Responses of Periphyton to Fe2O3 Nanoparticles: A Physiological

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Responses of Periphyton to Fe2O3 Nanoparticles - a Physiological and Ecological Basis for Defending Nanotoxicity Jun Tang, Ningyuan Zhu, Yan Zhu, Junzhuo Liu, Chenxi Wu, Philip G. Kerr, Yonghong Wu, and Kwan Sing Paul Kwan-Sing LAM Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02012 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Environmental Science & Technology

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Responses of Periphyton to Fe2O3 Nanoparticles - a Physiological and

2

Ecological Basis for Defending Nanotoxicity

3 4

Jun Tanga,e, Ningyuan Zhua,e, Yan Zhua,e, Junzhuo Liua, Chenxi Wub, Philip Kerrc,

5

Yonghong Wua*, Paul K. S. Lamd a

6 7

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Sciences, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China

8 9

b

10 11

c

12 13

d

State Key Laboratory of Marine Pollution, City University of Hong Kong, Hong Kong SAR, China

14 15

e

16

*Corresponding author:

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of sciences, Wuhan 430072, China School of Biomedical Sciences, Charles Sturt University, Boorooma St, Wagga Wagga, NSW 2678, Australia.

College of Resource and Environment, University of Chinese Academy of Sciences, Beijing 100049, China

17 18

Yonghong Wu

19

Address: 71, Beijing Dong Lu, Nanjing, P. R. China, 210008

20

Tel: +86-25-86881330

21

Fax: +86-25-86881000

22

Email : [email protected]

23 1

ACS Paragon Plus Environment


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ABSTRACT:

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The toxic effects of nanoparticles on individual organisms have been widely

26

investigated, while few studies have investigated the effects of nanoparticles on

27

ubiquitous multi-community microbial aggregates. Here, periphyton as a model of

28

microbial aggregates, was employed to investigate the responses of microbial

29

aggregates exposed continuously to Fe2O3 nanoparticles (5.0 mg L-1) for 30 days.

30

The exposure to Fe2O3 nanoparticles results in the chlorophyll (a, b and c) contents

31

of periphyton increasing and the total antioxidant capacity decreasing. The

32

composition of the periphyton markedly changes in the presence of Fe2O3

33

nanoparticles and the species diversity significantly increases. The changes in the

34

periphyton composition and diversity were due to allelochemicals, such as

35

3-methylpentane, released by members of the periphyton which inhibit their

36

competitors. The functions of the periphyton represented by metabolic capability and

37

contaminant (organic matter, nitrogen, phosphorus and copper) removal were able to

38

acclimate to the Fe2O3 nanoparticles exposure via self-regulation of morphology,

39

species composition and diversity. These findings highlight the importance of both

40

physiological and ecological factors in evaluating the long-term responses of

41

microbial aggregates exposed to nanoparticles.

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KEYWORDS: Periphyton; Fe2O3 nanoparticles; Microbial aggregates; Functional

43

acclimation; Fe2O3 NPs-nanotoxicology.

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INTRODUCTION

47

Over the past few decades, engineered nanoparticles (NPs) have been increasingly

48

used for commercial purposes such as fillers, opacifiers, catalysts, semiconductors,

49

cosmetics, microelectronics, and drug carriers.1, 2 Among these commercialized NPs,

50

iron oxide nanoparticles (IONPs) have great potential in areas such as drug delivery

51

vehicles,3 contrast agents for magnetic resonance imaging,4 and environmental

52

remediation.5 IONPs are the most widely used magnetic NPs in biomedical and

53

biotechnological

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compatibility.3 However, IONPs also have the ability to enhance the generation of

55

reactive oxygen species (ROS), which stimulates the peroxidation of cell membrane

56

lipids and causes DNA damage.6, 7 Thus, the physiological risks of IONPs exposure

57

have been widely evaluated. 8, 9

fields

due

to

their

super-paramagnetism

and

biological

58 59

In addition to many terrestrial animal and plant studies, much research has looked

60

to evaluate the toxic effects of IONPs on single species in aquatic ecosystems.8, 10, 11

61

For example, the toxic effects of IONPs on the fresh water alga Mougeotia sp. were

62

mainly due to the increased production of ROS which in turn exhausted the

63

antioxidant defense system including catalase, glutathione reductase, and superoxide

64

dismutase.12 Meanwhile, at least one study showed that IONPs demonstrated

65

antimicrobial activity against Escherichia coli with the mechanism of action being

66

due to oxidative stress resulting from the generation of ROS with the interplay of 3



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oxygen with reduced iron species.13 However, the studies focusing on the

68

investigation of a single species exposed to IONPs ignored the interaction of different

69

members of microbial species in microbial aggregates (i.e. periphyton) that commonly

70

exist on submerged surfaces, potentially leading to different physiological and

71

ecological results.

72 73

It is known that the assimilation of NPs not only causes toxic effects on the

74

organism itself but also can be transported to other species via the food chain.14 The

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physiological responses and resistance to toxicity of each species exposed to NPs are

76

dissimilar.15-17 When aggregates comprised of different species of microbes form, the

77

“integrative” response or resistance to toxicity of the microbial aggregates might

78

differ from the sum of the responses of each species. Therefore, it is necessary to

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investigate the toxic effects of IONPs at the ‘microbial aggregate’ level. Periphyton is

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a microbial aggregate attaching to submerged surfaces in aquatic ecosystems,

81

comprising of a range of microalgae, bacteria, fungi and protozoa.18 Periphyton are

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distributed at the interface of sediments and overlying water and act as an important

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sink (or source) for materials such as phosphorus and heavy metals (Cu, Cd, Ag,

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Fe).19, 20 Meanwhile, periphyton comprising of different communities are relatively

85

robust to external environmental stresses such as the toxic effects of heavy metals

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(Cu2+ and Cd2+) via community composition regulation, and inter- and intra-species

4


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interactions.21 Thus, periphyton was chosen as a model of microbial aggregates in this

88

study.

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To date, one study investigating the toxic effects of silver NPs on stream periphyton

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has been reported; with experiments investigating photosynthetic yield, respiration

91

potential, and the activity of three extracellular enzymes conducted over very

92

short-term (2 h) exposures.22 Such a short time might underestimate the acclimation

93

and resistance of periphyton to NPs, therefore reflecting incomplete results.

94

Moreover, microbial aggregates offer an elegant solution to toxicity due to the diverse

95

species composition, which could maintain the microbial ecosystem in a functionally

96

sustainable manner.23 Accordingly, to investigate the responses of periphyton to long

97

term IONPs exposure on an ecological basis, fundamentals such as morphology,

98

composition, species diversity and function need to be addressed.

99 100

In the present study, a long-term exposure (30 d) experiment of IONPs to

101

periphyton was conducted to investigate (i) the toxic effects of IONPs on

102

physiological properties of periphyton including cell structure, total antioxidant

103

capacity (T-AOC) and chlorophyll content, (ii) the ecological response reflected by

104

changes in microbial diversity and community composition of periphyton, (iii) the

105

changes in the carbon metabolism, and (iv) the contaminant (organic matter, nitrogen,

106

phosphorus and copper) removal by the periphyton. The results are expected to

5



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provide valuable insight into evaluating the effects on periphyton exposed to IONPs

108

which differ from those of an individual organism.

109 110

111

Characterization of NPs and the preparation of NPs suspensions

MATERIALS AND METHODS

112

Fe2O3 NPs (60 nm, 99.9% purity) were purchased from Aladdin (Shanghai, China).

113

The IONPs were dispersed into Milli-Q water by sonication for 30 min (100 W, 25

114

KHz, 25 °C) to a concentration of 100.0 mg L-1 as stock. IONPs suspensions were

115

prepared by diluting the stock to the required concentration with modified Woods

116

Hole culture medium (WC medium) 24 (composition in supporting information, SI).

117

To compare our studies with those examining IONPs toxic effects on individuals, the

118

initial exposure concentrations of IONPs suspensions were set at 5.0 mg L-1.11, 25

119 120

The morphology of the IONPs was determined by transmission electron microscopy

121

(TEM) (HT-7700, Hitachi, Japan). The hydrodynamic diameter of the IONPs

122

dispersed in Milli-Q water and WC medium for 48 h at 5 mg L-1 was determined

123

using a Zetasizer (90PLUS PALS, Brookhaven, USA). The phase composition and

124

crystal structure of the NPs was determined by a powder X-ray diffractometer (XRD)

125

with Cu Kα radiation (X’Pert PRO, Philips, Netherlands). Raman spectra were

126

obtained on an FT-Raman spectrometer (Nexus, Nicolet, USA) to characterize the

6


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crystallinity of IONPs. Iron ions released from IONPs in solution were separated

128

using a 3 kDa centrifugal ultrafiltration filter (Amicon Ultra-15, Millipore, USA) and

129

then determined by inductively coupled plasma atomic emission spectroscopy

130

(ICPAES, Optima 8000, PerkinElmer, USA).

131

Periphyton collection and cultivation

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Periphyton was collected by peeling from stone surfaces using a silicone spatula

133

sterilized by 0.1 M HCl for 2 h from Xuanwu Lake, East China (the lake water

134

parameters: TN = 1.90 mg L−1; TP = 0.1 mg L−1; pH = 7.8; ammonia = 0.53 mg L−1;

135

and nitrate = 0.73 mg L−1). Industrial soft carriers (ISC, polyurethane, length × width

136

× height = 9 cm × 2 cm × 1 cm, Jineng Environmental Protection Company of

137

YiXing, China) were used as solid substrates for periphyton attachment. The collected

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periphyton was cultured in WC medium with ISC in an incubator with light/dark

139

regime of 12 h/12 h, and illuminated at 2800 Lux with an air temperature of 28 ± 1 °C,

140

under shaking conditions (120 r min-1).26 To maintain nutrient supply, 0.1 mL stock

141

solutions (1000 times concentration of the culture medium) of WC medium were

142

added to every 100 mL culture medium each week. When dense periphyton formed

143

after 60 d (the thickness of periphyton exceeding 5 mm), it was peeled off using a

144

sterilized silicone spatula for the following experiments. The ratio of dry mass to wet

145

mass of periphyton was 0.0532 ± 0.0085 (n = 10). Thus, a 5% ratio was selected as a

7



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standard for dry weight (DW) conversion of periphyton. The periphyton mass in the

147

whole study was expressed as DW.

148

Exposure experiment

149

The exposure experiments were conducted as follows: putting 0.05 g periphyton

150

into flasks filled with 100 mL IONP suspensions, followed by periphyton culture in

151

an incubator under the same experimental conditions of periphyton culture mentioned

152

above. To better gauge the responses of the periphyton over the long-term, the

153

exposure experiment was performed for 30 d. No IONPs solution was added into the

154

control (CK). The exposure treatment and the control were performed in triplicate.

155 156

The distribution of the IONPs in the exposed periphyton and the morphologies of

157

exposed versus non-exposed periphyton cells were determined by transmission

158

electron microscope (TEM) (HT-7700, Hitachi, Japan). Surface morphology, integral

159

structure and element distribution on the surfaces of periphyton were observed by

160

scanning electron microscopy (SEM) (SU3500, Hitachi, Japan) and Energy

161

Dispersive Spectrometry (EDS) (Aztec, Oxford, UK).

162 163

The chlorophyll of periphyton was extracted for 24 h in 90% acetone solution and

164

measured with a UV spectrophotometer.27 The T-AOC of the periphyton was

165

determined using a T-AOC assay Kit (Beyotime, Nanjing, China)28 and the details are 8


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presented in the SI. To investigate the carbon metabolic capacity of periphyton,

167

Biolog analysis was employed and the detailed process is described in a previous

168

study.29 Because the periphyton reached a stable state at 96 h, the Biolog data at this

169

time were selected to indicate the carbon metabolic capacity, and analyzed according

170

to Guckert’s method.30 To investigate the responses of the multiple community

171

microorganisms to IONPs, 16S rDNA high-throughput sequencing by Illumina MiSeq

172

was employed to investigate the changes in periphyton microbial composition and

173

diversity. The details of high-throughput sequencing are described in the SI.

174

Isolation and identification of effective compounds from periphyton exposed

175

to IONPs

176

To examine the allelochemicals of the periphyton, the compounds in the periphyton

177

water were extracted, isolated and identified following Wu’s method.31 The processes

178

involved in Wu’s method and GC-MS analysis information are provided in the SI.

179

After GC-MS analysis, the experiment testing the effect of the allelochemicals (i.e.

180

3-methylpentane) on the community composition and structure of periphyton was

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conducted as follows. A total of 0.075 g periphyton were cultured in 250 mL flasks

182

containing 150 mL of WC medium. Then 3-methylpentane (0.01% and 0.1% v/v) was

183

added directly to the cultures. No 3-methylpentane was added to the control. All

184

cultures were incubated under the same experimental conditions as the periphyton

185

culture described previously. The experiment was performed in triplicate. Considering 9



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the heterogeneous physical structure of the periphyton, the chlorophyll fluorescence

187

(F0) test sample was collected from 12 positions in each sample every two days, and

188

the F0 was measured using an AquaPen-P AP-P100 hand-held fluorometer (Photon

189

Systems Instruments, Brno, Czech Republic) after 25 min dark adaption.

190

Contaminant removal experiments by periphyton after IONPS exposure

191

To evaluate the influence of NPs on periphyton function, a comparison of the

192

organic matter, copper, nitrogen and phosphorus removal by CK and IONPs treated

193

periphyton was conducted after exposure to NPs for 30 days. When the exposure

194

experiment was completed, the periphyton in the CK and IONPs treatment were

195

removed from their flasks by centrifuging at 3000 g for 5 min, rinsing with 100 mL

196

Milli-Q water three times by shaking (200 r min-1) in a centrifuge tube for 2 min,

197

followed again by centrifuging at 3000 g for 5 min. Then, 0.05 g periphyton were

198

added into flasks with 100 mL wastewater. The main parameters of wastewater are as

199

follows: COD = 159.26 ± 3.12 mg L-1; TN = 3.92 ± 0.19 mg L-1; TP = 1.15 ± 0.11 mg

200

L-1; NO3- = 2.76 ± 0.21 mg L-1; NH4+ = 0.52 ± 0.09 mg L-1; PO43- = 0.76 ± 0.08 mg

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L-1; Cu2+ = 2.95 ± 0.12 mg L-1; Ca2+ = 2.6 ± 0.19 mg L-1; and Zn2+ = 0.25 ± 0.02 mg

202

L-1. The pH was adjusted to 7.0 by using 0.1 M NaOH or HCl solution as required.

203

The flasks were placed into an incubator with the same experimental conditions for

204

the periphyton culture mentioned above. This contaminant removal experiment was

205

performed in triplicate. 10


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Total phosphorus (TP) in the wastewater was measured colorimetrically by the

207

persulfate digestion-molybdophosphate reaction method. Total nitrogen (TN) was

208

measured by the persulphate digestion and oxidation-double wavelength (220 nm and

209

275 nm) method. Procedures were standard methods.32 Chemical oxygen demand

210

(COD, representing organic matter level) in the wastewater was determined according

211

to the standard potassium dichromate digestion method (GB11914-89) of the Ministry

212

of Environmental Protection of China.33 The concentration of Cu2+ in the wastewater

213

was measured by ICP-MS (7700x, Agilent, America) after filtration and acidification

214

into 1% nitric acid solution.

215

Data analyses and statistics

216

Assays in this study were conducted in triplicate and the results were expressed as

217

mean ± standard deviation. Statistical analyses were performed using SPSS version

218

19.0 (International Business Machines Corporation, New York) and assay data were

219

tested using ANOVA. The significance of differences between means was evaluated

220

at the significance level p < 0.05. The figures were drawn using Sigmaplot 12.0.

221 222

223

Characterisations of IONPs

RESULTS

224

Before the experiments, the IONPs were characterized (Figure 1). In WC medium,

225

most of them were spherical, with relatively uniform size and a diameter of 62.8 ± 9.8 11



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nm (Figure 1a). The average hydrodynamic diameter of IONPs dispersed in Milli-Q

227

water after 48 h was 75.5 ± 11.3 nm, which was similar with the results from WC

228

medium (78.9 ± 11.1) nm, indicating that the IONPs was did not tended to

229

agglomerate within 48 h obviously at 5 mg L-1 concentration dispersed in both water

230

and WC medium (Figure 1b). Figure 1c shows that all the diffraction peaks of the

231

IONPs clearly indicated a pure rhombohedral phase [space group: R-3c (167)] of

232

α-Fe2O3 (JCPDS No. 89-0597, a = 5.039 Å, c = 13.77 Å). There are seven

233

Raman-active vibration modes in α-Fe2O3 crystalline structure, namely two A1g

234

modes (225 and 498 cm-1) and five Eg modes (247, 293, 299, 412 and 613 cm-1).34

235

The Raman bands at 223 and 496 cm-1 in our present work belong to two A1g modes,

236

while those at 287, 410, and 608 cm-1 to Eg modes (Figure 1d). This implies that the

237

IONPs were α-Fe2O3, crystallized NPs.

238

The characterizations of aged IONPs in WC medium after incubation for 30 d were

239

also determined (Figure S1). After dispersal in WC medium for 30 d, the IONPs

240

presented an obvious increase in hydrodynamic diameter from 78.9 ± 11.1 nm to

241

122.5 ± 9.8 nm, indicating a significant aggregation of IONPs after aging for 30 d.

242

The shift in crystalline structure of IONPs in the aging is not obvious. A certain

243

amount of dissolved iron ions (0.25 mg L-1) from the IONPs solution could be

244

detected. The TEM image of aged IONPs showed that more IONPs with smaller

12


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diameters could be observed (Figure S1), which implied that an obvious disintegrating

246

by dissolution existed in the aged IONPs in WC medium.

247

Distribution of IONPs by periphyton

248

Figure 2 shows the distribution of IONPs in the microbial cells of the periphyton

249

after 30 d of exposure. Some NPs were observed in the TEM images of microbial

250

cells exposed to IONPs including spherical cyanobacteria (Nostoc), filamentary

251

cyanobacteria (Leptolyngbya) and bacteria (Gemmatimonadetes) (Figure 2b, e and h).

252

The morphologies of these NPs observed in TEM images were approximately

253

spherical, similar to the original IONPs. The diameters of these NPs were smaller than

254

the original IONPs observed under TEM, ranging from 20 to 30 nm (Figure 2c, f and

255

i). Unlike the in vitro situation in water, where the IONPs tend to agglomerate, in vivo

256

they disperse, distributing throughout the cytoplasm of the organisms. By

257

distinguishing these NPs with organelles and other natural particles in cells from

258

morphology, diameter and distribution (Figure 2), it was possible that these NPs

259

observed in the microbial cells of periphyton were IONPs.

260 261

In addition, the distributions of the IONPs in the extracellular matrix and on cell

262

surfaces were also investigated by SEM and EDS (Figure S3). The iron content

263

covering the periphyton surfaces in the treatment of IONPs (wt% = 2.83, Table S1)

13



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was markedly higher than that in the CK (wt% = 0, Table S1), implying that IONPs

265

had been trapped on the surface of periphyton.

266

Changes in the physiological properties of periphyton

267

The cyanobacterial (Nostoc and Leptolyngbya) and bacterial (Gemmatimonadetes)

268

cells in the CK were intact (Figure 2a, d and g). In the presence of IONPs, the

269

lamellar structure of the cyanobacterial cell was destroyed and the nucleoid regions

270

disappeared (Figure S2a and b). In addition, the cyanobacterial cell membrane was

271

destroyed and cytoplasm leaked out in the presence of IONPs. The cell membranes of

272

bacteria were obviously detached from the cytoplasm and the morphology became

273

deformed (Figure S2c). In the case of periphyton exposed to IONPs (Figure 3b), the

274

extracellular space was filled more with extracellular polymeric substances (EPS)

275

than in the control (Figure 3a). The periphyton exposed to IONPs tended to disperse

276

with filamentous algae, producing more EPS compared to the CK.

277

278

T-AOC, a sensitive and reliable marker to detect changes due to oxidative stress in

279

vivo, was assayed to determine the level of oxidative damage by IONPs on periphyton

280

(Figure 3c). The T-AOC of the periphyton after 30 d exposure to IONPs were

281

significantly reduced, by 43.5% compared with the CK. To estimate the effects of

282

IONPs on periphyton photosynthesis, the chlorophyll (a, b and c) contents were

283

determined (Figure 3d). The chlorophyll (a, b and c) contents in the periphyton treated 14


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with IONPs were significantly higher than those in the CK (p < 0.05). Increases of

285

9.6%, 9.7% and 9.7% were observed, respectively.

286

Microbial community composition of periphyton

287

The Chao 1 richness of the periphyton exposed to IONPs was significantly lower

288

than that in the CK (p < 0.05) while the Shannon diversity index of the periphyton in

289

the presence of IONPs was higher than that in the CK (p < 0.05) (Table 1). These

290

results imply that the periphyton species richness decreased and the species alpha

291

diversity of periphyton increased after the exposure to IONPs.

292 293

In all, 41 classes were detected in the CK and IONPs samples including

294

Cyanobacteria, Bacilli, Gemmatimonadetes, Sphingobacteria, Alphaproteobacteria,

295

Planctomycetes and Spirochaetes with the abundances of only 14 classes higher than

296

1% (Figure 4a). Obviously, exposure to IONPs increased the abundance of

297

Cyanobacteria and Sphingobacteria while the proportion of Bacilli decreased sharply.

298

Specifically, in the CK, Bacillus, Gemmatimonadetes and Cyanobacteria were the

299

dominant species, with their abundances accounting for 41.8%, 22.0% and 10.8%,

300

respectively.

301

Sphingobacteria were the dominant species, with abundances of 30.3%, 19.1% and

302

14.0%, respectively.

In

the

IONPs

exposed

sample,

Cyanobacteria,

Bacilli and

303 15



304

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It is noteworthy that the proportion of Gemmatimonadetes in the periphyton

305

decreased

from

25%

to

2%

after

exposure

to

IONPs,

which

implies

306

Gemmatimonadetes is sensitive to IONPs. The proportions of the three most abundant

307

classes (Cyanobacteria, Bacilli and Gemmatimonadetes) decreased from 75% in the

308

CK to 50% in the IONP samples, meaning that the proportion of other classes (e.g.

309

Sphingobacteriia, Verrucomicrobia and Apirochaetes) had increased and that the

310

species richness was becoming more even.

311

Compounds released by periphyton

312

To investigate whether there was competition among components of periphyton via

313

the release of compounds in the presence of IONPs, compounds in the periphyton

314

water extracts were isolated and identified by GC-MS analysis (Figure S4). There

315

were 12 peaks in the spectrum of CK, with three identified as naturally-produced

316

compounds. Twenty-nine peaks were detected in the IONPs periphyton water extract

317

and five were identified as naturally-produced compounds (Table S2). It should be

318

noted that the compound 3-methylpentane, a common allelochemical, was detected in

319

periphyton exposed IONPs but not in the CK.

320

Effects of 3-methylpentane on periphyton composition and structure

321

To test the allelopathic effects and evaluate whether the changes in periphyton

322

composition were due to the release of allelochemicals, a common allelochemical,

323

3-methylpentane, was selected and the periphyton composition and structural changes 16


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324

were determined in the presence and absence of 3-methylpentane. Due to the

325

differences in the chlorophyll types and contents in different types of phototrophs,35

326

chlorophyll fluorescence (F0) was used for detecting the shifts in community

327

composition. The F0 values of both treatments (0.01% and 0.1%) were markedly

328

different from the CK, with a significant difference in the F0 values between the

329

0.01% treatment and the CK (p < 0.05) (Figure 4b).

330

Metabolic capacity of periphyton and contaminant removal by periphyton

331

To characterize the metabolic capacity of periphyton, carbon sources metabolic

332

versatility indices (Simpson, Shannon, McIntosh and Pielou indices) were calculated

333

based on AWCD values (Figure 4c). There were no significant differences (p > 0.05)

334

between these four versatility indices for periphyton exposed to IONPs and the CK.

335

Except for carboxylic acid and polymer, there were no significant differences in the

336

utilization of the other carbon sources and thus the metabolic capacity of the

337

periphyton between the CK and the IONPs exposed periphyton (p > 0.05) (Figure 4d).

338

This means that both the periphyton in the CK and exposed to IONPs had similar

339

carbon metabolic versatilities and capacities.

340 341

Periphyton plays an important role in removing pollutants from wastewater. Thus,

342

the changes in the COD, TN, TP and Cu2+ in the presence of periphyton exposed to

343

IONPs and the CK were studied (Figure 5). The decreases in COD, Cu2+, TP and TN 17



Page 18 of 40

344

concentrations of the wastewater in the IONPs treatment and CK over time were not

345

significantly different (p > 0.05), indicating that the ability of periphyton to affect

346

these typical contaminants in wastewater was not adversely altered by their its

347

exposure to IONPs.

348 349

DISCUSSION

350

Much progress has been made in the last decade, especially in regard to

351

investigations of bioaccumulation, fate and distribution of NPs, such as CeO2 and

352

CuO, in individuals.36-38 Studies found that the presence of NPs such as IONPs caused

353

DNA damage, increased ROS production and protein misfolding.7, 9 Although these

354

investigations of IONP nanotoxicity represent a step forward, the potential effects of

355

IONPs on microbial aggregates are still unknown. Thus, our study is valuable in that

356

it advances the investigation of the effects of IONPs from a simple community to a

357

complicated aggregation that is close to the ‘real world’ microbial environment.

358 359

Similar to the results found for the toxic effects of IONPs on the green alga

360

Chlorella vulgaris11 and the bacterium Escherichia coli,13 some NPs were observed in

361

cells of Cyanobacteria and Gemmatimonadetes in the periphyton, though these cells

362

were aggregated and wrapped with abundant EPS. Cell structure damage, such as

363

plasmolysis, cell membrane and organelle destruction, induced by the direct

364

penetration and the oxidative damage of ROS is one of the main toxicity effects of 18


Page 19 of 40


365

NPs exposure on cells.39 In the present study, cell damage such as plasmolysis and

366

cell membrane damage was observed in the microbial cells of periphyton. It is

367

possible that some IONPs penetrate into the cells of microbes in periphyton and

368

caused the damage. However, compared with the results of previous studies on cells

369

of planktonic algae (Chlorella pyrenoidosa)40 and bacterium (Escherichia coli)13, the

370

damage to cells in periphyton is much less. This was mainly due to the protection

371

from the densely packed structure and abundant EPS of periphyton, which have been

372

demonstrated to minimize cell damage in the research on the interactions of Ag NPs

373

with Escherichia coli in planktonic and biofilm forms.41

374 375

It is noteworthy that the size of IONPs observed in the cyanobacterial and bacterial

376

cells (20-30 nm) were smaller than the original IONPs (62.8 ± 9.8 nm). Previous

377

studies have measured the dissolution of metal-based NPs (Fe2O3, CuO, ZnO and

378

Ag), showing that dissolution may be a critical fate of metal-based NPs in water and it

379

is the dissolved metal ions that cause the physiological toxicities such as oxidative

380

stress and DNA damage.9, 42-44 In this study, the obvious disintegration of IONPs and

381

many IONPs with small diameter (20-30 nm) could be observed in the aging IONPs

382

in WC medium. Therefore, partially dissolving of IONPs in extracellular region might

383

be one of the reasons for the smaller NPs observed in cells. Meanwhile, Fe ions could

384

be also reduced to form NPs in the cells by the metabolic activity of microorganisms

19



Page 20 of 40

385

such as some magnetotactic bacteria,45 which might also explain the observed NPs in

386

cells with smaller size. Thus, the internalization processes and chemical forms

387

resolving of IONPs in microbial cells of periphyton needs further investigation.

388

Although the exposure to IONPs affected the physiological properties of some

389

microorganisms, such as Cyanobacteria, Gemmatimonadetes and Tintinnopsis

390

tutuformis, similar to the findings from investigations based on individuals, the

391

ecological functioning (such as the removal of contaminants including organic matter,

392

nitrogen, phosphorus and copper) was sustained. This might be mainly attributed to

393

the shifts in community composition, structure and interactions between various

394

members of the periphyton. The dominant species maintain the overall ability of the

395

community to remove the range of contaminants.

396 397

Compared to a single species community, a periphyton comprising of multiple

398

species including producers, consumers and decomposers is of strong resistance to

399

negative effects such as exposure to IONPs.18, 46 The community composition of the

400

periphyton changed on exposure to the toxic effects of the IONPs, an acclimation

401

process,47 leading to the adapted microbial community’s survival and the formation of

402

a new and stable microbial periphyton ecosystem. Such an ecosystem has the

403

capability of alleviating and transforming toxicity and can resist stress.48 As a result,

20


Page 21 of 40


404

in this study the carbon metabolic capacity and the removal of contaminants in the CK

405

and the exposure samples were similar.

406 407

It is well known that periphyton with a more densely packed structure has more

408

sites for adsorption.49, 50 EPS plays an important role in periphyton resistance to NP

409

toxicity because the EPS is able to combine with metal-NPs such as Ag-NPs and

410

TiO2-NPs.51, 52 In our study, the periphyton exposed to IONPs led to a more highly

411

compacted structure. Accordingly, IONPs were observed in the algal, bacterial and

412

protozoan intercellular and intracellular compartments as well as in the EPS, which is

413

different from studies based on single entity populations. This intracellular space

414

within the periphyton (including EPS) might play a role of “buffer” to mitigate the

415

toxic effects of the IONPs.

416 417

Periphyton produces some allelochemicals, such as indole, for intraspecific and

418

interspecific competition in the community31. Under IONPs exposure, it is possible

419

that the periphyton might have produced more allelochemicals, as seen in more peaks

420

in the GC-MS spectrum, compared to the CK. Due to the limitation of current

421

technologies,

422

allelochemical releases in periphyton communities are difficult to determine.31,

423

Thus, only a single effective compound in the periphyton water extracts was tested

the complex interactions of microorganisms

responsible for 53

21



Page 22 of 40

424

for. The presence of the 3-methylpentane brought about shifts in periphyton

425

composition and structure. Although the 3-methylpentane released by members of the

426

periphyton caused the changes in periphyton community composition and structure,

427

identifying the specific members responsible for allelochemical release needs further

428

exploration.

429 430

Under the prolonged exposure to IONPs, some sensitive species in the periphyton,

431

such as Bacilli and Gemmatimonadetes, may be inhibited, leading to a decrease in

432

Chao 1 richness. The growth of some acclimation-developed species, such as

433

Cyanobacteria may be promoted resulting in higher species evenness, which could be

434

reflected by the increased microbial diversity.54, 55 High species diversity can buffer

435

against the effects of environmental variation and maintain function despite changes

436

in the community.56, 57 This implies that the increased species diversity might protect

437

against the IONPs toxic effects. The species-sorting concept of the meta-community

438

framework assumes that microbial communities are able to acclimatize rapidly to new

439

environmental conditions by enhancing the relative importance of anti-stressed

440

species.58 Microbial communities can exhibit strong functional plasticity.59 The

441

results of this study can be extrapolated to suggest that the enhanced diversity induced

442

by IONPs exposure was beneficial to the functional acclimation of the periphyton,

443

such as the sustained contaminant (COD, TN, TP and Cu2+) removal efficiencies. 22


Page 23 of 40


444 445

In summary, although IONPs exposure affects cell structure, photosynthesis and

446

antioxidant ability, the periphyton maintains stable carbon metabolism and

447

contaminant removal abilities via the shifts of community composition, structure and

448

interactions between various members of the periphyton. Interactions between various

449

community members and the increased microbial diversity enhance the ability of

450

periphyton to adapt to chronic exposure to IONPs. By combining physiological and

451

ecological aspects to investigate periphyton responses, this study provides a valuable

452

reference for understanding the interactions between NPs and microaggregates in

453

natural environment. This study also provides a new approach to control NPs in the

454

environment by using periphyton attached to artificial substrates due to its strong

455

ability to resist nanotoxicity and abundant EPS for entrapping the NPs. However, due

456

to the complexity of the reactions among members in microbial aggregates, the

457

specific biochemical reactions involving NPs, and the mitigation and transformation

458

of the NPs in the extracellular and intercellular compartments, further research is

459

needed to reveal the complete mechanism behind the periphyton defense against

460

nanotoxicity.

461 462

ACKNOWLEDGEMENTS

23



Page 24 of 40

463

This work was supported by the State Key Basic Research Program of China

464

(2015CB158200), the National Natural Science Foundation of China (41422111) and

465

the Natural Science Foundation of Jiangsu Province, China (BK20150066).

466 467

468

DECLARATION OF INTEREST The authors declare no competing financial interests.

469 470

471 472 473

The composition of Woods Hole culture medium, characterization of aged IONPs in WC medium, details of T-AOC, high-throughput sequencing and GC-MS analyses, Figures S1 to S4 and Tables S1 and S2.

SUPPORTING INFORMATION

474 475

476 477 478

1. Mihalache, R.; Verbeek, J.; Graczyk, H.; Murashov, V.; van Broekhuizen, P. Occupational exposure limits for manufactured nanomaterials, a systematic review. Nanotoxicology 2017, 11 (1), 7-19.

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2. Wang, P.; Menzies, N. W.; Dennis, P. G.; Guo, J.; Forstner, C.; Sekine, R.; Lombi, E.; Kappen, P.; Bertsch, P. M.; Kopittke, P. M. Silver nanoparticles entering soils via the wastewater-sludge-soil pathway pose low risk to plants but elevated Cl concentrations increase Ag bioavailability. Environ. Sci. Technol. 2016, 50 (15), 8274-8281.

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3. Liu, G.; Gao, J. H.; Ai, H.; Chen, X. Y. Applications and potential toxicity of magnetic iron oxide nanoparticles. Small 2013, 9 (9-10), 1533-1545.

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4. Vargo, K. B.; Al Zaki, A.; Warden-Rothman, R.; Tsourkas, A.; Hammer, D. A. Superparamagnetic iron oxide nanoparticle micelles stabilized by recombinant oleosin for targeted magnetic resonance imaging. Small 2015, 11 (12), 1409-1413.

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667

31



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668

Figure Captions

669

Figure 1. Characterizations of IONPs. (a) TEM image of IONPs. (b) Hydrodynamic

670

diameter of IONPs dispersed in Milli-Q water and WC medium for 48 h at 5 mg L-1.

671

(c) XRD patterns of IONPs. (d) Raman spectra of IONPs.

672

Figure 2. TEM images of periphyton in the CK and the IONPs treatment. (a), (d) and

673

(g) TEM images of spherical cyanobacteria, filamentary cyanobacteria and bacteria in

674

periphyton of CK; and (b), (e), (h) in periphyton of IONPs treatment; (c), (f) and (i)

675

the magnified images of the region marked by the red boxes in (b), (e) and (h). CW:

676

cell wall; CM: cell membrane; Th: thylakoids; NR: nucleoid regions; Cy:

677

cyanophycin; SG: starch grains; LD: Lipid droplets; S: sheath; NPs: nanoparticles.

678

Figure 3. The physiological characteristics of periphyton in the CK and the IONPs

679

treatment. The SEM images of the aggregate structure of periphyton in CK (a) and

680

IONPs treatment (b). Changes in the total antioxidant capacity of periphyton (c) and

681

the chlorophyll concentrations of periphyton in the CK and the IONPs treatment (d). *

682

statistically significant (p < 0.05).

683

Figure 4. (a) Microbial community composition at phylum level of periphyton in CK

684

and IONPs treatment. (b) Changes of chlorophyll fluorescence (F0) of periphyton with

685

time in CK, 0.01% and 0.1% 3-methylpentane treatments in testing the effect of

686

allelochemical on the community composition and structure of periphyton. (c)

687

Functional diversity of periphyton in CK and IONPs treatment. (d) The metabolic 32


Page 33 of 40


688

capability of six main types of carbon sources by periphyton in CK and IONPs

689

treatment on the Biolog ECO microplate.

690

Figure 5. The decreases in the organic matter (represented by COD), copper (Cu),

691

total phosphorus (TP) and total nitrogen (TN) from wastewater in the presence of

692

periphyton in CK and IONPs treatment.

693 694

33



Page 34 of 40

695

696 697

Figure. 1

698

34


Page 35 of 40


699

700 701

Figure. 2

702 703

35



Page 36 of 40

704 705

Figure. 3

706

36


Page 37 of 40


707 708

Figure. 4

709

37



Page 38 of 40

710

711 712

Figure. 5

38


Page 39 of 40


713 714

715

Table 1. Statistical summary for pyrosequencing and microbial diversity analysis. Sample

Reads

OTU

Chao 1 richness

Shannon diversity

CK

42012

329

365 (347, 401)

3.02 (3.00, 3.04)

IONPs

41572

255

281 (266, 314)

3.44 (3.42, 3.46)

*Note: Interzones in brackets show the 95% confidence intervals.

716 717 718

39



Graphical abstract


Page 40 of 40

Responses of Periphyton to Fe2O3 Nanoparticles: A Physiological

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