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Remediation and Control Technologies

Photocatalytic Reactive Ultrafiltration Membrane for Removal of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes from Wastewater Effluent Shaojie Ren, Chanhee Boo, Ning Guo, Shu-Guang Wang, Menachem Elimelech, and Yunkun Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01888 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Environmental Science & Technology

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Photocatalytic Reactive Ultrafiltration Membrane for Removal of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes from Wastewater Effluent

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Environmental Science & Technology 10 11

Shaojie Ren,† Chanhee Boo,‡ Ning Guo,† Shuguang Wang,† and Menachem Elimelech,‡* Yunkun Wang,†‡*

12 13 14 15 16 17 18 19 20 21

†

Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Qingdao 266237, China ‡

Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520-8286, United States

22 23 24 25 26 27 28 29

‡*Corresponding

author: email: [email protected]; Tel: +1 (203) 432-2789

†‡*Corresponding

author: email: [email protected]; Tel: +86 (532) 58630960

1

ACS Paragon Plus Environment


ABSTRACT 30

Biological wastewater treatment is not effective in removal of antibiotic resistant

31

bacteria (ARB) and antibiotic resistance genes (ARGs). In this study, we fabricated a

32

photocatalytic reactive membrane by functionalizing polyvinylidene fluoride (PVDF)

33

ultrafiltration (UF) membrane with titanium oxide (TiO2) nanoparticles for the removal

34

of ARB and ARGs from a secondary wastewater effluent. The TiO 2-modified PVDF

35

membrane provided complete retention of ARB and effective photocatalytic

36

degradation of ARGs and integrons. Specifically, the total removal efficiency of ARGs

37

(i.e., plasmid-mediated floR, sul1, and sul2) with TiO2-modified PVDF membrane

38

reached ~98% after exposure to UV irradiation. Photocatalytic degradation of ARGs

39

located in the genome was found to be more efficient than those located in plasmid.

40

Excellent removal of integrons (i.e., intI1, intI2, and intI3) after UV treatment indicated

41

that the horizontal transfer potential of ARGs was effectively controlled by the TiO 2

42

photocatalytic reaction. We also evaluated the antifouling properties of the TiO 2-UF

43

membrane to demonstrate its potential application in wastewater treatment.

44 45 46 47 48 49

TOC Art

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51 52 53

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INTRODUCTION

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Widespread use of antibiotics results in the emergence of antibiotic resistant bacteria

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(ARB) and antibiotic resistance genes (ARGs) in diverse environmental matrices,

57

including soil, drinking water resources, and wastewater effluents. Stringent control of

58

ARB and ARGs release to the environment is of critical importance because these

59

microbial contaminants can potentially have a large negative impact on the

60

environment and human health.1,

61

(WWTPs) are not designed to eliminate microbial contaminants resistant to antibiotics,

62

but rather become the main source for proliferation of ARB and ARGs due to the high

63

concentration of activated sludge employed during biological wastewater treatment. 3,

64

4

65

2

Currently, most wastewater treatment plants

Advanced oxidation processes such as chlorination, ozonation, and ultraviolet (UV)

66

disinfection have recently been employed to control antibiotic resistant bacteria and

67

genes from wastewater effluents. However, the effectiveness of these advanced

68

oxidation treatment technologies for inactivation of ARB and ARGs is still

69

controversial.5 For instance, Zhang et al. have demonstrated inactivation of

70

tetracycline-resistant bacteria (TRB) containing tetC and tetA genes by sodium

71

hypochlorite,6 whereas Munir et al. reported conflicting results indicating that

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chlorination is not effective in inactivating these ARB and ARGs.7 In another study,

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residual chlorine has been shown to trigger the emergence of ARGs by acting like an

74

antibiotic.8 Inactivation of ARB by ozone oxidation is possible, but the release of free

75

ARGs from the bacteria cells decreases oxidation efficiency.5 UV treatment provides

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a promising solution for ARB removal by directly damaging the cell nuclei acid. 9

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However, employing high-intensity UV continuously during wastewater treatment

78

remains a challenge.10-12 Moreover, the genes fragmented by oxidation can be

79

integrated with other pathogens in the wastewater effluent, thereby requiring

80

post-treatment.13, 14 Thus, there is a critical need to develop alternative approaches

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for inactivation of ARB and further degradation of ARGs from wastewater effluents.

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Membrane technology has the potential to provide an effective solution for 3



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removal of ARB and ARGs from wastewater effluents.15-17 However, fouling by

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attachment of microbes on the membrane surface significantly deteriorates

85

membrane performance.18,

86

promoted within the fouling layer (i.e., biofilm) due to high bacteria density. 20,

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Incorporating photocatalytic nanomaterials on the membrane surface can control

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biofouling during treatment of wastewater effluents contaminated by ARB and

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ARGs.22 Titanium dioxide (TiO2) provides a promising platform for the development of

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a photocatalytic reactive membrane, which enables inactivation of ARB and ARGs 23,

91

24

92

(ROS) under UV irradiation.25, 26

19

Furthermore, horizontal transfer of ARGs can be 21

and reduces fouling potential through the generation of reactive oxygen species

93

The proliferation of ARGs is driven by vertical and horizontal gene transfers that

94

are closely related to the types of bacteria host and mobilome, respectively. 27 It is well

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known that both genome and plasmid (i.e., mobile genetic element) in the bacteria

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cell contain ARGs and integrons.28, 29 The ARGs carried by the genome are relatively

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stable in terms of their heredity. Thus, proliferation of these ARGs is mainly driven by

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vertical gene transfer. In contrast, horizontal transfer of ARGs takes place through

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transformation, transduction, or plasmid conjugation more easily and frequently.

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Integrons are considered an indicator of the potential for horizontal gene transfer

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due to their ability to capture exogenous gene cassettes.30,

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transfer mechanism of integrons is site-specific.32 For example, integrons located in

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plasmids have more chances for inter-species transmission, resulting in an increased

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ability to integrate with other bacteria. Likewise, degradation of integrons is expected

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to be dependent on their location in the bacteria cell, but studies of relevant

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mechanisms are still scarce. Therefore, fundamental understanding of the removal

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mechanisms of ARGs and integrons is critical for the development of effective control

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strategies.

31

As with ARGs, the

109

In this study, a photocatalytic reactive membrane was fabricated via a facile and

110

scalable surface modification technique to remove antibiotic resistant bacteria (ARB)

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and antibiotic resistance genes (ARGs) from secondary wastewater effluent. A 4


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polyvinylidene fluoride (PVDF) ultrafiltration membrane was functionalized with TiO 2

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nanoparticles to enable photocatalytic degradation of ARB and ARGs. Removal

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efficiency and degradation mechanisms of ARB and ARGs were investigated. We

115

also demonstrated the antifouling performance of the TiO2 functionalized PVDF

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membrane by evaluating fouling reversibility with secondary wastewater effluent.

117

Implications of the results for the use photocatalytic reactive membranes to remove

118

antibiotic resistant bacteria and genes in wastewater effluent are evaluated and

119

discussed.

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MATERIALS AND METHODS

121

Materials and Chemicals. PVDF ultrafiltration membrane (Sepro, Carlsbad, CA) with

122

a molecular weight cut-off of 100 KDa was used as a substrate. Before use,

123

membranes were immersed in ethanol for 30 min, followed by three 30-min periods of

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rinse with deionized (DI) water to remove residual ethanol. Titanium oxide

125

nanoparticles (TiO2, P25) were acquired from Evonik Industries AG. Tris-HCl solution

126

(1 M, pH 9) and dopamine hydrochloride were purchased from Sinopharm Chemical

127

Reagent Co., Ltd. The secondary wastewater effluent samples were collected from a

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WWTP located in Jinan City, China (36°42'11"N，117°02'10"E).

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Functionalization of PVDF Membrane with TiO2. Pre-wetted PVDF membrane

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coupons were cut to fit a custom-made rectangular frame and held tightly by a soft

131

rubber spacer using a clamp with the active surface layer facing up. A 10 mM Tris

132

buffer was prepared by diluting the Tris-HCl solution (1 M, pH 9) in DI water and

133

adjusting the pH to 8.5 using hydrochloric acid (HCl, 0.1 M). A freshly prepared 2

134

mg/mL dopamine solution in 10 mM Tris buffer (pH 8.5) was carefully poured onto the

135

membrane surface. After 6 h, the polydopamine (PDA)-deposited membrane was

136

removed from the frame and thoroughly rinsed with DI water three times. TiO2 (P25,

137

100 mg) was dispersed in 20 mL DI water, followed by 10 min sonication in ultrasonic

138

bath. The PDA-coated membrane was in contact with the TiO2 nanoparticle

139

suspension (5 mg/mL) for 24 h. All surface modification steps were performed on a

5



140

rocking platform at 30 °C. A schematic description of the PVDF membrane

141

modification with TiO2 nanoparticles and the subsequent use of this membrane for

142

ARB/ARGs filtration followed by UV photodegradation is presented in Figure S1.

143

Membrane Characterization. Membrane morphology was characterized by

144

scanning electron microscopy (SEM, Hitachi SU8010, Japan). Before imaging,

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samples were coated with a platinum layer to enhance membrane conductance.

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Membrane surface roughness was evaluated by atomic force microscopy (AFM,

147

Dimension Icon, Veeco Instruments Inc., USA) in tapping mode. Membrane samples

148

were dried in a vacuum desiccator for 24 h at 50 °C before AFM measurements. The

149

water contact angle of the membranes was measured by a goniometer (Shanghai

150

Zhongchen Digital Technology Apparatus Co. Ltd) using the sessile drop method. A

151

2-μL water droplet was placed on the surface of the pre-dried membrane and

152

photographed using a digital camera. The measurements were conducted on a

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minimum of two random locations with three different membrane samples, and the

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data were averaged.

155

Membrane porosity and water uptake were estimated gravimerically.33 A 1.5 cm 

156

1.5 cm membrane sample was stored in DI water for 24 h and weighed carefully after

157

removing excess water from the surface. Then, the sample was dried in a vacuum

158

oven for 24 h and the weight was reevaluated. Porosity () and water uptake values

159

were calculated using

160

 (%)  

161

 W  Wd Water uptake (%)   w  Wd

162

where Ww and Wd are the weights of the wet and dry membrane samples,

163

respectively, ρw is the density of water, S is the membrane area, and l is the

164

membrane thickness determined using a micrometer.

 Ww  Wd  w Sl

 100 

(1)

 100 

(2)

165

Filtration and UV Photocatalytic Degradation Experiment. The separation and

166

antifouling performance of the TiO2-modified PVDF membrane were evaluated in a 6


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dead-end UF system with a cell volume of 10 mL and an effective membrane area of

168

4.1 cm2 (Amicon stirred cell, Model 8010), which was connected to a dispensing

169

reservoir with a nitrogen gas supply. The secondary wastewater effluent was

170

pre-filtered through a filter paper (pore size of 80–120 μm) to remove large particulate

171

and suspended matters. To determine the retention performance, 250 mL secondary

172

effluent was filtrated through the membrane at a pressure of 1.4 bar (20 psi). Then,

173

100 μL sterile DI water was carefully dispensed on the membrane surface to wet the

174

fouling layer, followed by membrane exposure to UV254 for 1 h. The distance between

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the membrane surface and UV lamp (QiPaiWells Company, Shanghai) was

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maintained at 10 cm to obtain a consistent luminous intensity of 12 μW/cm 2.

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Fouling reversibility was evaluated to investigate the antifouling property of the

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TiO2-modified PVDF membrane. Before fouling experiments, a new membrane

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coupon was compacted for 3 h under a pressure of 1.4 bar using DI water as the feed.

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After compaction, the membrane was further stabilized for 20 min to obtain a constant

181

permeate water flux. Fouling experiments were conducted for 1 h using wastewater

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effluent as the feed at a pressure of 1.4 bar. After gentle rinsing with DI water, the

183

fouled membrane was exposed to UV for 1 h. Last, the pure water flux was

184

re-evaluated at 1.4 bar and the water flux recovery was calculated by comparing the

185

value to that obtained with the fresh membrane coupon.

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After filtration and UV treatment, bacteria with intact and compromised cell

187

membrane on the fouled membrane surface were quantified using confocal laser

188

scanning microscopy (CLSM, FV1000-IX81, Olympus, Japan). The samples were

189

stained with a solution of SYTO 9 and propidium iodide (PI) for 15 min in the dark,

190

according to the reagent manual (LIVE/DEAD® BacLight™ Bacterial Viability Kit,

191

Thermo Fisher Scientific, USA). After each staining step, the membrane samples

192

were rinsed with sterile phosphate buffered saline (PBS, pH 7.2).

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Confocal images at random locations on the membrane surface were captured

194

using a CLSM equipped with 20 flat field half apochromatic objective lens

195

(UPLSAPO20). SYTO 9 was excited with an argon laser at 488 nm while PI was 7



196

excited using a diode-pumped solid state (DPSS) 561 nm laser. Emissions were

197

observed at 528 nm for live bacteria (stained with SYTO 9) and 645 nm for dead

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bacteria (stained with PI). SYTO 9 and PI were excited separately using two

199

independent scans. FV10-ASW and Image-Pro® Plus (version 6.0) software were

200

used to analyze the confocal images.

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Plasmid and Genome DNA Extraction. For plasmid and genomic DNA

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extraction, each wastewater effluent sample (40 mL) collected before and after the

203

UF experiments was centrifuged for 10 min at 10,000 rpm. Plasmid and genomic DNA

204

were then extracted from the precipitates using the TIANpure Mini Plasmid Kit

205

(TIANGEN) and TIANamp Bacteria DNA Kit (TIANGEN), respectively, following the

206

instructions from the manufacturer.

207

To extract plasmid and genomic DNA from the bacteria retained on the membrane

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surface, membrane coupons (4.1 cm2, before or after UV treatment) were immersed

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in 5 mL sterile PBS and ultrasonicated for 20 min. Next, 2 mL aliquots of the

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ultrasonicated solution were placed in sterile centrifuge tubes for extracting the

211

genome and plasmid DNA. All DNA samples were stored at –20 °C before use. The

212

residual 1 mL ultrasonicated solution was stored at 4 °C for bacteria count.

213

Bacteria Count and qPCR Measurement. Bacteria abundance was determined

214

by a plate count.34 Dilutions of the cell suspension were plated on Luria broth (LB,

215

Thermo Fisher Scientific, USA) selective plates containing different antibiotics

216

(chloramphenicol, tetracycline, and sulfadiazine) to select the bacteria resistant to the

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corresponding antibiotic. The concentrations of chloramphenicol, tetracycline, and

218

sulfadiazine employed in the LB selective plates were 32, 16, and 512 mg/L,

219

respectively.35 After incubation for 24 h at 37 °C, the ARB were quantified via a plate

220

count.

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Quantitative PCR (qPCR) was performed to determine the abundances of various

222

ARGs using a real-time PCR System (LightCycler®480, Roche, Applied Science,

223

Switzerland). The primers used in this work were listed in Table S1. The DNA of target

224

gene was purified by the Qiagen Gel Extraction Kit (Qiagen, Germany) and ligated 8


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into a pMD 19-T vector (Takara, Japan) and cloned into E. coli DH5 competent cell

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(Takara, Japan). The transformed cells were grown in an LB selective medium

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containing 100 mg/L of ampicillin to determine the positive clones. A series of 10-fold

228

dilutions of plasmid standards ranging from 109 to 104 copies L-1 were conducted to

229

generate a standard curve.7 Quantitative PCR (qPCR) was conducted in a 96-well

230

plate containing 2 μL plasmid or genomic DNA template, 10 μL of SYBR Premix Ex

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Taq (Takara, Japan), 0.4 μL of forward and reverse primers, and 7.2 μL of DNA-free

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water. All samples were run in triplicates in the qPCR reactions. Details on the qPCR

233

measurements, including the sequences of ARGs and integrons, standard cures, and

234

detection limits are presented in the Supporting Information (Table S2 and Table S3).

235

Gene copy number was calculated from the corresponding standard curve. To clarify

236

the removal mechanism, the Spearman correlation analysis between degradation

237

efficiency of ARGs and integrons and a relative proportion of guanine and cytosine to

238

the total nucleotides sequences (GC%) was performed using the Statistical Package

239

for Social Science software (SPSS) based on a 5% significance level.

240

RESULTS AND DISCUSSION

241

Characteristics of TiO2-Modified PVDF Membrane. Surface morphology and

242

structure of the TiO2-modified PVDF membrane were investigated by SEM. As shown

243

in Figure 1a, the pristine PVDF membrane has pores with sizes in the range of 20–50

244

nm in diameter. Surface morphology did not change much after a relatively long 6-h

245

PDA coating as shown in Figure 1b. TiO2 nanoparticles were anchored to the

246

PDA-coated PVDF membrane utilizing the catechol/quinone groups of PDA. Previous

247

studies have reported that catechol/quinone groups enable homogeneous surface

248

functionalization with TiO2 nanoparticles by preventing particle aggregation.36, 37 We

249

also evaluated the amount of TiO2 incorporated in the PVDF membranes using

250

thermogravimetric analysis (TGA). Results from TGA indicate that TiO 2 accounts for

251

1.5 wt % of the TiO2-modified PVDF membrane (Figure S2a). Images from SEM-EDS

252

elemental mapping revealed evenly distributed TiO2 nanoparticles on the membrane

253

surface (Figures S2b and S2c in the Supporting Information), consistent with SEM 9



254

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images of the surface of the TiO2-modified PVDF membrane (Figure 1c).

255

Figure 1

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The average pore size of the PVDF membranes before and after surface

257

modification

was

determined

by

the

Brunauer–Emmett–Teller

(BET)

and

258

Barrett–Joyner–Halenda (BJH) analyses using a gas sorptometer (SSA-4300, Builder,

259

China) (Figure S3). We observed that surface modification with PDA and TiO 2

260

reduced the average pore size of the PVDF membranes (pristine PVDF, 17 nm;

261

PDA-coated PVDF, 15 nm; and TiO2-modified PVDF, 6 nm). AFM analysis indicates

262

that coating of the PVDF membrane with PDA as well as with TiO2 nanoparticles had

263

very little influence on the overall surface roughness (Figures 1d-f), although SEM

264

images reveal a change in the overall surface morphology after TiO2 surface

265

modification.

266

Water contact angles for the pristine and surface modified PVDF membranes

267

were measured to evaluate the surface hydrophilicity. As shown in Figure 1g, the

268

water contact angle of the PVDF membrane decreased after PDA modification,

269

indicating that –OH and –NH2 functional groups of PDA render the membrane surface

270

hydrophilic.38 Surface hydrophilicity was further increased after functionalization with

271

TiO2 as evidenced by a water contact angle of the TiO2-modified PVDF membrane

272

lower than that of the PDA-coated membrane, likely due to the hydroxyl groups of the

273

TiO2 clusters.39. In contrast to surface hydrophilicity, the TiO2-modified PVDF

274

membrane exhibited the lowest water uptake. The observed lower water uptake of the

275

TiO2-modified PVDF membrane compared to the pristine PVDF membrane is

276

attributed to the reduction of membrane porosity (Figure 1h). Porosity translates to

277

void volume within the membrane, which may impact water permeability and retention

278

behaviors. The reduced pore size and more compact structure of the TiO2-modified

279

PVDF membrane compared to the pristine membrane is beneficial to the removal of

280

bacteria as discussed in the following subsection.

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Removal and Inactivation of Total and Antibiotic Resistant Bacteria. Both

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membrane filtration (Figure 2a, left panel) and UV treatment (Figure 2a, right panel) 10


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provided respectively near complete removal and inactivation of total bacteria,

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especially with the TiO2-modified PVDF membrane. The TiO2-modified PVDF

285

membrane exhibited over 99.9% retention of total bacteria, higher than the pristine

286

PVDF membrane (98.9%). The higher total bacteria removal efficiency of the

287

TiO2-modified PVDF membrane compared to that of the pristine PVDF membrane

288

was further confirmed by the reduced abundance of 16S rRNA in the permeate

289

(Figure S4a). It is noteworthy that a small amount of bacteria passed through both the

290

pristine and TiO2-modified PVDF membranes, although bacteria (size of 0.1 – 2 μm,

291

refer to inset of Figure 2b) are larger than the size of membrane pores (several

292

tens-of-nanometer, Figures 1a and 1c and Figure S3). Previous studies have reported

293

that bacteria permeation through microporous membranes depends on the structural

294

properties of bacteria, including size and shape40,

295

conditions.42 The incomplete removal of bacteria by the investigated UF membranes

296

is likely because bacteria deformed under the hydraulic pressure (1.4 bar) pass

297

through the membrane despite their much larger intrinsic size than membrane

298

pores.41

41,

and hydrodynamic filtration

299

Figure 2

300

The TiO2-modified PVDF membrane exhibited much higher retention of

301

tetracycline-resistant bacteria (TRB) and chloramphenicol-resistant bacteria (CRB)

302

than the pristine PVDF membrane, while the retention of sulfadiazine-resistant

303

bacteria (SRB) displayed an opposite result. To explain the observed different ARB

304

retention behaviors, ARB from the wastewater sample were isolated and examined

305

by optical microscopy (insets of Figure 2b). Results indicate that TRB and CRB are

306

mostly coccus with a spherical or round shape and much smaller size compared to

307

SRB, which is a rod-shaped bacillus. Such structural properties of TRB and CRB

308

result in a lower retention by the pristine PVDF membrane compared to that by the

309

TiO2-modified PVDF membrane which has smaller pores than the pristine PVDF

310

membrane (Figure 1c). It can be inferred from this observation that TRB and CRB in

311

wastewater effluent are more challenging to remove than SRB by the UF membrane. 11



312

The TiO2-modified PVDF membrane showed significant improvement in

313

inactivation of total bacteria after UV treatment compared with the pristine PVDF

314

membrane (Figure 2a, right panel). Similarly, the TiO2-modified PVDF membrane

315

exhibited a higher photocatalytic degradation efficiency of ARB than the pristine

316

PVDF membrane (Figure 2b). To better evaluate the effect of TiO2 photocatalytic

317

reaction on bacteria inactivation, the live/dead bacteria retained on the membrane

318

surface were quantified via CLSM analysis. As Figure 3 shows, more dense

319

population of dead cells (shown red) was observed on the TiO2-modified PVDF

320

membrane (Figures 3c and 3d) compared to that on the pristine PVDF membrane

321

(Figures 3a and 3b) after UV treatment. We further verified the strong photocatalytic

322

degradation efficiency of TiO2 by the reduced abundance of the 16S rRNA genes in

323

the permeate obtained from UF experiments with the TiO2-modified PVDF membrane

324

compared to that obtained with the pristine PVDF membrane (Figure S4b). It is

325

interesting to note that some bacteria are destroyed during filtration by the

326

TiO2-modified PVDF membrane (compared the Figures 3a and 3c). In contrast,

327

photocatalytic inactivation of bacteria retained on the pristine PVDF membrane was

328

incomplete after UV treatment (Figures 3b and 3d), suggesting that surface

329

functionalization with TiO2 is critical to achieve high inactivation rate of bacteria.

330

Figure 3

331

Retention of Antibiotic Resistance Genes and Integrons. The abundance of

332

chloramphenicol resistance gene (floR), tetracycline resistance genes (tetC, tetW,

333

and tetQ), sulfonamides resistance genes (sul1 and sul2), and three integrons in the

334

feed (i.e., secondary wastewater effluent) and in the permeate measured after

335

filtration with the pristine and TiO2-modified PVDF membranes is presented in Figure

336

4. Compared with the pristine PVDF membrane, the TiO2-modified membrane

337

showed increased retention of most ARGs and integrons because the attached TiO 2

338

nanoparticles form a denser membrane structure. Notably, approximately several

339

tens to hundreds of copies of tetQ gene in plasmid were measured in the feed and

340

permeate obtained with both the pristine and TiO 2-modified PVDF membranes, while

12


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these genes were not detectable in the genome, indicating that tetQ gene transfer

342

was mainly carried through the plasmid (Figures 4a and 4c). This result also implies

343

that ARGs have distinctive locational distribution in the microorganisms which exist in

344

the effluent of wastewater treatment plants. All three integrons (intl1, intl2, and intl3)

345

were found in the secondary wastewater effluent. Specifically, IntI1 accounted for the

346

major portion (over 103 copies/mL in genome and plasmid), while intI2 was detected

347

at low very concentration (less than 5 copies/mL) in both genome and plasmid

348

(Figures 4b and 4d).

349

Figure 4

350

As shown in Figures S5a-5d, removal efficiencies of ARGs and integrons by the

351

pristine and TiO2-modified PVDF membranes differ substantially, depending on their

352

locations in the bacteria cell. Genome size is reported to be in the range of ~130 kbp

353

to ~14 Mbp, while plasmid has relatively small sizes ranging from ~1 to ~100 kbp. 43, 44

354

Thus, ARGs located in the genome were better retained by the pristine and

355

TiO2-modified PVDF membranes than those located in the plasmid (Figures 4a and

356

4c). The ARGs carried by the genome are more stable in terms of their heredity than

357

those carried by plasmid, and thus propagate mainly through vertical gene transfer.45

358

A relatively high removal efficiency of ARGs in the genome by both the pristine and

359

TiO2-modified PVDF membranes indicates that the potential of vertical ARG transfer

360

can effectively be controlled by membrane filtration. We also found that removal of

361

integrons in the genome was more efficient than those in plasmid by the pristine and

362

TiO2-modified PVDF membranes (Figures 4b and 4d).

363

Degradation of Retained Antibiotic Resistance Genes and Integrons. To

364

determine the photocatalytic degradation of ARGs and integrons retained by the

365

membrane, we exposed the membrane surface after filtration experiments to UV

366

irradiation for 1 h. As shown in Figure 5, ARGs and integrons located in the genome

367

had a higher photocatalytic degradation efficiency compared to those in plasmid. This

368

observation indicates that degradation of ARGs and integrons by UV photocatalysis

369

depends on their location in the bacteria cell. Compared with the relatively small size

370

plasmid, the larger genome can easily be attacked by reactive oxygen species (ROS) 13



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371

produced by TiO2 under UV irradiation. The TiO2-modified PVDF membrane showed

372

higher degradation efficiencies for most ARGs and integrons located in both the

373

genome and plasmid than the pristine PVDF membrane after UV treatment (Figures

374

5a-5d), in accordance with the previous results on total and antibiotic resistant

375

bacteria inactivation (shown earlier in Figure 2). The degradation efficiencies of floR,

376

tetC, sul1, and intI1 in the plasmid by the TiO2-modified PVDF membrane were

377

97.82%, 20.66%, 99.45%, and 93.67%, respectively, which are higher than those by

378

the pristine PVDF membrane (93.07%, 15.06%, 99.29%, and 88.86%, respectively)

379

after UV treatment. Although these improvements were not substantial (P-value of

380

0.016, 0.047, 0.016, and 0.305 for the floR, tetC, sul1, and intI1, respectively), the

381

results indicate that the degradation of ARGs and integrons was enhanced by the

382

photocatalytic activity of TiO2.

383

Figure 5

384

Photocatalytic degradation efficiency of tetracycline resistance genes (tetC, tetW,

385

and tetQ) was lower than that of sulfonamides resistance genes (sul1 and sul2)

386

located in both the plasmid and genome. The degradation efficiencies of tetC, tetW,

387

and tetQ in the plasmid were only 20.6%, 27.2%, and 2.0%, respectively, for the

388

TiO2-modified PVDF membrane after UV treatment, which is much lower than those

389

of sul1 (99.3%) and sul2 (98.8%). This finding is in good agreement with results from

390

previous studies showing that tetracycline resistance genes are more difficult to

391

degrade by UV disinfection than sulfonamides resistance genes (Figure 5c).46

392

Oxidants generated by an indirect photolysis pathway, such as reactive oxygen

393

species (ROS), preferentially react with guanine bases in DNA. 47,

394

strong correlation between the guanine-cytosine content (or GC%) and DNA

395

degradation efficiency of the investigated ARGs and integrons by UV treatment; this

396

finding indicates that ROS generated via TiO2 photocatalytic reaction was the major

397

contributor for degradation of ARGs and integrons located in both the genome and

398

plasmid (Table S4). The GC% of tetracycline resistance genes, especially tetQ (46%),

399

was lower than that of sulfonamides resistance genes (Table S2), which explains the

14


48

We found a

Page 15 of 27


400

previously observed lower degradation efficiency of tetracycline resistance genes

401

located in both the genome and plasmid by UV irradiation.

402

Anti-fouling Performance of TiO2-Modified PVDF Membrane. To investigate

403

the antifouling performance of the TiO2-modified membrane, we conducted dead-end

404

UF experiments to evaluate water flux recovery after fouling. Rapid water flux decline

405

was observed for both the pristine and TiO2-modified PVDF membranes with a

406

secondary wastewater effluent feed solution (Figure 6). A substantial fouling layer is

407

likely to form on the membrane surface due to the high foulant loading in the

408

wastewater effluent, causing a sharp decrease of water flux. After the fouling

409

experiments, membranes were exposed to UV irradiation for 1 h to degrade ARB and

410

ARGs retained on the membrane surface. We observed a much higher flux recovery

411

for the TiO2-modified PVDF membrane compared to the pristine PVDF membrane

412

after UV treatment. Furthermore, long-term UF operation with several filtration cycles

413

indicated that the recovery of water flux for the TiO2-modified PVDF membrane

414

remained high and exceeded significantly that of the pristine PVDF membrane

415

(Figure S6).

416

Figure 6

417

The stability of the TiO2 surface coating during filtration was evaluated by

418

analyzing the titanium concentration in the permeate after UF experiments. Even after

419

48-h long term UF experiments, titanium concentration in the permeate was only ~0.3

420

ng/L (Figure S7 in the Supporting Information), substantially lower compared to the

421

amount of TiO2 deposited on the membrane. Hence, the amount of TiO2 leaching

422

from the membrane surface during filtration is negligible and will not compromise

423

membrane performance and permeate water quality.

424

The observed antifouling property of the TiO2-modified PVDF membrane is

425

attributed to the ROS produced by the TiO2 nanoparticles under UV irradiation, which

426

effectively inactivate the ARB and ARGs, thereby disrupting the fouling layer.

427

Previous studies have reported that the PDA intermediate layer could act as a

428

free-radical scavenger to prevent the substrate from damage by UV irradiation.49 15



Page 16 of 27

429

Thus, the PDA layer coated on the PVDF substrate also contributes to the almost

430

complete water flux recovery of the TiO2-modified membrane after UV treatment. The

431

observed membrane stability and antifouling performance of the TiO2-modified PVDF

432

membrane with UV treatment demonstrate the potential application of photocatalytic

433

reactive membranes in treatment of wastewater effluents contaminated by ARB and

434

ARGs.

435

ASSOCIATED CONTENT

436

The Supporting Information is available free of charge on the ACS Publication

437

website at DOI:

438

Scheme

439

removal/degradation mechanisms of antibiotic resistant bacteria (ARB) and antibiotic

440

resistance genes (ARGs) (Figure S1); Amount of TiO2 incorporated in the PVDF

441

membrane (Figure S2); Nitrogen adsorption-desorption isotherms and pore size

442

distribution of membranes (Figure S3); Abundance of 16S rRNA in the feed, permeate,

443

and from bacteria retained on the membrane (Figure S4); Removal efficiencies of

444

ARGs and integrons by UF (Figure S5); Antifouling performance in long-term UF

445

operation (Figure S6); Amount of TiO2 leaching during filtration (Figure S7);

446

Information on primers used for qPCR (Table S1); Annealing temperatures and

447

sequences of ARGs (Table S2); Standard curves and LOD95 of ARGs and integrons

448

(Table S3); Spearman’s correlation coefficients between degradation efficiency of

449

ARGs and integrons and a proportion of guanine and cytosine to the total nucleotides

450

sequences (GC%) (Table S4)

451

AUTHOR INFORMATION

452

Corresponding Author

453

‡*Phone:

+1 (203) 432-2789, e-mail: [email protected]

454

†*Phone:

+86 (532) 58630960, e-mail: [email protected]

455

ACKNOWLEDGEMENT

456

We acknowledge the support received from the NSFC (51508309), China

illustrating

the

functionalization

of

16


PVDF

membrane

and

Page 17 of 27


457

Postdoctoral Science Foundation (2015M570596 and 2017T100496) and the

458

Research Award Fund for Outstanding Young Scientists of Shandong Province

459

(BS2015HZ013).

460

17



461

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2017, 523, 446-455. 43. Bennett, G. M.; Moran, N. A., Small, smaller, smallest: the origins and evolution of ancient dual symbioses in a phloem-feeding insect. Genome. Biol. Evol. 2013, 5, (9), 1675-1688. 44. Slater, F. R.; Bailey, M. J.; Tett, A. J.; Turner, S. L., Progress towards understanding the fate of plasmids in bacterial communities. FEMS Microbiol. Ecol. 2008, 66, (1), 3-13. 45. Summers, D. K.; Sherratt, D. J., Multimerization of high copy number plasmids causes instability: ColE 1 encodes a determinant essential for plasmid monomerization and stability. Cell 1984, 36, (4), 1097-1103. 46. Auerbach, E. A.; Seyfried, E. E.; McMahon, K. D., Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Res. 2007, 41, (5), 1143-51. 47. Qiao, Z.; Wigginton, K. R., Direct and indirect photochemical reactions in viral RNA measured with RT-qPCR and mass spectrometry. Environ. Sci. Technol. 2016, 50, (24), 13371-13379. 48. Cadet, J.; Douki, T., Formation of UV-induced DNA damage contributing to skin cancer development. Photochem. Photobiol. Sci. 2018. 49. Feng, K.; Hou, L.; Tang, B. B.; Wu, P. Y., A self-protected self-cleaning ultrafiltration membrane by using polydopamine as a free-radical scavenger. J. Membr. Sci. 2015, 490, 120-128.

21


20

100

16

80 60 40 20 0

614 615 616 617 618

Porosity Water uptake

PDA

25

15 8

10

4

5 0

Pristine

TiO2-PDA

30

20

12

0

Pristine

613

Page 22 of 27

Water uptake (%)

120

Porosity (%)

Water Contact Angle ()


PDA

TiO2-PDA

Figure 1. SEM and AFM micrographs of pristine PVDF membranes (a and d), PDA-coated membranes (b and e), and TiO2-modified (c and f) membranes. (g) Water contact angle and (h) porosity and water uptake of the various membranes. Error bars represent standard deviations from three independent replicates.

619

22


Page 23 of 27


8

30

6

20

4

10

0

0.1 d e ed tin odifie s Fe i Pr -m 2 TiO

ARB (104 CFU/cm2)

30

621 622 623 624 625 626 627 628 629 630

0.2

Filtrate

2

(b)

620

Photocatalysis

Filtration

e V d V stin ine-U difie i r d-U o t P e i s if -m Pri od 2 -2 m TiO TiO

0

Total Bacteria (105 CFU/cm2)

Total Bacteria (103 CFU/mL)

(a)

Pristine Pristine-UV TiO2-modified

20 10

TiO2-modified-UV

0.2 0.1 0 SRB

TRB

CRB

Figure 2. (a, left) Total bacteria abundance in the feed and filtrate obtained from UF experiments. The secondary wastewater effluent was filtered by the pristine PVDF and TiO2-modified PVDF membranes until the permeate volume reached 250 mL, at a pressure of 1.4 bar (20 psi) and temperature of 25.0  0.5 °C. (a, right) Photocatalytic degradation of total bacteria on the surface of the pristine PVDF and TiO2-modified PVDF membranes before and after exposure to UV for 1 h. (b) CFU of antibiotic resistant bacteria (ARB) on the surfaces of the pristine PVDF membrane and TiO2-modified PVDF membrane, respectively, before and after exposure to UV irradiation, measured via spread plate method. Microscopic images of ARB are shown in the inset with a 2-μm scale bar.

631

23



632 633 634 635 636

Figure 3. CLSM images representing intact bacteria (green) and damaged bacteria (red) on (a) pristine PVDF, (b) pristine PVDF after UV treatment, (c) TiO2-modified PVDF, and (d) TiO2-modified PVDF membranes after UV treatment.

24


Page 24 of 27

Page 25 of 27


Feed

Permeate - Pristine PVDF

4

10

(a) Genome

Integrons (copies/mL)

ARGs (copies/mL)

10

4

103 102 101 ND

0

10

ND

102 101 100

Integrons (copies/mL)

ARGs (copies/mL)

104

(c) Plasmid

103 102 101 0

10

ND

floR tetC tetW tetQ sul1 sul2

(b) Genome

103


104

Permeate - TiO2-modified PVDF

ND

intI1

intI2

intI3

intI2

intI3

(d) Plasmid

103 102 101 100

intI1

Figure 4. Retention of ARGs and integrons by the pristine PVDF and TiO2-modified PVDF membranes. The UF experiments were conducted at 1.4 bar (20 psi) until cumulative permeate volume reached 250 mL. The feed and permeate were collected and analyzed for abundance of ARGs and integrons that position in genome (a, b) and plasmid (c, d).

25



10 10

5

10

Integrons (copies/cm2)

104 103 102 101

106

TiO2-modified before UV 6

(a) Genome

100

ARGs (copies/cm2)

Pristine after UV


105 104 103 102 101 100

(b) Genome

10

104 103 102 101 100 106

(c) Plasmid


TiO2-modified after UV

5

intI1

Integrons (copies/cm2)

ARGs (copies/cm2)

Pristine before UV 6

Page 26 of 27

intI2

intI3

intI2

intI3

(d) Plasmid

105 104 103 102 101 100

intI1

Figure 5. Photocatalytic degradation of ARGs and integrons on the surface of pristine PVDF and TiO2-modified PVDF membranes after UV treatment. ARGs and integrons in genome (a, b) and plasmid (c, d) were extracted using bacteria DNA kit and plasmid kit, respectively, and analyzed via quantitative PCR method.

26



Normalized Water Flux (J/J0)

Page 27 of 27

1.2 PVDF

1.0

TiO2-PVDF

0.8 0.6 0.4 0.2 0.0

0

20

40

60

80

100

Time (min) Figure 6. Anti-fouling performance of the pristine PVDF and TiO 2-modified PVDF membranes evaluated using a dead-end UF set-up. Before fouling runs, membranes were compacted for 3 h at 1.4 bar (20 psi) until a steady flux was obtained. Fouling experiments were conducted for 60 min using pretreated (by a filter paper with a pore size of 80-120 μm) secondary effluent from a wastewater treatment plant as a feed at applied pressure of 1.4 bar (20 psi) and temperature of 25.0  0.5 °C. Initial water fluxes of the pristine PVDF and TiO2-modified PVDF membrane were 1,225 and 1,925 L m-2h-1, respectively. After fouling experiments, fouled membranes were exposed to UV irradiation for 1 h and pure water flux was re-evaluated at an applied pressure of 1.4 bar (20 psi).

27


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