Response of Aerobic Granular Sludge to the Long ... - ACS Publications

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Response of aerobic granular sludge to the long-term presence of CuO NPs in A/O/A SBRs: nitrogen and phosphorus removal, enzymatic activity and the microbial community Xiao-ying Zheng, Dan Lu, Wei Chen, Yajie Gao, Gan Zhou, Yuan Zhang, Xiang Zhou, and Mengqi Jin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02768 • Publication Date (Web): 23 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Environmental Science & Technology

Page 1 of 20 1

Response of aerobic granular sludge to the long-term presence of CuO NPs in

2

A/O/A SBRs: nitrogen and phosphorus removal, enzymatic activity and the

3

microbial community

4

Xiao-ying Zheng1,2,*, Dan Lu2, Wei Chen1,2, Ya-jie Gao2, Gan Zhou2, Yuan Zhang2, Xiang Zhou 2, Meng-Qi Jin2

5 6 7

1. Ministry of Education Key Laboratory of Integrated Regulation and Resource

8

Development on Shallow Lakes, Hohai University, Nanjing 210098, PR China

9

2. College of Environment, Hohai University, Nanjing 210098, PR China.

10 11 12 13 14 15 16 17 18 19 20 21 22

*

23

86-25-83786707

Corresponding author (e-mail): [email protected] (Xiao-ying Zheng), Tel: 86-25-83786707, Fax:

ACS Paragon Plus Environment


Page 2 of 20 24

Abstract

25

The increasing use of cupric oxide nanoparticles (CuO NPs) has raised concerns

26

about their potential environmental toxicity. Aerobic granular sludge (AGS) is a

27

special form of microbial aggregates. In this study, the removal efficiencies of

28

nitrogen and phosphorus, enzyme activities and microbial community of AGS under

29

long-term exposure to CuO NPs (at concentrations of 5, 20, 50 mg/L) in

30

aerobic/oxic/anoxic (A/O/A) sequencing batch reactors (SBRs) were investigated. The

31

results showed the chronic toxicity caused by different concentrations of CuO NPs (5,

32

20, 50 mg/L) resulted in increases in the production of ROS of 110.37%, 178.64%,

33

188.93% and in the release of lactate dehydrogenase (LDH) of 108.33%, 297.05%,

34

335.94%, respectively, compared to the control. Besides, CuO NPs decreased the

35

activities of polyphosphate kinase (PPK) and exophosphatase (PPX), leading to lower

36

phosphorus removal efficiency. However, the NH4+-N removal rates remained stable,

37

and the removal efficiencies of TN increased due to the synthesis of nitrite and nitrous

38

oxide (N2O) reductases. In addition, CuO NPs at concentrations of 0, 5, 20 mg/L

39

increased the secretion of protein (PN) to 90, 91, 105 mg/gVSS, respectively, which

40

could alleviate the toxicity of CuO NPs. High-throughput sequencing showed that

41

CuO NPs increased the abundance of nitrogen-removal bacteria and reduced the

42

abundance of phosphorus-removal bacteria, which is consistent with the results of

43

pollutant removal upon long-term exposure to CuO NPs.

44

Key words: CuO NPs; AGS; nitrogen and phosphorus; environmental impact;

45

microbial community

46 47 48 49 50


Page 2 of 29

Page 3 of 29


Page 3 of 20 51

1. Introduction

52

Because of the unique physicochemical properties of cupric oxide nanoparticles

53

(CuO NPs), they have been widely utilized in various fields, such as in gas sensors,

54

wood preservation, antimicrobial textiles, batteries, catalytic processes, marine

55

antifouling, plastics and metallic coating

56

NPs on organisms and cell tissue injury have attracted extensive attention due to their

57

nanometer size and specific properties, for instance, strong adsorption capacity and

58

high chemical activities. CuO NPs also have shown evident toxicity in bacteria 1,

59

algae 4, yeast, protozoa 5, mammalian cells 6 and Daphnia magna 7. Some studies also

60

pointed out that CuO NPs can damage organisms at the cellular, protein and gene

61

levels 8. At the same time, nanomaterials can lead to significant pulmonary disease,

62

when people inhale them.

1,3,2

. However, the negative impacts of CuO

63

Meanwhile, CuO NPs are inevitably released into industrial and municipal

64

wastewater owing to the rapid increases in their production and applications 9. The

65

nanomaterials adsorbed by activated sludge will have a detrimental effect on the

66

growth of some microorganisms in wastewater treatment plants. Therefore, it is

67

necessary to explore the potential impacts of CuO NPs on biological wastewater

68

treatment systems with different types of activated sludge, which are crucial to a

69

normal ecosystem. In general, there are two different types of activated sludge in

70

sewage treatment: the flocculent form, such as flocculent sludge, and the aggregate

71

form, for instance, biofilms and granular sludge. Different types of sludge may

72

respond differently to the presence of nanoparticles due to their varying structures and

73

microbial properties. Hou et al.10 found that the presence of CuO NPs had an impact

74

on the flocculating ability of flocculent sludge because it affected the composition of

75

extracellular polymeric substances (EPS). Moreover, CuO NPs decreased the nitrogen

76

and phosphorus removal rates and microbial enzymatic activities of flocculent sludge

77

11

78

biofilm to CuO NPs exposure is different from that of flocculent sludge because of the

. Besides, studies have shown that the acute toxicity response mechanism of a



Page 4 of 20 79

denser and stronger microbial aggregate structure of a biofilm under short-term

80

exposure (24 h) 12. Researchers elsewhere 13 also pointed out that short-term exposure

81

(8 h) to 1 and 50 mg/L CuO NPs induced negligible effects on the nitrogen-removal

82

efficiency in a sequencing batch biofilm reactor (SBBR). On the contrary, aerobic

83

granular sludge (AGS) is rich in microbial populations with different functions due to

84

the different intraparticle oxygen environments, and has a dense structure that

85

enhances the internal bacterial resistance to external toxic substances. As far as we

86

know, little information is available that enables evaluation of the long-term effects of

87

CuO NPs on the performance (nitrogen- and phosphorus-removal rates), microbial

88

enzymatic activities and microbial community of AGS. Furthermore, previous studies

89

have rarely focused on the toxicity of CuO NPs toward AGS in an anaerobic–oxic–

90

anoxic (A/O/A) sequencing batch reactor (SBR).

91

Therefore, the major purposes of this study were: (i) to display the long-term

92

(90-d) effects of CuO NPs on the nitrogen and phosphorus removal, microbial

93

activities and microbial enzymatic activities of AGS in an A/O/A SBR; (ii) to

94

investigate the integrity of the cells and oxidative stress induced by CuO NPs via

95

measurements of lactate dehydrogenase (LDH) and reactive oxygen species (ROS);

96

(iii) to evaluate the variations in microbial richness and diversity in the activated

97

sludge at different CuO NPs concentrations through high-throughput sequencing. This

98

study will provide detailed information on the impacts of chronic CuO NPs exposure

99

on the special microbial aggregate of AGS.

100

2. Materials and methods

101

2.1. Preparation of CuO NPs suspension

102

CuO NPs were obtained from Shanghai Sigma-Aldrich Trading Co., Ltd. The

103

morphology of aggregated CuO NPs was observed by scanning electron microscopy

104

(SEM), which showed CuO NPs had an average particle diameter of about 50 nm (Fig.

105

S1). The preparation of a CuO NPs stock solution was carried out using the ultrasonic


Page 4 of 29

Page 5 of 29


Page 5 of 20 14

106

method

, and the steps can be summarized as follows: 500 mg CuO NPs were put

107

into 1 L of Milli-Q water and followed by 1 h of ultrasonication (25 °C, 120 W, 40

108

kHz) to obtain a 500 mg/L stock solution. Then the CuO NPs stock solution was

109

diluted to 5, 20, 50 mg/L prior to use.

110

2.2. Set-up and operation of the granular SBR

111

The inoculated AGS for the CuO NPs exposure experiments was obtained from a

112

laboratory culture of 2 months, and it had an average particle diameter of 1.50 mm.

113

The experiments were carried out in SBRs with inner diameters of 120 mm, heights of

114

700 mm and volume exchange ratios of 60%, giving an effective volume of 7 L. The

115

quantity of mixed liquor suspended solids (MLSS) of AGS in each reactor was 3 g/L.

116

The AGS was fed with synthetic wastewater, which was composed of (mg/L): COD

117

400 (sodium acetate), NH4+-N 50 (NH4Cl), PO43−-P 10 (KH2PO4), CaCl2 10, MgSO4

118

50 and 5 mL of a concentrated trace elements solution (mg/L) containing H3BO4 1.16,

119

FeSO4·7H2O 2.78, ZnSO4·7H2O 1.25, MnSO4·H2O 1.69, CuSO4·5H2O 0.38,

120

CoCl2·6H2O 0.15 and MoO3 0.10. Solutions with CuO NPs concentrations of 5, 20,

121

50 mg/L were added to three SBRs during the feeding period, while the control

122

reactor received no CuO NPs. These reactors were operated on a 6-h cycle, consisting

123

of feeding (10 min), anaerobic phase (90 min), aerobic phase (140 min), anoxic phase

124

(116 min), settling time (2 min) and effluent discharge periods (2 min). The air was

125

supplied by a fine-bubble aerator connected to the bottom of each reactor with an

126

airflow of 2 L/min, and the dissolved oxygen (DO) concentration was stable at about

127

5.0 mg/L during the aerobic stage. The temperature was controlled at 20±3 °C, and

128

the pH was maintained between 7 and 8.

129

2.3. Determination of key enzyme activities in the AGS

130

Biological nitrogen removal processes include ammonification, nitrification and

131

denitrification, and the latter two especially have pivotal roles in the process.



Page 6 of 29

Page 6 of 20 132

Generally,

autotrophic

ammonia

oxidizing

bacteria

(AOB)

uses

ammonia

133

monooxygenase (AMO) to catalyze ammonia oxidation, followed by the oxidation of

134

nitrite to nitrate via nitrite oxidoreductase (NOR) in nitrite oxidizing bacteria (NOB).

135

Finally, denitrification is catalyzed by nitrate reductase (NR) and nitrite reductase

136

(NIR). And exophosphatase (PPX) and polyphosphate kinase (PPK), which are

137

closely related to biological phosphorus removal, can hydrolyze and synthesize

138

phosphorus during the anaerobic and anoxic phases, respectively. Therefore, AMO,

139

NOR, NR, NIR, PPX and PPK play significant roles in biological nitrogen and

140

phosphorus removal, and the methods of measurement of the activities of these

141

enzymes have been described in the literature 15,16.

142

2.4. Determination of ROS production and LDH release in the AGS

143

The production of intracellular ROS can be used to evaluate the extent of

144

oxidative stress caused by CuO NPs. The amount of extracellular LDH released can

145

be used to evaluate the integrity of the cell membranes. Hence, ROS release and LDH

146

production can measure the toxic mechanisms of CuO NPs after long-term exposure.

147

The intracellular ROS production was determined according to the literature

148

short, AGS samples were first washed with phosphate buffer solution (PBS) three

149

times; then the particles (wet weight 15 mg) were resuspended in 0.1 M phosphate

150

buffer containing 20 M dichlorodihydrofluorescein acetate at 35±1 °C in the dark for

151

30 minutes; the particles were harvested by centrifugation and suspended in 0.1 M

152

phosphate buffer and inoculated in a 96-hole plate. The fluorescein DCF generated

153

was measured after 30 min using a microplate reader (Tecan InfiniteM200,

154

Switzerland) with 485 nm excitation and 520 nm emission filters. LDH is used to

155

characterize the integrity of the cell membrane, and the LDH level was determined

156

according to previous research 18.


17

. In

Page 7 of 29


Page 7 of 20 157

2.5. Evaluation of the microbial community in the AGS

158

First, triplicate samples were collected from each SBR reactor for

159

high-throughput sequencing in order to ensure the integrity of the AGS samples.

160

Genomic DNA of the mixed AGS sample was extracted directly with the E.Z.N.A.®

161

Tissue DNA kit (Omega Bio-tek, Norcross, GA, USA) according to the

162

manufacturer's instructions. The extracted DNA samples were stored at -20 °C until

163

use. Then, partial 16S rDNA based on high-throughput sequencing was used to

164

determine the microbial diversity and composition of each AGS sample. PCR

165

amplification was based on the primers 515F (50-GTGCCAGCAGCCGCGGTAA-30)

166

and 806R (50-GGACTACCAGGGTATCTAAT-30) in the V4 region of 16S rDNA.

167

Bacterial communities were measured by Illumina high-throughput sequencing

168

technology, which was conducted by Majorbio Bio-Pharm Technology Co., Ltd.

169

(Shanghai, China).

170

Operational units (OTUs) were clustered with a 97% similarity cutoff using

171

UPARSE (version 7.1 http://drive5.com/uparse/), and chimeric sequences were

172

identified and removed using UCHIME. The sequences were systematically classified

173

using the RDP classifier and assigned to different levels. Besides, the phylogenetic

174

relationships of each 16S rRNA gene sequence were analyzed by RDP classifier

175

against the silva (SSU115) 16S rRNA database using a confidence threshold of 70%.

176

Coverage, Shannon, Chao, ACE and Simpson indexes were generated in MOTHUR

177

for each AGS sample. A Venn diagram with shared and unique OTUs was used to

178

describe the similarities and differences between AGS samples at different CuO NPs

179

concentrations.

180

2.6. Other analytical methods and statistical analysis

181

The concentration of copper ions in the solution was measured by inductively

182

coupled plasma spectrometry (ICP-AES, Spectro Arcoss Eop, Germany). The granule

183

size was measured using a particle-size analyzer (Mastersizer 2000, England). The



Page 8 of 20 184

surface of the AGS was observed by scanning electron microscopy (SEM, S4800,

185

Hitachi, Japan). EPS was extracted with the cationic resin extraction method. In

186

addition, methods used to measure the soluble protein (PN) and polysaccharide (PS)

187

contents of the EPS extraction were the Coomassie Brilliant Blue and anthracene-Cu

188

methods, respectively. The procedures for determining COD, TN, NH4+-N, NO3−-N,

189

NO2−-N, TP, MLVSS and SVI are detailed in the Standard Methods (APHA, 2005).

190

The pH and DO were measured using a pHS-25 m and YSI5000 m. All assays were

191

performed in triplicate, an analysis of variance (ANOVA) was used to test the

192

significance of the results and p < 0.05 was considered to be statistically significant.

193

3. Results and discussion

194

3.1. Reactor performance and sludge properties during CuO NPs exposure

195

Some studies have shown that the important reasons for the effects of CuO NPs

196

on biological nitrogen and phosphorus removal are nano-size effect and the release of

197

copper ions (Cu2+) from CuO NPs 19. In our experiments, the average concentrations

198

of the released Cu2+ were 0.08, 0.19, 0.27 mg/L at CuO NPs concentrations of 5, 20,

199

50 mg/L, respectively (Fig. S2). The performance of the AGS reactors and sludge

200

properties in response to the long-term exposure to CuO NPs were determined, and

201

the results are presented in Fig. 1.

202

The average removal efficiencies of COD and ammonia nitrogen in the AGS

203

reactors at CuO NPs concentrations of 5, 20 and 50 mg/L were almost the same as the

204

control reactor throughout the whole operation period (Fig. 1a,b), remaining at about

205

97% and 91%, respectively, at the end of the exposure. The average removal

206

efficiency of total nitrogen (TN) at CuO NPs concentrations of 5 mg/L was almost the

207

same as that of the control reactor (Fig. 1c). However, with increased concentrations

208

of CuO NPs (20 and 50 mg/L), the TN removal rates increased to 80.62% and 81.68%,

209

respectively, when the removal rate of the control was 76.09%. It can be easily seen

210

that the increase in Cu2+ enhances the effects of CuO NPs on TN removal. It is known


Page 8 of 29

Page 9 of 29


Page 9 of 20 211

that copper is responsible for the positive synthesis and activation of nitrite reductase

212

(CuNIR) and N2O reductase. CuNIR folds into a homotrimeric structure with two

213

distinct Cu-binding sites and can catalyze the conversion of nitrite to nitric oxide

214

(NO2- to NO), and N2O reductase is the enzyme catalyzing the final step of bacterial

215

denitrification (i.e., reducing N2O to N2) 20. Therefore, when the growth conditions of

216

the sludge lacked Cu2+, the denitrifying process was inhibited, resulting in the

217

accumulation of NO2--N and N2O 21. However, it has been found that the impacts of

218

ZnO NPs and AgO NPs on the activities and functions of the bacteria in AGS reactors

219

are different from that of CuO NPs

220

suppressor effects on the AGS. At the end of operation (on day 90), the total

221

phosphorus (TP) removal efficiencies had dropped to 74.01%, 53.19% and 69.53%

222

because of the increased CuO NPs concentrations of 5, 20 and 50 mg/L, respectively,

223

much lower than the control (77.46%) (Fig. 1d). These data indicate that high CuO

224

NPs concentrations had chronic toxic effects on biological phosphorus removal.

22 23

. And ZnO NPs and AgO NPs show some

225

Fig. 1e demonstrates that the biomass at a low CuO NPs concentration of 5 mg/L

226

was basically the same as that of the control reactor, while the reactors fed with 20

227

and 50 mg/L CuO NPs had biomasses of 3.40 and 3.39 gMLVSS/L, respectively,

228

slightly lower than the control (3.89 gMLVSS/L). These data indicate that long-term

229

CuO NPs exposure brought down the biomass production in AGS reactors, which was

230

a similar trend to the effect of chronic toxicity of CuO NPs on biological phosphorus

231

removal.

232

The sludge volume index (SVI) is an important indicator of sludge settling

233

performance. With the increased concentrations of CuO NPs (5, 20 and 50 mg/L), the

234

SVI decreased to 28.81, 25.20 and 23.85 mL/g, respectively, as compared to the

235

control (29.13 mL/g) (Fig. 1f), which indicates an improvement in AGS settling

236

ability. The settling ability of the biofilm in response to exposure to CuO NPs was

237

enhanced, which was similar to the results of the AGS

238

concentrations of CuO NPs resulted in the deterioration of the settling ability of


12

. However, high


Page 10 of 20 239

flocculent sludge 11. And the average particle size of the AGS increased with increased

240

CuO NPs concentrations (5, 20 and 50 mg/L) to 1.92, 2.21 and 2.58 mm, respectively,

241

when the average particle size of the control was 1.59 mm (Fig. 1g). This

242

phenomenon can be explained by the following: more CuO NPs accumulated in the

243

AGS with the extension of exposure time, resulting in chronic toxicity toward the

244

microbial cells and more extracellular DNA production in the EPS matrix, which in

245

turn could induce more cells to be adsorbed and accumulate on the surface of the

246

AGS.

247

In general, the AGS maintained good endurance with long-term exposure to CuO

248

NPs, and preserved its particle size, shape, sedimentation and good effluent qualities

249

(especially TN).

250

3.2. Influences of CuO NPs on the transformations of nitrogen, phosphorus

251

The removal efficiencies of NH4+-N were nearly the same in the four reactors

252

during one cycle (Fig. 2a), which almost corresponded with the NH4+-N removal

253

efficiencies during the long-term (90-d) exposure. However, there were significant

254

differences in the variations of NO2--N, NO3--N and TP at different concentrations of

255

CuO NPs during one cycle. For example, with the increased concentrations of CuO

256

NPs (5, 20 and 50 mg/L), the NO2--N and NO3--N values were 0.26 and 8.61, 0.15 and

257

5.33, 0.07 and 4.85 mg/L, respectively, at the end of the anoxic stage, when the

258

concentrations of NO2--N and NO3--N were 0.32 and 12.19 mg/L in the control (Fig.

259

2a). These data indicate that the CuO NPs could reduce the accumulation of NO2--N

260

and NO3--N. Obviously, the positive effects of CuO NPs on the removal of TN mainly

261

occurred in the denitrification stage, which is consistent with the above results that the

262

copper was beneficial for the syntheses involving CuNIR and N2O reductase that

263

catalyze the conversion of NO2- to NO and N2O to N2, respectively 20.

264

Nevertheless, the different variations in TP during one cycle were mainly due to

265

the different amounts of phosphorus released during the anaerobic phase (Fig. 2b).


Page 10 of 29

Page 11 of 29


Page 11 of 20 266

The maximum amounts of phosphorus release dropped with increased CuO NPs

267

concentrations (5, 20 and 50 mg/L) to 46.01, 33.97 and 34.77 mg/L, respectively, at

268

the end of the anaerobic stage, when the phosphorus release of the control was 46.33

269

mg/L (Fig. 2b). Thus, CuO NPs had an obvious inhibitory effect on phosphorus

270

release in the anaerobic stage. A possible reason may be that CuO NPs can inhibit the

271

conversion of the carbon source to polyhydroxyalkanoates (PHA). In turn, lower

272

amounts of carbon source were consumed for phosphorus removal, and higher

273

amounts of carbon source would be left for denitrification, which would also be one

274

of the reasons why CuO NPs promotes TN removal.

275

3.3. Effects of CuO NPs on the microbial enzymatic activities of AGS

276

The performances of biological nitrogen and phosphorus removal are closely

277

related to the activities of some enzymes, for example AMO and NOR play a

278

significant role in nitrification, and NR and NIR are two important enzymes in

279

denitrification. Besides, the activities of PPX and PPK are related directly to

280

phosphorus removal. As shown in Fig. 3a, CuO NPs had no obvious impact on the

281

activities of AMO and NOR, which could explain why NH4+-N levels did not change

282

significantly during one cycle. However, CuO NPs had a positive role in the

283

production of NR and NIR, and caused the inhibition of PPX and PPK activities.

284

When the concentrations of CuO NPs were 0, 5, 20, 50 mg/L, NR and NIR increased

285

from 0.022 and 0.139 to 0.024 and 0.147, 0.028 and 0.167, 0.03 (µmol NO2--N/mg

286

protein·min) and 0.17 (µmol NO2--N/mg protein·min) (Fig. 3a), respectively. These

287

results show that CuO NPs could improve denitrification in the AGS by promoting the

288

synthesis of NR and NIR to decrease the accumulation of NO2--N and NO3--N.

289

Conversely, with the increased concentrations of CuO NPs (0, 5, 20, 50 mg/L), the

290

PPK and PPX activities decreased from 0.214 and 0.037 to 0.201 and 0.035, 0.143

291

and

292

p-nitrophenol/(min·mg protein)) (Fig. 3b), respectively. These data indicate that CuO

0.026,

0.093

(µmol

NADPH/

(min·mg

protein))


and

0.021

(µmol


Page 12 of 20 293

NPs were inversely related to the production of the PPK and PPX. The variational

294

tendencies of PPK and PPX were in accordance with the removal rates of TP during

295

the long-term exposure to CuO NPs.

296

3.4. Assessment of the toxicity of the CuO NPs towards the AGS

297

It is well known that poorly biodegraded and toxic substances, such as

298

nanomaterials, are removed by adsorption onto the AGS, leading to the accumulation

299

of nanomaterials on the surface of the AGS 24. Compared with the control reactor, the

300

surface of the AGS with a CuO NPs concentration of 50 mg/L added accumulated a

301

large amount of CuO NPs (Fig. S3). And EDX elemental analysis showed that the

302

amounts of CuO NPs absorbed by the AGS increased from 0.10% to 0.17%, 0.20%,

303

0.24% with increasing concentrations of CuO NPs of 0, 5, 20, 50 mg/L, respectively.

304

CuO NPs on the surface of the AGS could induce oxidative damage in the

305

microorganisms, leading to a loss of integrity of the cell membrane. And lactate

306

dehydrogenase (LDH) is a type of endoenzyme that can be released to the

307

extracellular region when the cytomembrane is inflicted with serious damage.

308

Therefore, the amounts of LDH release can be used to represent these injuries to the

309

cytomembrane. Compared to 0 mg/L, the LDH increased by 108.33%, 297.05%,

310

335.94% at 5, 20, 50 mg/L CuO NPs (Fig. 4), respectively. Obviously, high

311

concentrations of CuO NPs could destroy the cytomembrane integrity of

312

microorganisms in the AGS, releasing cellular contents into the extracellular region

313

and resulting in excessive production of reactive oxygen species (ROS). Meanwhile,

314

excessive production of ROS would destroy the balance of oxidation and

315

antioxidation within the cells, leading to excessive oxidation of DNA, lipids, proteins

316

and other molecules, and affecting the metabolic functions of the organisms. As

317

shown in Fig. 4, with the increased concentrations of CuO NPs (5, 20, 50 mg/L), the

318

ROS increased to 110.37%, 178.64%, 188.93%, respectively, compared to the control

319

(100%). And the variations in LDH and ROS of the biofilm and flocculent sludge in


Page 12 of 29

Page 13 of 29


Page 13 of 20 11,12

320

response to exposure to CuO NPs were consistent with our experimental results

.

321

These data show that high CuO NPs concentrations resulted in a serious imbalance

322

between the levels of oxidation and antioxidation in the AGS.

323

Bacteria secrete EPS as a barrier against toxic substances in response to

324

long-term exposure to NPs. Meanwhile, EPS plays a significant role in maintaining

325

the structure and stability of the AGS, which is composed of proteins (PN) and

326

polysaccharides (PS). Therefore, it is particularly important to analyze the variations

327

in EPS in the AGS. In this experiment, the contents of EPS secreted by the AGS were

328

measured under different concentrations of CuO NPs on day 90 after long-term

329

exposure. The concentrations of PS remained stable at about 71 ± 2 mg/gVSS at

330

different levels of CuO NPs exposure (0, 5, 20 mg/L), while the concentrations of PN

331

increased significantly to 90, 91, 105 mg/gVSS, respectively (Fig. 5). Thus, the ratio

332

of PN to PS (PN/PS) also grew dramatically. One possible explanation for the

333

elevated PN could be the induction of heat shock-like proteins as a defense

334

mechanism against high heavy metal ion concentrations 25. However, when the CuO

335

NPs concentration was 50 mg/L, the concentrations of PS and PN decreased

336

substantially to 36 and 74 mg/gVSS, respectively. This was the reason why the

337

toxicity caused by high concentrations of CuO NPs resulted in a large number of

338

cellular deaths in the AGS, which is consistent with the substantial release of LDH

339

and the overproduction of ROS.

340

3.5. Effects of CuO NPs on the microbial community of the AGS

341

Under the different concentrations of CuO NPs (0, 5, 20, 50 mg/L), the microbial

342

community was analyzed through high-throughput sequencing, and effective

343

sequences of the AGS in the four reactors were 49863, 42075, 34826, 37083 genomes,

344

respectively. Meanwhile, the effective sequences for the 0, 5, 20, 50 mg/L

345

concentrations of CuO NPs were divided into 2579, 2257, 2139, 1824 operational

346

taxonomic units (OUT), respectively, based on the similarities of the domain values



Page 14 of 29

Page 14 of 20 347

(0.97) (Tab. 1). The Good’s coverages of the four samples were all higher than 99%

348

(Tab. 1), indicating that the microbial diversities were basically covered by the

349

obtained sequence libraries. The richness and diversity of the microorganisms at

350

different concentrations of CuO NPs could be indicated by the Chao, Ace, Shannon

351

and Simpson indexes in Tab. 1. The results show that high CuO NPs concentrations

352

reduced the diversity and richness of the microbial community, which was contrary to

353

the decrease in microbial diversity of the biofilm in response to short-term exposure to

354

CuO NPs

355

communities were compared via a Venn diagram (Fig. S4).

12

. Moreover, the similarities and differences among the microbial

356

To better characterize the differences of the functional bacteria in nitrogen and

357

phosphorus removal among the four AGS samples, the microbial communities of the

358

AGS were analyzed at the genus level. The heat-maps of the genus levels showed that

359

the prevailing genera in the four AGS samples were Zoogloea (43.28–57.90%),

360

Nitrospira (2.22–8.80%), Dechloromonas (1.02–6.00%), Defluviimonas (0.31–8.13%),

361

Gemmatimonas (2.03–2.99%) and Tepidisphaera (1.05–2.82%) (Fig. 6). It can be

362

seen that Zoogloea was present in the largest proportion (about 50%), which was

363

associated with the resistance to heavy metal ions. Therefore, with the increase in

364

CuO NPs concentrations, AGS increased the resistance of the sludge system to the

365

CuO NPs toxicity, resulting in a slight increase in the relative abundance of the

366

Zoogloea. However, the relative abundance of the Zoogloea in the biofilm in response

367

to short-term exposure to CuO NPs decreased because of its weakly antitoxic activity

368

12

369

bacteria at the genus level (Fig. 7). Nitrosomonas and Nitrospira are closely related to

370

the nitrification process, and can, respectively, oxidize ammonia and nitrite into nitrite

371

and nitrate 26. Compared with the former, the latter was greatly affected by CuO NPs,

372

and the effect was positively related to the CuO NPs concentrations (Tab. S1).

373

Thermomonas and Flavobacterium are able to convert nitrite into N2

374

relative abundances increased from 0.73% and 0.27% to 1.68% and 0.62%, 2.84%

. Besides, there were significant differences in nitrogen- and phosphorus-removal


27

, and their

Page 15 of 29


Page 15 of 20 375

and 0.82%, 3.78% and 0.94%, respectively, with the increased concentrations of CuO

376

NPs (0, 5, 20, 50mg/L). In addition, anaerobic ammonium oxidizing bacteria

377

(Gemmata) were found in the reactors. It can be seen that the addition of CuO NPs

378

increased the abundance of nitrogen-removal bacteria, which also proves that CuO

379

NPs promote nitrogen removal from another angle. The genera Acinetobacter and

380

Pseudomonas, which have phosphorus-removal ability, displayed decreasing relative

381

abundances with increases in CuO NPs concentration, which were in accordance with

382

the TP removal rates under long-term exposure. It was also found that the relative

383

abundance of Dechloromonas, which are glycogen accumulating organisms (GAO),

384

was negatively correlated with CuO NPs concentration. And some studies showed that

385

GAO can produce N2O during denitrification 28, revealing that high CuO NPs could

386

reduce the generation of N2O.

387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402



Page 16 of 20 403

Acknowledgements

404

This work was supported by the National Natural Science Foundation of China

405

(Grant No. 51678214); the Jiangsu Province Natural Science Foundation (Grant No.

406

BK20161505); the Fundamental Research Funds for the Central Universities (Grant

407

No. 2005B16414); and a Project Funded by the Priority Academic Program

408

Development of Jiangsu Higher Education Institutions (PAPD)

409 410

411 412

Supporting Information Available

Table S1 and Figures S1~S4. This information is available free of charge via the internet at http://pubs.acs.org.

413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429


Page 16 of 29

Page 17 of 29


Page 17 of 20 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470

References: (1) Heinlaan, M.; Ivask, A.; Blinova, I.; Dubourguier, H.Kahru, A. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere. 2008, 71, 1308-1316. (2) Adam, N.; Vakurov, A.; Knapen, D.Blust, R. The chronic toxicity of CuO nanoparticles and copper salt to Daphnia magna. J. Hazard Mater. 2015, 283, 416-422. (3) Mortimer, M.; Kasemets, K.Kahru, A. Toxicity of ZnO and CuO nanoparticles to ciliated protozoa Tetrahymena thermophila. Toxicology. 2010, 269, 182-189. (4) Aruoja, V.; Dubourguier, H.; Kasemets, K.Kahru, A. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci Total Environ. 2009, 407, 1461-1468. (5) Kasemets, K.; Ivask, A.; Dubourguier, H.Kahru, A. Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae. Toxicol in Vitro. 2009, 23, 1116-1122. (6) Bondarenko, O.; Juganson, K.; Ivask, A.; Kasemets, K.; Mortimer, M.Kahru, A. Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review. Arch Toxicol. 2013, 87, 1181-1200. (7) Mwaanga, P.; Carraway, E. R.van den Hurk, P. The induction of biochemical changes in Daphnia magna by CuO and ZnO nanoparticles. Aquat Toxicol. 2014, 150, 201-209. (8) Weichenthal, S.; Dufresne, A.Infante-Rivard, C. Indoor ultrafine particles and childhood asthma: exploring a potential public health concern. Indoor Air. 2007, 17, 81-91. (9) Ganesh, R.; Smeraldi, J.; Hosseini, T.; Khatib, L.; Olson, B. H.Rosso, D. Evaluation of Nanocopper Removal and Toxicity in Municipal Wastewaters. Environ Sci Technol. 2010, 44, 7808-7813. (10) Hou, J.; Miao, L.; Wang, C.; Wang, P.; Ao, Y.Lv, B. Effect of CuO nanoparticles on the production and composition of extracellular polymeric substances and physicochemical stability of activated sludge flocs. Bioresource Technol. 2015, 176, 65-70. (11) Wang, S.; Li, Z.; Gao, M.; She, Z.; Ma, B.; Guo, L.; Zheng, D.; Zhao, Y.; Jin, C.; Wang, X.Gao, F. Long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial community and enzymatic activity of activated sludge in a sequencing batch reactor. J. Environ Manage. 2017, 187, 330-339. (12) Miao, L.; Wang, C.; Hou, J.; Wang, P.; Ao, Y.; Li, Y.; Yao, Y.; Lv, B.; Yang, Y.; You, G.; Xu, Y.Gu, Q. Response of wastewater biofilm to CuO nanoparticle exposure in terms of extracellular polymeric substances and microbial community structure. Sci Total Environ. 2017, 579, 588-597. (13) Hou, J.; You, G.; Xu, Y.; Wang, C.; Wang, P.; Miao, L.; Ao, Y.; Li, Y.; LV, B.Yang, Y. Impacts of CuO nanoparticles on nitrogen removal in sequencing batch biofilm reactors after short-term and long-term exposure and the functions of natural organic matter. Environ Sci Pollut R. 2016, 23, 22116-22125. (14) Keller, A. A.; McFerran, S.; Lazareva, A.Suh, S. Global life cycle releases of engineered nanomaterials. J. Nanopart Res. 2013, 15. (15) Wang, S.; Gao, M.; She, Z.; Zheng, D.; Jin, C.; Guo, L.; Zhao, Y.; Li, Z.Wang, X. Long-term effects of ZnO nanoparticles on nitrogen and phosphorus removal, microbial activity and microbial community of a sequencing batch reactor. Bioresource Technol. 2016, 216, 428-436.



Page 18 of 20 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506

(16) Wang, S.; Gao, M.; Li, Z.; She, Z.; Wu, J.; Zheng, D.; Guo, L.; Zhao, Y.; Gao, F.Wang, X. Performance evaluation, microbial enzymatic activity and microbial community of a sequencing batch reactor under long-term exposure to cerium dioxide nanoparticles. Bioresource Technol. 2016, 220, 262-270. (17) Mu, H.Chen, Y. Long-term effect of ZnO nanoparticles on waste activated sludge anaerobic digestion. Water Res. 2011, 45, 5612-5620. (18) Han, X.; Gelein, R.; Corson, N.; Wade-Mercer, P.; Jiang, J.; Biswas, P.; Finkelstein, J. N.; Elder, A.Oberdörster, G. Validation of an LDH assay for assessing nanoparticle toxicity. Toxicology. 2011, 287, 99-104. (19) Gao, J.; Youn, S.; Hovsepyan, A.; Llaneza, V. L.; Wang, Y.; Bitton, G.Bonzongo, J. J. Dispersion and Toxicity of Selected Manufactured Nanomaterials in Natural River Water Samples: Effects of Water Chemical Composition. Environ Sci Technol. 2009, 43, 3322-3328. (20) Nojiri, M.; Koteishi, H.; Nakagami, T.; Kobayashi, K.; Inoue, T.; Yamaguchi, K.Suzuki, S. Structural basis of inter-protein electron transfer for nitrite reduction in denitrification. Nature. 2009, 462, 117-120. (21) Granger, J.Ward, B. B. Accumulation of Nitrogen Oxides in Copper-Limited Cultures of Denitrifying Bacteria. 2003, 48, 313-318. (22) He, Q.; Yuan, Z.; Zhang, J.; Zhang, S.; Zhang, W.; Zou, Z.Wang, H. Insight into the impact of ZnO nanoparticles on aerobic granular sludge under shock loading. Chemosphere. 2017, 173, 411-416. (23) Quan, X.; Cen, Y.; Lu, F.; Gu, L.Ma, J. Response of aerobic granular sludge to the long-term presence to nanosilver in sequencing batch reactors: Reactor performance, sludge property, microbial activity and community. Sci Total Environ. 2015, 506-507, 226-233. (24) Kiser, M. A.; Ryu, H.; Jang, H.; Hristovski, K.Westerhoff, P. Biosorption of nanoparticles to heterotrophic wastewater biomass. Water Res. 2010, 44, 4105-4114. (25) Bradford, A.; Handy, R. D.; Readman, J. W.; Atfield, A.Muhling, M. Impact of silver nanoparticle contamination on the genetic diversity of natural bacterial assemblages in estuarine sediments. Environ Sci Technol. 2009, 43, 4530-4536. (26) Hoang, V.; Delatolla, R.; Abujamel, T.; Mottawea, W.; Gadbois, A.; Laflamme, E.Stintzi, A. Nitrifying moving bed biofilm reactor (MBBR) biofilm and biomass response to long term exposure to 1C. Water Res. 2014, 49, 215-224. (27) Adrados, B.; Sánchez, O.; Arias, C. A.; Becares, E.; Garrido, L.; Mas, J.; Brix, H.Morató, J. Microbial communities from different types of natural wastewater treatment systems: Vertical and horizontal flow constructed wetlands and biofilters. Water Res. 2014, 55, 304-312. (28) Lemaire, R.; Meyer, R.; Taske, A.; Crocetti, G. R.; Keller, J.Yuan, Z. Identifying causes for N2O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal. J. Biotechnol. 2006, 122, 62-72.

507 508


Page 18 of 29

Page 19 of 29


Page 19 of 20 509

Tables

510

Tab. 1. Similarity-based OTUs and species richness and diversity estimates for microbial

511

communities in SBR at different CuO NPs concentrations.

CuO NPs concentrations (mg/L)

OTUsa

Coverageb

Chaoc

Acec

Shannond

Simpsond

R1 (0)

2579

0.996

23597.12

53929.31

3.93

0.11

R2 (5)

2257

0.995

19908.92

44721.42

4.21

0.08

R3 (20)

2139

0.996

16372.78

32305.17

3.76

0.12

R4 (50)

1824

0.996

15704.21

31641.58

2.91

0.28

512

a

OTUs: operational taxonomic units.

513

b

Coverage: estimates the possibility that the next read will belong to a specific OTU.

514

c

Chao/Ace diversity estimator: the total amount of OTUs estimated by infinite sampling. A higher

515

number reflects more diversity.

516

d

517

represents higher richness.

Shannon/Simpson richness index: an index to characterize species richness. A higher level

518 519 520 521 522 523 524 525 526 527 528 529 530 531



Page 20 of 20 532

For Table of Contents Only

533


Page 20 of 29

Page 21 of 29


Fig. 1. The effects of CuO NPs on the removal of (a) COD (solide) and NH4+-N (hollow), (b) TN, (c) TP, (d) MLVSS, (e) SVI, (f) granule size in the AGS reactors. 85x106mm (300 x 300 DPI)



Fig. 2a. Effects of CuO NPs on the variations of (a) NH4+-N (color) and NO2--N (black), (b) TP (color) and NO3--N (black) during one cycle after long-term (90d) exposure. Error bars represent standard deviations of triplicate measurements. 85x59mm (300 x 300 DPI)


Page 22 of 29

Page 23 of 29


Fig. 2b. Effects of CuO NPs on the variations of (a) NH4+-N (color) and NO2--N (black), (b) TP (color) and NO3--N (black) during one cycle after long-term (90d) exposure. Error bars represent standard deviations of triplicate measurements. 85x61mm (300 x 300 DPI)



Fig. 3a. Microbial enzymatic activities of AGS at different CuO NPs concentrations. (a) AMO, NOR, NR and NIR; (b) PPK and PPX. Asterisks indicate statistical differences (p

Response of Aerobic Granular Sludge to the Long ... - ACS Publications

Recommend Documents