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Size-dependent effects of ZnO nanoparticles on performance, microbial enzymatic activity and extracellular polymeric substances in sequencing batch reactor
Journal Pre-proof Size-dependent effects of ZnO nanoparticles on performance, microbial enzymatic activity and extracellular polymeric substances in sequencing batch reactor Sen Wang, Mengchun Gao, Bingrui Ma, Min Xi, Fanlong Kong PII:
S0269-7491(19)33916-8
DOI:
https://doi.org/10.1016/j.envpol.2019.113596
Reference:
ENPO 113596
To appear in:
Environmental Pollution
Received Date: 19 July 2019 Revised Date:
1 November 2019
Accepted Date: 7 November 2019
Please cite this article as: Wang, S., Gao, M., Ma, B., Xi, M., Kong, F., Size-dependent effects of ZnO nanoparticles on performance, microbial enzymatic activity and extracellular polymeric substances in sequencing batch reactor, Environmental Pollution (2019), doi: https://doi.org/10.1016/ j.envpol.2019.113596. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
SBR Reactors
SBR Performance
Overflow port
Influent
Influence
microbial activity
Effluent
SOUR, SAOR, SNOR, SNIRR, SNRR, SPUR and SPRR
Air diffuser
Magnetic followers
15 nm
50 nm
90 nm
ZnO NPs with different size
microbial enzymatic activities DHA, AMO, NOR, NR, NIR, PPX and PPK.
Decrease the COD, N and P removal
Production and components of EPS
1
Size-dependent effects of ZnO nanoparticles on performance,
2
microbial enzymatic activity and extracellular polymeric
3
substances in sequencing batch reactor
4
Sen Wanga *, Mengchun Gaob, Bingrui Mab, Min Xia, Fanlong Konga
5 6
a. College of Environmental Science and Engineering, Qingdao University, Qingdao
7
266071, China
8
b. Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean
9
University of China, Qingdao 266100, China
10 11
*Corresponding author.
12
Address: College of Environmental Science and Engineering, Qingdao University of
13
China, No. 308 Ningxia Road, Qingdao, Shandong Province 266071, China.
14
Tel: +86 532 85953244; Fax: +86 532 85953244
15
E-mail: [email protected] (Sen Wang)
16
1
17
Abstract
18
ZnO nanoparticles (NPs) have been detected in various wastewater treatment
19
plants. It is widely assumed that size has a crucial effect on the NPs toxicity. Concerns
20
have been raised over probable size-dependent toxicity of ZnO NPs to activated
21
sludge, which could eventually affect the treatment efficiencies of wastewater
22
treatment facilities. The size-dependent influences of ZnO NPs on performance,
23
microbial activities, and extracellular polymeric substances (EPS) from activated
24
sludge were examined in sequencing batch reactor (SBR) in present study. Three
25
different sizes (15, 50, and 90 nm) and five concentrations (2, 5, 10, 30, and 60 mg
26
L‒1) were trialled. The inhibitions on COD and nitrogen removal were determined by
27
the particle size, and smaller ZnO NPs (15 nm) showed higher inhibition effect than
28
those of 50 and 90 nm, whereas the ZnO NPs with size of 50 nm showed maximum
29
inhibition effect on phosphorus removal among three sizes of ZnO NPs. After
30
exposure to different sized ZnO NPs, microbial enzymatic activities and removal rates
31
of activated sludge represented the same trend, consistent with the nitrogen and
32
phosphorus
33
concentration-dependent effects on EPS contents and components were also observed.
34
Compared with the absence of ZnO NPs, 60 mg L‒1 ZnO NPs with sizes of 15, 50,
35
and 90 nm increased the EPS contents from 92.5, 92.4, and 92.0 mg g‒1 VSS to 277.5,
36
196.8, and 178.2 mg g‒1 VSS (p < 0.05), respectively. The protein and polysaccharide
37
contents increased with the decreasing particle sizes and increasing ZnO NPs
38
concentrations, and the content of protein was always higher than that of
removal
efficiency.
In
2
addition,
apparent
size-
and
39
polysaccharide.
40
Keywords: ZnO nanoparticles; Size-dependent effect; Microbial enzymatic activity;
41
Extracellular polymeric substances.
42
Capsule： ：ZnO NPs exhibited size-dependent effects on the inhibition on COD and
43
nitrogen removal, as well as the production of EPS.
44
3
45
1. Introduction
46
ZnO NPs are increasingly applied to cosmetics, paints, textiles, and plastics due to
47
their specific physicochemical properties (Ma et al., 2013a). The increasing
48
application of ZnO NPs-containing products inevitably led to their release into water
49
environment (Gottschalk et al., 2009). Recently, ZnO NPs were detected in some
50
wastewater treatment plants (WWTPs), and large proportion were adsorbed onto
51
activated sludge in conventional biotreatment system (Kaegi et al., 2011). Previous
52
toxicological studies indicated that ZnO NPs had antibacterial abilities (Lallo da Silva
53
et al., 2019; Ma et al., 2013a). Concerns have therefore been raised over the probable
54
impacts of ZnO NPs on activated sludge containing various species of
55
microorganisms, which could eventually affect the treatment efficiencies of WWTPs.
56
Recent studies found that ZnO NPs could decrease microbial population, disturb
57
microbial diversity, and lead to a reduction in treatment efficiency of biotreatment
58
systems (Cheng et al., 2019; Zhang, et al., 2017). ZnO NPs had toxic effect on
59
nitrifying
60
ammonia-oxidizing bacteria was 13.1 mg Zn L‒1 (Liu et al., 2011). Exposure to 5 mg
61
L‒1 ZnO NPs slightly restrained the removal of COD and NH4+-N in SBR (Hou et al.,
62
2013), whereas significantly changed functional bacterial community (Chen et al.,
63
2014). High concentration ZnO NPs (20 and 50 mg L‒1) were proved to have
64
considerable negative impacts on nitrifying bacteria community of SBR (Wang et al.,
65
2015).
66
bacteria,
and
the
half
maximal
inhibitory
concentration
for
As a crucial material characteristic of NPs, particle size was one of the main 4
67
factors affecting their reactivity and cytotoxicity. The smaller NPs may exhibit greater
68
toxicity than the larger ones of the same substance due to their larger surface area per
69
unit of volume, high particle number-to-mass ratio, superior surface reactivity, and
70
easier penetration into cells (Elder et al., 2009; Sharifi et al., 2012). There were a few
71
comparative studies on the size-dependent effects of different NPs, such as Ag, Au,
72
CuO, Fe2O3, Fe3O4, Si and TiO2, on various cell lines (Carlson et al., 2008; Cui, et al.,
73
2017; Karlsson et al., 2009; Kim et al., 2012; Li et al., 2018). For ZnO NPs, its
74
antibacterial activities increased with the decreasing particle sizes (Chen et al., 2019;
75
Lallo da Silva et al., 2019; Padmavathy and Vijayaraghavan, 2008; Raghupathi et al.,
76
2011). ZnO<50 nm induced more toxicity on Daphnia magna than ZnO<100 nm,
77
with the EC50 of 3.1 and 1.9 mg L‒1, respectively (Santo et al., 2014). Wei et al. (2019)
78
observed that the binding of ZnO NPs onto the hydrophilic sludge EPS increased with
79
reducing ZnO NPs diameter from 50 nm to 30 nm, while the adsorption of the
80
hydrophobic sludge EPS declined with the increasing ZnO NPs diameter from 50 nm
81
to 100 nm. However, the size-dependent influence of ZnO NPs on performance and
82
microbial activity of SBR are still not clear.
83
The aims of this paper were to (a) examine the size-dependent influence of ZnO
84
NPs on performance, microbial activity and enzymatic activity of SBR; (b) research
85
the possible impacts of ZnO NPs size on production and component of EPS; and (c)
86
explore the potential size-dependent toxicity mechanisms of ZnO NPs.
5
87
2. Materials and methods
88
2.1. ZnO NPs and wastewater
89
The ZnO NPs of 15 nm (ZnO NPs-15) was obtained from Nanjing Emperor
90
Nanomaterials Co., Ltd. (Jiangsu, China). The ZnO NPs of 50 nm (ZnO NPs-50) and
91
90 nm (ZnO NPs-90) were obtained from Hangzhou Wanjing New Material Co., Ltd.
92
(Zhejiang, China). To prepare 0.5 g L‒1 stock suspensions, 0.5 g ZnO NPs was
93
dispersed in 1.0 L Milli-Q water by sonicating at 25 °C (1h, 250 W, 40 kHz) (Zheng et
94
al., 2011). The effective diameters and size distributions of ZnO NPs were examined
95
via TEM (JEM 1200-EX, JEOL Ltd., Japan). The average particle sizes in stock
96
suspension were detected by dynamic light scattering (Zetasizer Nano ZS90, Malvern
97
Instruments, UK).
98
The influent synthetic wastewater to SBRs comprised NaAc, NH4Cl, KH2PO4,
99
and K2HPO4, which were added into tap water daily. The characteristics of
100
wastewater were as follows: COD of 419±8 mg L‒1, NH4+‒N of 26.3±1.0 mg L‒1 and
101
soluble ortho-phosphorus (SOP) of 11.3±0.9 mg L‒1. The detailed composition of the
102
wastewater was described in the literature of Wang et al. (2016).
103
2.2. SBR set-up and operation
104
Three identical SBRs were used in this experiment. The SBR (effective volume,
105
7.7 L) was operated three cycles every day at 20‒30 °C. Each cycle contained: (i) 6
106
min influent feeding; (ii) 144-min anaerobic stage; (iii)) 240-min aerobic stage; (iv)
107
78 min settling stage; and (v) 12 min decanting stage. Except for the settling and
108
decanting periods, the SBR was constantly mixed using a magnetic stirring apparatus 6
109
((IKA RH basic 2, Staufen, Germany). The dissolved oxygen concentration level in
110
anoxic and aerobic phases were maintained less than 0.5 mg L‒1and over 2 mg L‒1,
111
respectively.
112
All SBRs were carried out with the return sludge from the secondary settling pond
113
in Licunhe WWTP (Qingdao, China) as seed sludge. The mixed liquor suspended
114
solids (MLSS) of SBRs in the beginning varied from 3300 to 3500 mg L‒1.
115
2.3. Analytical methods
116
COD, SOP, NH4+-N, NO2‒-N, NO3‒-N, and MLSS were performed following the
117
standard methods (Chinese SEPA., 2002). The EPS was collected according to the
118
method of Wang et al. (2013). The main components of EPS, such as protein (PN) and
119
polysaccharide (PS), were detected with modified Lowry method (using bovine serum
120
albumin for standard) and anthrone-sulfuric acid method (using glucose for standard),
121
respectively. The specific microbial activities of activated sludge including the
122
oxygen-utilizing rate (SOUR), ammonium-oxidizing rate (SAOR), nitrate-reducing
123
rate (SNRR), nitrite-oxidizing rate (SNOR), nitrite-reducing rate (SNIRR),
124
phosphorus uptaking rate (SPUR), phosphorus releasing rate (SPRR), and some key
125
enzymes activities including dehydrogenase (DHA), nitrate reductase (NR), ammonia
126
monooxygenase (AMO), nitrite reductase (NIR), nitrite oxidoreductase (NOR),
127
polyphosphate kinase (PPK) and exopolyphosphatase (PPX) were conducted in
128
accordance with the Text S1-S3 of Supplementary data.
129
2.4. Statistical analysis
130
Statistical analysis (one-way ANOVA and LSD tests) was performed through the 7
131
IBM SPSS Statistics 19.0 (IBM, USA) to evaluate the differences before and after
132
adding ZnO NPs. The difference was statistically significant at p < 0.05.
133
3. Results and discussion
134
3.1. Characterization of ZnO NPs
135
The ZnO NPs sizes (15, 50, and 90 nm) were measured as 18.3±5.2, 42.5±12.5,
136
and 99.1±22.3 nm by observing more than 200 particles randomly (Fig. 1). From
137
TEM images, ZnO NPs-15 and ZnO NPs-50 had an irregular spherical shape, while
138
ZnO NPs-90 showed a columnar shape. Homogeneous and narrow size distributions
139
were verified for three types of ZnO nanoparticles. The median values (first and third
140
quartile values, in brackets) were 17.2 nm (15.2 and 19.9 nm), 41.5 nm (34.3 and 49.6
141
nm) and 95.2 nm (85.2 and 110.9 nm) for ZnO NPs-15, ZnO NPs-50 and ZnO NPs-90,
142
respectively. From the results of the dynamic light scattering, the average diameters of
143
different sized ZnO NPs (15, 50, and 90 nm) in the stock suspensions were 538.3,
144
640.6, and 829.1 nm, respectively.
145
3.2. Size-dependent effects of ZnO NPs on SBR performance
146
Table S1 summarizes the variations of COD, nitrogen and phosphorus removals
147
exposed to ZnO NPs with different sizes and concentrations (2-60 mg L‒1). The COD
148
removal efficiencies had no significant change at low concentration ZnO NPs (2 and 5
149
mg L‒1). The reason might be heterotrophic bacteria adapted to the low concentration
150
ZnO NPs after long-term exposure (about 112 d) (Tan et al., 2015), indicating ZnO
151
NPs with environmentally relevant concentration would not affect COD removal in
152
SBR. This result corroborated the studies that 5 mg L‒1 ZnO NPs did not significantly 8
153
impact COD removal (Hou et al., 2013). When ZnO NPs concentration varied
154
between 10 and 60 mg L‒1, the apparent size- and concentration-dependent effects on
155
COD removal were observed. Compared to the absence of ZnO NPs, the average
156
COD removal efficiencies decreased about 4.7% for ZnO NPs-15, 3.1% for ZnO
157
NPs-50, and 2.3% for ZnO NPs-90 respectively, showing that the smaller ZnO NPs
158
had greater inhibition on COD removal.
159
The average NH4+-N removal efficiencies changed slightly at 0-10 mg L‒1 ZnO
160
NPs, ranged between 99% and 100%. In contrast to this result, it was reported that the
161
NH4+-N removal efficiencies were inhibited by 1 mg L‒1 ZnO NPs (over 62 d) or 5
162
mg L‒1 ZnO NPs (11 d) (Hou et al., 2013; Puay et al., 2015). The contradiction might
163
be caused by the different experimental conditions, such as exposure time or
164
wastewater used in the experiment. The NH4+-N removal efficiencies reduced
165
gradually as ZnO NPs concentrations were over 10 mg L‒1. The average NH4+-N
166
removal efficiencies exposure to 30 mg L‒1 ZnO NPs reduced about 1.1% for ZnO
167
NPs-15, 0.7% for ZnO NPs-50, and 0.5% for ZnO NPs-90 compared with the absence
168
of
169
average removal efficiencies decreased about 2.5% for ZnO NPs-15, 1.6% for ZnO
170
NPs-50 and 1.0% for ZnO NPs-90, respectively. Similarly, Wang et al. (2015)
171
reported nitrogen removal was not influenced by 10 mg L‒1 ZnO NPs, while NH4+-N
172
removal efficiencies respectively reduced about 75.7% and 100% after exposing to 20
173
and 50 mg L‒1 ZnO NPs for 5 d. Compared with the absence of ZnO NPs, ZnO
174
NPs-50 might produce slightly more inhibitory effects on the NO2--N oxidation
ZnO NPs, respectively. As ZnO NPs concentration reached 60 mg L‒1, the
9
175
during the nitrifying process or NO2--N reduction during the denitrifying process than
176
ZnO NPs-15 and ZnO NPs-90. The effluent NO3‒-N concentrations slightly increased
177
at low ZnO NPs concentration (0-5 mg L‒1), and then increased significantly as
178
increasing ZnO NPs concentrations. Size-dependent effect became significant at 60
179
mg L‒1 ZnO NPs. The effluent NO3‒-N concentrations increased about 17.4% for ZnO
180
NPs-15, 13.8% for ZnO NPs-50 and 12.8% for ZnO NPs-90 at 60 mg L‒1 ZnO NPs
181
compared with the absence of ZnO NPs. The above inhibition effects on nitrification
182
and denitrification of ZnO NPs agreed with the conclusion of Zheng et al. (2011),
183
which showed that NH4+-N removal wasn't obviously affected by ZnO NPs (1, 10 and
184
50 mg L‒1), but total nitrogen removal efficiencies declined from 81.5% (without
185
adding ZnO NPs) to 75.6% (10 mg L‒1) and 70.8% (50 mg L‒1).
186
The SOP removal efficiencies showed a slight increase trend after exposure to 2
187
and 5 mg L‒1 ZnO NPs. Compared with the absence of ZnO NPs, ZnO NPs-15, ZnO
188
NPs-50, and ZnO NPs-90 at 5 mg L‒1 increased the SOP removal efficiencies about
189
4.8%, 7.0% and 6.2%, respectively. Tan et al. (2015) found that 10 mg L‒1 ZnO NPs
190
increased the removal of phosphate in a membrane bioreactor, which was due to the
191
reaction between ZnO NPs and phosphate resulted in the formation of zinc-phosphate
192
and other larger phosphate complex substances (Qiu and Ting, 2014). However, a
193
relatively obvious decrease was observed when ZnO NPs concentration increased
194
from 10 to 60 mg L‒1 (Table S1). The average SOP removal efficiencies exposure to
195
60 mg L‒1 ZnO NPs respectively decreased about 8.8% for ZnO NPs-15, 9.5% for
196
ZnO NPs-50, and 6.4% for ZnO NPs-90 compared with the absence of ZnO NPs. This 10
197
agreed with a report where the phosphate release, uptake and removal were inhibited
198
by 10 and 50 mg L‒1 ZnO NPs (Zheng et al., 2011).
199
3.3. Toxicity of ZnO NPs on microbial activities
200
Fig. 2 presents the changes of SOUR after exposure to ZnO NPs with different
201
sizes and concentrations. The SOUR values had no obvious changes at low
202
concentration ZnO NPs (0-5 mg L‒1). As ZnO NPs concentration was over 5 mg L‒1,
203
the SOUR value decreased gradually (p < 0.05), indicating high concentrations of
204
ZnO NPs might suppress the respiration and growth of heterotrophic microorganism.
205
Compared with the absence of ZnO NPs, the SOUR exposure to 60 mg L‒1 reduced
206
about 20.0% for ZnO NPs-15, 14.4% for ZnO NPs-50, and 12.4% for ZnO NPs-90,
207
respectively. It was apparent tshat small sized ZnO NPs had stronger inhibition on
208
SOUR than the larger ones, consistent with the inhibition effects on COD removal. It
209
was probably attributed to the fact that as the particles’ size decreased, the surface
210
area exponentially increased and a greater proportion of the particles contacted with
211
microbial cells (Scown et al., 2010; Sharifi et al., 2012). Sibag et al. (2015) found
212
small silica NPs could be stronger inhibitor to the oxygen uptake of activated sludge
213
than larger ones.
214
The SAOR did not change obviously at 0-5 mg L‒1 ZnO NPs, whereas declined as
215
the increasing ZnO NPs concentrations (Fig. 3a). ZnO NPs-15, ZnO NPs-50, and ZnO
216
NPs-90 respectively reduced the SAOR about 32.6%, 27.5%, and 24.9% (p < 0.05)
217
when ZnO NPs concentration reached 60 mg L‒1. Three different sized ZnO NPs
218
inhibited the SNOR and SNIRR in a concentration and size-dependent manner (p < 11
219
0.05) (Fig. 3b and c). ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1
220
respectively reduced the SNOR about 31.9%, 30.2%, and 29.2%, and SNIRR about
221
37.1%, 36.8%, and 33.7% compared with the absence of ZnO NPs. The SNRR
222
increased slightly at 0-5 mg L‒1 ZnO NPs, whereas decreased at higher concentration
223
ZnO NPs (30 and 60 mg L‒1) (p < 0.05) (Fig. 3d). ZnO NPs-15, ZnO NPs-50, and
224
ZnO NPs-90 at 60 mg L‒1 respectively reduced SNRR about 20.2%, 17.0%, and
225
14.6% compared with the absence of ZnO NPs (p < 0.05). It was apparent that ZnO
226
NPs-15 showed more inhibition effects on SAOR, SNOR, SNIRR and SNRR than
227
ZnO NPs-50 and ZnO NPs-90.
228
The SPUR and SPRR show obvious decrease at 10-60 mg L‒1 ZnO NPs (p < 0.05)
229
(Fig. 3e and f). ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1 respectively
230
reduced the SPUR about 30.7%, 35.1%, and 27.6%, and the SPRR about 17.5%,
231
24.6%, and 15.1% compared with the absence of ZnO NPs,. In contrast to the
232
inhibition effect on COD and nitrogen removal, the ZnO NPs-50 showed more
233
inhibition effects on phosphorus removal than ZnO NPs-15 and ZnO NPs-90.
234
ZnO NPs showed size-dependent effects on microbial activities, such as SOUR,
235
SAOR, SNRR, SNOR, SNIRR, SPUR, and SPRR, due to the fact that smaller NPs
236
possessed higher specific surface area and number of individual NPs could contact
237
with microbial cells (Kyung et al., 2009; Napierska et al., 2009; Scown et al., 2010).
238
Padmavathy and Vijayaraghavan (2008) have demonstrated that the higher toxicity of
239
smaller ZnO NPs was because that more NPs were adsorbed on the cells surface and
240
formed abundant reactive oxygen species. The Zn2+ dissolution from ZnO NPs has 12
241
been reported as size dependent (Pasquet et al, 2014b), which also contributed to the
242
antimicrobial activity (Pasquet et al, 2015). Besides, the smaller NPs can penetrate the
243
bacterial membrane more easily due to their high interfacial area (Kim et al., 2010;
244
Sirelkhatim et al., 2015). Thus, the smaller ZnO NPs possessed higher antimicrobial
245
activity than the larger ones (Lallo da Silva et al., 2019; Pasquet et al., 2014a;
246
Raghupathi et al., 2011), resulting in more obvious decrease in microbial activities.
247
3.4. Effects of ZnO NPs on microbial enzymatic activities
248
The biological removal of organic compounds, nitrogen and phosphorus is
249
associated with some enzymatic activities. DHA is often applied to assess microbial
250
activity in degrading organic compounds (Yang et al., 2002). As shown in Fig. 4,
251
DHA changed slightly at less than 5 mg L‒1 ZnO NPs; above this concentration, it
252
decreased gradually (p < 0.05). The DHA exposure to 60 mg L‒1 ZnO NPs-15, ZnO
253
NPs-50, and ZnO NPs-90 decreased about 24.4%, 17.4%, and 15.3% compared with
254
the absence of ZnO NPs, respectively. This agreed with the variation of SOUR (Fig.
255
2). The above results indicated that ZnO NPs-15 showed more significant inhibition
256
on COD removal than ZnO NPs-50 and ZnO NPs-90.
257
The activities of AMO, NIR, NR, and NOR corresponded to the SAOR, SNIRR,
258
SNRR, and SNOR, indicating the abilities of activated sludge on nitrogen
259
transformation. As ZnO NPs concentration reached 10-60 mg L‒1, three sized ZnO
260
NPs obviously inhibited AMO activity (p < 0.05) (Fig. 5a). Compared with the
261
absence of ZnO NPs, ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 with concentration
262
of 60 mg L‒1 reduced the AMO activity about 25.4%, 21.8%, and 19.3%, respectively. 13
263
The activities of NOR and NIR decreased gradually at 5-60 mg L‒1 ZnO NPs (p <
264
0.05) (Fig. 5b and c). Compared with the absence of ZnO NPs, ZnO NPs-15, ZnO
265
NPs-50, and ZnO NPs-90 at 60 mg L‒1 reduced the NOR activity about 28.9%, 20.3%,
266
and 15.9%, and the NIR activity about 30.6%, 29.2%, and 23.4%, respectively. The
267
activities of NR increased slightly at low ZnO NPs concentration (0-5 mg L‒1) (p <
268
0.05), while decreased obviously at 30 and 60 mg L‒1 (p < 0.05) (Fig. 5d). ZnO
269
NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1 reduced NR activity about
270
12.4%, 7.5%, and 6.0% compared with the absence of ZnO NPs, respectively.
271
Apparently, the smaller sized ZnO NPs (15 nm) showed more inhibition effects on
272
enzymatic activities relevant to nitrogen removal than the bigger ones (50 and 90 nm).
273
ZnO NPs with size of less than 100 nm (20 and 50 mg L‒1) was proved to significant
274
inhibit the AMO and NOR activities (Wang et al., 2015). Nevertheless, Zheng et al.
275
(2011) declared 10 and 50 mg L‒1 ZnO NPs decreased NR activity, but did not
276
apparently affect the AMO, NOR, and NIR activities after short time exposure. These
277
results demonstrated that the ZnO NPs-15 was more toxic on nitrogen removal than
278
ZnO NPs-50 and ZnO NPs-90.
279
The PPK and PPX activities, corresponding to SPUR and SPRR respectively,
280
indicated the biological phosphorus removal capability (Lee et al., 2006). Fig. 5e and f
281
show that the PPK and PPX activities had slightly changes at low ZnO NPs
282
concentration (0-10 mg L‒1), and then declined obviously as increasing ZnO NPs
283
concentrations (p < 0.05). ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1
284
reduced PPX activity by 15.7%, 22.7%, and 14.5%, and PPK activity by 9.3%, 12.8%, 14
285
and 8.6% compared with the absence of ZnO NPs, respectively. Similarly, it was
286
reported that PPK and PPX activities in activated sludge were inhibited by 10 or 50
287
mg L‒1 ZnO NPs in SBR (Zheng et al., 2011). In this study, ZnO NPs-50 inhibited
288
PPX and PPK activities more obvious than ZnO NPs-15 and ZnO NPs-90, which
289
agreed with the changes of SPUR and SPRR (Fig. 3e and f), and the effluent
290
concentrations of SOP (Table S1).
291
3.5. Effect of ZnO NPs on EPS production and components
292
EPS, as the main constituent of activated sludge, exert critical roles in protecting
293
microbial cells from the toxic substance (Sheng et al., 2010). Thus, the influences of
294
ZnO NPs on EPS were investigated. EPS production increased in a size- and
295
concentration-dependent manner after exposure to ZnO NPs (Fig. 6a). Compared with
296
the absence of ZnO NPs, ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1
297
increased the EPS contents from 92.5, 92.4, and 92.0 mg g-1 VSS to 277.5, 196.8, and
298
178.2 mg g-1 VSS (p < 0.05), respectively. EPS can trap and reduce the NPs toxicity
299
to protect the cells (Kiser et al., 2010), so the microorganisms which produced more
300
EPS exhibited better tolerability to NPs (Sheng and Liu, 2011). These microorganisms
301
became predominant in SBR after exposing to ZnO NPs, which then led to the
302
increasing EPS content.
303
EPS mainly consists of PS, PN, and a little nucleic acids and lipids. In order to
304
examine the influences of different sized ZnO NPs on EPS production, the variations
305
of PS and PN contents were analyzed. As shown in Fig. 6b-d, the PS and PN contents
306
significantly increased with the increasing concentration (5-60 mg L‒1) as well as the 15
307
decreasing size of ZnO NPs (p < 0.05). The PN was the dominant component in EPS,
308
which was always more than the PS content. The PN and PS contents exposed to ZnO
309
NPs-15 increased more significantly than those of ZnO NPs-50 and ZnO NPs-90.
310
The smaller ZnO NPs showed more toxic than the larger ones because of the
311
larger surface area per volume, so the activated sludge exposed to ZnO NPs-15
312
produced more EPS to prevent the microorganisms within activated sludge from the
313
toxic effects of the incoming ZnO NPs. The increasing EPS (especially PS) could
314
accelerate the aggregation of NPs enlarging their hydrodynamic diameter (Ma et al.,
315
2013b), which would decrease the toxicity of NPs (Choi and Hu, 2008). Pletikapić et
316
al. (2012) found that after exposure to Ag NPs, silicified marine diatoms produced
317
amounts of EPS to trap the Ag NPs, and thus prevented the NPs from contacting and
318
damaging the cells.
319
4. Conclusion
320
The ZnO NPs showed size- and concentration-dependent effects on COD, and
321
nitrogen removal, and EPS contents. The removal efficiencies of COD, nitrogen, and
322
phosphorus had no obvious changes at lower ZnO NPs, while had obvious decrease as
323
ZnO NPs concentration was above 10 mg L‒1. The ZnO NPs-15 inhibited COD and
324
nitrogen removal more obviously than those of ZnO NPs-50 and ZnO NPs-90,
325
whereas ZnO NPs-50 showed more inhibition effects on phosphorus removal. The
326
smaller ZnO NPs produced more EPS to reduce NPs toxicity than ZnO NPs-50 and
327
ZnO NPs-90.
16
328
Acknowledgements
329
This project was supported by A Project of Shandong Province Higher
330
Educational Science and Technology Program (No. J18KA108) and Shandong
331
Provincial Natural Science Foundation, China (ZR2019MD042).
332 333
Abbreviation Abbreviation
Full name
AMO DHA EPS MLSS NIR NOR NPs NR PN PPK PPX PS SAOR SBR SNIRR SNOR SNRR SOP SOUR SPRR SPUR WWTPs SOUR SPRR SPUR WWTPs
Ammonia monooxygenase Dehydrogenase Extracellular polymeric substances Mixed liquor suspended solids Nitrite reductase Nitrite oxidoreductase Nanoparticles Nitrate reductase Protein Polyphosphate kinase Exopolyphosphatase Polysaccharide Specific ammonium-oxidizing rate Sequencing batch reactor Specific nitrite-reducing rate Specific nitrite-oxidizing rate Specific nitrate-reducing rate Soluble ortho-phosphorus Specific oxygen-utilizing rate Specific phosphorus releasing rate Specific phosphorus uptaking rate Wastewater treatment plants Specific oxygen-utilizing rate Specific phosphorus releasing rate Specific phosphorus uptaking rate Wastewater treatment plants
334
17
335
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2826‒2832.
24
60
a
50
a1
Frequency (%)
40
30
20
10
0 5
10
15
20
25
30
35
40
45
50
Diameter (nm)
20
b
b1
Frequency (%)
15
10
5
0 15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
Diameter (nm)
15
c
c1
Frequency (%)
10
5
0 60
80
100
120
140
160
Diameter (nm)
Fig. 1.
60
SOUR (mg O2 g-1 MLSS h-1)
15 nm
50 nm
90 nm
50
* ** 40
* **
* **
30 20 10 0 0
2
5
10
30
ZnO NPs addition (mg L-1)
Fig. 2.
60
7 15 nm
50 nm
b
90 nm
15 nm
6
4
* 3
**
* * *
***
2
SNOR (mg N g-1 MLSS h-1)
SAOR (mg N g-1 MLSS h-1)
a
50 nm
*
5
**
* **
* **
4
90 nm
* *
** **
3 2
1 1 0
0
14
c
15 nm
50 nm
90 nm
15
*
10
**
*
**
*
8
**
*
**
**
*
6 4
SNRR (mg N g-1 MLSS h-1)
SNIRR (mg N g-1 MLSS h-1)
12
d
15 nm
50 nm
**
**
90 nm
* * **
10
* *
*
5
2 0
0 20 15 nm
50 nm
f
90 nm
15 nm
50 nm
90 nm
15
*
15
*
*
*
*
* *
10
*
* *
*
* *
*
5
SPRR (mg P g-1 MLSS h-1)
SPUR (mg P g-1 MLSS h-1)
e
*
**
*
*
10
** *
** *
5
0
0
0
2 5 10 30 ZnO NPs addition (mg L-1)
0
60
2
5
10
30
ZnO NPs addition (mg
Fig. 3.
L-1)
60
15 DHA ((mg TF mg-1 MLSS h-1)
15 nm
50 nm
90 nm
* *
10
*
* **
* **
5
0 0
2
5
10
30
ZnO NPs addition (mg L-1 )
Fig. 4.
60
0.0003 AMO (mg NO2--N mg-1 protein min-1)
15 nm
50 nm
***
0.0002
90 nm
NOR (mg NO2--N mg-1 protein min-1)
0.0016
a
b
15 nm
*
0.0012
***
***
50 nm
*
90 nm
* *
** *
*
*
0.0008
0.0001
0.0004
0.0000
0.0000
c
15 nm
50 nm
90 nm
0.0060
*
***
*
**
**
* *
0.0040
**
0.0020
NR (mg NO2--N mg-1 protein min-1)
NIR (mg NO2--N mg-1 protein min-1)
0.0005 15 nm
0.0004
*
*
50 nm
90 nm
**
*
**
*
0.0003
*
0.0002
0.0001
0.0000 0.2000
·
0.1000
PPK（µmol NADPH mg-1 protein min-1 )
0.0000 PPX (µmol pnitrophenol mg-1 protein min-1)
d
e
15 nm
*
0.0800
*
50 nm
* * *
0.0600
90 nm
** *
*
**
0.0400
0.0200
f
15 nm
0.1500
50 nm
*
*
90 nm
* *
* *
*
0.1000
0.0500
0.0000
0.0000
0
2 5 10 30 ZnO NPs addition (mg L-1 )
0
60
2
5
10
30
ZnO NPs addition (mg L-1)
Fig. 5.
60
210
a
15 nm
300
50 nm
90 nm
*
EPS 250
* 200
*
150
*
* **
**
**
*
*
**
100 50
PS and PN contents ( mg g-1 VSS)
EPS contents ( mg g-1 VSS)
350
0
PN
*
15 nm 150
*
120
* 90 60
*
*
*
*
*
*
*
30
150
c
PS
PN
*
50 nm *
90
* * *
60
*
*
*
30
PS and PN contents ( mg g-1 VSS)
PS and PN contents ( mg g-1 VSS)
PS
0
150
120
b 180
d 120
PS
PN
90 nm
* *
90
* *
*
60
*
*
*
*
30
0
0
0
2 5 10 30 ZnO concentration (mg L-1 )
0
60
2
5
10
30
ZnO concentration (mg L-1 )
Fig. 6.
60
1
Highlights
2
• Size-dependent effects of ZnO NPs on the performance of SBR were studied.
3
• ZnO NPs had size- and concentration-dependent effects on COD and N removal.
4
• The ZnO NPs of 50 nm showed maximum inhibition effect on P removal.
5
• EPS contents increased in size- and concentration-dependent manner.
Figure captions Fig. 1. Characterization of ZnO NPs with different sizes. Size frequency of ZnO NPs of 15 nm (a), 50 nm (b) and 90 nm (c) as observed from TEM images. Typical TEM image of ZnO NPs of 15 nm (a1), 50 nm (b1) and 90 nm (c1). Fig. 2. Effects of ZnO NPs with different sizes on SOUR. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 3. Effects of ZnO NPs with different sizes on microbial activity of activated sludge in SBR. (a) SAOR, (b) SNOR, (c) SNIRR, (d) SNRR, (e) SPRR and (f) SPUR. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 4. Effects of ZnO NPs with different sizes on DHA. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 5. Effects of ZnO NPs with different sizes on microbial enzymatic activities of activated sludge in SBR. (a) AMO, (b) NOR, (c) NIR, (d) NR, (e) PPX and (f) PPK. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 6. Effects of ZnO NPs with different sizes on EPS contents and PN and PS contents from activated sludge. (a) EPS contents; (b) 15 nm ZnO NPs; (c) 50 nm ZnO NPs; (d) 90 nm ZnO NPs. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0269-7491(19)33916-8
DOI:
https://doi.org/10.1016/j.envpol.2019.113596
Reference:
ENPO 113596
To appear in:
Environmental Pollution
Received Date: 19 July 2019 Revised Date:
1 November 2019
Accepted Date: 7 November 2019
Please cite this article as: Wang, S., Gao, M., Ma, B., Xi, M., Kong, F., Size-dependent effects of ZnO nanoparticles on performance, microbial enzymatic activity and extracellular polymeric substances in sequencing batch reactor, Environmental Pollution (2019), doi: https://doi.org/10.1016/ j.envpol.2019.113596. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
SBR Reactors
SBR Performance
Overflow port
Influent
Influence
microbial activity
Effluent
SOUR, SAOR, SNOR, SNIRR, SNRR, SPUR and SPRR
Air diffuser
Magnetic followers
15 nm
50 nm
90 nm
ZnO NPs with different size
microbial enzymatic activities DHA, AMO, NOR, NR, NIR, PPX and PPK.
Decrease the COD, N and P removal
Production and components of EPS
1
Size-dependent effects of ZnO nanoparticles on performance,
2
microbial enzymatic activity and extracellular polymeric
3
substances in sequencing batch reactor
4
Sen Wanga *, Mengchun Gaob, Bingrui Mab, Min Xia, Fanlong Konga
5 6
a. College of Environmental Science and Engineering, Qingdao University, Qingdao
7
266071, China
8
b. Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean
9
University of China, Qingdao 266100, China
10 11
*Corresponding author.
12
Address: College of Environmental Science and Engineering, Qingdao University of
13
China, No. 308 Ningxia Road, Qingdao, Shandong Province 266071, China.
14
Tel: +86 532 85953244; Fax: +86 532 85953244
15
E-mail: [email protected] (Sen Wang)
16
1
17
Abstract
18
ZnO nanoparticles (NPs) have been detected in various wastewater treatment
19
plants. It is widely assumed that size has a crucial effect on the NPs toxicity. Concerns
20
have been raised over probable size-dependent toxicity of ZnO NPs to activated
21
sludge, which could eventually affect the treatment efficiencies of wastewater
22
treatment facilities. The size-dependent influences of ZnO NPs on performance,
23
microbial activities, and extracellular polymeric substances (EPS) from activated
24
sludge were examined in sequencing batch reactor (SBR) in present study. Three
25
different sizes (15, 50, and 90 nm) and five concentrations (2, 5, 10, 30, and 60 mg
26
L‒1) were trialled. The inhibitions on COD and nitrogen removal were determined by
27
the particle size, and smaller ZnO NPs (15 nm) showed higher inhibition effect than
28
those of 50 and 90 nm, whereas the ZnO NPs with size of 50 nm showed maximum
29
inhibition effect on phosphorus removal among three sizes of ZnO NPs. After
30
exposure to different sized ZnO NPs, microbial enzymatic activities and removal rates
31
of activated sludge represented the same trend, consistent with the nitrogen and
32
phosphorus
33
concentration-dependent effects on EPS contents and components were also observed.
34
Compared with the absence of ZnO NPs, 60 mg L‒1 ZnO NPs with sizes of 15, 50,
35
and 90 nm increased the EPS contents from 92.5, 92.4, and 92.0 mg g‒1 VSS to 277.5,
36
196.8, and 178.2 mg g‒1 VSS (p < 0.05), respectively. The protein and polysaccharide
37
contents increased with the decreasing particle sizes and increasing ZnO NPs
38
concentrations, and the content of protein was always higher than that of
removal
efficiency.
In
2
addition,
apparent
size-
and
39
polysaccharide.
40
Keywords: ZnO nanoparticles; Size-dependent effect; Microbial enzymatic activity;
41
Extracellular polymeric substances.
42
Capsule： ：ZnO NPs exhibited size-dependent effects on the inhibition on COD and
43
nitrogen removal, as well as the production of EPS.
44
3
45
1. Introduction
46
ZnO NPs are increasingly applied to cosmetics, paints, textiles, and plastics due to
47
their specific physicochemical properties (Ma et al., 2013a). The increasing
48
application of ZnO NPs-containing products inevitably led to their release into water
49
environment (Gottschalk et al., 2009). Recently, ZnO NPs were detected in some
50
wastewater treatment plants (WWTPs), and large proportion were adsorbed onto
51
activated sludge in conventional biotreatment system (Kaegi et al., 2011). Previous
52
toxicological studies indicated that ZnO NPs had antibacterial abilities (Lallo da Silva
53
et al., 2019; Ma et al., 2013a). Concerns have therefore been raised over the probable
54
impacts of ZnO NPs on activated sludge containing various species of
55
microorganisms, which could eventually affect the treatment efficiencies of WWTPs.
56
Recent studies found that ZnO NPs could decrease microbial population, disturb
57
microbial diversity, and lead to a reduction in treatment efficiency of biotreatment
58
systems (Cheng et al., 2019; Zhang, et al., 2017). ZnO NPs had toxic effect on
59
nitrifying
60
ammonia-oxidizing bacteria was 13.1 mg Zn L‒1 (Liu et al., 2011). Exposure to 5 mg
61
L‒1 ZnO NPs slightly restrained the removal of COD and NH4+-N in SBR (Hou et al.,
62
2013), whereas significantly changed functional bacterial community (Chen et al.,
63
2014). High concentration ZnO NPs (20 and 50 mg L‒1) were proved to have
64
considerable negative impacts on nitrifying bacteria community of SBR (Wang et al.,
65
2015).
66
bacteria,
and
the
half
maximal
inhibitory
concentration
for
As a crucial material characteristic of NPs, particle size was one of the main 4
67
factors affecting their reactivity and cytotoxicity. The smaller NPs may exhibit greater
68
toxicity than the larger ones of the same substance due to their larger surface area per
69
unit of volume, high particle number-to-mass ratio, superior surface reactivity, and
70
easier penetration into cells (Elder et al., 2009; Sharifi et al., 2012). There were a few
71
comparative studies on the size-dependent effects of different NPs, such as Ag, Au,
72
CuO, Fe2O3, Fe3O4, Si and TiO2, on various cell lines (Carlson et al., 2008; Cui, et al.,
73
2017; Karlsson et al., 2009; Kim et al., 2012; Li et al., 2018). For ZnO NPs, its
74
antibacterial activities increased with the decreasing particle sizes (Chen et al., 2019;
75
Lallo da Silva et al., 2019; Padmavathy and Vijayaraghavan, 2008; Raghupathi et al.,
76
2011). ZnO<50 nm induced more toxicity on Daphnia magna than ZnO<100 nm,
77
with the EC50 of 3.1 and 1.9 mg L‒1, respectively (Santo et al., 2014). Wei et al. (2019)
78
observed that the binding of ZnO NPs onto the hydrophilic sludge EPS increased with
79
reducing ZnO NPs diameter from 50 nm to 30 nm, while the adsorption of the
80
hydrophobic sludge EPS declined with the increasing ZnO NPs diameter from 50 nm
81
to 100 nm. However, the size-dependent influence of ZnO NPs on performance and
82
microbial activity of SBR are still not clear.
83
The aims of this paper were to (a) examine the size-dependent influence of ZnO
84
NPs on performance, microbial activity and enzymatic activity of SBR; (b) research
85
the possible impacts of ZnO NPs size on production and component of EPS; and (c)
86
explore the potential size-dependent toxicity mechanisms of ZnO NPs.
5
87
2. Materials and methods
88
2.1. ZnO NPs and wastewater
89
The ZnO NPs of 15 nm (ZnO NPs-15) was obtained from Nanjing Emperor
90
Nanomaterials Co., Ltd. (Jiangsu, China). The ZnO NPs of 50 nm (ZnO NPs-50) and
91
90 nm (ZnO NPs-90) were obtained from Hangzhou Wanjing New Material Co., Ltd.
92
(Zhejiang, China). To prepare 0.5 g L‒1 stock suspensions, 0.5 g ZnO NPs was
93
dispersed in 1.0 L Milli-Q water by sonicating at 25 °C (1h, 250 W, 40 kHz) (Zheng et
94
al., 2011). The effective diameters and size distributions of ZnO NPs were examined
95
via TEM (JEM 1200-EX, JEOL Ltd., Japan). The average particle sizes in stock
96
suspension were detected by dynamic light scattering (Zetasizer Nano ZS90, Malvern
97
Instruments, UK).
98
The influent synthetic wastewater to SBRs comprised NaAc, NH4Cl, KH2PO4,
99
and K2HPO4, which were added into tap water daily. The characteristics of
100
wastewater were as follows: COD of 419±8 mg L‒1, NH4+‒N of 26.3±1.0 mg L‒1 and
101
soluble ortho-phosphorus (SOP) of 11.3±0.9 mg L‒1. The detailed composition of the
102
wastewater was described in the literature of Wang et al. (2016).
103
2.2. SBR set-up and operation
104
Three identical SBRs were used in this experiment. The SBR (effective volume,
105
7.7 L) was operated three cycles every day at 20‒30 °C. Each cycle contained: (i) 6
106
min influent feeding; (ii) 144-min anaerobic stage; (iii)) 240-min aerobic stage; (iv)
107
78 min settling stage; and (v) 12 min decanting stage. Except for the settling and
108
decanting periods, the SBR was constantly mixed using a magnetic stirring apparatus 6
109
((IKA RH basic 2, Staufen, Germany). The dissolved oxygen concentration level in
110
anoxic and aerobic phases were maintained less than 0.5 mg L‒1and over 2 mg L‒1,
111
respectively.
112
All SBRs were carried out with the return sludge from the secondary settling pond
113
in Licunhe WWTP (Qingdao, China) as seed sludge. The mixed liquor suspended
114
solids (MLSS) of SBRs in the beginning varied from 3300 to 3500 mg L‒1.
115
2.3. Analytical methods
116
COD, SOP, NH4+-N, NO2‒-N, NO3‒-N, and MLSS were performed following the
117
standard methods (Chinese SEPA., 2002). The EPS was collected according to the
118
method of Wang et al. (2013). The main components of EPS, such as protein (PN) and
119
polysaccharide (PS), were detected with modified Lowry method (using bovine serum
120
albumin for standard) and anthrone-sulfuric acid method (using glucose for standard),
121
respectively. The specific microbial activities of activated sludge including the
122
oxygen-utilizing rate (SOUR), ammonium-oxidizing rate (SAOR), nitrate-reducing
123
rate (SNRR), nitrite-oxidizing rate (SNOR), nitrite-reducing rate (SNIRR),
124
phosphorus uptaking rate (SPUR), phosphorus releasing rate (SPRR), and some key
125
enzymes activities including dehydrogenase (DHA), nitrate reductase (NR), ammonia
126
monooxygenase (AMO), nitrite reductase (NIR), nitrite oxidoreductase (NOR),
127
polyphosphate kinase (PPK) and exopolyphosphatase (PPX) were conducted in
128
accordance with the Text S1-S3 of Supplementary data.
129
2.4. Statistical analysis
130
Statistical analysis (one-way ANOVA and LSD tests) was performed through the 7
131
IBM SPSS Statistics 19.0 (IBM, USA) to evaluate the differences before and after
132
adding ZnO NPs. The difference was statistically significant at p < 0.05.
133
3. Results and discussion
134
3.1. Characterization of ZnO NPs
135
The ZnO NPs sizes (15, 50, and 90 nm) were measured as 18.3±5.2, 42.5±12.5,
136
and 99.1±22.3 nm by observing more than 200 particles randomly (Fig. 1). From
137
TEM images, ZnO NPs-15 and ZnO NPs-50 had an irregular spherical shape, while
138
ZnO NPs-90 showed a columnar shape. Homogeneous and narrow size distributions
139
were verified for three types of ZnO nanoparticles. The median values (first and third
140
quartile values, in brackets) were 17.2 nm (15.2 and 19.9 nm), 41.5 nm (34.3 and 49.6
141
nm) and 95.2 nm (85.2 and 110.9 nm) for ZnO NPs-15, ZnO NPs-50 and ZnO NPs-90,
142
respectively. From the results of the dynamic light scattering, the average diameters of
143
different sized ZnO NPs (15, 50, and 90 nm) in the stock suspensions were 538.3,
144
640.6, and 829.1 nm, respectively.
145
3.2. Size-dependent effects of ZnO NPs on SBR performance
146
Table S1 summarizes the variations of COD, nitrogen and phosphorus removals
147
exposed to ZnO NPs with different sizes and concentrations (2-60 mg L‒1). The COD
148
removal efficiencies had no significant change at low concentration ZnO NPs (2 and 5
149
mg L‒1). The reason might be heterotrophic bacteria adapted to the low concentration
150
ZnO NPs after long-term exposure (about 112 d) (Tan et al., 2015), indicating ZnO
151
NPs with environmentally relevant concentration would not affect COD removal in
152
SBR. This result corroborated the studies that 5 mg L‒1 ZnO NPs did not significantly 8
153
impact COD removal (Hou et al., 2013). When ZnO NPs concentration varied
154
between 10 and 60 mg L‒1, the apparent size- and concentration-dependent effects on
155
COD removal were observed. Compared to the absence of ZnO NPs, the average
156
COD removal efficiencies decreased about 4.7% for ZnO NPs-15, 3.1% for ZnO
157
NPs-50, and 2.3% for ZnO NPs-90 respectively, showing that the smaller ZnO NPs
158
had greater inhibition on COD removal.
159
The average NH4+-N removal efficiencies changed slightly at 0-10 mg L‒1 ZnO
160
NPs, ranged between 99% and 100%. In contrast to this result, it was reported that the
161
NH4+-N removal efficiencies were inhibited by 1 mg L‒1 ZnO NPs (over 62 d) or 5
162
mg L‒1 ZnO NPs (11 d) (Hou et al., 2013; Puay et al., 2015). The contradiction might
163
be caused by the different experimental conditions, such as exposure time or
164
wastewater used in the experiment. The NH4+-N removal efficiencies reduced
165
gradually as ZnO NPs concentrations were over 10 mg L‒1. The average NH4+-N
166
removal efficiencies exposure to 30 mg L‒1 ZnO NPs reduced about 1.1% for ZnO
167
NPs-15, 0.7% for ZnO NPs-50, and 0.5% for ZnO NPs-90 compared with the absence
168
of
169
average removal efficiencies decreased about 2.5% for ZnO NPs-15, 1.6% for ZnO
170
NPs-50 and 1.0% for ZnO NPs-90, respectively. Similarly, Wang et al. (2015)
171
reported nitrogen removal was not influenced by 10 mg L‒1 ZnO NPs, while NH4+-N
172
removal efficiencies respectively reduced about 75.7% and 100% after exposing to 20
173
and 50 mg L‒1 ZnO NPs for 5 d. Compared with the absence of ZnO NPs, ZnO
174
NPs-50 might produce slightly more inhibitory effects on the NO2--N oxidation
ZnO NPs, respectively. As ZnO NPs concentration reached 60 mg L‒1, the
9
175
during the nitrifying process or NO2--N reduction during the denitrifying process than
176
ZnO NPs-15 and ZnO NPs-90. The effluent NO3‒-N concentrations slightly increased
177
at low ZnO NPs concentration (0-5 mg L‒1), and then increased significantly as
178
increasing ZnO NPs concentrations. Size-dependent effect became significant at 60
179
mg L‒1 ZnO NPs. The effluent NO3‒-N concentrations increased about 17.4% for ZnO
180
NPs-15, 13.8% for ZnO NPs-50 and 12.8% for ZnO NPs-90 at 60 mg L‒1 ZnO NPs
181
compared with the absence of ZnO NPs. The above inhibition effects on nitrification
182
and denitrification of ZnO NPs agreed with the conclusion of Zheng et al. (2011),
183
which showed that NH4+-N removal wasn't obviously affected by ZnO NPs (1, 10 and
184
50 mg L‒1), but total nitrogen removal efficiencies declined from 81.5% (without
185
adding ZnO NPs) to 75.6% (10 mg L‒1) and 70.8% (50 mg L‒1).
186
The SOP removal efficiencies showed a slight increase trend after exposure to 2
187
and 5 mg L‒1 ZnO NPs. Compared with the absence of ZnO NPs, ZnO NPs-15, ZnO
188
NPs-50, and ZnO NPs-90 at 5 mg L‒1 increased the SOP removal efficiencies about
189
4.8%, 7.0% and 6.2%, respectively. Tan et al. (2015) found that 10 mg L‒1 ZnO NPs
190
increased the removal of phosphate in a membrane bioreactor, which was due to the
191
reaction between ZnO NPs and phosphate resulted in the formation of zinc-phosphate
192
and other larger phosphate complex substances (Qiu and Ting, 2014). However, a
193
relatively obvious decrease was observed when ZnO NPs concentration increased
194
from 10 to 60 mg L‒1 (Table S1). The average SOP removal efficiencies exposure to
195
60 mg L‒1 ZnO NPs respectively decreased about 8.8% for ZnO NPs-15, 9.5% for
196
ZnO NPs-50, and 6.4% for ZnO NPs-90 compared with the absence of ZnO NPs. This 10
197
agreed with a report where the phosphate release, uptake and removal were inhibited
198
by 10 and 50 mg L‒1 ZnO NPs (Zheng et al., 2011).
199
3.3. Toxicity of ZnO NPs on microbial activities
200
Fig. 2 presents the changes of SOUR after exposure to ZnO NPs with different
201
sizes and concentrations. The SOUR values had no obvious changes at low
202
concentration ZnO NPs (0-5 mg L‒1). As ZnO NPs concentration was over 5 mg L‒1,
203
the SOUR value decreased gradually (p < 0.05), indicating high concentrations of
204
ZnO NPs might suppress the respiration and growth of heterotrophic microorganism.
205
Compared with the absence of ZnO NPs, the SOUR exposure to 60 mg L‒1 reduced
206
about 20.0% for ZnO NPs-15, 14.4% for ZnO NPs-50, and 12.4% for ZnO NPs-90,
207
respectively. It was apparent tshat small sized ZnO NPs had stronger inhibition on
208
SOUR than the larger ones, consistent with the inhibition effects on COD removal. It
209
was probably attributed to the fact that as the particles’ size decreased, the surface
210
area exponentially increased and a greater proportion of the particles contacted with
211
microbial cells (Scown et al., 2010; Sharifi et al., 2012). Sibag et al. (2015) found
212
small silica NPs could be stronger inhibitor to the oxygen uptake of activated sludge
213
than larger ones.
214
The SAOR did not change obviously at 0-5 mg L‒1 ZnO NPs, whereas declined as
215
the increasing ZnO NPs concentrations (Fig. 3a). ZnO NPs-15, ZnO NPs-50, and ZnO
216
NPs-90 respectively reduced the SAOR about 32.6%, 27.5%, and 24.9% (p < 0.05)
217
when ZnO NPs concentration reached 60 mg L‒1. Three different sized ZnO NPs
218
inhibited the SNOR and SNIRR in a concentration and size-dependent manner (p < 11
219
0.05) (Fig. 3b and c). ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1
220
respectively reduced the SNOR about 31.9%, 30.2%, and 29.2%, and SNIRR about
221
37.1%, 36.8%, and 33.7% compared with the absence of ZnO NPs. The SNRR
222
increased slightly at 0-5 mg L‒1 ZnO NPs, whereas decreased at higher concentration
223
ZnO NPs (30 and 60 mg L‒1) (p < 0.05) (Fig. 3d). ZnO NPs-15, ZnO NPs-50, and
224
ZnO NPs-90 at 60 mg L‒1 respectively reduced SNRR about 20.2%, 17.0%, and
225
14.6% compared with the absence of ZnO NPs (p < 0.05). It was apparent that ZnO
226
NPs-15 showed more inhibition effects on SAOR, SNOR, SNIRR and SNRR than
227
ZnO NPs-50 and ZnO NPs-90.
228
The SPUR and SPRR show obvious decrease at 10-60 mg L‒1 ZnO NPs (p < 0.05)
229
(Fig. 3e and f). ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1 respectively
230
reduced the SPUR about 30.7%, 35.1%, and 27.6%, and the SPRR about 17.5%,
231
24.6%, and 15.1% compared with the absence of ZnO NPs,. In contrast to the
232
inhibition effect on COD and nitrogen removal, the ZnO NPs-50 showed more
233
inhibition effects on phosphorus removal than ZnO NPs-15 and ZnO NPs-90.
234
ZnO NPs showed size-dependent effects on microbial activities, such as SOUR,
235
SAOR, SNRR, SNOR, SNIRR, SPUR, and SPRR, due to the fact that smaller NPs
236
possessed higher specific surface area and number of individual NPs could contact
237
with microbial cells (Kyung et al., 2009; Napierska et al., 2009; Scown et al., 2010).
238
Padmavathy and Vijayaraghavan (2008) have demonstrated that the higher toxicity of
239
smaller ZnO NPs was because that more NPs were adsorbed on the cells surface and
240
formed abundant reactive oxygen species. The Zn2+ dissolution from ZnO NPs has 12
241
been reported as size dependent (Pasquet et al, 2014b), which also contributed to the
242
antimicrobial activity (Pasquet et al, 2015). Besides, the smaller NPs can penetrate the
243
bacterial membrane more easily due to their high interfacial area (Kim et al., 2010;
244
Sirelkhatim et al., 2015). Thus, the smaller ZnO NPs possessed higher antimicrobial
245
activity than the larger ones (Lallo da Silva et al., 2019; Pasquet et al., 2014a;
246
Raghupathi et al., 2011), resulting in more obvious decrease in microbial activities.
247
3.4. Effects of ZnO NPs on microbial enzymatic activities
248
The biological removal of organic compounds, nitrogen and phosphorus is
249
associated with some enzymatic activities. DHA is often applied to assess microbial
250
activity in degrading organic compounds (Yang et al., 2002). As shown in Fig. 4,
251
DHA changed slightly at less than 5 mg L‒1 ZnO NPs; above this concentration, it
252
decreased gradually (p < 0.05). The DHA exposure to 60 mg L‒1 ZnO NPs-15, ZnO
253
NPs-50, and ZnO NPs-90 decreased about 24.4%, 17.4%, and 15.3% compared with
254
the absence of ZnO NPs, respectively. This agreed with the variation of SOUR (Fig.
255
2). The above results indicated that ZnO NPs-15 showed more significant inhibition
256
on COD removal than ZnO NPs-50 and ZnO NPs-90.
257
The activities of AMO, NIR, NR, and NOR corresponded to the SAOR, SNIRR,
258
SNRR, and SNOR, indicating the abilities of activated sludge on nitrogen
259
transformation. As ZnO NPs concentration reached 10-60 mg L‒1, three sized ZnO
260
NPs obviously inhibited AMO activity (p < 0.05) (Fig. 5a). Compared with the
261
absence of ZnO NPs, ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 with concentration
262
of 60 mg L‒1 reduced the AMO activity about 25.4%, 21.8%, and 19.3%, respectively. 13
263
The activities of NOR and NIR decreased gradually at 5-60 mg L‒1 ZnO NPs (p <
264
0.05) (Fig. 5b and c). Compared with the absence of ZnO NPs, ZnO NPs-15, ZnO
265
NPs-50, and ZnO NPs-90 at 60 mg L‒1 reduced the NOR activity about 28.9%, 20.3%,
266
and 15.9%, and the NIR activity about 30.6%, 29.2%, and 23.4%, respectively. The
267
activities of NR increased slightly at low ZnO NPs concentration (0-5 mg L‒1) (p <
268
0.05), while decreased obviously at 30 and 60 mg L‒1 (p < 0.05) (Fig. 5d). ZnO
269
NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1 reduced NR activity about
270
12.4%, 7.5%, and 6.0% compared with the absence of ZnO NPs, respectively.
271
Apparently, the smaller sized ZnO NPs (15 nm) showed more inhibition effects on
272
enzymatic activities relevant to nitrogen removal than the bigger ones (50 and 90 nm).
273
ZnO NPs with size of less than 100 nm (20 and 50 mg L‒1) was proved to significant
274
inhibit the AMO and NOR activities (Wang et al., 2015). Nevertheless, Zheng et al.
275
(2011) declared 10 and 50 mg L‒1 ZnO NPs decreased NR activity, but did not
276
apparently affect the AMO, NOR, and NIR activities after short time exposure. These
277
results demonstrated that the ZnO NPs-15 was more toxic on nitrogen removal than
278
ZnO NPs-50 and ZnO NPs-90.
279
The PPK and PPX activities, corresponding to SPUR and SPRR respectively,
280
indicated the biological phosphorus removal capability (Lee et al., 2006). Fig. 5e and f
281
show that the PPK and PPX activities had slightly changes at low ZnO NPs
282
concentration (0-10 mg L‒1), and then declined obviously as increasing ZnO NPs
283
concentrations (p < 0.05). ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1
284
reduced PPX activity by 15.7%, 22.7%, and 14.5%, and PPK activity by 9.3%, 12.8%, 14
285
and 8.6% compared with the absence of ZnO NPs, respectively. Similarly, it was
286
reported that PPK and PPX activities in activated sludge were inhibited by 10 or 50
287
mg L‒1 ZnO NPs in SBR (Zheng et al., 2011). In this study, ZnO NPs-50 inhibited
288
PPX and PPK activities more obvious than ZnO NPs-15 and ZnO NPs-90, which
289
agreed with the changes of SPUR and SPRR (Fig. 3e and f), and the effluent
290
concentrations of SOP (Table S1).
291
3.5. Effect of ZnO NPs on EPS production and components
292
EPS, as the main constituent of activated sludge, exert critical roles in protecting
293
microbial cells from the toxic substance (Sheng et al., 2010). Thus, the influences of
294
ZnO NPs on EPS were investigated. EPS production increased in a size- and
295
concentration-dependent manner after exposure to ZnO NPs (Fig. 6a). Compared with
296
the absence of ZnO NPs, ZnO NPs-15, ZnO NPs-50, and ZnO NPs-90 at 60 mg L‒1
297
increased the EPS contents from 92.5, 92.4, and 92.0 mg g-1 VSS to 277.5, 196.8, and
298
178.2 mg g-1 VSS (p < 0.05), respectively. EPS can trap and reduce the NPs toxicity
299
to protect the cells (Kiser et al., 2010), so the microorganisms which produced more
300
EPS exhibited better tolerability to NPs (Sheng and Liu, 2011). These microorganisms
301
became predominant in SBR after exposing to ZnO NPs, which then led to the
302
increasing EPS content.
303
EPS mainly consists of PS, PN, and a little nucleic acids and lipids. In order to
304
examine the influences of different sized ZnO NPs on EPS production, the variations
305
of PS and PN contents were analyzed. As shown in Fig. 6b-d, the PS and PN contents
306
significantly increased with the increasing concentration (5-60 mg L‒1) as well as the 15
307
decreasing size of ZnO NPs (p < 0.05). The PN was the dominant component in EPS,
308
which was always more than the PS content. The PN and PS contents exposed to ZnO
309
NPs-15 increased more significantly than those of ZnO NPs-50 and ZnO NPs-90.
310
The smaller ZnO NPs showed more toxic than the larger ones because of the
311
larger surface area per volume, so the activated sludge exposed to ZnO NPs-15
312
produced more EPS to prevent the microorganisms within activated sludge from the
313
toxic effects of the incoming ZnO NPs. The increasing EPS (especially PS) could
314
accelerate the aggregation of NPs enlarging their hydrodynamic diameter (Ma et al.,
315
2013b), which would decrease the toxicity of NPs (Choi and Hu, 2008). Pletikapić et
316
al. (2012) found that after exposure to Ag NPs, silicified marine diatoms produced
317
amounts of EPS to trap the Ag NPs, and thus prevented the NPs from contacting and
318
damaging the cells.
319
4. Conclusion
320
The ZnO NPs showed size- and concentration-dependent effects on COD, and
321
nitrogen removal, and EPS contents. The removal efficiencies of COD, nitrogen, and
322
phosphorus had no obvious changes at lower ZnO NPs, while had obvious decrease as
323
ZnO NPs concentration was above 10 mg L‒1. The ZnO NPs-15 inhibited COD and
324
nitrogen removal more obviously than those of ZnO NPs-50 and ZnO NPs-90,
325
whereas ZnO NPs-50 showed more inhibition effects on phosphorus removal. The
326
smaller ZnO NPs produced more EPS to reduce NPs toxicity than ZnO NPs-50 and
327
ZnO NPs-90.
16
328
Acknowledgements
329
This project was supported by A Project of Shandong Province Higher
330
Educational Science and Technology Program (No. J18KA108) and Shandong
331
Provincial Natural Science Foundation, China (ZR2019MD042).
332 333
Abbreviation Abbreviation
Full name
AMO DHA EPS MLSS NIR NOR NPs NR PN PPK PPX PS SAOR SBR SNIRR SNOR SNRR SOP SOUR SPRR SPUR WWTPs SOUR SPRR SPUR WWTPs
Ammonia monooxygenase Dehydrogenase Extracellular polymeric substances Mixed liquor suspended solids Nitrite reductase Nitrite oxidoreductase Nanoparticles Nitrate reductase Protein Polyphosphate kinase Exopolyphosphatase Polysaccharide Specific ammonium-oxidizing rate Sequencing batch reactor Specific nitrite-reducing rate Specific nitrite-oxidizing rate Specific nitrate-reducing rate Soluble ortho-phosphorus Specific oxygen-utilizing rate Specific phosphorus releasing rate Specific phosphorus uptaking rate Wastewater treatment plants Specific oxygen-utilizing rate Specific phosphorus releasing rate Specific phosphorus uptaking rate Wastewater treatment plants
334
17
335
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2826‒2832.
24
60
a
50
a1
Frequency (%)
40
30
20
10
0 5
10
15
20
25
30
35
40
45
50
Diameter (nm)
20
b
b1
Frequency (%)
15
10
5
0 15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
Diameter (nm)
15
c
c1
Frequency (%)
10
5
0 60
80
100
120
140
160
Diameter (nm)
Fig. 1.
60
SOUR (mg O2 g-1 MLSS h-1)
15 nm
50 nm
90 nm
50
* ** 40
* **
* **
30 20 10 0 0
2
5
10
30
ZnO NPs addition (mg L-1)
Fig. 2.
60
7 15 nm
50 nm
b
90 nm
15 nm
6
4
* 3
**
* * *
***
2
SNOR (mg N g-1 MLSS h-1)
SAOR (mg N g-1 MLSS h-1)
a
50 nm
*
5
**
* **
* **
4
90 nm
* *
** **
3 2
1 1 0
0
14
c
15 nm
50 nm
90 nm
15
*
10
**
*
**
*
8
**
*
**
**
*
6 4
SNRR (mg N g-1 MLSS h-1)
SNIRR (mg N g-1 MLSS h-1)
12
d
15 nm
50 nm
**
**
90 nm
* * **
10
* *
*
5
2 0
0 20 15 nm
50 nm
f
90 nm
15 nm
50 nm
90 nm
15
*
15
*
*
*
*
* *
10
*
* *
*
* *
*
5
SPRR (mg P g-1 MLSS h-1)
SPUR (mg P g-1 MLSS h-1)
e
*
**
*
*
10
** *
** *
5
0
0
0
2 5 10 30 ZnO NPs addition (mg L-1)
0
60
2
5
10
30
ZnO NPs addition (mg
Fig. 3.
L-1)
60
15 DHA ((mg TF mg-1 MLSS h-1)
15 nm
50 nm
90 nm
* *
10
*
* **
* **
5
0 0
2
5
10
30
ZnO NPs addition (mg L-1 )
Fig. 4.
60
0.0003 AMO (mg NO2--N mg-1 protein min-1)
15 nm
50 nm
***
0.0002
90 nm
NOR (mg NO2--N mg-1 protein min-1)
0.0016
a
b
15 nm
*
0.0012
***
***
50 nm
*
90 nm
* *
** *
*
*
0.0008
0.0001
0.0004
0.0000
0.0000
c
15 nm
50 nm
90 nm
0.0060
*
***
*
**
**
* *
0.0040
**
0.0020
NR (mg NO2--N mg-1 protein min-1)
NIR (mg NO2--N mg-1 protein min-1)
0.0005 15 nm
0.0004
*
*
50 nm
90 nm
**
*
**
*
0.0003
*
0.0002
0.0001
0.0000 0.2000
·
0.1000
PPK（µmol NADPH mg-1 protein min-1 )
0.0000 PPX (µmol pnitrophenol mg-1 protein min-1)
d
e
15 nm
*
0.0800
*
50 nm
* * *
0.0600
90 nm
** *
*
**
0.0400
0.0200
f
15 nm
0.1500
50 nm
*
*
90 nm
* *
* *
*
0.1000
0.0500
0.0000
0.0000
0
2 5 10 30 ZnO NPs addition (mg L-1 )
0
60
2
5
10
30
ZnO NPs addition (mg L-1)
Fig. 5.
60
210
a
15 nm
300
50 nm
90 nm
*
EPS 250
* 200
*
150
*
* **
**
**
*
*
**
100 50
PS and PN contents ( mg g-1 VSS)
EPS contents ( mg g-1 VSS)
350
0
PN
*
15 nm 150
*
120
* 90 60
*
*
*
*
*
*
*
30
150
c
PS
PN
*
50 nm *
90
* * *
60
*
*
*
30
PS and PN contents ( mg g-1 VSS)
PS and PN contents ( mg g-1 VSS)
PS
0
150
120
b 180
d 120
PS
PN
90 nm
* *
90
* *
*
60
*
*
*
*
30
0
0
0
2 5 10 30 ZnO concentration (mg L-1 )
0
60
2
5
10
30
ZnO concentration (mg L-1 )
Fig. 6.
60
1
Highlights
2
• Size-dependent effects of ZnO NPs on the performance of SBR were studied.
3
• ZnO NPs had size- and concentration-dependent effects on COD and N removal.
4
• The ZnO NPs of 50 nm showed maximum inhibition effect on P removal.
5
• EPS contents increased in size- and concentration-dependent manner.
Figure captions Fig. 1. Characterization of ZnO NPs with different sizes. Size frequency of ZnO NPs of 15 nm (a), 50 nm (b) and 90 nm (c) as observed from TEM images. Typical TEM image of ZnO NPs of 15 nm (a1), 50 nm (b1) and 90 nm (c1). Fig. 2. Effects of ZnO NPs with different sizes on SOUR. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 3. Effects of ZnO NPs with different sizes on microbial activity of activated sludge in SBR. (a) SAOR, (b) SNOR, (c) SNIRR, (d) SNRR, (e) SPRR and (f) SPUR. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 4. Effects of ZnO NPs with different sizes on DHA. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 5. Effects of ZnO NPs with different sizes on microbial enzymatic activities of activated sludge in SBR. (a) AMO, (b) NOR, (c) NIR, (d) NR, (e) PPX and (f) PPK. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements. Fig. 6. Effects of ZnO NPs with different sizes on EPS contents and PN and PS contents from activated sludge. (a) EPS contents; (b) 15 nm ZnO NPs; (c) 50 nm ZnO NPs; (d) 90 nm ZnO NPs. Asterisks indicate statistical differences (p < 0.05) from the microbial activities of 0 mg L-1 ZnO NPs. Error bars represent standard deviations of triplicate measurements.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: