S1

Supporting Information 1

2

Biofouling Mitigation in Forward Osmosis 3

using Graphene Oxide Functionalized Thin-Film 4

Composite Membranes 5

6

Francois Perreault,*,†,‡,§ Humberto Jaramillo,†,§ Ming Xie,†,∥ Mercy Ude,† Long D. 7

Nghiem┴, and Menachem Elimelech†,§ 8

9

10 †Department of Chemical and Environmental Engineering, Yale University, New 11

Haven, Connecticut 06520-8286, United States 12

13 ‡ § School of Sustainable Engineering and the Built Environment, Arizona State 14

University, Tempe, AZ, 85287-3005. 15

16 § Nanosystems Engineering Research Center for Nanotechnology-Enabled Water 17

Treatment (NEWT), Rice University, 6100 Main St., MS 6398, Houston, TX 77005 18

19 ∥ Institute for Sustainability and Innovation, College of Engineering and Science, 20

Victoria University, PO Box 14428, Melbourne, Victoria 8001, Australia 21

22 ┴

Strategic Water Infrastructure Laboratory, School of Civil, Mining and 23

Environmental Engineering, University of Wollongong, Wollongong, NSW 2522, 24

Australia 25

26 * Corresponding author: Email: francois.perreault@asu.edu; Phone: 480-965-4028; Fax: 480-965-0557. 27 28

29

Summary 30

Cover page S1 31

Materials and Methods S2-S7 32

Figure S1 S8 33

Figure S2 S9 34

Figure S3 S10 35

Figure S4 S11 36

Figure S5 S12 37

Figure S6 S13 38

Table S1 S14 39

40

S2

MATERIALS AND METHODS 41

Materials and Chemicals. N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride 42

(EDC, 98%), ethylenediamine (BioXtra), N-hydroxysuccinimide (NHS, 98%), HEPES 43

(>99.5%), and MES monohydrate (>99.0%, BioXtra) were obtained from Sigma-Aldrich (St. 44

Louis, MO) and used as received. Polyamide TFC FO membranes were provided by Oasys 45

Water, Inc. (Boston, MA). Graphite powder (99%, -300 mesh, Alfa Aesar) was obtained from 46

VWR (Radnor, PA). Sodium chloride (NaCl, crystals, ACS reagent) was obtained from J.T. 47

Baker (Phillipsburg, NJ). The LIVE/DEAD Baclight bacterial viability kit, containing propidium 48

iodide (PI) and SYTO 9, and Concanavalin A, Alexa Fluor® 633 Conjugate, was obtained from 49

Thermo Fisher Scientific (Molecular Probes®, Grand Island, NY). Unless specified, all 50

chemicals were dissolved in deionized (DI) water obtained from a Milli-Q ultrapure water 51

purification system (Millipore, Billerica, MA). 52

Graphene Oxide Synthesis. GO was produced from graphite powder according to a 53

previously described method.32 Briefly, graphite (1.5 g) was added to a 9:1 mixture of 54

H2SO4:H3PO4 (200 mL) and bath sonicated (26 W L-1, FS60 Ultrasonic Cleaner, Fisher Scientific 55

Co., Pittsburgh, PA) for 5 min. The reaction vessel was placed in an ice bath and KMnO4 (9 g) 56

was added under constant stirring. The solution was brought to 50 oC and stirred for 12 h. 57

Subsequently, the reaction was cooled to room temperature and poured on ice (~400 mL). H2O2 58

(3 mL) was then added to reduce the excess KMnO4. The reaction was diluted to 2 L with DI 59

water and the solid fraction collected on a 5 µm PTFE filter. The material was washed twice with 60

100 mL of DI water, 100 mL of 1:10 HCl and 100 mL of DI water. For each washing step, the 61

mixture was centrifuged (4000 × g, 4 h) and the supernatant decanted away. Finally, the material 62

was purified by dialysis for 72 h, filtered on a 0.45 µm PTFE filter, and vacuum dried. 63

Graphene Oxide Characterization. Spectroscopic characterization was performed on dry 64

powders. Raman spectroscopy was performed on a Horiba Jobin Yvon HR-800 Raman 65

Microscope with 532 nm excitation. Fourier-Transformed Infrared (FTIR) spectra were collected 66

using a Thermo Nicolet 6700 FTIR spectrometer equipped with a Diamond Attenuated Total 67

Reflectance cell. X-ray photoelectron spectroscopy (XPS) was performed on a ThermoScientific 68

ESCALAB 250 with a monochromatized AI X-ray source (150 eV for survey scans, 20 eV for 69

composition scans, 500 µm spot size) at the University of Oregon CAMCOR facility. 70

S3

For microscopy analysis, 3 µL of a dilute GO suspension was drop casted on a 1 cm2 71

silicon wafer previously cleaned for 20 min by UV-ozone treatment (UV/Ozone ProCleaner, 72

BioForce Nanosciences, Ames, IA). Atomic Force Microscopy analysis of GO sheets was 73

performed in tapping mode with a Bruker Multimode (Digital Instruments, Plainview, NY) AFM 74

equipped with a Tap300Al-G cantilever (BudgetSensors, Sofia, Bulgaria). For SEM analysis, 75

GO sheets were imaged with a Hitachi SU-70 microscope at a 5 kV acceleration voltage. 76

Antimicrobial Activity of Graphene Oxide. P. aeruginosa (ATCC 27853TM) was grown 77

in Lysogeny Broth (LB) overnight at 37 °C. The cultures were then diluted in fresh LB and 78

grown until log phase (~2 hours), verified by the optical density at 600 nm (OD600). Bacterial 79

cells were washed three times with sterile 0.9% NaCl solution before being diluted to 107 80

colony-forming units (CFU) mL-1 in sterile saline solution. 81

A GO suspension (100 µg mL-1) was filtered through a 0.22 µm black polycarbonate filter 82

(Millipore, Billerica, MA) until formation of a homogeneous GO layer. Then, 3 mL of diluted 83

bacterial suspension were slowly added on the GO layer. Cells were kept in contact with GO for 84

1h, after which the suspension was removed and the GO layer washed with sterile 0.9% NaCl 85

suspension to remove unattached cells. Cells adhered to the GO layer were stained with 3.34 µM 86

SYTO 9 and 20 µM PI in 0.9% saline solution (Thermo Fisher Scientific, Molecular Probes, 87

Grand Island, NY) for 30 min. The staining solution was removed before mounting the sample 88

on a microscopic slide. Ten pictures per replicate were taken with an Axiovert 200M 89

epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY, USA). Live (green) and dead 90

(red) cells were counted using Image J (National Institutes of Health, MD). 91

Membrane Functionalization. GO sheets were covalently bound to FO TFC membranes 92

by an amide coupling reaction as previously described.31 Briefly, the membranes were first 93

wetted in 25% isopropanol for 20 min and rinsed in DI water for 3 h, changing the water every 94

30 min. Membrane coupons were placed on a glass plate and covered with a frame to leave the 95

active surface exposed. The native carboxyl groups on the membrane active layer were converted 96

to amine-reactive esters by reaction with a solution of 4 mM EDC, 10 mM NHS, 0.5 M NaCl in 97

10 mM MES buffer, pH 5. After 1 h, the membrane was washed twice with DI and reacted for 30 98

min with a solution of 10 mM ethylenediamine, 0.15 mM NaCl in 10 mM HEPES buffer at pH 99

7.5. This step attached ethylenediamine to the activated carboxyl groups of the membrane. The 100

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membrane was then washed twice with DI to remove unlinked ethylenediamine. GO (10 mg) 101

was dispersed in 30 mL of 10 mM MES buffer, pH 6, and reacted with 2 mM EDC and 5 mM 102

NHS for 15 min. The pH of the GO suspension was adjusted to 7.2 with NaOH and contacted 103

with the ethylenediamine -modified membranes for 3 h. During this step, GO was covalently 104

bound to the membrane via amide coupling between the amine group of ethylenediamine and the 105

ester-activated carboxyl group of GO. After 3 h, the GO-functionalized membranes were rinsed 106

abundantly with DI water to remove unbound GO and stored in DI at 4o C until use. 107

Membrane Characterization. For membrane characterization, 1 cm2 membrane coupons 108

were dried overnight in a desiccator at room temperature. Raman spectra of control and GO-TFC 109

were collected on a Horiba Jobin Yvon HR-800 Raman Microscope using a 532 nm laser 110

excitation. For SEM imaging, membrane samples were sputter-coated with chromium and 111

imaged with a Hitachi SU-70 microscope at a 10 kV acceleration voltage. Membrane 112

hydrophilicity was evaluated by the sessile drop method using a Theta Lite Optical Tensiometer 113

TL100 (Attension, Espoo, Finland). Results are presented as an average of 30 measurements 114

taken on three different membrane coupons, using a drop volume of 5 µL. Surface roughness 115

was measured by AFM imaging in tapping mode, using a Dimension Icon AFM equipped with a 116

SNL-10 silicon nitride cantilever (Bruker, Santa Barbara, CA). 117

The membrane water permeability, A, salt permeability, B, and structural parameter, S, 118

were determined in a bench-scale FO unit according to a method previously described.33 Draw 119

solution concentrations of approximately 0.2, 0.4, 0.7, and 1.2 M NaCl, and DI as feed solution 120

were used for the different characterization steps. 121

Bacterial Adhesion and Viability. Membrane coupons of ~ 3.5 cm2 were punched from 122

each membrane and placed in plastic holders leaving only the active layer exposed to bacteria. A 123

3 mL volume of P. aeruginosa cell suspension (~108 CFU mL-1), which was rinsed three times to 124

remove cell debris and re-suspended in sterile wastewater media, was contacted with the 125

membrane for 1 h at room temperature. The suspension was then discarded and the membranes 126

were washed with fresh media to remove non-attached cells. Cell viability was determined by 127

staining the cells with 3.34 µM SYTO 9 and 20 µM PI for 30 min in the dark. The staining 128

solution was removed and the membranes were rinsed twice before mounting on a microscopic 129

slide for epifluorescence microscopy. Ten pictures per replicate were taken with an Axiovert 130

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200M epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY). Live (green) cells, dead 131

(red) cells, and total number of cells were counted with the Image J Cell Counter Plugin 132

(National Institutes of Health, MD). 133

AFM Adhesion Force Measurements. Adhesion forces between the membrane and a 134

carboxylated latex particle (4.0 μm diameter, carboxyl content 19.5 μeq g-1, Life Technologies, 135

Eugene, OR), simulating the adhesion of colloidal foulants, were measured on a Dimension Icon 136

AFM (Bruker, Santa Barbara, CA). Particle-functionalized AFM probes were prepared according 137

to a procedure previously described.34 Force measurements were collected in synthetic 138

wastewater media using a trigger force of 1 nN, a ramp size of 1 µm, and a ramp rate of 0.5 Hz. 139

Adhesion forces were quantified for more than 300 individual force measurements, taken on 140

random locations over three different membrane coupons. The cantilever deflection sensitivity 141

and spring constant were determined before each experiment using the thermal noise method.59 142

Adhesion forces were determined using the Peak Analysis function of Nanoscope Analysis v1.5 143

(Bruker). 144

Membrane Biofouling Experiments. Biofouling experiments were carried out in a 145

bench-scale FO unit. Membrane cell dimensions were 7.7 cm × 2.6 cm × 0.3 cm (length, width, 146

and height, respectively), with an active membrane surface area of 20.0 cm2. Gear pumps 147

(Micropump, Cole-Parmer, Vernon Hills, IL) were used to circulate the feed and draw solutions 148

in a closed-loop configuration. Before each experiment, the system was cleaned and disinfected 149

by flushing sequentially with 10% bleach, 5 mM EDTA, and 95% ethanol for 1 h. Then, the 150

system was rinsed three times with DI water to thoroughly remove the cleaning reagents. 151

An artificial secondary wastewater medium, with an ionic strength of 16 mM and pH of 152

7.6 ± 0.2, was used as a feed solution (see Table S1 for medium composition).35 A draw solution 153

was prepared using NaCl and the concentration adjusted to achieve an initial water flux of 20 ± 1 154

L m-2 h-1 (~ 1 M NaCl). A baseline FO run was conducted without bacteria for both Ctrl and GO-155

TFC membranes using synthetic wastewater as a feed and NaCl as a draw solution. The baseline 156

runs were used to account for the dilution of the draw solution during FO experiments. 157

P. aeruginosa cultures were grown in sterile LB overnight at 37 °C. The cultures were 158

diluted with fresh LB and cultivated to an OD600 of 0.6. A 50 mL culture volume was then 159

centrifuged for 15 min at 4,000 rpm, under a controlled temperature of 25 °C, and the cell pellet 160

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was re-suspended in 20 mL of sterile wastewater media. Biofouling experiments were performed 161

using feed and draw volumes of 2 L. The permeate flux was allowed to stabilize at 20 ± 1 L m-2 162

h-1 before addition of bacteria. Feed solution was inoculated with 20 mL of P. aeruginosa to 163

obtain an initial bacteria concentration of ~6.0 ± 0.5 × 107 CFU L-1. The FO system was operated 164

for 24 hours at a flow rate of 8.5 cm/s and the permeate flux was continuously monitored using 165

an electric balance (Denver Instrument, Bohemia, NY). Temperature in the system was 166

maintained at 25 ± 1o C using a chiller/heater (Thermo Scientific, Vernon Hills, IL). At the end 167

of the biofouling experiment, membrane coupons were cut and the biofilm was characterized. 168

Biofilm Characterization. Membrane coupons (1 cm2) were cut from the center of the 169

biofouled membrane and stained with 3.34 µM SYTO 9, 20 µM PI, and 0.5 µM concavalin A 170

(Con A) for 30 min, as previously described.36 Samples were rinsed three times with sterile 171

wastewater to remove unbound stains and mounted in a custom-made characterization chamber 172

for confocal laser scanning microscopy (CLSM) imaging.36 Confocal images were captured 173

using a Zeiss LSM 510 (Carl Zeiss, Inc., Thornwood, NY) equipped with a Plan-Apochromat 174

20×/0.8 numerical aperture objective. Three sets of lasers were used for the excitation of SYTO 175

9, PI, and Con A: 488 nm argon, 561 nm diode-pumped solid state, and 633 nm helium-neon 176

laser. A minimum of three Z stack random fields (635 µm × 635 µm) were collected for each 177

sample, with a slice thickness of 2.3 µm, to obtain a representative biofilm ortho image. Biofilm 178

dimensions were analyzed by capturing a minimum of ten random Z stack regions (90 µm × 90 179

µm), with a slice thickness of 1.2 µm, for each sample. Confocal image analysis was performed 180

using Auto-PHLIP-ML, ImageJ software, and MATLAB. Thickness and biovolume were 181

determined for the live and dead cells (SYTO 9 and PI staining), and EPS (Con A staining) 182

components of the biofilm. Total biovolume and thickness were calculated by summing live, 183

dead, and EPS components. 184

Quantitative analysis of the biofilm composition was performed by measuring the total 185

protein and organic carbon (TOC) content of the biofilm. For protein analysis, membrane 186

coupons were re-suspended in 1 mL Lauber buffer (50 mM HEPES (pH 7.3), 100 mM NaCl, 187

10% sucrose, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1 propanesulfonate, 10 mM 188

dithiothreitol). Samples were probe-sonicated on ice (2 × 30 s, two times) with an ultra-cell 189

disruptor (MISONIX Inc., Farmingdale, NY). Cell extracts were centrifuged 10 min at 12,000 190

rpm and the supernatant collected for protein quantification using the BCA Protein Assay Kit 191

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(Thermo Scientific, Rockford, IL). Protein concentrations were normalized according to the 192

membrane surface area. For TOC analysis, membrane coupons were placed in acid-cleaned glass 193

vials with 20 mL DI water and 40 µL of 1 M HCl. Samples were probe-sonicated as just 194

described, and TOC content was analyzed using a TOC analyzer (TOC-V, Shimadzu Corp., 195

Japan). TOC values were normalized by the membrane surface area. 196

197

S8

198

199

Figure S1. Representative SEM micrograph of GO sheets deposited on a silicon wafer. 200

201

S9

202 203

Figure S2. Antimicrobial activity of the produced GO sheets. (a-b) P. aeruginosa cells deposited 204

on a control (Ctrl) polycarbonate filter (a) and on a pure GO layer formed by filtration of GO 205

sheets (b) were stained with SYTO 9 (green) and PI (red) for “live” and “dead” cells, 206

respectively, and imaged by epifluorescence microscopy. (c) Cell viability of P. aeruginosa 207

deposited to a Ctrl and GO-coated filter after 1 h of contact. Cell viability was quantified by 208

image analysis of the epifluorescence microscopy pictures using Image J. 209

210

S10

0

1

2

3

300

400

500 Ctrl

GO-TFC

S

[m]

B

[L m-2 h

-1]

A, B

, or

S

A

[L m-2 h

-1 b

-1]

211

Figure S3. Membrane transport properties of Ctrl and GO-functionalized TFC membranes, 212

where A is the water permeability coefficient, B is the salt (NaCl) permeability coefficient, and S 213

the structural parameter of the membrane. 214

215

216

S11

217

218

Figure S4. Representative atomic force microscopy imaging of Ctrl and GO-TFC membrane 219

surface. The 3-D height image shows the native polyamide structure that is still observable due 220

to the thin layer of GO sheets on the surface. Images were obtained in tapping mode using a 221

SNL-10 silicon nitride cantilever on a Dimension Icon AFM, using a scan rate of 0.5 Hz. 222

223

S12

224

225

226

227

Figure S5. SEM micrograph of the carboxylated-particle attached on a tipless silicon nitride 228

cantilever. Cantilever was chromium-sputtered for visualization. 229

230

S13

3 4 5 6 7 8 9-20

-15

-10

-5

0

5

10

Zeta

pote

ntial (m

V)

pH

Ctrl

GO-TFC

231

Figure S6. Zeta potential of Ctrl and GO-TFC membranes. Zeta potential was measured at 1.1 232

mM ionic strength (0.1 mM KHCO3 and 1.0 mM KCl) at different pH (adjusted with KOH or 233

HCl) on an electro kinetic analyzer (EKA, Brookhaven Instruments) equipped with an 234

asymmetric clamping cell. 235

236

237

238

239

S14

240

Table S1: Synthetic wastewater composition used in biofouling experiment 241

242

Chemical Concentration (mM)

Glucose 0.6

NH4Cl 0.4

KHPO4 0.2

CaCl2 0.1

NaHCO3 0.5

NaCl 8

MgSO4 0.15

243

244