation

236

reaction

237

demonstrated that only hydrolysis degradation of penicillin antibiotics occurred, when

238

Zn2+ or Fe3+ ions existed in solution.4,8,10 Therefore, the coexistence of Zn and

239

goethite catalyzes the rapid degradation of penicillins dominantly via hydrolysis

240

rather than oxidation. Interestingly, the degradations of both AMX and PG were

241

completely suppressed by addition of EDTA (Figure S10), which could be explained

in

the

AMX(PG)/Zn/goethite

system.

Similarly,

12


previous

studies

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242

by the strongly competitive complexation of EDTA against penicillins for the reactive

243

sites in Zn/goethite system. Hence, the primary interaction between penicillin

244

antibiotics and Zn/goethite is prerequisite for the hydrolysis reaction.

245

Furthermore, the effects of Zn concentration, goethite dosage and system pH on

246

AMX and PG hydrolysis were also investigated. As shown in Figure 3, PG was more

247

facile to undergo hydrolysis than AMX in Zn/goethite system, in agreement with the

248

regular hydrolysis pattern in homogeneous solution.4,40 The hydrolysis rates of both

249

AMX and PG increased with the increasing dosage of adsorbed Zn. Moreover, the

250

remarkable correlation between hydrolysis rates (k, min-1) and amounts of adsorbed

251

Zn (Figure 3a) suggested that the degradation of AMX (R2 = 0.94, p < 0.01) and PG

252

(R2 = 0.96, p < 0.01) was dependent on the adsorbed Zn rather than the aqueous Zn. In

253

addition, less enhancement for both PG and AMX degradation was observed with

254

high adsorbed Zn (>24 mg g-1, Figure 3a). As the initial concentration of Zn ion

255

was >30 µM, nearly maximum adsorption capacity (24 mg g-1) on goethite (50 mg

256

L-1) could be achieved (Figure S12a). Meanwhile, the Zn ion in solution also

257

significantly increased (Figure S12b), which would complex with penicillins in

258

homogenous solution and prevent the formation of ternary intermediate. This result

259

further confirmed the important role of adsorbed Zn species in penicillin hydrolysis in

260

Zn/goethite system. Similarly, Figure S13 showed that the rates of AMX and PG

261

hydrolysis also increased as more goethite was added, presumably due to the creation

262

of additional reactive sites for hydrolysis as Zn ions were increasingly adsorbed.

263

However, the excessive goethite (dosage above 0.2 g L-1) slightly suppressed both 13



264

AMX and PG hydrolysis in Figure S13. When the amount of goethite was over 0.2 g

265

L-1, all the Zn ions were adsorbed, and the excessive goethite could compete against

266

Zn species to form the reactive ternary complex, finally resulting in the decreased

267

rates of hydrolysis.4 Moreover, system pH also played an essential role in hydrolysis

268

with the optimal condition at pH 8.5 (Figure 3b). As the pH changed from 5.5 to 10, a

269

few parameters in penicillin/Zn/goethite system would be altered, including the

270

adsorption amount of Zn, species of adsorbed Zn and penicillin antibiotics, and

271

surface charge of goethite. Specifically, due to the multiple pKa values of AMX (2.4,

272

7.4 and 9.6) and PG (2.7),41 AMX would exist as zwitterion AMX, AMX- and

273

AMX2-, whereas anionic PG- was the dominant species of PG in this pH range (Figure

274

S14). Compared to PG hydrolysis, the similar trend for AMX degradation was

275

observed (Figure 3b), suggesting that the varied charge of AMX might have little

276

influence on its hydrolysis process. Figure 3b also showed that the adsorption of Zn

277

ion on goethite reached the maximum at pH 8.5, since higher pH promoted the

278

deprotonation of the active surface and the surface-mediated hydrolysis of Zn.42 As

279

the pH increased to 8.5, the increased amounts of adsorbed Zn would provide more

280

reactive sites and penicillin hydrolysis would be accelerated.43 However, at a pH >

281

8.5, the surface charge of goethite would alter from positive to negative, as the point

282

of zero charge (pHPZC) of goethite was measured as ~9 (Figure S19). The electrostatic

283

repulsion between negatively charged goethite and penicillin molecules would

284

strongly inhibit their interactions, finally suppressing the hydrolysis process. In

285

addition, as the change of system pH, the speciation of adsorbed Zn also changed. 14


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286

However, it is quite difficult to distinguish the importance between the speciation of

287

adsorbed Zn and the adsorption amount for the enhanced hydrolysis of penicillin,

288

since the two variables changed simultaneously. More discussion for the reactive Zn

289

species on goethite surface was provided below.

290

FTIR and Zn K-edge XANES Analysis. In this study, FTIR spectroscopy was

291

applied to investigate the interactions between penicillin antibiotics and Zn/goethite.

292

Due to the low absorption intensity of PG, AMX served as the model compound

293

(Figure S15). As shown in Figure 4, the absorption bands of AMX solution at 1766,

294

1674, 1602, 1518, 1457, 1402 and 1268 cm-1 referred to the stretching vibration of

295

ketone in β-lactam ring (νC=O), stretching vibration of ketone in amide bond (νC=O,

296

amide),

297

phenolic hydroxyl (νOH), bending vibration of C-N-C in amide group (νC-N-C,

298

symmetric stretching vibration of carboxyl (νCOOs) and bending vibration of C-N in

299

β-lactam ring (νC-N), respectively. The FTIR spectra of AMX binding to the sole Zn

300

ion (Figure 4a), goethite (Figure 4b) and coexistence of Zn and goethite (Figure 4c)

301

were also recorded for comparison. It was reported that the difference Δν (Δν = νCOOas

302

- νCOOs) between asymmetric and symmetric stretching vibrations of carboxyl could

303

be used to identify the binding mode of the carboxylic group with metals.25 When the

304

Δν of the free carboxyl was greater than that of the carboxyl-metal coordination, it

305

suggested a bidentate complex in carboxyl-metal chelate, otherwise a monodentate

306

complex.

307

asymmetric stretching vibration of carboxyl (νCOOas), stretching vibration of amide),

At pD 7.0, the Δν of AMX/Zn (232 cm-1) was greater than that of AMX (201 15



308

cm-1), indicating a monodentate complex between the carboxylic group and Zn ion in

309

solution (Figure 4a). Together with the blue shift of C-N group from 1269 to 1283

310

cm-1 in β-lactam ring and red shift of carboxyl group from 1402 to 1372 cm-1,

311

aqueous Zn ion was indicated to complex with AMX via carboxyl group and ternary

312

nitrogen, which was in good agreement with a previous study.4 Furthermore, the

313

slight red shifts (4 and 3 cm-1) at 1518 and 1457 cm-1 in AMX/Zn system suggested

314

that the active coordination sites of AMX might also involve the phenolic hydroxyl

315

and amide nitrogen. However, the absence of an obvious change at 1766 cm-1 ruled

316

out the coordination of carbonyl group in β-lactam ring with aqueous Zn ion (Figure

317

4a).

318

In contrary, unlike AMX/Zn system, the carbonyl bonds in both β-lactam ring

319

and amide group shifted to 1752 and 1656 cm-1 upon the interaction with goethite,

320

respectively (Figure 4b). These widened and red-shifted peaks in AMX/goethite

321

system evidenced the formation of hydrogen bonds between carbonyl groups and

322

hydroxyl groups on goethite surface.25,44,45 Furthermore, the decreased Δν (191 cm-1)

323

in AMX/goethite system supported the bidentate coordination between carboxylic

324

group and surface iron, which was different from the monodentate complex in

325

AMX/Zn system. The unchanged absorption at 1268 cm-1 indicated that the C-N

326

group in β-lactam ring was not involved in the interaction between AMX and goethite

327

(Figure 4b).

328

The coordination bonds between the goethite surface and the β-lactam ring

329

determined for AMX/Zn/goethite were confirmed by the similarities of peak shifts 16


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330

compared with the AMX/goethite and AMX/Zn systems (Figure 4c). Among them,

331

carboxyl group of AMX on Zn/goethite preferentially formed the bidentate complex

332

with surface iron rather than Zn species, evidenced by the less Δν of AMX (190 cm-1).

333

The coordination of AMX in Zn/goethite system was further confirmed at different Zn

334

concentrations. As shown in Figure 4d, the absorption peaks of AMX at 1752 and

335

1658 cm-1 did not increase accordingly, suggesting that the carbonyl groups were

336

primarily coordinated with goethite. Meanwhile, the increasing intensity of IR peak at

337

1272 cm-1 supported the coordination between Zn species and nitrogen in β-lactam

338

ring. In addition, the increase in the Δν of AMX from 176 cm−1 to 193 cm−1 was

339

consistent with a change in AMX complexation, from a bidentate ligand with surface

340

iron to a monodentate ligand with the excess of Zn species. Together with all the

341

evidence above, we could conclude that the specific interactions in AMX/Zn/goethite

342

system involved: (i) the coordination between adsorbed Zn species and nitrogen in

343

β-lactam ring, (ii) bidentate coordination between carboxyl group and surface iron,

344

and (iii) hydrogen bonds between carbonyl groups and surface hydroxyl groups.

345

Therefore, the enhancement of AMX hydrolysis can be mostly attributed to the

346

specific ternary coordination between AMX and Zn/goethite. A similar mechanism

347

for the abiotic hydrolysis of penicillins in Zn2+/tris(hydroxymethyl)aminomethane

348

(Zn2+/Tris) system was also reported.43 The chelating effect of Tris in a ternary

349

coordination of penicillin/Zn2+/Tris was shown to be responsible for stabilization,

350

with the Zn-bound hydroxyl group of Tris serving as a powerful nucleophile to attack

351

the β-lactam carbonyl group. An analogous mechanism was described for hydrolytic 17



352

in metallo-β-lactamase enzymes,41 in which two dominant metals were located in

353

their active sites. One is essential for catalysis and the other for stabilizing the

354

reaction intermediate.15-18,46 In AMX/Zn/goethite system, the abundance of hydroxyl

355

groups on the crystallographic surface of goethite offer the sufficient reactive sites to

356

form hydrogen and coordination bonds, which in turn stabilize the ternary

357

intermediates. The positive correlation between the degradation rates (k, min-1) and

358

amounts of adsorbed Zn (R2>0.94, Figure 3a) suggests that the adsorbed Zn species

359

may act as the strong nucleophile to attack the β-lactam ring, leading to rapid

360

cleavage of the C-N bond. In contrary, in penicillin/goethite system, the complexation

361

forms between carbonyl group and surface hydroxyl group without a powerful

362

nucleophile on the reactive site. In this case, H2O or OH- in solution acts as the weak

363

nucleophile, which would explain the slow hydrolysis measured at neutral pH (Figure

364

S9). Similarly, the slow hydrolysis in penicillin/Zn system is attributed to the low

365

concentrations of stabilizer. In summary, the enhanced hydrolysis determined in

366

penicillin/Zn/goethite system can be attributed to the synergistic effects of both

367

goethite surface and adsorbed Zn species. Furthermore, goethite surface may offer

368

sufficient reaction sites to concentrate both nucleophile and penicillin molecule,

369

thereby increasing the possibility of their direct contact.

370

Since the hydrolysis reaction was strongly affected by the species of adsorbed

371

Zn, we analyzed the morphology of Zn at different pH values using Zn K-edge

372

XANES analysis. As shown in Figure 5, the normalized absorptions could be fitted by

373

standard references, including franklinite, smithsonite, willenite, Zn(OH)2 and ZnO at 18


Page 18 of 33

Page 19 of 33


374

pH 5.5, 7 and 10 (Figure S16). According to the linear combination fitting shown in

375

Table 1, franklinite was the predominant Zn species at pH 5.5 (41.4%), followed by

376

Zn(OH)2 (26.8%). As the pH increased to 7, 61.5% of the adsorbed Zn species was in

377

the form of Zn(OH)2, while ZnO accounted for 67.3% of the Zn species and Zn(OH)2

378

decreased to 7.1% at pH 10 on the goethite surface (Table 1). Based on these results,

379

hydrolysis reaction is mostly related to the Zn adsorption in the presence of

380

zinc-hydroxide species such as Zn(OH)2, which could mediate the rapid hydrolysis of

381

penicillin antibiotics. Similarly, in homogeneous Zn/penicillin reaction, zinc-bound

382

hydroxyls could act as the main nucleophile to catalyze the hydrolysis of penicillins.43

383

In summary, Zn ion can interact with goethite surface in penicillin/Zn/goethite

384

system. The pKa of Zn-bound water decreases via polarization effect to form the

385

adsorbed zinc-hydroxides on goethite surface, which act as the Lewis acid to complex

386

with functional groups in penicillin.43 The generation of zinc-hydroxide would

387

account for its role as the main nucleophile in penicillin hydrolysis.

388

Effects of Different Surface Hydroxyl Groups on Penicillin Hydrolysis.

389

Based on the FTIR results (Figure 4), the hydrogen bonds between the surface

390

hydroxyl groups and AMX are crucial for hydrolysis. However, three different

391

hydroxyl groups, including singly (≡FeOH), doubly (≡Fe2OH) and triply (≡Fe3OH)

392

coordinated forms, co-exist on goethite surface.47 To differentiate the effects of

393

different hydroxyl groups on hydrolysis reaction, goethite surface was modified by

394

phosphate or fluoride following the Wei’s method (Supporting Information).48

395

Excessive Na2HPO4 (1 mM) and NaF (10 mM) were utilized in modification to 19



396

ensure the complete OH-substitution on surface. After modification, hydroxyl groups

397

were coordinated by fluoride or phosphate, as the hydroxyl stretching vibration (3132

398

cm-1) for both phosphate and fluoride modified goethite underwent the remarkable red

399

shifts (4 cm-1) in Figure S17.49,50 However, previous study indicated that fluoride and

400

phosphate ions preferentially exchanged with singly coordinated hydroxyls, while the

401

doubly and triply coordinated groups remained intact.47 Therefore, the modification

402

mainly changed the singly coordinated hydroxyls, rather than the doubly or triply

403

complexed counterparts. Interestingly, as shown in Figure S18, phosphate modified

404

goethite exhibited the strong inhibition for AMX hydrolysis, while the negligible

405

effect was observed for fluoride modification. In the reaction solution, neither

406

phosphate nor fluoride ion was detected (data not shown), suggesting that the

407

disturbance of aqueous phosphate or fluoride ion on the adsorption of Zn could be

408

excluded. Our results demonstrate that lack of singly coordinated hydroxyls in

409

fluoride modified goethite does not result in the remarkable decrease of hydrolysis

410

rate, indicating that the carbonyl group in β-lactam ring might prefer to complex with

411

doubly and triply coordinated hydroxyl groups to form the surface hydrogen bond.

412

The inhibition by phosphate modified goethite might be explained by the change of

413

surface charge on goethite. After phosphate modification, the pHPZC of goethite

414

decreased from 9.2 to 6.7, while the change of pHPZC for fluoride modified goethite

415

(8.9) was negligible (Figure S19). The repulsions between negatively charged AMX

416

and phosphate modified goethite would obstruct the coordination at pH 7, finally

417

inhibiting the AMX hydrolysis. Therefore, the doubly or triply coordinated hydroxyl 20


Page 20 of 33

Page 21 of 33


418

groups on goethite surface might be the dominant sites for penicillin complexation.

419

Environmental Significance

420

This study demonstrates that penicillin antibiotics can be rapidly hydrolyzed in a

421

Zn/goethite system under environmentally relevant conditions (neutral pH, room

422

temperature and environmental concentration). The enhanced hydrolysis is likely due

423

to the synergetic effects of goethite and the adsorbed Zn species, in which goethite

424

stabilizes the reaction intermediates and zinc-hydroxide acts as a powerful

425

nucleophile. This finding reveals a new abiotic degradation route for penicillin

426

antibiotics, and thereby improves the understanding of environmental fate for both

427

penicillin antibiotics and their transformation products in soil. Moreover, the

428

degradation mechanism may be also relevant to the hydrolysis of other β-lactam

429

antibiotics (e.g. cephalosporins and monobactams) in the environmental matrices,

430

containing metal ions and minerals, which would provide valuable information for

431

developing abiotic degradation strategies for antibiotics. More importantly, our results

432

suggest that the potential effects of naturally occurring ions (K+, Ca2+, Na+ and heavy

433

metals) on the adsorption and transformation processes of common antibiotics in soil

434

should not be ignored and need more investigations in future researches.

435

Acknowledgements

436

This work was financially supported by National Science Foundation of China

437

(21777066), the Natural Science Foundation of Jiangsu Province (BK20170634) and

21



438

the Collaborative Innovation Center for Regional Environmental Quality.

439

Supporting Information Available

440

Parameters of HPLC and HPLC-QTOF-MS methods for 6-APA, PG, AMP and

441

AMX antibiotics; degradation of PG in the presence of goethite in control treatment;

442

degradation of 6-APA, PG, AMP and AMX antibiotics in control treatments;

443

degradation of AMX and PG with quenching reagents or under ambient and anoxic

444

conditions; AMX and PG degradation products analysis by HPLC-QTOF-MS ；

445

degradation of PG and AMX in different concentration of goethite; distribution

446

fractions of PG and AMX molecules in pH (1 ~ 12); different molecular structures of

447

PG and AMX; Zn K-edge XANES spectra of standard reference compounds; FTIR

448

spectra of goethite, phosphate-modified and fluoride-modified goethite; hydrolysis of

449

AMX with original and different modified goethite; zeta potentials of original and

450

different modified goethite from pH 5.5 to 10.

22


Page 22 of 33

Page 23 of 33


452

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alga-containing water environment: a mechanism and kinetic study. Environ. Sci. Pollut. R. 2019, 26, 9184-9192. 2.

Zhang, H.; Huang, C. H. Adsorption and oxidation of fluoroquinolone antibacterial agents and

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Kümmerer, K. Antibiotics in the aquatic environment - A review Part II. Chemosphere 2009, 75,

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Chen, J.; Wang, Y.; Qian, Y.; Huang, T. Fe(III)-promoted transformation of β-lactam antibiotics:

Hydrolysis vs oxidation. J. Hazard. Mater. 2017, 335, 117-124. 5.

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hydrolysis of three β-lactam antibiotics: Ampicillin, cefalotin and cefoxitin. Sci. Total. Environ. 2014, 466-467, 547-555. 7.

Kümmerer, K. Antibiotics in the aquatic environment - A review Part I. Chemosphere 2009, 75,

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dinuclear zinc(II) complexes: Functional mimics of metallo-β-lactamases. J. Am. Chem. Soc. 2000, 122, 6411-6422. 19. Hsu, M. H.; Kuo, T. H.; Chen, Y. E.; Huang, C. H.; Hsu, C. C.; Lin, A. Y. C. Substructure reactivity affecting the manganese dioxide oxidation of cephalosporins. Environ. Sci. Technol. 2018, 52, 9188-9195. 20. Pan, M.; Chu, L. M. Fate of antibiotics in soil and their uptake by edible crops. Sci. Total. Environ. 2017, 599-600, 500-512. 21. Liu, Q.; Li, X.; Tang, J.; Zhou, Y.; Lin, Q.; Xiao, R.; Zhang, M. Characterization of goethite-fulvic acid composites and their impact on the immobility of Pb/Cd in soil. Chemosphere 2019, 222, 556-563. 22. Xu, J.; Marsac, R.; Costa, D.; Cheng, W.; Wu, F.; Boily, J. F.; Hanna, K. Co-binding of pharmaceutical compounds at mineral surfaces: Molecular investigations of dimer formation at goethite/water interfaces. Environ. Sci. Technol. 2017, 51, 8343-8349. 23. Krumina, L.; Lyngsie, G.; Tunlid, A.; Persson, P. Oxidation of a dimethoxyhydroquinone by ferrihydrite and goethite nanoparticles: Iron reduction versus surface catalysis. Environ. Sci. Technol. 2017, 51, 9053-9061. 24. Benacherine, M. E. M.; Debbache, N.; Ghoul, I.; Mameri, Y. Heterogeneous photoinduced degradation of amoxicillin by goethite under artificial and natural irradiation. J. Photoch. Photobio. A. 2017, 335, 70-77. 25. Gu, X.; Tan, Y.; Tong, F.; Gu, C. Surface complexation modeling of coadsorption of antibiotic ciprofloxacin and Cu(II) and onto goethite surfaces. Chem. Eng. J. 2015, 269, 113-120. 26. Zhao, Y.; Geng, J.; Wang, X.; Gu, X.; Gao, S. Adsorption of tetracycline onto goethite in the presence of metal cations and humic substances. J. Colloid Interf. Sci. 2011, 361, 247-251. 27. Carabante, I.; Grahn, M.; Holmgren, A.; Kumpiene, J.; Hedlund, J. Influence of Zn(II) on the adsorption of arsenate onto ferrihydrite. Environ. Sci. Technol. 2012, 46, 13152-13159. 28. Wei B. G.; Yang L. S. A review of heavy metal contaminations in urban soils, urban road dusts and agricultural soils from China. Microchem. J. 2010, 94, 99-107. 29. Singh, B. and Gilkes, R. J. Properties and distribution of iron oxides and their association with minor elements in the soils of south-western Australia. J. Soil Sci. 1992, 43, 77-78. 30. Manceau, A.; Tamura, N.; Celestre, R. S.; MAcdowell, A. A.; Geoffroy, N.; Sposito, G.; Padmore, H. A. Molecular-scale speciation of Zn and Ni in soil ferromanganese nodules from loess soils of the Mississippi Basin. Environ. Sci. Technol. 2003, 37, 75-80. 31. Pan, L. B.; Ma, J.; Wang, X. L.; Hou, H. Heavy metals in soils from a typical county in Shanxi Province, China: Levels, sources and spatial distribution. Chemosphere 2016, 148, 248-254. 32. Holmgren, G. G. S.; Meyer, R. L.; Chaney, R. L.; Daniels R. B. Cadmium, lead, zinc, copper and nickel in agricultural soils of the United States of America. J. Environ. Qual. 1993, 22, 335-348. 33. Facchinelli., A.; Sacchi., E.; Mallen., L. Multicariate statistical and GIS-based approach to identify heavy metal sources in soils. Environ. Pollut. 2001, 114, 313-324. 34. Forbes, E. A.; Posner, A. M.; Quirk, J. P. The specific adsorption of divalent Cd, Co, Cu, Pb, and Zn on goethite. J. Soil Sci. 1976, 27, 154-166. 35. Buerge-Weirich, D.; Hari, R.; Xue, H.; Behra, P.; Sigg, L. Adsorption of Cu, Cd and Ni on goethite in the presence of natural groundwater ligands. Environ. Sci. Technol. 2002, 36, 328-336. 36. Swedlund, P. J.; Webster, J. G.; Miskelly, G. M. Goethite adsorption of Cu(II), Pb(II), Cd(II), and Zn(II) in the presence of sulfate: Properties of the ternary complex. Geochim. Cosmochim. Ac. 2009, 24


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73, 1548-1562. 37. Alcacio, T. E.; Hesterberg, D.; Chou, J. W.; Martin, J. D.; Beauchemin, S.; Sayers, D. E. Molecular scale characteristics of Cu(II) bonding in goethite-humate complexes. Geochim. Cosmochim. Ac. 2001, 65, 1355-1366. 38. Grossl, P. R.; Sparks, D. L.; Ainsworth, C. C. Rapid kinetics of Cu(II) adsorption/desorption on goethite. Environ. Sci. Technol. 1994, 28, 1422-1429. 39. Chen, J.; Sun, P.; Zhang, Y.; Huang, C. H. Multiple roles of Cu(II) in catalyzing hydrolysis and oxidation of β-lactam antibiotics. Environ. Sci. Technol. 2016, 50, 12156-12165. 40. Hirte, K.; Seiwert, B.; Schüürmann, G.; Reemtsma, T. New hydrolysis products of the beta-lactam antibiotic amoxicillin, their pH-dependent formation and search in municipal wastewater. Water Res. 2016, 88, 880-888. 41. Vijaya Bhaskar Reddy, A.; Yusop, Z.; Jaafar, J.; Jamil, N. H.; Majid, Z. A.; Aris, A. B. Development and validation of capillary electrophoresis method for simultaneous determination of six pharmaceuticals in different food samples combining on-line and off-line sample enrichment techniques. Food Anal. Method. 2018, 11, 533-545. 42. Mustafa, G.; Singh, B.; Kookana, R. S. Cadmium adsorption and desorption behaviour on goethite at low equilibrium concentrations: Effects of pH and index cations. Chemosphere 2004, 57, 1325-1333. 43. Diaz, N.; Sordo, T. L.; Suarez, D.; Mendez, R.; Martin-Villacorta, J. Zn2+ catalysed hydrolysis of β-lactams: Experimental and theoretical studies on the influence of the β-lactam structure. New J. Chem. 2004, 28, 15-25. 44. Fang, Y.; Zhou, W.; Tang, C.; Huang, Y.; Johnson, D. M.; Ren, Z. J.; Ma, W. Brönsted catalyzed hydrolysis of microcystin-LR by siderite. Environ. Sci. Technol. 2018, 52, 6426-6437. 45. Paul, T.; Machesky, M. L.; Strathmann, T. J. Surface complexation of the zwitterionic fluoroquinolone antibiotic ofloxacin to nano-anatase TiO2 photocatalyst surfaces. Environ. Sci. Technol. 2012, 46, 11896-11904. 46. Hu, Z.; Periyannan, G.; Bennett, B.; Crowder, M. W. Role of the Zn1 and Zn2 sites in metallo-β-lactamase L1. J. Am. Chem. Soc. 2008, 130, 14207-14216. 47. Ding, X.; Song, X.; Boily, J. F. Identification of fluoride and phosphate binding sites at FeOOH surfaces. J. Phys. Chem. C 2012, 116, 21939-21947. 48. Li, W.; Zhang, S.; Shan, X. Q. Surface modification of goethite by phosphate for enhancement of Cu and Cd adsorption. Colloid. Surface. A 2007, 293, 13-19. 49. Wei, S.; Tan, W.; Zhao, W.; Yu, Y.; Liu, F.; Koopal, L. K. Microstructure, interaction mechanisms and stability of binary systems containing goethite and kaolinite. Soil Sci. Soc. Am. J. 2012, 76, 389-398. 50. Weckler, B.; Lutz, H. D. Lattice vibration spectra. Part XCV. Infrared spectroscopic studies on the iron oxide hydroxides goethite(α) akaganéite(β) lepidocrocite(γ) and feroxyhite(δ). Eur. J. Solid State Inorg. Chem. 1998, 35, 531-544.

25



Page 26 of 33

578

Table 1 Relative proportion of Zn species on goethite at different pH values (5.5,

579

7 and 10) as determined by Zn XANES linear combination fitting (%) Sample

Franklinite

Smithsonite

Willenite

Zn(OH)2

ZnO

R-factor

pH 5.5

41.4

11.4

21.3

26.8

NA

0.0025

pH 7.0

10.4

4.9

23.2

61.5

NA

0.0014

pH 10

10.6

NA

15.0

7.1

67.3

0.0024

580

Franklinite: ZnFe2O4, Smithsonite: ZnCO3, Willenite: Zn2SiO4.

581

NA: Not available.

582

R-factor: Residual factor.

26


Page 27 of 33


584

Figure legends

585

Figure 1. (a) Degradation kinetics of PG in the presence of goethite and different

586

metal ions, including Co2+, Ni2+, Cu2+, Pb2+ and Zn2+; (b) Degradation kinetics of

587

6-APA, PG, AMP and AMX in the presence of goethite and Zn ion. The initial

588

concentrations of penicillin antibiotic, goethite, metal salt were 50 µM, 50 mg L-1 and

589

3 µM, respectively. The reaction pH was controlled at 7.0 ± 0.1 by 10 mM HEPES

590

buffer.

591

Figure 2. Molecular structures of 6-APA, PG, AMP and AMX.

592

Figure 3. (a) The degradation constants (k, min-1) of PG (red) and AMX (black)

593

plotted against the adsorption amounts of Zn ion (mg g-1) on goethite. The linear

594

fitting equations, determination coefficient (R2) and significance (p) were shown.

595

Reaction pH was controlled at 7.0 ± 0.1 by 10 mM HEPES. (b) Degradation constants

596

(k, min-1) of PG and AMX plotted against the different pH values (black). Adsorption

597

amounts of Zn on goethite plotted against the different pH values (blue). The initial

598

concentrations of Zn, AMX, PG and goethite were 3 µM, 50 µM, 50 µM and 50 mg

599

L-1, respectively. The pH values ranged from 5.5-9.5 were stabilized in 10 mM MES,

600

HEPES and CHES buffers.

601

Figure 4. ATR-FTIR spectra of (a) AMX in Zn solution; (b) AMX on goethite film;

602

(c) AMX on goethite film in the presence of Zn. The spectra were recorded every 10

603

min within 30 min. (d) FTIR spectra of AMX on goethite film with different initial Zn

604

concentrations (1, 10 and 100 µM) after 30 min equilibrium. The ionic strength and

605

pD were 10 mM NaCl and 7.0 ± 0.1, respectively. The initial concentration of AMX

606

was 500 µM. The spectrum of AMX (5 mM) in D2O at pD 7.0 was shown in black

607

line.

608

Figure 5. Zn K-edge XANES spectra of adsorbed Zn species on goethite at different

609

pH of 5.5, 7.5 and 10. Normalized absorption and fitting absorption at different pH

610

were represented in solid line and dots, respectively.

27



612

613 614

Figure 1

615

28


Page 28 of 33

Page 29 of 33


616 617

Figure 2

618

29



619

620 621

Figure 3

622 623

30


Page 30 of 33

Page 31 of 33


624

625

31



626

627 628

Figure 4

32


Page 32 of 33

Page 33 of 33


630 631 632 633

Figure 5

33


Rapid Hydrolysis of Penicillin Antibiotics Mediated by Adsorbed Zinc

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