Nanoparticles and UV Radiation on Extracellular Enzyme Activity of

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Effect of TiO2 Nanoparticles and UV radiation on Extracellular Enzyme Activity of Intact Heterotrophic Biofilms Hannah Schug, Carl William Isaacson, Laura Sigg, Adrian A. Ammann, and Kristin Schirmer Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 10 Sep 2014 Downloaded from http://pubs.acs.org on September 14, 2014

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Environmental Science & Technology

159x85mm (150 x 150 DPI)

ACS Paragon Plus Environment


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1

Effect of TiO2 Nanoparticles and UV radiation on

2

Extracellular Enzyme Activity of Intact Heterotrophic

3

Biofilms

4

Hannah Schug1,2,#, Carl W. Isaacson1,#,5, Laura Sigg1,3, Adrian A. Ammann1, and Kristin

5

Schirmer1,3,4,*

6 7

1

8

2

University of Constance, 78467 Constance, Germany

9

3

ETH Zürich, Swiss Federal Institute of Technology, Institute of Biogeochemistry and Pollutant Dynamics, 8092

Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland

10

Zürich, Switzerland

11

4

EPF Lausanne, School of Architecture, Civil and Environmental Engineering, 1015 Lausanne, Switzerland

12

5

Present address: Bemidji State University, Department of Environmental Science, Bemidji, MN, USA

13 14

# Authors contributed equally to this work

15 16 * Address correspondence to

17 18 19 20 21

Prof. Dr. Kristin Schirmer Head of Department - Environmental Toxicology Eawag, Swiss Federal Institute of Aquatic Science and Technology

22 23 24 25

Überlandstrasse 133 P.O. Box 611 8600 Dübendorf Switzerland

26

Phone: +41 (0)58 765 5266

27

Fax: +41 (0)58 765 53 11

28

email: [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT

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When introduced into the aquatic environment, TiO2 NP are likely to settle from the water

31

column, which results in increased exposure of benthic communities. Here, we show that the

32

activity of two extracellular enzymes of intact heterotrophic biofilms, β – glucosidase (carbon

33

– cycling) and L – leucin aminopeptidase (nitrogen – cycling), was reduced following

34

exposure to surface functionalized TiO2 NP and UV radiation, depending on the particles’

35

coating. This reduction was partially linked to ROS production. Alkaline phosphatase

36

(phosphorus – cycling) activity was not affected, however in contrast, an alkaline

37

phosphatase isolated from E.coli was strongly inhibited at lower concentrations of TiO2 NP

38

than the intact biofilms. These results indicate that enzymes present in the biofilm matrix are

39

partly protected against exposure to TiO2 NP and UV radiation. Impairment of extracellular

40

enzymes which mediate the uptake of nutrients from water may affect ecosystem function.

41 42 43 44 45 46 47 48 49 50

Keywords: nanoparticle coating, photocatalytic activity, reactive oxygen species (ROS), β –

51

glucosidase (Glu), L-Leucine Aminopeptidase (LAP), Alkaline Phosphatase (AP)



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INTRODUCTION

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In freshwater environments, most submerged surfaces are colonized by complex microbial

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communities known as biofilms. They are taxonomically diverse and consist of a variety of

55

bacteria, fungi and algae embedded in a self-synthesized matrix of extracellular polymeric

56

substances (EPS).1 The EPS mediates the adhesion and protects the organisms against

57

external threats and environmental stressors. It contains extracellular enzymes, which

58

hydrolyze dissolved high molecular weight compounds into smaller biomolecules. Only these

59

can subsequently be taken up by microorganisms.2,

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fundamental link between nutrients dissolved in the water column and higher trophic levels

61

and are essential for nutrient cycling in freshwater ecosystems.4 Further, viability of biofilms

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is of environmental concern, because they serve an important role in the degradation of

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natural and anthropogenic pollutants.

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As extracellular enzymes are located outside the cell, they might represent a first site of

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interaction with potential stressors, such as TiO2 nanoparticles (TiO2 NP). The input of TiO2

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NP to aquatic environments has been modeled 5 and measured as a result of abrasion from

67

tiles 6, washout from exterior facades 7, and release from cosmetics, especially sunscreen.8

68

However, the effluents of production facilities and wastewater treatment plants were reported

69

to be the main point source.9,

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from the water column, which leads to entrainment and accumulation in the biofilm over

71

time.11-13 Yet, there is little knowledge about the effects of TiO2 NP exposure to intact

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heterotrophic biofilms. The majority of ecotoxicological effect studies of TiO2 NP focused on

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pelagic organisms and only two studies, with a focus on technical applications, dealt with

74

suspended biofilms14 and less complex monoculture biofilms.15

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Bulk TiO2 is generally regarded as being of low toxicity, however, nano-sized TiO2 has been

76

shown to produce toxic effects.9, 16-18 These were often attributed to photo-activation of TiO2

77

NP and the production of reactive oxygen species (ROS).19,

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advantageous in controlled processes, e.g. wastewater purification and disinfection

10

3

Therefore, biofilms represent a

When TiO2 NP enter the aquatic environment, they settle

20

While these can be 21

, they 3


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might be harmful in natural freshwater environments. The UV intensity of sunlight has been

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reported to be sufficient to photo-activate TiO2 NP and induce bacterial cell death.22 By

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entrainment of TiO2 NP in the biofilm, they may come into close contact with microbial

82

organisms and components of the matrix, like the extracellular enzymes, affecting their

83

functionality.

84

In this study we selected three hydrolytic enzymes important for essential nutrient cycling: β-

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glucosidase, carbon cycling; L-leucin aminopeptidase, nitrogen cycling and alkaline

86

phosphatase, phosphorus cycling. Enzymes of the major nutrients are of high importance

87

with regard to the freshwater ecosystem. Biofilms are a major site of nutrient uptake since

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they are the only biological population capable of altering dissolved and particulate organic

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matter.

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surface coated with molecules selected based on their environmental relevance and the

91

resulting physicochemical properties (SI Table 1). The coatings mimic the variety of

92

engineered and naturally occurring surface modifications and allow to determine how surface

93

chemistry influences their behavior in natural waters and the effect on enzymatic activity. All

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particles were characterized for their behavior in stream water and for their ability to produce

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ROS upon exposure to UV radiation. As biological test system, we used intact heterotrophic

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biofilms, cultured from natural stream water, and a pure alkaline phosphatase isolated from

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E. coli.

1, 2, 11

TiO2 NP included conventional P25 and self-synthesized TiO2 NP, which were

98 99



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MATERIALS AND METHODS

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Colonization of heterotrophic biofilms

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Heterotrophic biofilms were cultivated in flow cells (for dimensions and experimental set up

103

please see SI Figure 2) at 15 °C with a constant water flow (flow rate = 2 cm s-1) over a

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membrane support (Regenerated cellulose acetate, PM UC030, Microdyn Nadir, Wiesbaden,

105

Germany). The culture conditions were based on the development of β glucosidase (Glu)

106

activity which was arbitrarily chosen as a representative. For cultivation, water from the

107

Chriesbach river was filtered through a 1.5 µm glass fiber filter. This treatment showed

108

highest Glu activity (see SI Figure 3) while suspended natural colloids and biofilm predators,

109

like larger zooplankton and benthic invertebrates, were removed. The water was changed

110

weekly. Biofilms were sampled for effect studies after three weeks of cultivation, since from

111

then on no major increase in Glu activity was detected (see SI Figure 3).

112

Exposure setup and control experiments

113

For exposure experiments, biofilm disks (0.8 cm2) were cut, placed in 48-well plates and

114

exposed to 1 ml of TiO2 NP (10 mg l-1). To simulate a more realistic environmental exposure

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scenario, exposures were conducted in filter sterilized Chriesbach water buffered with 10 mM

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Tris Base at pH 8.5 to maintain a constant pH. Biofilms were exposed to TiO2 NP for four

117

hours in the presence and absence of simulated solar radiation. For each type of

118

nanoparticle – enzyme combination, the biofilms for three technical replicates were taken

119

from the same flow cell. The exposure time of four hours was chosen to balance between

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naturally occurring enzymatic activity loss in unexposed biofilm samples and a sufficient time

121

to allow for NP sedimentation.

122

The influence of UV intensity alone on enzymatic activity was assessed. Biofilm disks were

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irradiated with three different UV intensities (SI Table 2) and the activity of the three

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extracellular enzymes was compared to the dark control. Since no effect on enzymatic

125

activity was found, the UV intensity used for further exposures ranged between the medium

126

and high UV scenario as is stated below. 5 ACS Paragon Plus Environment

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The effect of chemicals used for surface functionalization of TiO2 NP on the extracellular

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enzyme activity was assessed to discriminate NP effects from the effect of the coating itself.

129

Here, a similar exposure setup as for TiO2 NP exposure was used. Exposures where the

130

coated NP did not decease enzymatic activity but the chemical used for coating did, e.g. rutin

131

(see SI Figure 8), were not further evaluated.

132

Extracellular enzyme assays

133

The effects of TiO2 NP on the extracellular enzyme activity were assayed by determining the

134

utilization rate of fluorescent linked substrates. The three extracellular enzymes were chosen

135

as representatives of macronutrient cycling processes: β–glucosidase (E.C. 3.2.1.21) –

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hydrolyzing β-linked polysaccharides (carbon cycling), L–leucine aminopeptidase (E.C.

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3.4.11.1) – cleaving peptides and amino acids (nitrogen cycling) and alkaline phosphatase

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(E.C. 3.1.3.1-2) – breaking organophosphoric esters (phosphorus cycling). Activity was

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measured using fluorescently linked substrates (4–Methylumbelliferyl (MUF) β–D–

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glucopyranoside

141

hydrochloride for the L–leucine aminopeptidase and 4–Methylumbelliferyl (MUF) phosphate

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disodium salt for alkaline phosphatase (Sigma Aldrich, Buchs, Switzerland). To test the effect

143

of TiO2 NP on enzyme activity in the absence of the extracellular matrix of the biofilm, pure

144

alkaline phosphatase, isolated from Escherichia coli, was purchased from Sigma Aldrich

145

(Buchs, Switzerland). At 100 mg l-1, tannic acid coated TiO2 NP interfered with the

146

fluorescence of the enzyme assay. This value was excluded from the graph (see Figure 2

147

panel B).

148

The substrate was added as saturating concentrations (1 mM for β–glucosidase and L–leucin

149

aminopeptidase, and 2 mM for alkaline phosphatase). Conversion of the fluorescent linked

150

substrate was measured over at least 30 min at an excitation/emission wavelength of λex/λem

151

370/441 nm using a multiwell plate reader (Tecan, Infinite M200; Männedorf, Switzerland).

152

Exposures and measurements were performed with intact heterotrophic biofilms.

for

β–glucosidase,

L–leucine–7–amid-4–Methylcoumarin

(AMC)



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Final enzyme activities were determined by converting the linear regression slope of the

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substrate conversion rate to concentration MUF or AMC per biofilm total protein and time

155

(µmol gprotein-1 s-1). TiO2 NP coated with rutin and tannic acid decreased fluorescence counts

156

by 18 ± 14 % and 22 ± 4 %, respectively by interfering with the fluorophore MUF and AMC,

157

which was determined by three technical replicates. Therefore a calibration curve was

158

established which was used to normalize the fluorescence data to determine final enzyme

159

activities.

160

The mean of the replicates was calculated and normalized using the untreated dark control,

161

allowing for better comparison between independent experiments. In total, three biological

162

replicates with the value of a single independent experiment being the average of at least

163

three technical replicates were measured. Total protein content per cm2 was determined to

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be 18.2 ± 1.7 µg cm -2 of three biological replicates. Statistical significance was determined by

165

performing a two-way–ANOVA followed by a Bonferroni post – test.23 P values less than 0.05

166

were considered as significant. Individual p-values are presented in two tables, one for TiO2

167

NP

and

one

for

the

NP

coatings

(SI

Table

3

and

4

respectively).


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Titanium dioxide nanoparticles (TiO2 NP)

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A variety of TiO2 NP were used in this study: flame synthesized TiO2 NP doped with 1 %

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atomic niobium (Nb) made by the Swiss Federal Laboratories for Materials Science and

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Technology (Empa, Dübendorf, Switzerland)24, Degussa P-25 received from Evonik

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Industries (Essen, Germany) and solution synthesized TiO2 NP produced by reaction of TiCl4

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with ethanol, followed by condensation in benzyl alcohol and subsequently surface coated

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with molecules having an enediol moiety.25 Niobium doped TiO2 NP were previously shown

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to have enhanced photocatalytic activity compared to P-25 TiO2. Chemi-sorption of enediol

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molecules to the surface of the TiO2 NPs was previously extensively described24, 25 (see also

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SI methods).

178

Surface coatings were selected based on their environmental relevance and the

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physicochemical properties of the functionalized particles. Coatings included strong and

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weak

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Dopamine, Ascorbic Acid), nonpolar organic coating (Catechol), environmentally (Rutin

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trihydrate and tannic acid) and biologically relevant molecules (Dopamine, 3,4- Dihydroxy –

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DL – Phenylalanine, Ascorbic Acid) and molecules with different chromophores (Alizarin

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complexone, Alizarin SO4, Alizarin, Gallocyanine, Dihydroxycoumarin) (SI Table 1). These

185

coatings mimic the variety of engineered and naturally occurring surface modifications and

186

allow for the determination of the mechanism by which particle behavior is affected and how

187

it might affect enzymatic activity.

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Concentrations of TiO2 NP are expressed as TiO2 mass determined by micro digestion with

189

hydrofluoric acid followed by quantification by ICP-MS.

190

Nanoparticle characterization

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Nanoparticle stock suspensions were characterized at 10 mg l-1 TiO2 in the experimental

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medium of 0.2 µm filtered water from the Chriesbach river (a small stream in Dübendorf,

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Switzerland; DOC: 3.7 mg C l-1, Ionic strength (free ions): 8.1 mM, Cl-1: 1.0 mM, NO3- : 0.4

acids

and

bases

(1,3–Benzenedisulfonic

acid,

3,4–Dihydroxybenzhydrazide,



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mM, SO42-: 0.3 mM, K+: 0.1 mM, Na+: 0.8 mM, Ca2+: 2.6 mM, Mg2+: 0.6 mM) and 10 mM Tris

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Base buffer (pH 8.5).

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Dry particle sizes were determined by applying the Scherrer Equation to the X-ray diffraction

197

(XRD) spectra and by TEM measurements. Hydrodynamic diameter and zeta – potential (ζ –

198

potential) were measured via Dynamic Light Scattering (DLS) using the Zetasizer (Zetasizer

199

Nano Series, Malvern Instruments). Optical properties were evaluated by recording the UV –

200

visible (UV-vis) spectra of TiO2 NP diluted in Chriesbach river water at a final concentration

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of 5 mg l-1 using an UV–vis spectrometer (UVIKON 930, Kontron Instruments).

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Sedimentation rates were determined over 8 hours by tracking the absorbance at 260 nm.

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Briefly, particles were suspended at 10 mg l-1 and samples were collected and analyzed at 0,

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0.5, 1, 2, 4, and 8 hours. Samples were measured as four technical replicates and final

205

sedimentation rates were calculated from the regression line of ln(C/C0) vs. time as apparent

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first order sedimentation rate constant ksed [h-1] ± SD of the slope.

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Simulated sunlight setup

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For simulated solar irradiation, two fluorescent lamps (CLEO Compact 25W-S-R, Philips,

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Germany) and one daylight lamp (BIOLUX, L 15W/72, Osram, Switzerland) were installed.

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The height of the lamps was adjusted to set the radiation intensity to UVA 3.6 mW cm-2, UVB

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0.19 mW cm-2, PAR 75 µE m-2s-1, which represents environmentally realistic UV irradiation

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reaching the water surface for a summer day in Dübendorf in Switzerland. (Measurements

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conducted in the beginning of March 2013: UVA 1.3 – 2.8 mW cm-2 and UVB 0.07 – 0.1 mW

214

cm-2.)26

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Determination of reactive oxygen species (ROS) production

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The ability of the TiO2 NP to produce ROS was determined via methylene blue (MB)

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oxidation. Photo-oxidation rates were determined by placing 10 mg l-1 nanoparticles and

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3 mg l-1 MB in 96–well plates, then illuminating them under the simulated solar irradiation

219

setup described above. Absorbance at 660 nm was tracked over four hours.


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To determine the influence of different wavelength regions of the simulated solar radiation on

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TiO2 NP photoactivity, an acrylic plate was used to filter out most UV radiation (93.3 % UVA

222

and 98.9 % UVB removed) while leaving the photosynthetically active radiation (PAR) intact

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(only 4 % PAR is removed). Control experiments in the dark were conducted simultaneously

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to determine any MB losses not resulting from photo-oxidation. Samples were measured as

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three technical replicates and the average was used for determining the apparent first order

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oxidation rate constant kox [h-1] ± SD, calculated from the regression line of ln(C/C0) vs. time.

227



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RESULTS AND DISCUSSION

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Characterization of TiO2 NP in Chriesbach water

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The dry particle size for P25 was 28 nm (XRD, SI Figure 4), while for the solution

231

synthesized TiO2 NP, the size was between 3.1 - 10 nm (XRD, SI Figure 4; TEM, SI Figure

232

5). When suspended in Chriesbach river water, a small stream in Dübendorf, Switzerland,

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TiO2 NP agglomerated to 890 – 5400 nm in size, with the exception of rutin coated particles,

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which were 70 nm (Figure 1, panel A, SI Table 5). Zeta potential for all coated TiO2 NP and

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P25 was –17 ± 1 mV (SI Table 5).

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The surface coating influences the colloidal stability of TiO2 NP in Chriesbach river water,

237

which in turn affects the fate of the NP in the water body. Colloidal stability was assessed by

238

the sedimentation rate and TiO2 NP suspensions were classified as: stable (ksed < 0.02 h-1;

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rutin, alizarin red, alizarin and coumarin coated NP), moderately stable (ksed = 0.02 – 0.1 h-1;

240

tannic acid, dihydroxyybenzhydrazide, catechol, alizarin complexone, gallocyanine, Nb –

241

doped and P25 TiO2 NP) and unstable suspensions (ksed > 0.1 h-1, phenylalanine, dopamine,

242

ascorbic acid, benzenedisulfonic acid coated NP) (Figure 1B, SI Table 5).

243

In general, electrostatic forces did not stabilize functionalized TiO2 NP in the high ionic

244

strength water of the Chriesbach river (2.6 mM Ca2+, 0.6 mM Mg2+) while steric forces

245

seemed to be more important for stabilizing TiO2 NP in this water.

246

The functionalization of TiO2 NP resulted in different colors, which were observed in the UV–

247

vis spectra and by visible inspection (Figure 1, panel C, inset). TiO2 NP strongly absorbed

248

radiation of less than 400 nm in wavelength when suspended in Chriesbach river water

249

(Figure 1C), indicating photocatalytic activity. Previous reports showed that absorption of

250

radiation less than 388 nm by TiO2 NP produced ROS

251

(between 3.2 and 3.0 eV) of TiO2 NP.28

252

These results highlight that a significant portion of the particles settles from the water

253

column, which indicates that benthic organisms, like heterotrophic biofilms, are likely to be 11

27

, due to the wide band gap energy


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more exposed to TiO2 NP than pelagic organisms.29, 30 Additionally, biofilms will mostly be

255

exposed to micro size agglomerates instead of individual TiO2 NP. For larger agglomerated

256

particles, mechanisms related to small particle size, like facilitated uptake or the possibility to

257

particle induced toxicity at sites which are unavailable for larger particles

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Other modes of action may still cause adverse effects, such as oxidative stress from the

259

generation of ROS produced by the photocatalytic activity of TiO2 NP.19,

260

sediment, they might become trapped within the EPS which allows for close contact to the

261

extracellular enzymes. ROS produced by the TiO2 NP may oxidize the extracellular enzymes,

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resulting in an alteration of enzyme structure and function.31

263

Photocatalytic activity of coated and uncoated TiO2 NP

264

Effect of different UV spectra and intensities on the photocatalytic activity of TiO2 NP

265

Control experiments with P25 showed that upon irradiation with environmentally realistic UV

266

spectra and intensities, methylene blue (MB) was significantly oxidized (SI Figure 6, panel A;

267

SI Table 6) with an apparent first order oxidation rate constant (kox) of 0.35 ± 0.06 h-1, similar

268

to previous reports.32 Control experiments in the dark with and without P25 showed no

269

degradation of MB, while exposure of MB to UV radiation alone led to an average

270

degradation rate of kox = 0.05 ± 0.01 h-1, which was set as the effective threshold of

271

photocatalytic activity. After applying a UV-filter to remove radiation of less than 400 nm in

272

wavelength, the remaining 4 % of the UV radiation (here referred to as photosynthetically

273

active radiation - PAR, SI Figure 6, panel A) still activated catalytic MB oxidation by P25 (kox

274

= 0.06 ± 0.01 h-1). This indicates that low intensities of UV radiation are effective in photo-

275

activating TiO2 NP. This is of particular interest for freshwater environments, like the

276

Chriesbach river, since it is a rather shallow stream with an average depth of approximately

277

60 cm and some deeper parts reaching depths of maximal 1 m. In Chriesbach river water,

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these minor intensities can still be found at 54 cm depths for UVA and 21 cm depth for UVB

279

radiation (SI Table 7).

18

, may be reduced.

20

After TiO2 NP



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280

In general, the natural sun light intensity decreases with increasing water depth due to the

281

presence of UV quenching materials, e.g. dissolved organic matter (DOC), colloid particles,

282

and pelagic organisms. The risk of increased ROS production due to photo-activation of TiO2

283

NP is therefore dependent on the water characteristics and the depths of the water body. In

284

streams deeper than the Chriesbach river, biofilm grown in shallow regions, e.g. closer to the

285

shore line, could be exposed to UV intensities sufficient to activate TiO2 NP.

286

Effect of different TiO2 NP concentration on MB degradation

287

MB oxidation was dependent on the TiO2 NP concentration with the highest MB degradation

288

found at 10 mg l-1 TiO2 NP (SI Figure 6, panel B, SI Table 8). At higher concentrations,

289

shading by the NP in solution reduced photocatalytic degradation of MB. Based on this

290

observation, the TiO2 NP concentration for the biofilm exposure was set to 10 mg l-1. For

291

short exposures, this concentration might still be a realistic exposure concentration even

292

though it exceeds the predicted environmental concentration (PEC) of 0.7 – 16 µg l-1 for

293

surface waters in Switzerland 33: accumulation of TiO2 NP in the biofilm over time can lead to

294

much higher concentrations compared to the water phase

295

time.

296

Effect of surface modification of TiO2 NP on the MB degradation

297

Surface functionalization resulted in different photocatalytic efficiencies of different TiO2 NP,

298

determined by comparison of MB oxidation rates, kox (SI Figure 6, panel C, SI Table 5). P25

299

was most effective in degrading MB followed by Nb-doped TiO2, followed by the coated

300

particles. This is consistent with the two flame synthesized particles being more crystalline

301

and therefore more photo catalytically active.35

302

Five types of coated TiO2 NP were chosen for biofilm exposure. Phenylalanine, alizarin red

303

and catechol coated NP were selected because of their increased MB degradation rates

304

when compared to the uncoated TiO2 NP. These coating chemicals possibly act as

305

photosensitizers, extending the catalytic response of TiO2 NP in the longer wavelength area.

306

Rutin and tannic acid coated NP were chosen for their environmental relevance; they most

34

and a much longer exposure


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closely mimic coatings which are similar to natural organic molecules in environmental

308

waters. Their reduced photoactivity may result from direct absorption of the energetic UV

309

radiation without the creation of free electrons in the TiO2 core or the scavenging of electrons

310

by the coating, which could reduce ROS formation.

311

Effect of TiO2 NP on an isolated alkaline phosphatase

312

To test the efficiency of photo-inactivation of TiO2 NP towards a simplified biological test

313

system, the enzyme alkaline phosphatase (AP), isolated from E. coli, was exposed to TiO2

314

NP in the absence and presence of UV radiation. Unlike extracellular enzymes in the biofilm,

315

alkaline phosphatase is present freely in solution, allowing maximal accessibility of the TiO2

316

NP to affect the enzyme.

317

TiO2 NP addition in the dark slightly decreased the enzymatic activity of the phosphatase

318

compared to the unexposed dark control (Figure 2, panel A). Non-UV-induced radical

319

reactions at the surface of TiO2 NP, which produce minor portions of ROS, have been

320

reported 36 and could affect enzymatic function. In contrast to other studies 37, 38, UV radiation

321

alone did not alter the activity of the phosphatase, whereas TiO2 NP addition under UV

322

radiation completely inhibited phosphatase activity. The hypothesized mode of inhibition is

323

the interference of generated ROS with enzyme structure, e.g. amino acid chains, which

324

leads to a loss of function.39

325

Dose response curves were measured for P25 and TiO2 NP coated with rutin, tannic acid,

326

phenylalanine, catechol and alizarin red under UV radiation (Figure 2, panel B). When

327

comparing the IC50 values and the corresponding 95 % confidence intervals (CI) (SI Table 9),

328

there were no differences between the TiO2 NP tested, except for tannic acid coated TiO2

329

NP, where exposure resulted in only slight reduction of enzyme activity. Assuming that the

330

underlying mechanism of inhibition is due to the generation of ROS, P25 was expected to

331

have the greatest effect on enzyme activity. Therefore, oxidation through reactions with ROS

332

is unlikely to be the dominant mechanism of inactivation for the isolated enzyme. Another 14 ACS Paragon Plus Environment


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333

possible mode of action is through adsorption of the phosphatase to the particle surface. As

334

soon as enzymes bind to the surface of NP they undergo conformational changes, which in

335

some cases leads to a lack of functionality.40 However, in the dark there was only a minor

336

measurable decrease in activity, which indicates that UV radiation still seems to be a

337

significant factor for the inactivation of the isolated AP, with an yet undetermined mode of

338

action.

339

Effect of TiO2 NP on enzymatic activity of intact heterotrophic biofilms

340

In initial experiments with intact heterotrophic biofilms, activity of the three enzymes was

341

tested to be unaffected under three different UV exposure intensities (SI Table 2) when

342

compared to a dark control (SI Figure 7). However, in later exposures with TiO2 NP and UV

343

radiation, evaluated by two-way ANOVA, UV radiation alone significantly decreased the

344

enzymatic activity of alkaline phosphatase (AP) (Figure 3, panel C, F, SI Table 3) and β-

345

glucosidase (Glu) (Figure 3, panel G, SI Table 3) in some cases. This decrease was not

346

further amplified by combined exposure with TiO2 NP (SI Table 3). The loss of activity likely

347

does not result from UV sensitivity of the enzymes themselves, based on the previously

348

described results (SI Figure 7), but rather from variable UV tolerance of the biofilm, since

349

exposures to TiO2 NP and UV radiation were done at a later time point than the UV control

350

experiments. As biofilms were cultured directly from the Chriesbach river, they represent the

351

actual heterotrophic microbial community in the river at the given time and season. The

352

varying microbial composition can lead to varying matrix thickness and composition, which

353

influences the ability to protect against UV radiation.

354

Toxicity of the coatings itself was excluded. Tannic acid, catechol and alizarin red, which

355

decreased enzyme activity when used as a particle coating, themselves did not alter the

356

enzymatic activity (SI Figure 8, Panel D-F, J-L, M-O, SI Table 4). Exposures where the

357

coated NP did not decrease enzymatic activity but the chemical used for coating did, e.g.

358

rutin (SI Figure 8, Panel A, B), were not further evaluated.


Page 17 of 26


359

When exposing intact heterotrophic biofilms with one of the five selected coated TiO2 NP or

360

P25 in the presence and absence of UV radiation, P25 and tannic acid coated TiO2 NP

361

affected enzymatic activity in most cases (Figure 3, Panel A-F). In general, the three

362

enzymes responded differently to the combined exposure of TiO2 NP and UV radiation and to

363

UV radiation alone: activity of Glu was significantly decreased in more cases compared to L-

364

leucine aminopeptidase (LAP). The activity of the AP was not impaired by the exposure to

365

TiO2 NP and UV radiation in any of the tested biofilms. These differences in sensitivity might

366

result from different localization patterns of the three enzymes. Enzymes in the biofilm can be

367

either freely diluted in the matrix, associated with the insoluble fraction, e.g. microbial cells or

368

organic particles or be localized intracellularly.41 Previous studies show that a major fraction

369

of secreted AP and LAP are associated with the insoluble fraction,42, 43 whereas Glu appears

370

mainly located in the soluble fraction (SI Figure 9). Glu might therefore be more accessible

371

for TiO2 NP and ROS when compared to AP and LAP, which is in support of the higher

372

incidence of decreased enzyme activity. Another potential explanation concerns changes in

373

the community profile due to antimicrobial activities of TiO2 NP which could lead to an

374

alteration of enzymatic activity by means of lowered production and secretion of these

375

enzymes.

376

Exposure to P25 led to reduced enzymatic activity for Glu and LAP (Figure 3, panel A, B, SI

377

Table 3). The deleterious effect observed following the biofilm exposure is less distinct when

378

compared to the exposure of the isolated dissolved enzyme, where activity was completely

379

abolished (Figure 2, panel A). This indicates that the intact matrix acts as a partial barrier,

380

which protects not only against desiccation, oxidizing biocides, antibiotics and natural

381

predators3, but similarly against toxicity induced by UV and TiO2 NP.37 However there is still a

382

measureable loss of functionality which may limit the biofilms’ ability to process essential

383

macronutrients.

384

Exposure to tannic acid coated TiO2 NP and UV radiation significantly decreased enzymatic

385

activity of LAP, when compared to the dark control (Figure 3, panel E, SI Table 3). Since the 16 ACS Paragon Plus Environment


Page 18 of 26

386

tannic acid coated particles were determined to be ineffective photo-oxidizers (SI Figure 6,

387

panel C), no ROS induced impact on activity is expected. Further, reduced enzyme activity of

388

Glu and AP was measured even in the absence of light (Figure 3, panel F, SI Table 3). Since

389

any deleterious effect of the tannic acid coating alone was ruled out (SI Figure 8, Panel D-F),

390

the observed reduction in enzyme activity must result from a tannic acid coated particle

391

specific effect independent from UV radiation.

392

For exposures to either catechol or alizarin red coated TiO2 NP and UV radiation, only Glu

393

activity was decreased (Figure 3, Panel M, P, SI Table 3) when compared to the dark control

394

whereas the activity of LAP and AP remained unchanged (Figure 3, Panel N,O,Q,R). Even

395

though TiO2 NP coated with rutin and phenylalanine were shown to be efficient in generating

396

ROS, there was no measurable decrease in enzymatic activity upon exposure, either in the

397

presence or in absence of UV radiation (Figure 3, G-L). This indicates that the components

398

of the biofilm matrix scavenge ROS produced and maintain extracellular enzymatic activity.

399

In summary the results show that the activity of the extracellular enzymes β-glucosidase and

400

L-leucin aminopeptidase of intact heterotrophic biofilms is decreased by exposure to TiO2 NP

401

in combination with environmentally relevant intensities of UV radiation, dependent on the

402

particle coating. The exposure reflects realistic environmental scenarios in shallow

403

freshwater streams, where TiO2 NP input and simultaneous exposure to UV radiation of

404

sunlight will frequently occur. This loss of function for enzymes associated with biofilms likely

405

occurs as a consequence of ROS mediated protein oxidation, where ROS are generated

406

through the photocatalytic activity of TiO2 NP. In other cases, e.g. tannic acid coated TiO2

407

NP, the effect seems to occur based on a NP specific mode of inhibition which cannot yet be

408

explained. This NP specific mode of action is of even greater concern since it occurred

409

independently of UV radiation and tannic acid represents frequently occurring components of

410

natural freshwaters. The strong inhibition of the freely diluted alkaline phosphatase was also

411

independent of ROS production and was attributed to an effect following NP binding.

412

However UV radiation seemed to play an important role. The intact extracellular matrix, 17 ACS Paragon Plus Environment

Page 19 of 26


413

which encapsulates the microbial organisms of the biofilm, reduces but does not abolish this

414

effect. Overall, the significant decrease in activity of β-glucosidase and L-leucin

415

aminopeptidase may adversely affect nutrient acquisition in the biofilm and might have

416

implications for nutrient cycling and the degradation of pollutants in environmental systems.

417



Page 20 of 26

418

Conflicts of interest

419

The authors declare no competing financial interest.

420

Supporting information available

421

Detailed Methods, XRD spectra and TEM images of TiO2 NP; characterization of TiO2 NP

422

and methylene blue oxidation rate, penetration depths of solar UVA and UVB radiation in

423

Chriesbach river water, effect of radiation exposure on extracellular enzyme activity, ,

424

extracellular enzyme activities exposed to five coated TiO2 NP and P25, localization of

425

enzyme activities in phototrophic biofilms, effect of coating chemicals on extracellular

426

enzyme activity, Chemicals used for surface functionalization of TiO2 NP, numeric p-values

427

for two-way-ANOVAs, Experimental setup for the cultivation of heterotrophic biofilms,

428

photocatalytic activity of TiO2 NP, β glucosidase activity to determine cultivation conditions.

429

Acknowledgment

430

We acknowledge Ralf Kaegi for TEM analysis of TiO2 NP and Andreas Vögelin for XRD

431

spectra of TiO2 NP. We thank Niko Derlon for support in the biofilm cultivation setup and

432

Carmen Gil-Allué for introduction and assistance with the extracellular enzyme assays and

433

for data on the localization of the enzymatic activity in phototrophic biofilms. We are grateful

434

to Heike Hildebrand, Stefan Schymura and Karsten Franke for fruitful discussions and a

435

lively collaboration.

436

This study was financially supported by the German Federal Ministry of Education and

437

Research within the NanoNature initiative (project NanoTrack, support code 03X0078A).


Page 21 of 26 438 REFERENCES

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Environmental Science & Technology Page 22 of 26 22. Wei, C.; Lin, W. Y.; Zainal, Z.; Williams, N. E.; Zhu, K.; Kruzic, A. P.; Smith, R. L.; Rajeshwar, K., Bactericidal Activity of Tio2 Photocatalyst in Aqueous-Media - toward a Solar-Assisted Water Disinfection System. Environmental science & technology 1994, 28, 934-938. 23. Neter, J.; Wasserman, W.; Kutner, M. H., Applied linear statistical models: regression, analysis of variance, and experimental designs Homewood, Illinois, Irwin: 1990; Vol. 3rd. ed. , p 1181. 24. Michalow, K. A.; Flak, D.; Heel, A.; Parlinska-Wojtan, M.; Rekas, M.; Graule, T., Effect of Nb doping on structural, optical and photocatalytic properties of flame-made TiO2 nanopowder. Environmental science and pollution research international 2012, 19, 3696-708. 25. Kotsokechagia, T.; Cellesi, F.; Thomas, A.; Niederberger, M.; Tirelli, N., Preparation of ligand-free TiO2 (anatase) nanoparticles through a nonaqueous process and their surface functionalization. Langmuir 2008, 24, 6988-6997. 26. Hader, D. P.; Lebert, M.; Schuster, M.; del Campo, L.; Helbling, E. W.; McKenzie, R., ELDONET - A decade of monitoring solar radiation on five continents. Photochem Photobiol 2007, 83, 1348-1357. 27. Whang, T. J.; Huang, H. Y.; Hsieh, M. T.; Chen, J. J., Laser-induced silver nanoparticles on titanium oxide for photocatalytic degradation of methylene blue. International journal of molecular sciences 2009, 10, 4707-18. 28. Wetchakun, N.; Wetchakun, K.; Phanichphant, S.; Inceesungvorn, B.; Pongwan, P., Highly Efficient Visible - Light - Induced Photocatalytic Activity of Fe - doped TiO2 Nanoparticles. Engineering Journal 2012. 29. Sharma, V. K., Aggregation and toxicity of titanium dioxide nanoparticles in aquatic environment--a review. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering 2009, 44, 1485-95. 30. Ferry, J. L.; Craig, P.; Hexel, C.; Sisco, P.; Frey, R.; Pennington, P. L.; Fulton, M. H.; Scott, I. G.; Decho, A. W.; Kashiwada, S.; Murphy, C. J.; Shaw, T. J., Transfer of gold nanoparticles from the water column to the estuarine food web. Nature nanotechnology 2009, 4, 441-4. 31. Cabiscol, E.; Tamarit, J.; Ros, J., Oxidative stress in bacteria and protein damage by reactive oxygen species. International microbiology : the official journal of the Spanish Society for Microbiology 2000, 3, 3-8. 32. Marsolek, M. D.; Torres, C. I.; Hausner, M.; Rittmann, B. E., Intimate coupling of photocatalysis and biodegradation in a photocatalytic circulating-bed biofilm reactor. Biotechnology and bioengineering 2008, 101, 83-92. 33. Mueller, N. C.; Nowack, B., Exposure modeling of engineered nanoparticles in the environment. Environmental science & technology 2008, 42, 4447-4453. 34. Yeo, M. K.; Nam, D. H., Influence of different types of nanomaterials on their bioaccumulation in a paddy microcosm: A comparison of TiO2 nanoparticles and nanotubes. Environ Pollut 2013, 178, 166-172. 35. Bickley, R. I.; Gonzalezcarreno, T.; Lees, J. S.; Palmisano, L.; Tilley, R. J. D., A Structural Investigation of Titanium-Dioxide Photocatalysts. J Solid State Chem 1991, 92, 178-190. 36. Fenoglio, I.; Greco, G.; Livraghi, S.; Fubini, B., Non-UV-Induced Radical Reactions at the Surface of TiO2 Nanoparticles That May Trigger Toxic Responses. Chem-Eur J 2009, 15, 4614-4621. 37. Espeland, E. M.; Wetzel, R. G., Complexation, stabilization, and UV photolysis of extracellular and surface-bound glucosidase and alkaline phosphatase: Implications for biofilm microbiota. Microbial ecology 2001, 42, 572-585. 38. Santos, A. L.; Oliveira, V.; Gomes, N. C. M.; Coelho, F. J. R. C.; Almeida, A.; Cunha, A., Bacterial Extracellular Enzymatic Activity in Globally Changing Aquatic Ecosystems. Technology and Education Topics in Applied Microbiology and Microbial Biotechnology 2010, 125-135. 39. Turcotte, P. Enzyme inactivation by photoexcited titanium dioxide (TiO₂) and prevention by encapsulation. Concordia University, http://spectrum.library.concordia.ca/2171/, 2003. 40. Zhaochun, W.; Bin, Z.; Bing, Y., Regulation of Enzyme Activity through Interactions with Nanoparticles. International journal of molecular sciences 2009, 10. 41. Wetzel, R., Extracellular Enzymatic Interactions: Storage, Redistribution, and Interspecific Communication. In Microbial Enzymes in Aquatic Environments, Chróst, R., Ed. Springer New York: 1991; pp 6-28. 42. Rego, J. V.; Billen, G.; Fontigny, A.; Somville, M., Free and Attached Proteolytic Activity in Water Environments. Mar Ecol Prog Ser 1985, 21, 245-249. 43. Dimkpa, C. O.; Calder, A.; Gajjar, P.; Merugu, S.; Huang, W. J.; Britt, D. W.; McLean, J. E.; Johnson, W. P.; Anderson, A. J., Interaction of silver nanoparticles with an environmentally beneficial bacterium, Pseudomonas chlororaphis. J Hazard Mater 2011, 188, 428-435.


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Figure 1 Characterization of TiO2 NP

547

Panel A: Size of TiO2 NP measured by dynamic light scattering (DLS) in Chriesbach river water. Panel B: Sedimentation

548

rate constants were determined over 6 time points (0, 0.5, 1, 2, 4, 8 h; N=4). Rate constants are shown as ksed [h ] ± SD.

549

Dotted lines highlight the ranges for unstable, moderately stable and stable colloidal suspensions. Numerical values for

550

NP size and for sedimentations rates are shown in SI Table 2. Panel C: Absorption of UV – visible light by TiO2 NP (5 mg

551

l ) in Chriesbach river water, measured from 190 to 800 nm. In the inset, the visible color of a representative selection of

552

TiO2 NP in suspension is shown for rutin, phenylalanine, tannic acid, dihydroxybenzhydrazide, benzenedisulfonic acid,

553

ascorbic acid, catechol, alizarin complexone and gallocyanine coated TiO2 NP (from left to right).

-1

-1

554 22 ACS Paragon Plus Environment


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555

556

Figure 2 Effect of TiO2 NP on an isolated alkaline phosphatase (AP)

557

Effect of coated TiO2 NP (rutin, tannic acid, phenylalanine, alizarin red and catechol) and P25 on the activity of an

558

alkaline phosphatase (AP) enzyme isolated from E. coli. Panel A: AP activity following P25 exposure in the presence and

559

absence of UV radiation. Enzyme activity data are shown as mean ± SD (N=4). Means with different letters were

560

determined to be statistically different via two – way ANOVA followed by a Bonferroni post test (p < 0.05). Panel B: Dose

561

response relationship of AP activity exposed to coated TiO2 NP and P25 under UV radiation. The response at the highest

562

concentration of tannic acid TiO2 NP was removed from evaluation because, due to interference with the fluorescence of

563

the enzyme assay, a very high value was obtained (4.7). IC50 ± CI were calculated for the individual exposures and are

564

summarized in Table 1.

565 566 567


Alizarin Red coated TiO2 NP

Catechol coated TiO2 NP

Phenylalanine coated TiO2 NP

Rutin coated TiO2 NP

Tannic Acid coated TiO2 NP

P25 TiO2 NP

Page 25 of 26

Glu activity Environmental Science & Technology LAP activity


AP activity

568

24


Page 26 of 26

569 570

Figure 3. Effect of TiO2 NP and UV radiation on extracellular enzyme activity

571

presence of UV radiation. Activity of β glucosidase (Glu), L – leucin aminopeptidase (LAP) and alkaline phosphatase

572

(AP) was measured following exposure to P25 (A-C), coated TiO2 NP: tannic acid (D-F) rutin (panel G-I), phenylalanine

573

(panel J-L), catechol (panel M-O) and alizarin red (panel P-R). Each enzyme – TiO2 NP combination represents a single

574

experiment. Activity data are normalized to the dark control and shown as mean ± SD of three technical replicates.

575

Numeric values are shown in SI Table 7. Means with different letters were determined to be statistically different by two –

576

way ANOVA followed by a Bonferroni post test comparing all individual bars (p < 0.05).

Measured enzyme activity data following exposure of intact heterotrophic biofilms to TiO2 NP in the absence and

577 578


Nanoparticles and UV Radiation on Extracellular Enzyme Activity of

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