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Short Communication

Fate of cerium dioxide (CeO2) nanoparticles in municipal wastewater duringactivated sludge treatment

Francisco Gmez-Rivera, James A. Field, Dustin Brown, Reyes Sierra-Alvarez

Department of Chemical and Environmental Engineering, University of Arizona, P.O. Box 21011, Tucson, AZ 85721, United States

a r t i c l e i n f o

Article history:

Received 19 October 2011

Received in revised form 14 December 2011

Accepted 15 December 2011

Available online 3 January 2012

Keywords:

Aerobic wastewater treatment

Nanoparticle removal

Biosorption

Aerobic inhibition

a b s t r a c t

This study investigated the fate of nano-CeO2 during municipal wastewater treatment using a laboratory-

scale activated sludge (A/S) system fed with primarily-treated municipal wastewater and nano-CeO2(55.0 mg Ce/L). Nano-CeO2 was highly removed during A/S treatment (96.6% total Ce). Extensive removal

of CeO2

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water. CeO2 was selected due to its importance in nanotechnology

and its low background in wastewater, which minimizes interfer-

ence of natural or anthropogenic occurring CeO2. The stability of

CeO2 NPs in demineralized water and wastewater, as well as their

sorption to biomass, was studied to gain insight on the dominant

mechanisms of CeO2 removal.

2. Methods

2.1. Nanoparticles

Nano-CeO2 (50 nm) was obtained from SigmaAldrich (St Louis,

MO) as a dry powder (Supplementary Fig. 1). Stock dispersions of

CeO2 were prepared in 1 mM HCl to ensure acidic conditions that

promote CeO2 dispersion. The pH of the CeO2 stock suspensions

was 3.1.

2.2. Lab-scale secondary wastewater treatment

The treatment system consisted of an aeration tank (1.19 L) and

a settler (0.66 L) (Supplementary Fig. 2) operated at 27.5 2.0 C.

The reactor feed was prepared by mixing primarily-treatedmunicipal wastewater with either demineralized (DI) water or a

nano-CeO2 dispersion, depending on the period of operation, at a

volumetric ratio of 10:1 (v/v). The target concentration of CeO2in the influent was 67.6 mg/L (55.0 mg Ce/L). The CeO2 stock was

mixed using a magnetic stirrer to minimize NP agglomeration.

The (waste)water and the NP stock were supplied at the inlet of

the bioreactor using two separate peristaltic pumps to minimize

NP aggregation prior to treatment. The average wastewater pH

was 7.4. The total chemical oxygen demand (COD) concentration

of the wastewater was 248 50 mg/L, and the soluble COD fraction

averaged 64.6% of the total COD.

The aeration tank was seeded with 3.5 g volatile suspended sol-

ids (VSS)/L of activated sludge that was collected from a local

WWTP. Two air pumps provided air (430 L air/d) and mixing forthe bioreactor and recirculation of the settled sludge, respectively.

The aeration tank was operated at a hydraulic retention time of

10.50 0.96 h. To maintain a fairly constant concentration of bio-

mass in the bioreactor, 90 mL of mixed liquor were withdrawn

every 3 d, which were replaced with wastewater. Samples were

collected periodically from the influent of the bioreactor and the

effluent of the settler for analysis of total and soluble COD, VSS, to-

tal and filtered Ce, and pH.

2.3. Stability of CeO2 nanoparticle dispersions

Assays were conducted using both municipal wastewater and

synthetic wastewater. The synthetic wastewater was formulated

according to guidelines of the Organization for Economic Co-oper-ation and Development (OECD, 2001). It contained (g/L): peptone

(0.22), meat extract (0.15), urea (0.01), K2HPO4 (0.008), and NaH-

CO3 (0.4). The final pH of the wastewater was set to 7.0. Stock sus-

pensions of CeO2 (1 mL) were diluted with 10 mL of municipal

wastewater, synthetic wastewater, or DI water in 15-mL test tubes

to a final concentration of 73 mg Ce/L. The pH of the samples was

adjusted to 3.1 or 7.1 using HCl or NaOH, as needed. Prior to dilu-

tion, wastewater samples were membrane filtered (25 nm) to min-

imize interference by suspended and colloidal materials. The

samples were homogenized by vortexing and, then incubated at

27.5 2.0 C for 24 h under static conditions. Next, the supernatant

of each sample was analyzed for particle size distribution (PSD),

zeta potential, and CeO2. An aliquot of the sample containing

CeO2 in DI water (pH 3.1) was collected prior to incubation to as-sess PSD at the start of the assay.

2.4. Batch adsorption experiments

The contribution of activated sludge-NP interactions to the re-

moval of nano-CeO2 was studied in batch sorption experiments

at 27.5 2.0 C. The sludge was washed four times with DI water

and then centrifuged (4000 rpm, 30 min). The rinsed, dewatered

sludge was used in the adsorption tests. Assays were set up at

pH 3.0 and 6.0 in duplicated 50-mL test tubes supplied with DI

water, or DI water with washed sludge (3.5 g VSS/L), and spiked

with nano-CeO2 (81.687.6 mg Ce/L). The tubes were shaken at

150 rpm for 15.2 h, and then incubated for 4.8 h under stationary

conditions. The amount of CeO2 sorbed onto the sludge was esti-

mated from the difference of the concentrations of Ce added and

Ce in the supernatant of each treatment.

2.5. Analytical procedures, particle size distribution, and zeta potential

measurements

Cerium was analyzed using inductively coupled plasmaoptical

emission spectroscopy (Optima 2100 DV, Perkin Elmer, Waltham,

MA) at a wavelength of 413 nm. The CeO2 fraction

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dispersions of CeO2 in water are expected to have a low apparent

surfacechargeand show a high tendency to aggregateat circumneu-

tral pH values, whichis in agreement with the high average particle

size, lowzetapotentialvalues, andlowresidual concentrationof dis-persed CeO2 found in experiments performed in DI water at pH 7.1

(Fig. 1). Under strongly acidic conditions, on the other hand, CeO2NPs are stabilized by electrostatic repulsion of the positively

chargedoxidesurface, which alsoagrees with thefindings of thesta-

bility tests in pH-3.1 DI water.

The stabilizing effect observed for dispersions of CeO2 in syn-

thetic wastewater (pH 7.1) may be related to the presence of high

concentrations of proteins and peptides in the model water which

main organic components were peptone and meat extract. Several

reports have confirmed that some proteins can increase the stabil-

ity of nano-sized inorganic oxide (NIOx) dispersions, and protein

supplementation has been proposed as an effective approach to re-

duce NP aggregation in cytotoxity testing (Sager et al., 2007). On

the other hand, the negative effect of municipal wastewater onCeO2 dispersion stability is also likely due to NPs interactions with

organic and/or inorganic compounds in the wastewater. Although

wastewaters can contain surfactants and organic constituents that

can increase NP stability, significant aggregation of NIOx disper-

sions following addition of proteins and humic substances has

been reported (Hotze et al., 2010).

In conclusion, the results of the stability tests confirmed that

CeO2 NP dispersions tend to aggregate at circumneutral pH values

which are typically found in municipal wastewaters. Organic and/

or inorganic constituents in the real wastewater also contributed

to promote the aggregation of the nano-CeO2. Furthermore, the re-

sults obtained indicate that the synthetic wastewater utilized did

not adequately simulate the complex chemical composition of real

municipal wastewater and its impact on the state of aggregation ofCeO2 NPs.

3.2. Biosorption of CeO2 nanoparticles by activated sludge

Partitioning of NPs onto sludge biomass has been proposed to

be a major mechanism causing removal of NPs during biological

wastewater treatment (Kiser et al., 2009; Limbach et al., 2008).

In this study, the affinity of A/S for CeO2 NPs was investigated in

batch experiments using rinsed sludge to avoid interference by

wastewater constituents. Extensive removal of CeO2

(94.097.1%)

was observed in assays spiked with A/S after 20 h of incubation

in DI water at pH 3.0 and pH 6.0, confirming CeO2 biosorption by

the sludge. Removal of CeO2 at pH 6.0 was partly due to particle

destabilization and agglomeration as evidenced by the large de-

crease observed in the concentration of suspended CeO2 in the ab-

sence of sludge (45.0% removal). At pH 3.0, however, CeO2elimination was almost completely due to biosorption and only

5.3% of the CeO2 settled out of dispersion in the absence of sludge.

The mechanisms responsible for adsorption of NPs to bacterial sur-

faces are not well understood, but electrostatic interactions be-

tween microbial cells and NPs are thought to play a crucial role

(Kiser et al., 2010; Thill et al., 2006). Removal of NPs by biosorption

onto sewage sludge appears to vary widely depending on the type-

and surface functionality of the NPs. In a study considering inor-

ganic and carbon-based NPs, NP removal by partitioning onto A/S

sludge ranged from 12 to 84% depending on the nature of the

NPs (Kiser et al., 2010).

3.3. Removal of CeO2 NPs from municipal wastewater during activated

sludge treatment

The fate of CeO2 NPs in municipal wastewater during secondary

treatment was studied using a bench-scale A/S system operated for

70 d. Initially, the reactor was fed with wastewater without adding

CeO2 to allow stabilization of the system. CeO2 NPs (55.0 12.8 mg

Ce/L) were spiked into the influent from day 7 onwards. Over the

course of the experiment, an average 96.6% of the total CeO2 was

removed indicating the high efficiency of the A/S process to elimi-

nate CeO2 from municipal wastewater (Fig. 2A). In spite of the hightreatment efficiency, significant levels of total Ce (1.8 1.4 mg Ce/

L) escaped in the effluent of the reactor system.

The effectiveness of A/S treatment for eliminating nano-scale

particles of CeO2 in the wastewater was evaluated by sample filtra-

tion through a 200-nm pore size membrane. The results obtained

showed that NPs underwent significant aggregation following dilu-

tion into the wastewater. The average concentration of CeO2

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Ce, respectively. Moreover, Ce was highly eliminated from the

synthetic wastewater even in the presence of surfactants. Althoughthese studies provide insights on the fate of NIOx during secondary

wastewater treatment, they could fail in accurately representing

the behavior of the NPs during actual treatment. In particular,

the use of synthetic media in the earlier studies is a concern

since the present results showed that the stability of CeO2 NP in

synthetic- and municipal-wastewater differ greatly. Moreover,

the short duration of both studies (418 d) did not allow establish-

ing whether the measured removal efficiencies could be sustained

over extended time periods.

3.4. COD removal during secondary treatment

The average removal of soluble and total COD attained during

the reactor start-up period averaged 81.1 0.1% and 83.5 0.1%,respectively (Fig. 2C and D). After CeO2 NPs were supplied, the re-

moval of soluble and total COD averaged 65.4 0.1 and 65.9 0.1%,

respectively. The observed drop in efficiency was most likely due

to decrease of the average influent COD and not to deterioration

in effluent quality. Additional results from batch toxicity assays

suggested that microbial inhibition by CeO2 NPs at the concentra-

tion present in the reactor influent (55 mg Ce/L) is unlikely. Batch

toxicity assays showed that CeO2 NPs only displayed significant

inhibition of O2 uptake by the A/S when present at much higher

levels than those fed to the bioreactor (50% inhibition at 950 mg

CeO2/L; Supplementary Fig. 4). The inhibitory potential of CeO2 to-

wards microorganisms appears to vary widely. Nano-CeO2 inhib-

ited growth of E. coli and Bacillus subtilis, but not Shewanella

oneidensis (Pelletier et al., 2010; Thill et al., 2006). Interestingly,the use of a culture medium rich in organic constituents was

shown to negate the inhibitory effect of CeO2 towards E. coli (Thill

et al., 2006), suggesting that physico-chemical interactions be-tween NPs and media components may play a crucial role in deter-

mining nanotoxicity.

4. Conclusions

Nano-CeO2 was effectively removed by A/S treatment. Results

of a laboratory-scale study fed primarily-treated municipal waste-

water spiked with nano-CeO2 (55 mg Ce/L) confirmed that high re-

moval of total Ce (96.6%) was maintained over 63 d with effluent

concentrations of CeO2 particles

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