1 

Supporting text 1 

2 

Dual roles of capsular extracellular polymeric substances in the 3 

photocatalytic inactivation of Escherichia coli: Comparison of 4 

Escherichia coli BW25113 and its isogenic mutants 5 

6 

Guocheng Huang,a Dehua Xia,a Taicheng An,b*, Tsz Wai Ng,a Ho Yin Yip,a Guiying Li,b Huijun 7 

Zhao,c Po Keung Wonga* 8 

9 

School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, 10 

Chinaa; 11 

State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese 12 

Academy of Sciences, Guangzhou 510640, Chinab; 13 

Centre for Clean Environment and Energy, Griffith School of Environment, Griffith University, 14 

Queensland 4222, Australiac 15 

16 

*Corresponding Authors: 17 

Prof. Taicheng An, Tel: +86-20-85291501; Fax: +86-20-85290706; E-mail: antc99@gig.ac.cn 18 

Prof. Po Keung Wong, Tel: +852-39436383; Fax: +852-26035767; E-mail: pkwong@cuhk.edu.hk 19 

20 

2 

FT-IR Analysis. FT-IR was employed to probe the chemical and structural aspects of 21 

the bacteria as well as the changes induced by the photocatalytic treatment. A FTS-4000 22 

Varian Excalibur Series FT-IR spectrometer with attenuated total reflection (ATR) (Varian, 23 

Palo Alto, CA) was used to collect the infrared spectra. Spectra from 4000 to 800 cm-1 were 24 

collected with an interval of 2 cm-1, and the ordinate was express as absorbance. Each 25 

spectrum was an average of 256 scans with automatic baseline correction. Samples were 26 

prepared by the following procedure: the suspensions at different reaction times were 27 

evaporated by a freeze-drying method, then the dry residue was supported on KBr pellets for 28 

FT-IR measurement. Spectra were further analyzed in the amide I region (1700-1600 cm-1) to 29 

extract potential changes on protein secondary structures upon the photocatalytic treatment. 30 

The digitized amide I spectra was smoothed using Kubelka-Munk algorithm and converted 31 

into its second derivative using the Savitzky-Golay algorithm with 9-points smoothing. 32 

33 

Fluorescent Staining. In the non-partition system, the bacterial cells and TiO2 34 

photocatalyst before and after photocatalytic treatment were collected and stained with the 35 

dyes of LIVE/DEAD BacLightTM Bacterial Viability Kit (L7012, Molecular Probes, Inc., 36 

Eugene, OR) following the procedure recommended by the manufacturer. After being 37 

incubated at 25 °C in the dark for 15 min, the samples were transferred to the cover-slip and 38 

examined using a fluorescence microscopy (Nikon ECLIPSE 80i, Japan) equipped with a 39 

filter block NUV-2A consisting of excitation filter Ex 400-680 (Nikon, Japan) and Spot-K 40 

slider CCD camera (Diagnostic instruments. Inc., USA). In addition, to get quantitative data 41 

on the relative abundances of live and dead cells, the stained cells were also added to the 42 

3 

wells of a microplate and mix thoroughly. Then the microplate was incubated at 25 °C in the 43 

dark for 15 min, followed by fluorescence detection using a microplate reader (TECAN 44 

Magellan, Tecan Group Ltd.). The excitation/emission wavelengths for live (green) and dead 45 

(red) bacteria were 485/530 nm and 485/630 nm, respectively. The green-to-red fluorescence 46 

ratio was calculated and a calibration curve was obtained by using bacterial mixtures with 47 

known percentages of live cells. 48 

49 

Scanning Electron Microscope (SEM). The samples of bacteria cells interacted with 50 

TiO2 in dark were collected and transferred onto acid-washed and poly-lysine coated 51 

cover-slips. After pre-fixation in 5% glutaraldehyde solution for 1 h, the specimen was 52 

washed twice by 0.1 M PBS at pH 7 and then post-fixed with 2% osmium tetraoxide (E.M. 53 

grade, Electron Microscopy Sciences, Fort Washington, PA, USA) in dark for 1 h. Then the 54 

specimens were washed twice by distilled water before further soaked and dehydrated in a 55 

graded series of ethanol (50, 60, 70, 80, 90, 95 and 100%, and each for 10 min) and then 56 

critical point dried within CO2 atmosphere. Then the dried specimens were mounted on stubs 57 

and sputter-coated with gold and palladium. Finally, the prepared samples were observed on a 58 

scanning electron microscope with Energy Dispersive X-Ray (EDX) detector. 59 

60 

Extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) modeling. To model the 61 

interaction between bacteria and TiO2 particles, free energy curves were generated based on 62 

extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory, considering the total energy 63 

of adhesion as the sum of the electrostatic double layer (EL) interaction energy, Lifshiitz-van 64 

4 

der Waals (LW) interaction energy, and the Lewis acid-base (AB) (i.e., hydrophobic) 65 

interaction energy:1-4 66 

67 

ABLWELTOT GGGG (1) 68 

69 

The EL interaction energies between bacterial cells and TiO2 can be calculated from the 70 

surface potentials using the following equation: 71 

72 

)2exp(1ln(

)exp(1

)exp(1ln

2

)(4

)(G

22

220EL kd

kd

kd

rr

rr

tb

tb

tb

tbtbw

(2) 73 

74 

Where 0 (=8.854×10−12 CV−1 m−1) is the dielectric permittivity in vacuum, w (=79) 75 

is the relative permittivity of water; br and tr represent the equivalent radius of the bacteria 76 

cells and TiO2, which were calculated as

WL, where L and W represent the length 77 

and width of the bacteria cells and TiO2 agglomerates on SEM images; b and t are the 78 

surface potentials of the bacterial cells and TiO2, respectively; k is the inverse of Derby 79 

length with 2

12ii

9 ZC102.32 k where iC is the concentration of ion i , and iZ is 80 

its valence value; d is the distance between bacterial cells and TiO2. 81 

The LW interaction energies can be obtained using the following equations: 82 

83 

tb

tb

rr

rr

d

A

6G LW (3) 84 

85 

5 

LWw

LWt

LWw

LWbdA 2

024 (4) 86 

87 

Where A represents the Hamaker constant; 0d represents the minimum equilibrium 88 

distance between the bacterial cells and TiO2 (0.158 nm); LW is the dispersive parameter of a 89 

substance surface energy ( ), the subscripts of b , t and w represent the substances of 90 

bacteria cells, TiO2 and water, respectively. 91 

The AB interaction energies can be calculated by the following equations: 92 

0

00

AB exp2G0

dd

Grr

rr ABd

tbb

tb (5) 93 

94 

tbtb

wtbwwtbwABdG

)()(2

0 (6) 95 

96 

Where 0 is the characteristic decay length of AB interaction in water (0.6 nm); and 97 

ABdG

0 represents the AB interaction corresponding to 0d ; the indices and - 98 

represent the electron acceptor and the electron donor parameter of a substance surface 99 

tension ( ). 100 

The surface tension parameters of bacterial cells with TiO2 can be determined by 101 

measuring the contact angles using three different probe liquids, with known surface tension 102 

parameters and employing extended Young’s equation: 103 

104 

lsls2cos1 LW

lLWsl (7) 105 

106 

6 

Where is the measured contact angle; l represents the probe liquids (water, glycerol 107 

and diiodomethane), the surface tension parameters (i.e., LW , , and ) of the probe 108 

liquids can be found in Table S2; and s represent the bacterial cells or TiO2. The contact 109 

angles were measured with a contact angle meter (Kyowa Interface Science, CA- XP) using 110 

bacterial cells or TiO2 lawns produced by filtering bacterial cells or TiO2 onto a 0.22 μm filter 111 

paper. 112 

113 

7 

Table S1. The genetic information of wild type (E. coli BW25113) and its single-gene 114 

knock out mutants (E. coli JW2034 and E. coli JW5917) 115 

Strain Name Deleted Gene

CGSC mutation name

Gene function

E. coli BW25113 None No appropriable Nor appropriable

E. coli JW2034 cpsB cpsB747(del)::kan Colanic acid biosynthesis

E. coli JW5917 rcsC rcsC771(del)::kan Negative regulatory gene for

colanic acid synthesis

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Table S2 Surface tension parameters of the probe liquids in mJ/m-2 118 

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Table S3. Band assignments for secondary structures of E. coli from derivative FT-IR spectra 128 

Secondary structures Wavenumber (cm-1) Aggregated strands 1625-1610

β-sheet 1640-1630 unordered 1645-1640 α-helix 1657-1648

3-turn helix 1666-1659 Antiparallel β-sheet/aggregated strands 1680-1695

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Table S4. Contact angles 134 

135 

136 

LWL

L -L L

Water 21.8 25.5 25.5 72.8

Glycerol 34.0 3.9 57.4 63.9

Diiodomethane 50.8 0 0 50.8

TiO2 wild type cpsB- mutant rcsC- mutant

Water 11.4 25.4 14.1 24.6

Glycerol 12.6 23.2 22.1 17.3

Diiodomethane 57.9 65.9 60.4 73.9

8 

137 

 138 

Fig S1. Normalized FT-IR spectra of wild type, cpsB- mutant and rcsC- mutant. Bands in the 139 

spectral region (a) 3000-2600 cm-1 and (b) 1850-950 cm-1 in the integral absorbance were 140 

used as the normalization factors, respectively.141 

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142 

Fig S2. The control experiments in non-partition system. (a) Light control, (b) Dark control, 143 

and (c) Negative control. 144 

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145 

Fig S3. The control experiments in partition system. (a) Light control, (b) Dark control, and 146 

(c) Negative control. 147 

148 

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   149 

Fig S4. Inactivation efficiency of wild type, cpsB- mutant and rcsC- mutant in (a) Fenton 150 

reaction (10 mM H2O2, 0.2 mM Fe2SO4) system and (b) pure H2O2 (10 mM) system. Bars 151 

represent standard deviations (n=3) and data were statistically significant at p < 0.001 152 

calculated by one-way ANOVA. 153 

154 

12 

155 

Fig S5. (a) Effects of photocatalytic treatment on wild type, cpsB- mutant and rcsC- mutant by 156 

TiO2-UVA using microplate scale fluorochrome assay with the LIVE/DEAD BacLightTM 157 

Bacterial Viability Kit. Bars represent standard deviations (n=3) and data were statistically 158 

significant at p < 0.001 calculated by one-way ANOVA. (b) The standard curve of the assay. 159 

No change of standard curve was observed for all the three strains when TiO2 is added, 160 

indicating that TiO2 did not quench or enhance the fluorescence. 161 

162 

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163 

Fig S6. Fluorescence microscopy images of different bacteria without TiO2. (a) Wild type, (b) 164 

cpsB- mutant and (c) rcsC-. The scale bars are 20 µm. 165 

166 

14 

167 

Fig S7. SEM images of E. coli cells after 30 min treatment of contact with TiO2 P25 168 

nanoparticles in dark: (a) wild type, (b) cpsB- mutant and (c) rcsC- mutant; The EDX spectra 169 

(d, e and f) were obtained by scanning the red squares of corresponding SEM images (a, b 170 

and c). 171 

172 

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173 

174 

Fig S8. Zeta potential of TiO2, wild type , cpsB- mutant and rcsC- at different pH-value in 175 

0.9% saline solution (154 mM NaCl). The test pH for the photocatalytic inactivation was 6.5. 176 

Bars represent standard deviations (n=5). 177 

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References: 179 

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http://dx.doi.org/10.1016/S0021-9797(02)00242-4. 182 

2. Schwegmann H, Ruppert J, Frimmel FH. 2013. Influence of the pH-value on the 183 

photocatalytic disinfection of bacteria with TiO2 - Explanation by DLVO and XDLVO 184 

theory. Water Res. 47:1503-1511. http://dx.doi.org/10.1016/j.watres.2012.11.030. 185 

3. Chen G, Strevett KA. 2003. Microbial surface thermodynamics and interactions in 186 

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