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Effect of ZnO nanoparticles on biological

wastewater treatment in a sequencing batch
reactor (SBR)

Article in Journal of Cleaner Production · April 2014

DOI: 10.1016/j.jclepro.2014.03.081

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 Journal of Cleaner Production 88 (2015) 139e145

Contents lists available at ScienceDirect

Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Effect of Zinc oxide nanoparticles on biological wastewater

treatment in a sequencing batch reactor
Ni-Qing Puay, Guanglei Qiu, Yen-Peng Ting*
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore

a r t i c l e i n f o a b s t r a c t

Article history: The rapidly increasing use of engineered nanoparticles (NPs) in consumer products inevitably leads to
Received 29 November 2013 their greater presence in wastewater treatment plants. In this study, the effects of zinc oxide nano-
Received in revised form particles (ZnO-NPs) on system performance and bacterial community dynamics of biological wastewater
24 March 2014
treatment in a lab-scale sequencing batch reactor were evaluated, along with their fate within the
Accepted 24 March 2014
system. It was found that ZnO-NPs caused poor settleability of the activated sludge and a significant
Available online 4 April 2014
decrease in the removal of nitrogen and phosphorus over time. Denaturing gradient gel electrophoresis
analysis revealed that the bacterial community in the activated sludge became less diverse after exposure
Keywords:
Zinc oxide nanoparticles (ZnO-NPs)
to ZnO-NPs. In addition, the production of extracellular polymeric substances (EPS) increased, with the
Municipal wastewater treatment EPS forming a tight matrix to protect the cells from the NPs. The NPs were removed very effectively from
Sequencing batch reactor (SBR) wastewater, mainly via sorption to the sludge.
Nitrification Ó 2014 Elsevier Ltd. All rights reserved.
Extracellular polymeric substances (EPS)
Bacterial community dynamics

1. Introduction
Municipal wastewater treatment, typically consisting of primary
treatment using physical and chemical means, and secondary
treatment using activated sludge, is indispensable in any modern
city. A reduction in the treatment efficiencies in any of these
treatment processes would have adverse effects on the environ-
ment and the quality of life.
Due to their small size (with at least one dimension between 1
and 100 nm) and large specific surface area (>60 m2/cm3) (Kreyling
et al., 2010), nanoparticles (NPs) exhibit optical, electrical, and
chemical characteristics different from either their bulk or dis-
solved forms (Heithmar and Pergantis, 2010). Synthetically manu-
factured NPs, also known as engineered NPs, exploit these
properties and are becoming increasingly common in consumer
products (Bauer et al., 2008). In particular, zinc oxide nanoparticles
(ZnO-NPs) are widely used in semiconductors, sunscreens, pig-
ments and food additives (Wu et al., 2010). The possible substitu-
tion of nanoparticles for toxics materials in manufacturing holds
great promise, but also present many risks (Ellenbecker and Tsai,
2011); it has been well documented that these NPs can be haz-
ardous to human and ecological health (Reijnders, 2006). Earlier
* Corresponding author. Tel.: þ65 65162190; fax: þ65 67791936.
E-mail address: chetyp@nus.edu.sg (Y.-P. Ting).
studies have found that NPs can cause oxidative damage to bacte-
rial and mammalian cells through the production of reactive
oxidation species (Setyawatia et al., 2013). DNA damage caused by
NPs may also result in carcinogenesis in cells (Ng et al., 2011).
Indeed, there is urgent need for a major increase in nanomaterial
risk research, as well as the development of adequate regulatory
policies on nanomaterial risk management (Walsh et al., 2008).
As NPs enter wastewater streams and end up at the treatment
plants, they inhibit some bacterial species in the activated sludge
and result in a reduction in the efficiency in biological wastewater
treatment (Wang et al., 2012). Zheng et al. (2011) showed that long-
term exposure of NPs not only significantly reduced the population
of ammonia-oxidizing bacteria but also inhibited the activities of
ammonia monooxygenase and nitrite oxidoreductase. NPs report-
edly exert different effect on different bacteria species. For example,
slow growing bacteria and those that produce large amounts of
extracellular polymeric substances (EPS) were found to be more
resistant to NPs (Sheng and Liu, 2011). Liang et al. (2010) showed
that while NPs exposure did not affect the growth of heterotrophs,
the nitrifying bacteria population was significantly reduced.
Previous research has concluded that significant amounts of
ZnO-NPs pass through primary treatment to enter the secondary
biological treatment process (Hou et al., 2013). ZnO-NPs were also
observed to exhibit significant toxicity to the activated sludge at
lower concentrations compared to other NPs (Mu et al., 2011).
Unlike many other common NPs, the slight solubility of ZnO-NPs

http://dx.doi.org/10.1016/j.jclepro.2014.03.081
0959-6526/Ó 2014 Elsevier Ltd. All rights reserved.
140 N.-Q. Puay et al. / Journal of Cleaner Production 88 (2015) 139e145
may have contributed to its higher toxicity (Liu et al., 2011). How-
ever, not much research has been conducted on their effect on
biological wastewater treatment in the long term. As wastewater
treatment plants are the last barrier before the water is discharged
into the environment, it is important to understand the fate of NPs
in the treatment process and examine their removal from waste-
water. Studies have found that biomass may remove NPs from
wastewater by trapping them in the EPS or biofilm matrixes (Kiser
et al., 2010). However, there is a paucity of data on the removal of
ZnO-NPs during the biological wastewater treatment process, due
to the dearth of specific methods for quantification of ZnO-NPs in
wastewater samples and the lack of information regarding their
transformation during the wastewater treatment process (Lombi
et al., 2012).
In this work, long-term effects of ZnO-NPs on the biological
wastewater treatment (over 62 days) were studied in a sequencing
batch reactor (SBR) vis-a-vis the pollutant removal efficiencies,
activated sludge properties and bacterial community dynamics.
The removal and distribution behaviour of ZnO-NPs in the SBR
system were also investigated to illustrate the potential fate of ZnO-
NPs during biological wastewater treatment.
2. Material and methods
2.1. Sequencing batch reactor (SBR)
An SBR with an operating volume of 2.2 L and treating 1.54 L of
wastewater per cycle was used in this study. Each cycle consisted of
15 min fill, 120 min anaerobic react, 120 min aerobic (I) react,
90 min anoxic react, 60 min aerobic (II) react, 45 min settle, and
30 min decant. The influent fill stage was split into 11 min of
influent fill at the beginning, and 4 min of influent fill after the
aerobic (I) react stage to provide addition of organic material for
effective denitrification. In addition, 26.7 ml of mixed liquor was
withdrawn from the reactor at the end of the anoxic stage for each
cycle in the sludge wastage process (a total of 80 ml of mixed liquor
withdrawn per day). The SBR was operated with an initial sludge
retention time (SRT) and hydraulic retention time (HRT) of 16 days
and 11.7 h respectively, and was run for 56 days to achieve steady
state. From Day 57 onwards, ZnO-NPs were added to the synthetic
wastewater to simulate an environmentally relevant concentration
of 1 mg/L.
2.2. Wastewater and ZnO-NPs
Influent synthetic wastewater was prepared daily using tap
water supplied by the Public Utilities Board, Singapore. Sodium ac-
etate (CH3COONa), ammonium chloride (NH4Cl) and potassium
dihydrogen phosphate (KH2PO4) were added to give an influent
chemical oxygen demand (COD), nitrogen, and phosphorus con-
centrations of 650 mg/L, 40 mg/L, and 8 mg/L respectively. The
synthetic wastewater also contained 19.3 mg/L calcium chloride
dihydrate (CaCl2$2H2O), 71.0 mg/L magnesium sulphate heptahy-
drate (MgSO4$7H2O), 17.4 mg/L iron (II) sulphate heptahydrate
(FeSO4$7H2O), 0.07 mg/L copper (II) chloride dihydrate
(CuCl2$2H2O), 0.13 mg/L manganese (II) chloride tetrahydrate
(MnCl2$4H2O), 0.13 mg/L zinc sulphate heptahydrate (ZnSO4$7H2O),
0.03 mg/L sodium molybdate dihydrate (Na2MoO4$2H2O),
0.025 mg/L boric acid (H3BO3) and 0.033 mg/L potassium iodide (KI).
Commercially-produced ZnO-NPs as a suspension of 50 wt%
ZnO in water were purchased from Sigma Aldrich, USA. Analysis of
particle size and zeta potential were performed using a Malvern
Zetasizer Nano ZS (Malvern Instruments, USA). ZnO-NPs suspen-
sion was first diluted to 100 mg/L using Milli-Q water, followed by
sonication for 1 h using Elmasonic S30H (Elma GmbH & Co,
Germany). The particle size was determined to be 66.25  36.61 nm
for the freshly prepared suspension and 67.32  33.68 nm for the
suspension that have been left to stand for 24 h, with corre-
sponding zeta potentials of 47.2 mV and 45.6 mV respectively.
2.3. Analyses of wastewater quality and sludge properties
The concentration of wastewater pollutants and the character-
istics of the activated sludge were analysed in accordance to
Standard Methods (APHA, 1999). The concentration of total nitro-
gen in the wastewater was calculated as the sum of ammonia-
nitrogen, nitrite-nitrogen and nitrate-nitrogen concentrations in
the wastewater.
Wastewater analysis and sludge characterization were done at
least in duplicates, with the exception of the settled sludge volume
in the calculation of sludge volume index (SVI). The standard de-
viations of the measured values were calculated and represented by
the error bars in the figures. An analysis of variance (ANOVA) was
also performed using SPSS 13.0 for Windows (SPSS Inc, USA). A p-
value of less than 0.05 is taken to be statistically significant.
As ZnO-NPs are slightly soluble, both Zn2þ and total Zn con-
centrations in the influent and effluent wastewater were analysed.
Analysis was conducted using an Inductively Coupled Plasma -
Mass Spectrometer (ICP-MS) (Agilent Technologies 7500 series,
USA). Acid digestion of the sample wastewater was conducted in a
process similar to 3030E of the Standard Methods (APHA, 1999).
5 ml of the sample was acidified with 1 ml of trace metal grade
nitric acid and refluxed at 105  C for 2 h. The resultant solution was
filtered through a 0.45 mm filter membrane before analysis. The
released Zn2þ due to the dissolution of ZnO NPs was determined
according to the literature (Zheng et al., 2011).
In addition to the wastewater, Zn content in the activated sludge
was also analysed after acid digestion. 10 ml of mixed liquor was
first centrifuged at 5000 rpm for 5 min and the supernatant
removed before the sample was washed with Milli-Q water. 5 ml of
nitric acid was then added to the residue and refluxed at 105  C for
2 h, followed by filtration through a 0.45 mm filter membrane. The
resultant solution was diluted to a final volume of 10 ml using Milli-
Q water.
The amount of soluble microbial products (SMP) and EPS
generated by the bacteria were analysed as protein and poly-
saccharide concentrations. SMP were obtained by centrifuging the
mixed liquor at 12,000 rpm for 10 min and filtering the supernatant
through a 0.45 mm membrane filter (Antonelli et al., 2011). Milli-Q
water was then added to the sludge residue before the sample was
heated in a water bath at 80  C for 30 min. The resultant solution
was then centrifuged again at 12,000 rpm for 10 min and the su-
pernatant filtered through a 0.45 mm membrane filter to obtain the
EPS.
Protein concentration was analysed using a modified Lowry
procedure (Lowry et al., 1951), with Bovine Serum Albumin as
standard. Polysaccharide content was analysed using the phenol-
sulphuric acid method, with glucose as standard (Qiu and Ting,
2014).
To prepare the activated sludge for SEM analysis, sludge sample
from the SBR was first washed with phosphate buffer solution (PBS)
before being soaked overnight in 2.5 wt% glutaraldehyde. The
treated sample was then washed again with PBS and dehydrated
with an ethanol gradient (25%, 50%, 75%, 90% and 99%). The samples
were then resuspended in 99% ethanol before several droplets of
the suspension were added to the SEM specimen mount holder and
dried in the desiccator. The dried samples were coated with plat-
inum before SEM (Jeol JSM-5600LV, Japan) analysis. An EDX
elemental analysis of the sludge samples was performed using an
attached silicon drift detector (Oxford Instruments x-act, UK).
 N.-Q. Puay et al. / Journal of Cleaner Production 88 (2015) 139e145 141

2.4. Sludge sampling and DNA extraction 1000 4.0

10 ml mixed liquor were centrifuged (1000 g for 5 min), after 800

3.0

MLSS, MLVSS (g/L)

which the supernatant was decanted and the pellet was resuspended

SVI (ml/g-MLSS)
in 10 ml sterile TriseEDTA buffer (10.0 mM TriseHCl, 1.0 mM EDTA, 600
pH 8.0). All samples were immediately frozen after resuspension and
2.0
stored at 80  C until DNA extraction. DNA was extracted from the SVI
400
samples with a QIAamp DNA Mini Kit (Qiagen, Valencia, CA). To
MLSS
minimize variations in DNA extraction, templates used for poly- 1.0
200 MLVSS
merase chain reaction (PCR) were prepared by mixing the DNA that
was extracted in triplicate for each sample (Qiu et al., 2013a).
0 0.0
0 20 40 60 80 100 120
2.5. Polymerase chain reaction
Operation time, day

In order to increase the yield of PCR products and to facilitate the
denaturing gradient gel electrophoresis (DGGE) analyses, a nested
PCR technique was applied (Qiu and Ting, 2013). For the total bacterial
community, the 16S rRNA genes were amplified from the DNA ex-
tracts using universal primers 27F and 1492R, following a tempera-
ture cycling conditions: Pre-incubation at 95  C for 2 min, followed by
25 cycles of 95  C for 1 min, 62  C for 1.5 min, and 72  C for 1 min; and a
final elongation at 72  C for 10 min. A nested PCR was then performed
on the PCR products obtained from previously described primers with
a second primer pair 357F-Clamp and 518R, following a cycling pro-
gram: Pre-incubation at 95  C for 2 min, followed by 30 cycles of 95  C
for 1 min, 60  C for 45 s, and 72  C for 1 min; and a final elongation at
72  C for 10 min. All the PCR amplifications were carried out in a total
volume of 50 ml in 200 ml tubes using a DNA thermocycler (Bio Rad,
Philadelphia, PA). The PCR mixture contained 1.25 U of Taq poly-
merase (Promega, Madison, WI), 1  PCR buffer, 2 mM MgCl2,
0.5 mmol of each primer, each deoxynucleoside triphosphate at a
concentration of 200 mM, and 40 ng of template DNA.
Fig. 1. Effect of ZnO-NPs on SVI, MLSS, and MLVSS.
of sludge escaping with the effluent. Additionally, relative high
volumetric exchange ratio (70%) used in this study may have
aggravated the sludge escaping after the addition of ZnO-NPs. As a
result, the biomass in the SBR decreased considerably. The MLSS
and MLVSS decreased from 3.3 g/L and 2.9 g/L to approximately
0.65 g/L and 0.55 g/L respectively. The sludge retention time (SRT)
also decreased drastically from 16 days to about 2.2 days. The poor
settleability of the sludge is likely to be due to the adverse effect of
ZnO-NPs which altered the EPS and the floc structures of the acti-
vated sludge (which will be discussed later).
3.2. Treatment efficiencies
In addition to the changes in the activated sludge properties,
ZnO-NPs also significantly affected the treatment efficiency
(Fig. 2a). The removal of phosphorus and total nitrogen were

2.6. Denaturing gradient gel electrophoresis

a 100
DGGE was performed using a D-Code System (Bio Rad, Phila-
80
delphia, PA) maintained at a constant temperature of 60  C in 1  TAE
Removal efficiencies, %

buffer. PCR products were loaded onto 8% (w/v) polyacrylamide gels

(37.5:1, acrylamide/bisacrylamide) using a denaturing gradient 60 ZnO-NPs added
ranging from 35% to 65% denaturant (7 mol urea and 40% formamide
in the 1  TAE buffer constituted 100% denaturant). Gels were run at 40
80 V for 12 h and stained with SYBRÒ Gold nucleic acid stain (Invi- COD
trogen, Carlsbad, CA). The resultant image was analysed using Total nitrogen
20
Quantity One 4.6.2 software (Bio-Rad, Hercules, USA) to obtain the
Phosphorus
fingerprint patterns and to calculate the band similarities. Shannone
0
Wiener diversity index (H’) was used to evaluate the variation of
0 20 40 60 80 100 120
structural diversity and species richness of bacterial communities of Operation time, day
the different samples (Qiu et al., 2013b) as expressed in Eq. (1):
X
b 100 20
H0 ¼  pi ln pi (1) 90 18
80 16
Removal efficiencies, %

where pi is the ratio of i-th group of bacteria to total community. 70 14

SRT, day

60 12
Ammonia
3. Results and discussion 50 10
Total Nitrogen
40 8
3.1. Sludge characteristics
30 SRT 6
20 4
The addition of ZnO-NPs from Day 57 brought about significant
changes in SVI, mixed liquor suspended solids (MLSS) and mixed 10 ZnO-NPs added 2

liquor volatile suspended solids (MLVSS) of the sludge in the SBR 0 0

50 60 70 80 90 100 110 120
(Fig. 1). The SVI rose almost immediately after the addition of the
Operation time, day
NPs and reached a peak of over 900 mL/g-MLSS on Day 108. Indeed,
the height of the settled sludge at the end of the ‘settle’ stage was Fig. 2. Effect of ZnO-NPs on (a) pollutant removal efficiencies and (b) the changes in
above the effluent outlet of the SBR, resulting in significant amount nitrogen removal along with the SRT in the SBR.
142 N.-Q. Puay et al. / Journal of Cleaner Production 88 (2015) 139e145
inhibited, but the removal of COD from wastewater was not affected
significantly by the ZnO-NPs (p ¼ 0.084).
Analysis of the total nitrogen in the effluent wastewater
revealed that the decline in nitrogen removal is likely to be due to a
decrease in nitrification. A previous study has shown that total
nitrogen removal decreases with SRT. An SRT of lower than 5.9 days
would be insufficient for complete ammonia oxidation (Lee et al.,
2008). In the present study, ammonia removal efficiency
decreased steadily from 100% to 70% after the SRT dropped below 3
days (Fig. 2b). It was surprising that TN removal was not signifi-
cantly affected. A possible explanation would be that there were
relatively faster growing and slower growing species within the
nitrifiers and denitrifiers. The presence of relatively faster growing
nitrifiers and denitrifiers may result in high nitrification under
lower SRT. Additionally, the presence of certain amount of biofilm
on the bioreactor wall may also have contributed to TN removal
under lower SRT conditions. At the end of the experiment, the
removal of ammonia recovered to about only 87%, signifying that
the lower SRT played a role in the initial reduction in the treatment
efficiency of ammonia. In addition, the removal of ammonia from
wastewater started to increase slowly after Day 86 without a cor-
responding change in SRT. This suggests that ZnO-NPs inhibit the
ammonia-oxidising bacteria, and the increase in removal is due to
the recovery the bacteria from the effect of the NPs.
Fig. 3. (a) DGGE fingerprint pattern and (b) cluster analysis of the bacterial community in the SBR.
These results corroborate a study which found that short term
exposure of 1 mg/L of ZnO-NPs did not impact phosphorus and
nitrogen removal (Hou et al., 2013). However, inhibitory effect on
phosphorus and nitrogen removal became apparent after long term
exposure of more than 35 days and 10 days respectively.
3.3. Bacterial community dynamics
In addition to their effect on treatment efficiencies, ZnO-NPs were
also found to cause changes in the bacterial community in the acti-
vated sludge. DGGE fingerprint patterns (Fig. 3a) show bacteria
species that are tolerant (S5, S14, S22) and intolerant (S2, S6, S23) of
ZnO-NPs. One of the bands (S11) was present at a constant intensity
throughout the study, but became fainter after Day 94, indicating
that the bacteria tolerated exposure to ZnO-NPs for over a month
before being overwhelmed. This bacteria species may be involved in
phosphorus removal as the reduction in the band intensity corre-
sponded to the decrease in phosphorus removal after Day 94.
The ShannoneWiener diversity index is a quantitative measure
that reflects the number species in the sludge samples. Table 1
shows that the bacterial diversity in the activated sludge
decreased after exposure to ZnO-NPs. These results agree well with
the DGGE fingerprint patterns, where the sludge samples after the
addition of ZnO-NPs contain noticeably fewer bands.
 N.-Q. Puay et al. / Journal of Cleaner Production 88 (2015) 139e145 143

Table 1 embedded within the EPS matrix, and thus prevented the NPs from
ShannoneWiener diversity index of the bacterial community in the SBR. coming into contact with cell surface and damaging the cells
Sample Sludge Day 52 Day 56 Day 80 Day 94 Day 108 (Pletikapic et al., 2012). Alternatively, the increase in EPS may also
inoculum be due to a change in the bacterial community as discussed earlier.
Diversity index (H0 ) 2.907 2.724 2.758 2.696 2.627 2.584 As previous research has revealed that bacteria that produce more
EPS exhibit higher tolerance to NPs (Sheng and Liu, 2011), it is
possible that these organisms became dominant in the SBR after
the addition of ZnO-NPs and thus led to an increase in the EPS
300
ZnO-NPs added concentration.
Polysaccharide Changes in the morphology of the activated sludge were
250
revealed in SEM images of the sludge inoculum and the sludge at
Protein Day 56 and Day 106 (Fig. 5). A layer of EPS envelopes the sludge
EPS, mg/g-MLVSS

200
inoculum and the sludge from Day 56. However, much less EPS was
observed on the surface of the sludge from Day 106, in seeming
150
contradiction to Fig. 4, which showed enhanced EPS concentration
after exposure to ZnO-NPs. One likely reason is that instead of
100
enveloping the sludge in its aggregation to form flocs, the EPS
produced formed a dense matrix to protect the cells from the NPs.
50 The SEM images also revealed that the poor settleability of the
activated sludge was likely to be due to the lack of EPS enveloping
0 the sludge after exposure to ZnO-NPs. Its absence would result in
0 20 40 60 80 100 120
dispersed sludge that does not settle well.
Operation time, day

Fig. 4. Changes of EPS (in terms of polysaccharides and proteins) concentrations in the
SBR.
An unweighted pair group method with arithmetic mean cluster
analysis (Fig. 3b) shows two major shifts in the bacterial commu-
nity structure. The first was between the inoculum and the sludge
before the addition of ZnO-NPs, and the second was between the
sludge before and after the addition of the ZnO-NPs. The sludge
from days 94 and 108 showed more similarity to each other (84%
similarity) than to the sludge from day 80 (70% similarity), sug-
gesting that the change in bacterial community due to exposure to
ZnO-NPs occurred soon after the addition of the NPs and decreased
over time.
3.4. Soluble microbial products and extracellular polymeric
substances
To investigate the effect of ZnO-NPs on the sludge bacteria, the
soluble microbial products (SMP) and EPS produced were moni-
tored and quantified in terms of polysaccharide and protein con-
tent. The SMP concentration did not change significantly upon
exposure to the ZnO-NPs, indicating that the bacteria cells were not
damaged. However, both polysaccharide and protein content in the
EPS increased significantly after the addition of ZnO-NPs
(p < 0.001) (Fig. 4), indicating that the NPs may have induced a
higher production of EPS as a defence mechanism by the bacteria.
This corroborates an earlier study on Ag-NPs, which found that
exposure to NPs enhanced EPS production; the NPs were
3.5. Role of dissolution of ZnO-NPs
Previous studies suggested that the toxicity of ZnO-NPs may
arise from the released Zn2þ due to particles dissolution (Liu et al.,
2011). However, such effect was more obvious when Zn2þ was
present in high concentrations. Zheng et al. (2011) showed that the
effect of 0.12 mg/L of Zn2þ on activated sludge activities was not
detectable, while higher concentrations (0.45 mg/L) of Zn2þ were
inhibitory to biological nitrogen and phosphorus removal. Liu et al.
(2011) also suggested that the IC50 values of soluble Zn on activated
sludge endogenous respiration, BOD biodegradation, ammonia
oxidation, and nitrite oxidation were 2.2, 1.3, 0.8, and 7.3 mg-Zn/L,
respectively. In this study, after the addition of ZnO-NPs, the
measured Zn2þ concentration in the influent and effluent was
0.14  0.09 mg/L and 0.017  0.09 mg/L respectively, indicating a
low dissolution potential of ZnO-NPs in the system, a finding
consistent with a previous study (Zheng et al., 2011). Thus, in this
work, it is unlikely that the adverse effects of ZnO-NPs may be
attributed to the release of Zn2þ.
3.6. Fate of ZnO-NPs
The total Zn concentration in the influent, effluent and activated
sludge were analysed using an ICP-MS to determine the removal of
ZnO-NPs from wastewater.
Fig. 6 shows the percentage removal of ZnO-NPs from the
wastewater and the Zn content in the activated sludge, which is
largely due to sorption of ZnO-NPs onto the activated sludge. The

Fig. 5. SEM images (5000) of (a) sludge inoculum, (b) sludge on Day 56, and (c) sludge on Day 106.
144 N.-Q. Puay et al. / Journal of Cleaner Production 88 (2015) 139e145

100 5.0 loss of sludge in the effluent. Additional clarification would there-
fore be needed before discharge into the environment. Moreover,
Phase 1 Phase 2 Phase 3

Zn concentration in sludge
80 4.0 nitrogen and phosphorus removal efficiencies were also severely
Removal efficiencies, %

60
40
20
0 0.0
50 60 70 80 90 100
Operation time, day
Fig. 6. Percentage removal of total Zn, and Zn content in the activated sludge.
sorption of the ZnO-NPs occurred in three phases. During the first
phase, ZnO-NPs were sorbed onto the activated sludge, resulting in
very high removal of ZnO-NPs from the wastewater. During the
second phase (after Day 66) the amount of ZnO-NPs sorbed
decreased and this led to a further decrease in the removal of ZnO-
NPs from the wastewater. ZnO-NPs sorption in the sludge increased
again in the last phase, after Day 94, which resulted in an increase
in ZnO-NPs removal to almost 100%.
The activated sludge samples were also analysed using EDX to
quantify the amount of ZnO-NPs sorbed onto the sludge. Results
revealed that the Zn content in the sludge increased from 0.12
(sludge inoculum) and 0.16 (Day 56) to 0.5 wt % (Day 106) after the
addition of ZnO-NPs.
A mass balance of the total Zn loading in the influent, effluent
and in the reactor was performed. Fig. 7 revealed that the cumu-
lative ZnO-NPs added exceeded the sum of that present in the
effluent and that held in the wasted sludge and the mixed liquor,
thus suggesting the likelihood of metal sorption on the walls of the
reactor (which was made of poly (methyl methacrylate)). As the
walls of the reactor became saturated over time, this “unaccounted-
for” ZnO-NPs became relatively constant. In addition, it is evident
that most of the ZnO-NPs are removed through sorption to the
activated sludge, especially after Day 108.
mg Zn/mg MLSS
affected by ZnO-NPs over time, and strategies would need to be
3.0
developed to mitigate these effects.
Currently, knowledge on the concentrations of ZnO-NPs in
municipal wastewater is still scarce, due to the lack of specific
2.0
methods in distinguishing and quantifying it from naturally-
occurring nanoscale materials as well as its bulk materials.
Removal of Zn 1.0 Modelling results of Sun et al. (2014) suggested that the most
Zn in sludge pronounced flows of ZnO-NPs to wastewater treatment plants were
from their production, manufacturing, and consumption, due to
110 120
their major applications in cosmetics. The predicted ZnO-NPs
concentration in the effluent of the wastewater treatment plant is
up to 45 mg/L. As the removal efficiency of ZnO NPs used in the
calculation was about 91%, the predicted influent ZnO-NPs would
correspond to 0.5 mg/L, which is 7 fold of that predicted in 2009
(Gottschalk et al., 2009), due to the rapid growth in the production
of ZnO-NPs. Elevated concentration of ZnO-NPs in the sewage
wastewater seems inevitable in the near future. However, the po-
tential transformations of ZnO-NPs in the sewer systems need to be
considered in the assessment of the impacts of ZnO-NPs in the real
wastewater treatment facilities, since the microstructural trans-
formation of ZnO-NPs with the aid of phosphate (Lv et al., 2012) and
the sulfidization of ZnO-NPs under anaerobic conditions (Lombi
et al., 2012) may significantly change their availability and reduce
their toxicity.
Additionally, this study revealed that a large portion of the ZnO-
NPs was removed via sorption onto the activated sludge. Although
this raised the problem of safe disposal of ZnO-NPs-enriched waste
sludge, a study has suggested that considerable changes in the
speciation of the NPs may occur during anaerobic sludge digestion
which would significantly reduce the potential risk (Lombi et al.,
2012). Nonetheless, the conversion of ZnO-NPs may be signifi-
cantly retarded by surface coating or other characteristics in the
commercial formulation of the NPs. In view of the rapid growth in
the incorporation of ZnO-NPs in consumer products and the inev-
itable higher release into wastewater treatment system (Sun et al.,
2014), investigations are urgently needed for the safe handling and
disposal of this waste.

4. Conclusion
3.7. Discussion and implications
1 mg/L ZnO-NPs caused significant impact on activated sludge
In this study, 1.0 mg/L ZnO-NPs was found to be detrimental to
settling properties. Nitrogen and phosphorus removal efficiencies
the performance of the SBR. The most significant impact was the
were also severely affected by ZnO-NPs over time. Bacteria com-
poor settling properties of the activated sludge, which resulted in a
munity in the activated sludge was affected by ZnO-NPs; DGGE
analysis revealed changes in the bacterial community structure and
250 a reduction in the bacteria diversity, thus indicating that the NPs
Cumulative mass in
reduce the ability of the treatment process to handle changes in the
Cumulative mass out + in reactor influent wastewater conditions. A higher concentration of EPS was
200 produced by the bacteria after exposure to NPs. However, instead of
Cumulative mass out in effluent
Cumulative Zn, mg

Cumulative mass out in sludge loosely enveloping the sludge and enhancing agglomeration, the
150 EPS produced protected the sludge from NPs by forming a dense
Mass remaining in reactor
protective matrix around the cells.
ZnO-NPs were removed effectively from the wastewater. A mass
100
balance of total Zn loading in the influent, effluent and sludge
revealed that the main pathway for this removal was via sorption
50 onto the activated sludge.

0 Acknowledgement
55 65 75 85 95 105 115
Operation time, day This research was funded by the Singapore National Research
Foundation under its Competitive Research Program for the project
Fig. 7. Mass balance for total Zn in the SBR. entitled “Advanced FO Membranes and Membrane Systems for
 N.-Q. Puay et al. / Journal of Cleaner Production 88 (2015) 139e145
Wastewater Treatment, Water Reuse and Seawater Desalination”
(grant number R-279-000-338-281).
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