Simultaneous Supplement of Denitrifiers, Electron Donor Pool and

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A New Concept of Promoting Nitrate Reduction in Surface Waters: Simultaneous Supplement of Denitrifiers, Electron Donor Pool and Electron Mediators Ningyuan Zhu, Yonghong Wu, Jun Tang, Pengfei Duan, Lunguang Yao, Eldon Raj Rene, Po Keung Wong, Taicheng An, and Dionysios D. Dionysiou Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01605 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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Environmental Science & Technology

Table of Contents

ACS Paragon Plus Environment


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A New Concept of Promoting Nitrate Reduction in Surface Waters: Simultaneous

2

Supplement of Denitrifiers, Electron Donor Pool and Electron Mediators

3

Ningyuan Zhua,g, Yonghong Wua*, Jun Tanga,g, Pengfei Duanb, Lunguang Yaob, Eldon

4

R. Renec, Po Keung Wongd, Taicheng Ane, and Dionysios D. Dionysiouf

5

a

6

Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China

7

b

8

Mid-line of South-to-North Diversion Project, Nanyang Normal University, Nanyang,

9

473061, Henan, China

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Sciences,

Collaborative Innovation Center of Water Security for Water Source, Region of

10

c

11

for Water Education, Westvest 7, 2611 AX Delft, The Netherlands

12

d

13

Kong SAR, China

14

e

15

of Environmental Science and Engineering, Institute of Environmental Health and

16

Pollution Control, Guangdong University of Technology, Guangzhou, 510006, China

17

f

18

Research Center, University of Cincinnati, Cincinnati, OH 45221-0012 USA

19

g

20

Beijing 100049, China

Department of Environmental Engineering and Water Technology, IHE Delft Institute

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

Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, School

Department of Chemical and Environmental Engineering (ChEE), 705 Engineering

College of Resource and Environment, University of Chinese Academy of Sciences,

21 22 23

*

24

Dr. Yonghong Wu

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Tel: (+86)-25-8688 1330

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Fax: (+86)-25-8688 1000

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E-mail: [email protected]

Corresponding author:

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ABSTRACT

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The efficiency of biological nitrate reduction depends on the community composition of

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microorganisms, the electron donor pool and the electron mediators participating in the

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biological reduction process. This study aims at creating an in-situ system comprising of

32

denitrifiers, electron donors and electron mediators to reduce nitrate in surface waters.

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The ubiquitous periphytic biofilm in waters was employed to promote in-situ nitrate

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reduction in the presence of titanium dioxide (TiO2) nanoparticles (NPs). The nitrate

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removal rate in the periphytic biofilm and TiO2 NPs system was significantly higher than

36

the control (only periphytic biofilm or TiO2 NPs). TiO2 NPs optimized the community

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composition of periphytic biofilm for nitrate reduction by increasing the relative

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abundance of four dominant denitrifying bacteria. Periphytic biofilm showed a

39

substantial increase in extracellular polymeric substance, especially the humic acid and

40

protein content, due to the presence of TiO2 NPs. The synergistic action of humic acid,

41

protein, denitrifying bacteria of the periphytic biofilm and TiO2 NPs contributed to 80%

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of the nitrate reduction. The protein and humic acid, acting as electron mediators,

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facilitated the transfer of exogenous electrons from photoexcited TiO2 NPs to periphytic

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biofilm containing denitrifiers, which enhanced nitrate reduction in surface waters.

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Keywords: Nitrate reduction, periphytic biofilm, TiO2 nanoparticles, humic acid,

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protein

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INTRODUCTION

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Photocatalytic reduction of nitrate is an alternative transformative technology to address

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the continuous excessive discharge of nitrate into surface water which poses

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environmental risks such as eutrophication and harmful algal blooms

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our knowledge, very little information is available in the literature concerning in-situ

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nitrate reduction by photocatalysis in surface waters. This is because existing anionic

53

competitors, such as SO42- and CO32-, preferentially occupy the active catalytic sites

54

while inclusion hole scavengers (electron donor), such as formic acid, carry the risk of

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undesirable biofilm growth in surface water 3, 4. In consideration of the huge nitrate load

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of surface waters, it is very important to give priority to the development of in-situ

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technologies for nitrate treatment.

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Biological nitrate removal in surface waters depends on the combination of electron

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acceptors and electron donors which are usually provided by the organic matter present

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in water—represented by the chemical oxygen demand (COD) 4, 5. In the field of in-situ

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biological water treatment, the influent COD is capable of oxidizing the influent

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ammonium to nitrite and nitrate in the presence of electron acceptors, such as O2, that

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enter the water through photosynthesis or aeration 6. This process consumes large

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amounts of COD causing nitrate accumulation, while the remaining COD is insufficient

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for

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nitrifiers/denitrifiers and drive the denitrification step, an external carbon source should

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be added as the electron donor 7. However, supplementing organic electron donors as a

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COD source is expensive, carries a high risk of turbidity increase, and causes excess

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biomass growth and unwanted N2O emissions 8, 9.

70

It has been reported that microbial cells possess the ability to acquire electrons from

facilitating

denitrification.

To

maintain

the

3


growth

1, 2

. To the best of

and

activity

of

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10

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natural solid donors to reduce the electron acceptors

. The use of environmentally

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benign, low cost electron donors has attracted interest among the research community

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and environmental engineers. Titanium dioxide (TiO2) nanoparticles (NPs) have been

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extensively studied for photocatalytic denitrification in the presence of hole scavengers

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under ultraviolet radiation 11. The utilization of the photoexcited electrons derived from

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nanophotocatalysts, such as TiO2 NPs, for biological nitrate reduction is promising for

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the development of in-situ nitrate remediation technologies

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studies have demonstrated that nanophotocatalysts were toxic to single cells or pure

79

strains of microorganisms, such as E. coli 13, 14 or algae 15, 16.

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Fortunately, the ubiquitous periphytic biofilm in surface waters has a strong ability to

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grow successfully under a variety of environmental conditions, including those affected

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by the presence of nanoparticles

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microbial aggregates, is composed of heterotrophic and phototrophic microorganisms

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which are embedded in a self-produced mucilage matrix of extracellular polymeric

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substances (EPS)

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denitrifiers, defending the toxicity of nanoparticles, and are also capable of serving as

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electron mediators for electron transfer

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nitrate pollution in waterways in paddy fields or wetlands, a new concept of

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simultaneous supplement of denitrifiers and electron transfer mediators by periphytic

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biofilm, and the electron donor pool derived from TiO2 NPs to reduce the nitrate in

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surface water is proposed in this study.

92

The objectives of this study were: (i) to test the new concept of promoting in-situ nitrate

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reduction by simultaneous addition of denitrifiers, electron donor pool and electron

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mediators in surface waters, (ii) to study the responses of the periphytic biofilm in the

12

. However, previous

17

. Periphytic biofilm, as a mixture of multispecies

18

. EPS are able to provide refuge for microorganisms, such as

19, 20

. In consideration of the high loadings of

4



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presence of exogenous electron donor exciters, such as TiO2 NPs, and (iii) to explore the

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mechanism of nitrate reduction in simulated surface waters.

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MATERIALS AND METHODS

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Preparation of photocatalyst suspension and periphytic biofilm collection

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Analytical grade TiO2 NPs (Aladdin, China) was used in this study. The stock

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suspensions of TiO2 NPs were prepared by adding 1000 mg TiO2 NPs to 300 mL

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deionized water. The contents were agitated and mixed well for 30 min and thereafter

102

diluted to 1000 mL in an Erlenmeyer flask. This suspension was diluted to the required

103

concentrations for the individual experiments. The periphytic biofilms used in this study

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were collected from the authors biofilm culture system 21.

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Nitrate removal by periphytic biofilm in the presence of TiO2 NPs

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Bench scale experiments were performed for seven days to stimulate the growth of

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periphytic biofilm for nitrate removal. This seven days duration was based on the

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lifecycle of the periphytic biofilm used (21 days). Periphyton was collected and used in

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experiments after 14 days of growth. One g (w/w) of periphytic biofilm was added into

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250 mL flasks containing 100.0, 95.5, 95.0 and 90.0 mL Woods Hole (WC) media

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and sodium nitrate (see Supporting Information, SI). Either 0, 0.5, 5.0, or 10.0 mL TiO2

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NPs suspensions were added to the individual flasks to achieve a final volume of 100

113

mL. The initial concentrations of TiO2 NPs in the flasks were 0 (control), 5.0, 50.0 and

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100.0 mg L-1. The initial nitrate concentration was 124.0 mg L-1. All treatments and the

115

control were sealed with sterile sealing films in a standard light-dark cycle of 12 h

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illumination (150 W Xe-high pressure lamp, intensity 20 W m-2) followed by 12 h of

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total darkness at a temperature of 28 ± 1 ºC. On days 0, 1, 3, 5 and 7, the water was

118

sampled to analyze the pH and the residual concentrations of nitrate and nitrite.

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22

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To distinguish whether TiO2 NPs itself affects nitrate removal in the absence of the

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periphytic biofilm, another set of control experiment was conducted (details provided in

121

SI). To investigate the influence of the light response of TiO2 NPs on nitrate removal in

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this system, fluorescence lamp experiments (λ> 420 nm) were carried out in the

123

control and treatments while filtration UV experiments were conducted in the presence

124

of TiO2 NPs (details provided in SI).

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Characterization of periphytic biofilm and EPS

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Periphytic biofilm of the control and 100 mg L-1 TiO2 treatment were collected to

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analyze the activity of nitrate reductase (NaR) and catalase (CAT) and community

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composition on day 7. Nitrate reductase (NaR) and catalase (CAT) activities were

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determined using the reagent kit method (Nanjing Jiancheng Bioengineering Institute,

130

China). The periphytic biofilms were also collected on day 7 and sized after drying.

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Thereafter, TEM, scanning electron microscopy (SEM, JEOL Co, Ltd, Japan) and

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energy dispersive X-ray spectroscopy (EDX, Oxford Instruments Link ISIS) were

133

coupled to obtain the micrographs of periphytic biofilm, and determine the distribution

134

of NPs and corresponding elements on the biofilm surface. The diversity levels of the

135

bacterial communities within the periphytic biofilm matrix were analyzed on day 7 (the

136

end of the experiments) by MiSeq sequencing technology (details provided in SI).

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The EPS extraction procedure was based on the alkaline method

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content in the EPS was measured using the Coomassie brilliant blue staining method 24.

139

Polysaccharose content in the EPS was estimated using the Anthrone method 25. Humic

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acid in the EPS was analyzed by determining the absorbance at 260 nm, using a double

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UV-vis Spectrophotometer (UV-2450 Shimadzu, Japan) 26.

142

To evaluate whether the EPS played an important role as hole scavengers for nitrate

6


23

. The total protein


143

removal, the EPS were extracted from periphytic biofilm. The extract was then added to

144

nitrate solution containing TiO2 NPs under stimulated solar light irradiation (details

145

provided in SI). A single chamber three electrode electrochemical quartz cell system

146

was used to distinguish the role of EPS, TiO2 NPs and periphytic biofilm for nitrate

147

removal (details provided in SI).

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Characterization of TiO2 NPs

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Experiments were carried out to detect TiO2 NPs loading in solution and how they were

150

embedded in EPS (details provided in SI). The micrographs of TiO2 NPs were observed

151

under transmission electron microscope (TEM) (Zeiss 900, Japan). X-ray diffraction

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(XRD) patterns of the TiO2 NPs were measured using a Siemens D-501 diffractometer

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fitted with a Ni filter and a graphite Monochromator (LabX XRD-6100, Japan). All the

154

reagents used in this study were of analytical grade.

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Analytical techniques and statistical analyses

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Nitrate concentration was determined spectrophotometrically according to the

157

Brucine-sulfanil colorimetric method

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(UV-2450 Shimadzu, Japan). The pH was measured using a pH meter (PHSJ-4F Leici,

159

Shanghai, China). Nitrite concentration was determined by the Griess Assay reaction

160

which involves a diazo-coupling procedure

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N-(1-naphthyl) ethylenediamine) reacted with nitrite sequentially to form a

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diazo-compound which was detected at 530 nm using a double UV-vis

163

Spectrophotometer (UV-2450 Shimadzu, Japan).

164

Variance partition analysis (VPA) was used to identify the factors that play a major role

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in nitrate reduction by means of the R software. Statistically significant differences

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between the treatment and the control were evaluated using ANOVA. The probability p

27

, using a double UV-vis Spectrophotometer

28

. The Griess reagents (sulfanilamide and

7


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value was set at 0.05 for all analyses. All the figures were drawn using Origin 9.0 and R

168

software.

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RESULTS AND DISCUSSION

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The photocatalytic property of TiO2 NPs

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TiO2 NPs had cluster-shaped particles with the particle size distribution ranging

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between 10 and 40 nm (Figure S1a). The diffraction peaks were 2θ = 25.3, 37.8, 48.1,

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53.9 and 55.1 degrees, which correspond to the 101, 004, 200, 105 and 211 crystal

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planes, respectively (Figure S1b). These results clearly show that the crystal phase of

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the TiO2 NPs used in this study was the anatase phase.

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Nitrate removal in presence of TiO2 NPs

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The nitrate removal by the periphytic biofilm improved when the concentration of TiO2

178

NPs in suspension was higher than 50 mg L-1 (Figure 1A). When the TiO2 NPs dosage

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increased from 0 to 100 mg L-1, the maximum nitrate removal efficiency increased from

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58.1% to 95.2%, and the rate constant increased from 0.085 to 0.134 d−1 (Figure 1B).

181

The nitrate removal rate constant in the treatments with TiO2 NPs were significantly

182

faster than in the control (p < 0.05). To examine whether the TiO2 NPs directly affected

183

the nitrate removal, a separate experiment was conducted under Xe-lamp irradiation in

184

the absence of the periphytic biofilm (Figure S2a). The residual nitrate concentrations in

185

the control and the 5, 50, 100 mg L-1 TiO2 NPs treatments were not significantly

186

different during the experimental time of 14 h (p > 0.05). This observation clearly

187

indicates that the TiO2 NPs themselves did not affect the nitrate removal.

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The fluorescence lamp irradiation experiments showed no significant differences in

189

nitrate removal efficiency in the control (and treatment (Figure S2b). The nitrate

190

removal rate constants were 0.086 for control and 0.090 d-1 for treatment. Filtration of

8



191

UV light significantly decreased the nitrate removal efficiency by periphytic biofilm in

192

the presence of TiO2 NPs (p < 0.05). The nitrate removal rate constant was 0.136 d-1 in

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the UV treatment and 0.088 d-1 in the non-UV treatment. Nitrate removal efficiency of

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the UV treatment was significantly higher (33.8%) than that of the non-UV treatment in

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the presence of TiO2 NPs from day 3 onwards (p < 0.05) (Figure 1C). Thus, the

196

response of TiO2 NPs to light irradiation played an important role in promoting nitrate

197

removal.

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Nitrite concentration was also measured during the experimental period because nitrite

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is an important intermediate product during biological nitrate removal. The nitrite

200

concentration decreased with time in both the control (150 to 62 µg L-1) and the non-UV

201

treatment (149 to 56 µg L-1). Nitrite production in the UV treatment increased on the

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first day (150 to 178 µg L-1) and thereafter it decreased rapidly over the next six days

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(178 to 12 µg L-1). The addition of TiO2 NPs promoted nitrite production on the first day

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but nitrite production was inhibited for the rest of the experiment compared to the

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control and non-UV treatment (Figure 1D). These results show TiO2 NPs contributed to

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the periphytic biofilm by preventing the production of nitrite in the UV treatment.

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Previous research proved that high concentrations of nitrite may cause emissions of

208

NO/N2O

209

have a high selectivity for N2 because of the low concentration of nitrite. In biological

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nitrate removal, it is essential to provide external electron donors to convert nitrate to

211

nitrogen gas

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electrons for periphytic biofilm to reduce nitrate under Xe-lamp irradiation. With the

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external electron donor supply, periphytic biofilm could remove nitrate with high N2

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selectivity and few by-products. These results demonstrated the feasibility of combining

29, 30

. Nitrate removal in the presence of TiO2 NPs and periphytic biofilm may

31

. This result suggested that TiO2 NPs may provide their photoexcited

9


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a photocatalytic system and a periphytic biofilm for in-situ removal of nitrate from

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water.

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Response of periphytic biofilm to TiO2 NPs

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The growth of the periphytic biofilm in the absence (control) and in presence (treatment)

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of TiO2 NPs was very similar under the experimental conditions investigated in this

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study (Figure 2A and B). Bacterial biofilms are sensitive to NP exposure. Previous

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studies have reported that NPs have the capacity to modify the broader functions of the

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biofilm and significantly shift the bacterial community structure 32, 33. The results of the

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bacterial composition analysis (Table S1) showed that the Simpson index and Shannon

224

index of bacterial community were 4.79 and 0.81 for the control, respectively, and 5.30

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and 0.90 for the 100 mg L-1 TiO2 NPs treatment, respectively. This clearly demonstrates

226

that the invariability and abundance of the bacterial community increased

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simultaneously in the presence of TiO2 NPs.

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The microbial community composition of the periphytic biofilms at the phylum and

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family levels were determined. Figure 2A shows that 21 phyla were determined, mainly

230

comprising

231

Planctomycetes, and Proteobacteria (ranging from 92.7-98.0 %). Although the first

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three dominant communities at the phylum level (Cyanobacteria, Proteobacteria and

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Bacteroidetes) were the same in the control and the treatments, the proportion of these

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three communities changed (77.5 %, 17.7 % and 4.8 % for the control and 57.9 %, 29.6 %

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and 12.5% for the treatment, respectively. Compared to the sensitivity of single species

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bacteria to TiO2 under intense UV irradiation 34, the periphytic biofilms presented good

237

adaptation to TiO2 under intense UV irradiation. This is because multi-species

238

aggregates, such as periphytic biofilm, can resist unfavorable conditions including TiO2

of

the

phyla

Armatimonadetes,

Bacteroidetes,

10


Cyanobacteria,


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35

239

exposure because of their interspecies interactions and ‘collective function’

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addition, periphytic biofilm is able to produce more EPS and change their community

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composition to defend against the toxicity derived from NPs 36.

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Figure 2B compares the microbial composition in the control and treatment. The

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abundance of Sphingomonadaceae and Xanthomonadaceae (phylum Proteobacteria),

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and Chitinophagaceae and Cyclobacteriaceae (phylum Bacteroidetes) increased

245

significantly in the presence of TiO2 NPs compared to the control sample (p < 0.05).

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These results suggest that these four types of bacteria adapted to the TiO2 NPs

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conditions well. The Sphingomonadaceae, Xanthomonadaceae 37, Chitinophagaceae

248

39

249

are able to acquire electrons derived from natural solid donors to reduce electron

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acceptors 10. Researchers have proved that Sphingomonadaceae and Xanthomonadaceae

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could use the exogenous electrons derived from a semiconductor (e.g. pyrite) to

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accelerate denitrification

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composition of multi-species biofilm for specific biofilm-mediated bioremediation

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processes. These four types of denitrifying bacteria may contribute to nitrate reduction

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through denitrification or by using the photogenerated electrons produced by TiO2 NPs

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to catalyse nitrate reduction in the presence of TiO2 NPs.

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CAT is an important enzyme that indicates the activity of microorganisms against

258

unfriendly habitats

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the end of the experiment, i.e. after day 7 (Figure 3A). The CAT activity in the

260

treatments was significantly higher than that in the control (p < 0.05), further implying

261

that the periphytic biofilms were capable of growing well in the presence of TiO2 NPs.

262

The growth of the periphytic biofilm in the absence (control) and in the presence

and Cyclobacteriaceae

40

. In

38,

have been reported as autotrophic denitrifiers. Autotrophs

37

. TiO2 NPs were able to optimize the community

41

. Thus, the CAT activity of periphytic biofilm was determined at

11


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(treatment) of TiO2 NPs were almost similar under the experimental conditions

264

investigated in this study. The relative abundance of denitrifying bacteria increased

265

significantly in the presence of TiO2 NPs. However, the NaR also decreased slightly at

266

the end of the experiments (Figure 3A). It is noted that determining activity of NaR

267

required a dark environment which means that TiO2 will not provide its photoexcited

268

electrons under the circumstances. This denitrifying bacterium may require exogenous

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electron donor supply to function in denitrification.

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To investigate the changes in the composition of EPS, the humic acid, protein and

271

polysaccharose contents of the periphytic biofilm were determined (Figure 3B). The

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total EPS concentration (sum of humic acid, protein and polysaccharose) in the TiO2

273

NPs treatment was higher than that in the control (p < 0.05). The addition of TiO2 NPs

274

significantly enhanced the secretion of protein, from 42.2 mg L-1 in the control to 76.3

275

mg L-1 in the treatment, on day 7 (p < 0.05). After day 3, the concentration of humic

276

acid in the TiO2 NPs treatment was also significantly higher than that in the control (p
0.05), but a significant negative relationship between nitrate and humic acid in the

355

treatment (r = -0.992, p < 0.01) (Figure 6A &B). Similarly, a negative relationship was

356

observed between humic acid and nitrite production in the treatment (r = -0.935, p

Simultaneous Supplement of Denitrifiers, Electron Donor Pool and

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