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38
Fig. 4 (A)
UPGMA clustering tree based on Weighted unifrac distance (distribution of
phyla) and (B)
Percentage of representative abundance of genera in activated sludge.
A, B, C, D, and E represent the control, TiO
2
-A, aTiO
2
-A TiO
2
-R, and aTiO
2
-R
respectively.
(A)
(B)
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(T
iO
2
-A)
(C
o
ntr
o
l)
(a
T
iO
2
-A)
(T
iO
2
-R)
(a
T
iO
2
-R)
Merged-images
Live cell-images
Dead cell-images
(a)
(a1)
(a2)
(b)
(c)
(d)
(e)
(b1)
(c1)
(d1)
(e1)
(b2)
(c2)
(d2)
(e2)
(A)
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Fig. 5
(A) Confocal laser scanning microscope (CLSM) images of bacterial activity in
sludge
stained with doubling SYTO® 9 dye/Propidium iodide after exposure
experiment (50 mg/L). (B) Ultra-structural changes (TEM images) of bacterial cells
after 50 mg/L TiO
2
-NPs interaction with activated sludge with the EDS images of
their corresponding sludge. Green arrows, red arrows and purple arrows refer to living
cells, dead or erosive cells, TiO
2
-NPs or its aggregates respectively.
(a)
(b)
(c)
(d)
(e)
Ti
Ti
Ti
Ti
(b1)
(c1)
(d1)
(e1)
(B)
(Control)
(TiO
2
-A)
(aTiO
2
-A)
(TiO
2
-R)
(aTiO
2
-R)
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Fig. 6
(A) The LDH release in five different exposure groups were used to assess the
leakage of cytoplasmic membrane. (B) The crystalline-dependent extracellular and
intracellular titanium concentration in activate sludge. Error bars represent standard
deviations of triplicate measurements. Different letters indicate that there are
statistical differences between groups (
p
< 0.05).
(A)
(B)
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Table 1
Physicochemical parameters of TiO
2
-NPs used in this study.
TiO
2
-NP
samples
Crystalline phase (%)
a
Crystallite
size (nm)
a
Average particle
size (nm)
b
Surface area
(m
2
g
-1
)
c
Eg (eV)
d
Assay (trace
metal basis)
b
Milli-Q water (0 hour after ultrasound )
e
Hydrodynamic
diameter (nm)
Zeta potential
(mV)
TiO
2
-A
100% anatase
14.1
10.0
87.0
3.20
99.8%
42±16
16.2±5.2
aTiO
2
-A
97.8% anatase; 2.2%
others
14.1
25.8
71.2
3.24
97.1%
56±22
-25.1±7.5
TiO
2
-R
100% rutile
29.8
40.0
24.0
3.02
99.8%
89±35
22.4±6.8
aTiO
2
-R
98.5% rutile; 1.5%
others
29.8
54.3
23.1
2.97
98.2%
101±57
-28.2±10.2
Abbreviations: Eg, Band gap.
Others
represent the titanium metal (Fe, Al, Cu, etc.) oxides and halite (NaCl etc.), namely the inclusions.
a
:
were
confirmed
by XRD patterns.
b
:
were provided by the sample nameplate and ICP-MS and total organic carbon (
TOC)
analysis (He et al. 2016).
c
:
were obtained from BET.
d
:
were calculated from UV-Vis diffuse reflectance spectroscopy.
e
:
were determined via Dynamic Light Scattering (DLS) using Malvern Zetasizer Nano ZS90 equipment (10 mg/L NPs).
Reference
：
He, X., Sanders, S., Aker, W.G., Lin, Y., Douglas, J. and Hwang, H.-m. (2016) Assessing the effects of surface-bound humic acid on the phototoxicity of anatase and rutile
TiO2 nanoparticles in vitro. Journal Of Environmental Sciences-China 42, 50-60.
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Table 2
Effects of TiO
2
-NPs on the transformation of
PHA/glycogen and key enzyme activities during anaerobic and aerobic stages.
The data of
PHA and Glycogen (mg/g biomass) and PPX (mg P-Nitrophenol/g protein min) and PPK (nmol NADP/mg protein min) are given as the
averages and standard deviations of triplicate measurements.
a,b
0.1 mg/L
50 mg/L
Control
TiO
2
-A
aTiO
2
-A
TiO
2
-R
aTiO
2
-R
Control
TiO
2
-A
aTiO
2
-A
TiO
2
-R
aTiO
2
-R
Anaerobic
stage
PHA (synthesis)
25.2±1.3 24.8±2.1 25.6±1.6
25.4±1.6 24.6±2.4
25.2±1.3
16.8±1.5
b
24.5±2.1
17.9±0.5
b
22.4±1.7
b
Glycogen (degradation)
10.4±0.8 11.1±0.5 10.2±0.4
9.9±0.6
9.8±0.7
10.4±0.8
4.8±0.4
b
7.1±0.3
b
5.2±0.7
b
6.8±0.9
b
PPX
15.4±1.4 16.1±2.2 15.7±0.5
15.3±0.6 15.9±2.4
15.4±1.4
6.5±0.8
b
10.2±1.0
b
7.2±0.3
b
9.2±0.4
b
PPK
210±34
215±26
214±30
209±21
212±23
210±34
166±27
b
198±12
b
161±41
b
182±28
b
Aerobic
stage
PHA (degradation)
24.8±1.7 23.5±2.7 24.1±2.3
24.5±1.3 24.0±1.5
24.8±1.7
15.4±0.8
b
20.1±2.4
b
16.5±1.7
b
19.5±2.7
b
Glycogen (synthesis)
11.5±0.3 10.8±0.8 11.2±0.2
10.6±0.7 10.9±0.4
11.5±0.3
5.2±0.7
b
8.6±0.9
b
5.6±0.3
b
8.2±0.8
b
PPX
8.2±0.8
8.3±0.6
9.0±0.7
8.8±0.3
7.9±0.5
8.2±0.8
2.5±0.7
b
5.9±0.6
b
2.8±0.3
b
5.1±0.7
b
PPK
214±24
216±18
211±12
215±16
216±25
214±24
108±14
b
148±25
b
115±31
b
152±23
b
a
The transformation of polymers during the anaerobic or aerobic period was calculated as the difference in values between anaerobic or aerobic
end (beginning) and anaerobic or aerobic beginning (end).
b
The data reported are statistical differences (p<0.05) from the control.
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Table 3
Phosphorus fractionation and their contents in sludge cells and EPS at the end of anaerobic (2 h) and aerobic stage (6 h)
during the last
cycle of SBRs. Data are the averages and their standard deviations, determined from triplicate measurements, in units of mg P/g biomass.
a
imply the statistical difference (
p
< 0.05) compared to the control.
Sampling
times
Samples
(50 mg/L)
PCA extracts
NaOH extracts
CER extracts
TP
sludge
complex P
orthoP
complex P
orthoP
complex P
orthoP
2 h
Control
43.2±3.3
5.2±0.6
11.2±0.2
4.2±0.4
3.8±0.7
1.2±0.5
69.5±4.5
TiO
2
-A
39.5±4.1
a
3.2±0.3
a
8.1±0.7
a
3.5±0.3
a
5.2±0.4
a
9.7±0.6
a
73.4±5.3
a
aTiO
2
-A
42.5±5.7
4.8±0.4
10.5±0.8
4.0±0.1
4.1±0.9
3.2±0.3
a
69.9±6.5
TiO
2
-R
40.5±2.5
a
3.7±0.5
a
8.7±0.4
a
3.8±0.6
a
4.5±0.7
a
7.9±0.8
a
72.1±2.8
a
aTiO
2
-R
42.3±6.2
4.5±0.7
10.1±0.2
4.1±0.4
4.2±0.8
3.8±0.3
a
70.2±3.4
6 h
Control
54.2±2.6
6.3±0.7
21.2±1.1
5.4±0.5
4.8±0.1
2.1±0.4
101.3±7.1
TiO
2
-A
47.5±3.5
a
5.8±0.8
a
16.2±1.7
a
4.7±0.2
a
6.2±0.6
a
11.1±0.1
a
94.5±6.5
a
aTiO
2
-A
51.7±1.1
a
6.1±0.5
a
19.5±2.1
a
5.2±0.8
a
5.5±0.4
a
4.9±0.2
a
98.8±4.2
a
TiO
2
-R
48.7±5.3
a
5.9±0.4
a
17.4±0.8
a
4.8±0.3
a
6.4±0.8
a
8.7±0.7
a
94.4±3.4
a
aTiO
2
-R
51.1±2.8
a
6.0±0.6
a
19.2±1.2
a
5.1±0.6
a
5.6±0.2
a
5.0±0.3
a
98.1±2.4
a
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Table 4
Richness and diversity estimators of the microbial phylotypes in the five SBRs systems after 50 mg/L TiO
2
-NPs exposure.
Sample ID
OTUs
Chao
a
Shannon
b
Coverage
c
Control
638
724
6.2
0.998
TiO
2
-A
429
548
5.3
0.997
aTiO
2
-A
591
624
6.1
0.998
TiO
2
-R
489
517
5.8
0.998
aTiO
2
-R
549
612
5.4
0.997
a
Chao richness estimator: the total number of OTUs estimated by infinite sampling. A higher number indicates higher richness.
b
Shannon index: an index to characterize species diversity. A higher value represents more diversity.
c
Good’s coverage: estimated probability that the next read will belong to an OTU that has already been found.
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