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Impairment of Biofilm Formation by TiO2 Photocatalysis through Quorum Quenching Xiang Xiao, Wen-Wen Zhu, Qiu-Yue Liu, Hang Yuan, Wen-Wei Li, Lijun Wu, Qian Li, and Han-Qing Yu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03134 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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Environmental Science & Technology

Impairment of Biofilm Formation by TiO2 Photocatalysis through Quorum Quenching

Xiang Xiaoa,c,*, Wen-Wen Zhua, Qiu-Yue Liua, Hang Yuanb, Wen-Wei Lic, Li-Jun Wub,*, Qian Lia, Han-Qing Yuc,* a

School of The Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, China b

Key Laboratory of Ion Beam Bioengineering, Institute of Technical Biology &

Agriculture Engineering, Chinese Academy of Sciences, Hefei, 230031, China c

CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science & Technology of China, Hefei 230026, China

1

ACS Paragon Plus Environment


1

ABSTRACT

2

The release of nanomaterials into the environment, due to their massive

3

production and application today, has caused ecological and health safety concerns.

4

Semiconductor photocatalysts like TiO2 exhibit cytotoxicity to bacterial cells when

5

exposed to UV irradiation. However, information about their impacts on individual or

6

group bacterial behaviors is limited. In this work, the biofilm formation of

7

Escherichia coli K12 in the presence of TiO2 with and without UV irradiation was

8

investigated, and the biofilm formation was found not to be affected under the sole

9

application of TiO2 or UV irradiation. However, the biofilm development was

10

substantially delayed by TiO2 under UV irradiation, although no obvious cytotoxicity

11

to cell growth was observed. The reactive oxygen species photogenerated by TiO2

12

were found to quench the autoinducer 2 (AI-2) signals secreted by E. coli K12. As a

13

result, the initiation of quorum sensing for biofilm formation activated by AI-2 was

14

restrained. The expressions of two biofilm-formation-related genes, motA and rcsB,

15

were also suppressed. Dose of an AI-2 precursor, 4,5-dihydroxy-2,3-pentanedione,

16

effectively restored the biofilm development. These results show that the photoexcited

17

TiO2 could suppress biofilm formation through quenching AI-2 signals. This work

18

may facilitate a better understanding about the ecological effects of increasingly

19

released nanomaterials and provide implications for development of anti-fouling

20

membranes.

2


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INTRODUCTION

22 23

Biofilm is the most common form of microbial growth in natural and engineering

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environments. In biofilm, the aggregate of bacteria and other microorganisms are

25

embedded in self-synthetic extracellular polymeric substances (EPS) and adhered to

26

the surface of abiotic materials.1 Owing to the dense structure, biofilm can not only

27

adapt to the harsh environment, but also utilizes nutrients and transport metabolites.

28

Biofilm plays an important role in the microbial degradation of pollutants, wastewater

29

treatment, and ecological restoration.2 Generally, biofilm formation is controlled by

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quorum sensing signals. 3 Most bacteria can depend on the secretion and perception of

31

different chemical signals termed autoinducer (AI) to communicate and regulate their

32

population density-dependent behaviors.3 When the concentration of signal molecules

33

reaches a certain threshold value, the expression of specific genes will be triggered,

34

resulting in expression of virulence factors, production of antibiotics, secretion of EPS,

35

bioluminescence.4 Among these group behaviors, the formation of biofilm has been

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studied extensively and is confirmed to be mediated by various quorum-sensing signal

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molecules.5 Autoinducer 2 (AI-2) is a universal quorum-sensing signal molecule

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found in both gram-negative and gram-positive bacteria, playing essential roles in

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intraspecies and interspecies communication.6 Thus, quenching AI-2 signals by

40

adding chemical agents such as competitive inhibitors7 and trapping agents8 can

41

inevitably affect biofilm formation and other microbial group behaviors.

3



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In recent years, the rapidly increasing production and application of

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nanomaterials have led to their massive release into the environment.9-12 These

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nanomaterials would affect the biofilm formation of environmental microorganisms

45

and their pollutant degradation activities.13, 14 Especially, metal oxide semiconductors,

46

including of ZnO15 and CeO2,16 etc., have attracted wide attentions due to their

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superior photocatalytic properties. Among them, TiO2 is the most commonly used

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semiconductor photocatalyst. It can generate reactive oxygen species (ROS) under

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UV irradiation and cause microbial inactivation.17,

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antimicrobial

51

microorganisms.19 Up to now, studies about the microbial toxicity of photocatalytic

52

nanoparticles have been focused on the acute toxicity of ROS at a high level.

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However, in actual environments, light intensity is susceptible to weather and aquatic

54

conditions, leading to a significant decline in photoexcitation efficiency and reduced

55

production of ROS. Thus, it is unclear whether photocatalytic nanomaterials can still

56

impair biofilm formation at a low ROS level. Especially, it is interesting to know

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whether the photogenerated ROS by TiO2 can regulate the bacterial behaviors and

58

physiological metabolisms by acting as a quencher of AI-2 signals.

properties

contribute

to

18

confining

Its strong photocatalytic biofilm

formation

of

59

This work aims to answer the above two questions. For this purpose, a model

60

bacterial strain, Escherichia coli K12, was used for the microbial growth and biofilm

61

formation tests. The three-dimensional microstructures of the biofilm were observed,

62

and the AI-2 activities under the photoexcitation of TiO2 were measured. Meantime,

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the gene expression mediated by quorum sensing was also evaluated. This work might 4


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facilitate a better understanding about the multiple environmental effects of

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nanomaterials and exploiting their potential roles in the environment.

66 67

MATERIALS AND METHODS

68 69

Characterization of TiO2 Nanoparticles. Nano-TiO2 has three crystal forms, i.e.,

70

rutile, brookite, and anatase. In general, anatase TiO2 exhibits the highest

71

photocatalytic activity. Thus, anatase TiO2, purchased from Wanjing New Material

72

Co., China, was used in our work. The morphology of the TiO2 nanoparticles was

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imaged by using high resolution transmission electron microscopy (HRTEM,

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JEM-2100, Jeol Co., Japan). The nanoparticles were found to be uniform with a

75

25-nm average size (Fig. S1a). The crystal phase was characterized by X-ray

76

diffraction (XRD) patterns using a D8 instrument (Rigaku Co.,, Japan) with Cu K

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radiation (λ=1.54718 Å) in the 2θ range from 10o to 80o. As shown in Fig. S1b, all of

78

the diffraction peaks can be perfectly indexed to the cubic phase of anatase (JCPDS

79

card No. 65-5714).

80

ROS Assay. The photogenerated ROS were detected by using an electron

81

paramagnetic resonance spectrometer (EPR, A300-10/12, BRUKER Co., Germany).

82

TiO2 nanoparticles dispersed in methanol and water were prepared to detect O2•- and

83

•OH respectively. The TiO2 suspensions were then mixed with the spin trap

84

5,5-dimethyl-1-pyrolline-N-oxide (DMPO, Sigma-Aldrich Co., USA). The mixed

85

solutions were transferred to a quartz flat cell in the cavity of the ESR spectrometer to 5



86

detect ROS in dark and with UV irradiation for 1 min. The DMPO-O2•- and

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DMPO-•OH adducts were then monitored.

88

To evaluate the ROS production in the TiO2 photocatalytic process,

89

2',7'-dichlorofluorescin diacetate (DCFH-DA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT) were respectively used as the ROS-detection

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agent to monitor the generation of •OH and O2•-.20, 21 Aliquots of 200 µL mixing fluid

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containing ROS-detection agent with or without TiO2 (100 mg/L) were added into

93

each well of 96-well microtiter plates. The microplates were then exposed to UV (150

94

µW/cm2) for 0, 2, 4, and 8 h, respectively. The DCFH fluorescence of each well was

95

measured at an emission wavelength of 525 nm and an excitation wavelength of 488

96

nm using a Fluorescence/Multi-Detection Microplate Reader (Synergy 2, BioTek

97

Instrument, Inc., USA). The MTT supernatant was removed and the resultant

98

formazan was dissolved in 200 µl DMSO. The absorption value of formazan was

99

measured at 570 nm by the microplate reader.

100

Assessment of Bacteria Growth and Biofilm Formation. If not mentioned

101

otherwise, TiO2 suspension (100 mg/L) was prepared with fresh Luria-Bertani (LB)

102

medium and sonicated for 30 min. E. coli K12 cells grown overnight were collected

103

after centrifugation at 6000 rpm for 5 min, washed three times with 1% NaCl solution,

104

and then resuspended in fresh LB medium or TiO2 suspensions with a initial

105

concentration of 107 CFU/mL. Aliquots of 200 µL mixing fluid were added into each

106

well of 96-well microtiter plates. All plates were incubated at 37 oC. UV at a

107

wavelength 365 nm with an intensity of 150, 300, and 450 µW/cm2 was respectively 6


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used as the light source for the photoexcitation treatment because of its weak harmful

109

effect on bacteria. After 10-h incubation, the cell growth was evaluated by the plate

110

colony-counting method and biomass assay of biofilm was performed by staining

111

with crystal violet.22 Biofilm formation assays were performed with the following

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steps: when broth was removed, the wells were rinsed gently three times with 200 µL

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PBS buffer and air dried. 200 µL of 99% methanol was put into each well. After

114

treated for 15 min, methanol was removed. When the plate was air dried, each well

115

was stained with 200 µL of 1% crystal violet for 15 min. The dye solution was then

116

discarded and the wells were rinsed three times with 200 µL distilled water. After air

117

dried for at least 1 h, adhered crystal violet was re-dissolved in 200 µL of 33% acetic

118

acid. Finally, the OD value of each well was record using a microplate reader.

119

The E. coli K12 strain containing a plasmid pUC18-GFP (Fig. S2) with the

120

expression of green fluorescent protein was used to monitor the biofilm formation.

121

Sterilized polystyrene slices were inserted into the wells of 96-well plates vertically.

122

After 10-h incubation at 37 oC, these slices were observed using a laser scanning

123

confocal microscope (CLSM, TCS SP5 II, Leica Co., Germany) at an excitation

124

wavelength of 488 nm.

125

Analysis of AI-2. 200 µL TiO2 suspensions (100 mg/L) prepared with the

126

culture supernatants of E. coli K12 (after 8-h incubation) were added into the wells of

127

96-well plates. The microplates were then exposed with UV (150 µW/cm2) for 0, 0.5,

128

1, 2, 4, and 8 h, respectively, to analyze the degradation of AI-2. The AI-2 activities

129

coupled with biofilm formation under TiO2 photocatalysis were determined using the 7



130

reporter strain Vibrio harveyi BB170 as described previously.23 The luminescence of V.

131

harveyi BB170 was measured with a luminometer (GloMax, Promega Co., USA). The

132

supernatants were diluted at a ratio of 1:9 in the fresh autoinducer bioassay medium

133

containing V. harveyi BB170 to obtain a final volume of 200 µL in a 1.5-mL

134

Eppendorf tube. LB medium was added as the control group. The solution was shaken

135

at 180 rpm and 30 °C. The luminescence was determined at a 1-h interval. The

136

detection was terminated when the decreased luminescence of the control group

137

increased again. The AI-2 activity was measured as the fold induction of

138

bioluminescence of the treatment groups over the bioluminescence of the control

139

group.

140

4,5-dihydroxy-2,3-pentanedione (DPD), which is spontaneously rearranged into

141

AI-2,6, 24 was added to the cultures, which were subjected to UV irradiation and TiO2

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with a concentration gradient of 0, 1, 10, and 100 nM. After 10-h incubation, the

143

activities of DPD in restoring E. coli K12 biofilm development through compensating

144

the loss of AI-2 caused by TiO2 photoexcitation were evaluated.

145

Total RNA Isolation, cDNA Generation and qRT-PCR Analysis. Total RNA of

146

E. coli K12 cells incubated for 4 h was extracted with bacterial total RNA Extraction

147

Kit (Sangon Biotech Co., China). cDNA was synthesized according to the

148

manufacturer’s suggestions with M-MLV reverse transcription Kit (Invitrogen Inc.,

149

USA). Gene expression of motA (encoding the proton exchange conductor for

150

flagellum movement) and rcsB (affecting colanic acid capsular polysaccharide

151

synthesis), which were involved in the biofilm formation of E. coli K12, was analyzed 8


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by real-time quantitative reverse-transcription polymerse chain reaction (qRT-PCR,

153

Bio-Rad Inc., USA) to evaluate the impact of quorum quenching of AI-2 signal.

154

Primers

155

AGCCTTGGAGCACTCTATC-3’ and 5’-TTGGAGCGACGAAACAGC-3’, and

156

primers

157

5’-TCACCGTAACCACCAGCA-3’ (forward and reverse primers, respectively).

158

Gene expression was normalized to the housekeeping gene rpoA (forward primer

159

5’-GGAAACCAACGGCACAATC-3’;

160

5’-GCAGTTAGCAGAGCGGACAG-3’).25,

161

using 2× iTaq Universal SYBR green Supermix (Bio-Rad Inc., USA) with the

162

following components in a 10-µL reaction system: 5 µL SYBR PremixEx TaqTM buffer,

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0.4 µL forward primer (10 µmol/L), 0.4 µL reverse primer (10 µmol/L), 1 µL cDNA

164

and 3.2 µL ddH2O, and on a CFX96™ Real-Time PCR with the following parameters:

165

initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation at 94 °C

166

for 15 s and a final annealing/elongation at 55.7 °C for 30 s. Before the qRT-PCR

167

analysis, the amplification efficiency of each gene was determined by making a

168

standard curve. Expressions were normalized to rpoA and the relative expression

169

levels were calculated according to the 2–∆∆CT method.27

designed

for

to

rscB

amplify

were

motA

of

E.

coli

K12

were

5’-CCATTTCCCAAGCCTGTC-3’

reverse 26

5’-

and

primer

qRT-PCR analysis was performed

170 171

RESULTS AND DISCUSSION

172 173

Impacts of TiO2 Photoexcitation on Biofilm Formation. The impacts of TiO2 and 9



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UV irradiation on the biofilm formation and cell proliferation were evaluated (Fig. 1).

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In the absence of UV irradiation, the biofilm formation of E. coli K12 was not

176

affected by TiO2 (Fig. 1A), possibly attributed to the weak bactericidal toxicity of

177

TiO2. TiO2 did not affect the cell growth of E. coli K12 even at the highest

178

concentration of 200 mg/L (Fig. 1B). However, under UV irradiation of 150 µW/cm2,

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the biofilm biomass decreased significantly when TiO2 was present, but the cell

180

growth was not affected (Fig. 1B). This result suggests that the inhibition to biofilm

181

formation was not attributed to the acute cytotoxicity caused by TiO2 photoexcitation.

182

Under

183

concentration-dependent, with the occurrence of the strongest inhibitory in the

184

presence of 100 mg/L TiO2. Excessive TiO2 might affect the photoexcitation

185

efficiency,28 which reduced the inhibitory effect on biofilm formation.

UV irradiation,

the

impact

of

TiO2

on

biofilm

formation

was

186

The impact of UV intensity on cell growth in the absence of TiO2 was also

187

evaluated. UV irradiation alone did not exert significant cytotoxicity to cell growth

188

(Fig. 1D). With a low-intensity irradiation (150 µW/cm2), the biofilm formation of E.

189

coli K12 was not inhibited (Fig. 1C). However, a further increase in the UV intensity

190

led to retarded biofilm formation. This result clearly indicates that UV irradiation

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could interfere with the biofilm formation, rather than hinder cell proliferation. The

192

presence of 100-mg/L TiO2 substantially enhanced the inhibitory effect of UV on

193

biofilm formation, and the biofilm biomass was reduced by 43.5% under UV

194

irradiation of 150 µW/cm2 (Fig. 1C). However, when the UV irradiation reached 300

195

and 400 µW/cm2, 43.3% and 45.3% of biofilm biomass were kept, respectively. This 10


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result indicates that in the TiO2 treatment, biofilm biomass did not exhibit an obvious

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dose effect coupled with the increase in UV intensity.

198

Generally, nano-TiO2 has a strong chemical stability. In the present study, the

199

XRD analysis shows the crystallinities of TiO2 did not change in all treatments (Fig.

200

S3), though adsorption of organic matters was observed (Fig. S4). Because of no

201

release of toxic ions and production of intracellular ROS, the nano-TiO2 is exclusively

202

phototoxic.29 Thus, no cytotoxic effect of TiO2 was observed in dark (Fig. 1B). TiO2

203

excitation by UV can produce ROS, and high-dose ROS can lead to cell membrane

204

damage, lipid peroxidation, and even cell death.30 However, under our experimental

205

conditions, TiO2 photoexcitation did not cause any reduction in cell viability (Fig. 1B).

206

This should attribute to the significantly lower dosage of TiO2 and UV intensity, by

207

which the level of the extracellular ROS is too low to exert any bactericidal activity.

208

This makes it possible to explore the ecological effects of TiO2 under nontoxic stress

209

conditions.

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The biofilm development of E. coli K12 under the stress of TiO2 photoexcitation

211

was monitored for an entire growth cycle of 24 h at a TiO2 concentration of 100 mg/L

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and UV intensity of 150 µW/cm2. The color of crystal violet gradually enhanced over

213

time, indicating the increase in biofilm biomass of E. coli K12 (Fig. 2A). Application

214

of UV or TiO2 alone did not restrain the biofilm development, and the biofilm

215

formation reached its stationary-phase after 10-h incubation (Fig. 2B). But the

216

combined applications of UV and TiO2 severely delayed the biofilm development.

217

The biofilm biomass in the treatment of TiO2 irradiated by UV was reduced by 42.6% 11



218

at 10-h incubation, compared with the control.

219

The three-dimensional microstructure of biofilm of E. coli K12 containing

220

plasmid pUC18-GFP was imaged and reconstructed using a CLSM. The images in Fig.

221

3 clearly reveal that UV irradiation or TiO2 addition alone did not influence the

222

biofilm development, and that mature biofilm formation was observed after 10-h

223

incubation. However, when exposed to TiO2 nanoparticles and UV irradiation

224

simultaneously, the intensity and thickness of biofilm substantially decreased,

225

indicating that the biofilm development was impaired (Fig. 3D). It is noteworthy that

226

TiO2 excited by UV at 150 µW/cm2 could not stop the biofilm development, and a

227

mature biofilm was ultimately formed. After 18-h incubation, similar biofilms were

228

formed in the different treatments (Fig. 2B). These results clearly show that TiO2

229

photoexcitation could delay the biofilm development of E. coli K12, rather than cause

230

complete suppression. Since no obvious bactericidal toxicity of TiO2 was observed

231

there, a reasonable explanation was that the initiation of biofilm formation was

232

disturbed by TiO2 photoexcitation.

233

Biofilm Development Delayed by TiO2 Photoexcitation via Quorum

234

Quenching. The E. coli K12 biofilm formation is substantially affected by quorum

235

sensing via AI-2 signal.31, 32 Blockage of AI-2 quorum sensing pathways will result in

236

the failure of mature biofilm formation.31, 33 Competitive inhibitors can also inhibit

237

biofilm formation by interfering with the signal recognition of AI-2.34, 35 Thus, it is

238

necessary to verify whether quorum-sensing regulation with AI-2 signals was

239

involved in the impairment of biofilm formation under TiO2 photoexcitation. 12


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As shown in Fig. 4, a remarkable AI-2 activity was detected using the reporter

241

strain V. harveyi BB170 in the culture supernatant of E. coli K12. In the initial 10-h

242

incubation, the AI-2 activity increased significantly over time. The overall changing

243

tendency of AI-2 activity was consistent with that of biofilm development (Fig. 2),

244

suggesting a potential role of AI-2 signal in biofilm formation. No obvious

245

differences were observed among the treatments of the control, UV irradiation alone,

246

and TiO2 addition alone (Fig. 4). However, a substantial decrease in AI-2 activity was

247

found for the treatment with both UV and TiO2 nanoparticles. This clearly indicates

248

that the AI-2 activity was affected by TiO2 photoexcitation. After 12-h incubation, the

249

AI-2 activities in all treatments decreased rapidly. For E. coli K12, the AI-2

250

concentration varied in its growth cycle (Fig. S5). The AI-2 activity peaked when the

251

cell growth entered the late exponential phase, but then decreased gradually in the

252

stationary phase. This should contribute to the degradation of AI-2 mediated by the

253

cell metabolism.36

254

AI-2 Degradation by the Photogenerated ROS from TiO2 Photoexcitation.

255

Since both TiO2 photoexcitation and UV irradiation can produce ROS,37,

256

interesting to know whether ROS were involved in the degradation of AI-2 signals

257

secreted by E. coli K12 and delayed the biofilm development or not. Under UV

258

irradiation, TiO2 can generate electrons and holes, which can react with O2 and OH- to

259

form various ROS.39 Thus, the photoexcitation efficiency of TiO2 was measured (Fig.

260

S6). In the absence of UV irradiation, no obvious ROS formation was detected. In

261

contrast, the characteristic DMPO-O2•- spin adducts and DMPO-•OH spin adducts 13


38

it is


262

with six and four resolved peaks were observed under UV irradiation, indicating a

263

photoexcitation of TiO2 by UV irradiation.

264

To further explore the formation kinetics of ROS in the photocatalytic process,

265

DCFH-DA and MTT were used to detect •OH and O2•- respectively. As shown in Fig.

266

S7A, a strong background fluorescence produced by DCFH-DA itself was detected. In

267

this case, no obvious fluorescence enhancement was observed for the treatments with

268

TiO2 or UV alone. But for the treatment of combined application of UV and TiO2, a

269

considerable increase in fluorescence value was detected. The fluorescence value

270

increased gradually over time, indicating considerable production and accumulation

271

of •OH. In addition, formation of violet formazan, which indicates the presence of

272

O2•-, was found to closely related with TiO2 photoexcitation (Fig. S7B). These results

273

clearly indicate that TiO2 photoexcitation could result in ROS production under the

274

experimental conditions, which contribute to the degradation of AI-2 signals.

275

Due to the high oxidative activity of ROS such as O2•- and •OH, TiO2-mediated

276

photocatalysis has been widely used in the degradation of organic pollutants.40, 41

277

Thus, it is assumed that the ROS produced by TiO2 photoexcitation might be able to

278

degrade AI-2 molecules and thus impair the biofilm development of E. coli K12.

279

However, since the UV intensity of 150 µW/cm2 used in the experiment is just one

280

tenth of that of solar UV radiation,42 ROS at a low level only were produced under the

281

experimental conditions. Thus, the stability of AI-2 produced by E. coli K12 under the

282

stress of low-level ROS should be evaluated in vitro. E. coli K12 was cultured with

283

LB medium until the maximum AI-2 activity was reached (Fig. S5). The supernatant 14


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284

was collected and the AI-2 activities in different treatments were analyzed. As shown

285

in Fig. S8A, AI-2 was degraded spontaneously and slightly in the culture supernatant

286

of the control. Duncan's multiple-range test results indicate that compared with the

287

control, no significant difference in the AI-2 activity was observed for UV irradiation

288

or TiO2 treatment alone. In contrast, the combined application of UV irradiation and

289

TiO2 substantially reduced the AI-2 activity. A further test with the dose of H2O2 also

290

exhibited a similar trend (Fig. S8B), clearly indicating that TiO2 photoexcitation

291

contributed to the quorum-quenching of AI-2.

292

Restoration of Biofilm Development Suppressed by TiO2 Photoexcitation

293

through Dosing DPD. To further validate whether the AI-2 quenching led to the

294

delay of biofilm formation of E. coli K12, we added difference concentrations of DPD,

295

a product of S-ribosylhomocysteine catabolism that can spontaneously cyclize to form

296

AI-2.43 The biofilm biomass of E. coli K12 was determined after 10-h incubation (Fig.

297

5). As expected, the biofilm biomass increased with the increasing DPD concentration,

298

indicating that dose of DPD relieved the stress of TiO2 photoexcitation and promoted

299

the biofilm development. When the DPD concentration reached 100 nM, no obvious

300

difference was observed between the photoexcitation treatment and the control. Such

301

a concentration-dependent restoration of biofilm growth further confirms that the

302

impairment of biofilm development was attributed to the interference of

303

AI-2-mediated quorum sensing by TiO2 photoexcitation.31, 43

304

Although TiO2 photoexcitation was proven to degrade AI-2, the increase in AI-2

305

activity during the biofilm development was not completely suppressed (Fig. 4). This 15



306

should be attributed to the vigorous growth of E. coli K12 in LB medium. With the

307

increase in cell intensity, the production rate of AI-2 secreted by E. coli K12 increased

308

rapidly. When the AI-2 production rate exceeded its degradation rate by TiO2

309

photoexcitation, the signal molecule accumulated gradually, which eventually

310

triggered the perception of AI-2 signal. Thus, TiO2 photoexcitation resulted in the

311

delay, rather than blockage, of the quorum sensing for biofilm development.

312

Considering the slow reproductive rate of bacteria in actual aqueous environments, it

313

is possible for TiO2 photoexcitation to completely suppress the quorum sensing

314

activated by AI-2 signal. But this warrants further investigations.

315

Expression of the Genes Related to Biofilm Formation. The biofilm formation

316

and architecture of E. coli are also affected by metabolic activities, such as flagellar

317

motility44 and EPS synthesis.45 Thus, the expressions of flagellar motor gene motA

318

and polysaccharide synthesis regulatory genes rcsB in the biofilm formation exposed

319

to TiO2 photoexcitation were evaluated by qRT-PCR. As shown in Fig. 6, the

320

expression levels of motA and rcsB were not affected by UV irradiation or TiO2 alone.

321

But in the treatment with the presence of both TiO2 and UV, the gene expressions of

322

motA and rcsB were obviously suppressed, and their mRNA levels decreased by 53.5%

323

and 41.6%, respectively, compared to the control. Such a tendency is consistent with

324

the biofilm development (Fig. 6). Since both motA and rcsB are regulated positively

325

by AI-2 signal,46, 47 the decline of their expression levels suggests that the quorum

326

sensing regulation activated by AI-2 was weak. Thus, ROS generated by TiO2

327

photoexcitation quenched the AI-2 signals and resulted in the delay of activation of 16


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328

quorum-sensing. The expressions of the genes mediated by AI-2 were repressed,

329

which ultimately affected the biofilm development.

330

In summary, this work reveals a non-toxic impact of TiO2 nanomaterials on the

331

microbial biofilm formation. TiO2 exposed to the UV significantly suppressed the

332

biofilm formation of E. coli K12 without inhibiting the growth of individual cells. The

333

photoexcited TiO2 nanoparticles produced ROS, mainly O2•- and •OH, to quench the

334

AI-2 signals secreted by E. coli K12, resulting in delaying the activation of quorum

335

sensing. The reduced AI-2 activity decreased the expression of the biofilm-formation

336

related genes. Dose of an exogenous AI-2 precursor, DPD, effectively restored the

337

biofilm formation activity against the AI-2 quenching caused by TiO2 photoexcitation.

338

In this work, we found, for the first time, that TiO2 photoexcitation could influence

339

the group behavior of microbes through quorum quenching under environmental

340

conditions. These results may contribute to a better understanding on the

341

environmental toxicity of nanomaterials and provide implications for the

342

development of anti-fouling membranes.

343 344 345 346

AUTHOR INFORMATION *Corresponding authors: Prof. Han-Qing Yu, Fax: +86-551-63601592; E-mail: [email protected]; Prof. Li-Jun Wu, Fax: +86-511-65591602; E-mail: [email protected]

347 348

ACKNOWLEDGEMENTS

17



349

This study was partially supported by the National Basic Research Program of

350

China (2013CB934302) and the National Natural Science Foundation of China

351

(51478208, 21477120 and 21590812).

352 353

ASSOCIATED CONTENT

354

Supporting Information Available. TEM image and XRD spectra of TiO2 (Figure

355

S1), pUC18-GFP plasmid (Figure S2), XRD analysis of TiO2 before and after

356

treatment (Figure S3), FTIR spectra of the TiO2 samples before and after treatment

357

(Figure S4), cell growth of E. coli K12 and AI-2 activity during the growth (Figure

358

S5), DMPO spin-trapping ESR spectra recorded for DMPO-O2•- and DMPO-•OH

359

(Figure S6)，impact of TiO2 photoexcitation on the generation of •OH and O2•- (Figure

360

S7)，and impacts of TiO2 photoexcitation and H2O2 on the AI-2 activities in vitro

361

(Figure S8). This information is available free of charge via the Internet at

362

http://pubs.acs.org/.

363 364

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Figure legends Fig. 1 Biofilm development and cell proliferation of E. coli K12 with the impact of TiO2 and/or UV irradiation. A and B, different concentrations of TiO2 (0, 50, 100, and 200 mg/L) with or without UV irradiation (150 µW/cm2); C and D, different intensities of UV (0, 150, 300 and 450 µW/cm2) with or without TiO2 (100 mg/L). All values represent mean±SD. Fig. 2 Impact of TiO2 photocatalysis on the biofilm development of E. coli K12 under UV irradiation. (A) Biofilm analyzed by crystal violet staining; and (B) Biofilm development of E. coli K12 under stress of TiO2 photoexcitation. All values represent mean±SD. Fig. 3 Three-dimensional morphology of biofilm formed by E. coli K12 after 10-h incubation. (A) The control; (B) Treated with TiO2; (C) Treated with UV; and (D) Treated with TiO2 by UV irradiation. Fig. 4 AI-2 activity changing profiles with the biofilm formation of E. coli K12. All values represent mean±SD. Fig. 5 DPD-concentration-dependent changes in biofilm formation stressed by TiO2 photoexcitation. The CK refers to the treatment without TiO2 photoexcitation. The concentrations of added DPD were 0, 1, 10, and 100 nM, respectively. All values represent mean±SD. Fig. 6 Expression levels of motA and rcsB under the stress of TiO2 photoexcitation by UV. All values represent mean±SD.

26


Page 26 of 33


A 0.4

0 50 100 200

OD630

0.3 0.2 0.1

B

0

10 log(CFU/mL)

Page 27 of 33

50

100

200

8 6 4 2

0.0

0 dark

UV

C 0.4

dark

D without TiO2

log(CFU/mL)

OD630

0.3 0.2 0.1

without TiO2

10

with TiO2

UV with TiO2

8 6 4 2

0.0

0 0

0

150 300 450 2 Illumination (µW/cm )

150 300 2 Illumination (µW/cm )

450

Fig. 1 Biofilm development and cell proliferation of E. coli K12 with the impact of TiO2 and/or UV irradiation. A and B, different concentrations of TiO2 (0, 50, 100, and 200 mg/L) with or without UV irradiation (150 µW/cm2); C and D, different intensities of UV (0, 150, 300 and 450 µW/cm2) with or without TiO2 (100 mg/L). All values represent mean±SD

27



B

Control

0.4

TiO2 UV UV+TiO2

0.3 OD630

Page 28 of 33

0.2 0.1 0.0

2

4

6

10 8 Time (h)

12

18

24

Fig. 2 Impact of TiO2 photocatalysis on the biofilm development of E. coli K12 under UV irradiation. (A) Biofilm analyzed by crystal violet staining; and (B) Biofilm development of E. coli K12 under stress of TiO2 photoexcitation. All values represent mean±SD

28


Page 29 of 33


Fig. 3 Three-dimensional morphology of biofilm formed by E. coli K12 after 10-h incubation. (A) The control; (B) Treated with TiO2; (C) Treated with UV; and (D) Treated with TiO2 by UV irradiation

29



Control

50 Fold induction of luminescence

Page 30 of 33

TiO2 UV

40

TiO2+UV

30 20 10 0 2

4

6

10 8 Time (h)

12

18

24

Fig. 4 AI-2 activity changing profiles with the biofilm formation of E. coli K12. All values represent mean±SD

30


Page 31 of 33


0.4

OD630

0.3 0.2 0.1 0.0

CK

0

1

10

100

Fig. 5 DPD-concentration-dependent changes in biofilm formation stressed by TiO2 photoexcitation. The CK refers to the treatment without TiO2 photoexcitation. The concentrations of added DPD were 0, 1, 10, and 100 nM, respectively. All values represent mean±SD

31



Relative mRNA level

1.5

1.0

Control TiO2 UV UV+TiO2

0.5

0.0 motA

rcsB

Fig. 6 Expression levels of motA and rcsB under the stress of TiO2 photoexcitation by UV. All values represent mean±SD

32


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Table of Contents (TOC) art

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Impairment of Biofilm Formation by TiO2 ... - ACS Publications

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