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Long-term effect of ZnO nanoparticles on waste activatedsludge anaerobic digestion

Hui Mu, Yinguang Chen*

State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University,

1239 Siping Road, Shanghai 200092, China

a r t i c l e i n f o

Article history:

Received 9 May 2011

Received in revised form

10 August 2011

Accepted 14 August 2011

Available online 23 August 2011

Keywords:

Zinc oxide nanoparticles

Waste activated sludge

Anaerobic digestion

Mechanisms

* Corresponding author. Tel.: þ86 21 6598126E-mail address: yinguangchen@yahoo.co

0043-1354/$ e see front matter ª 2011 Elsevdoi:10.1016/j.watres.2011.08.022

a b s t r a c t

The increasing use of zinc oxide nanoparticles (ZnO NPs) raises concerns about their

environmental impacts, but the potential effect of ZnO NPs on sludge anaerobic digestion

remains unknown. In this paper, long-term exposure experiments were carried out to

investigate the influence of ZnO NPs on methane production during waste activated sludge

(WAS) anaerobic digestion. The presence of 1 mg/g-TSS of ZnO NPs did not affect methane

production, but 30 and 150 mg/g-TSS of ZnO NPs induced 18.3% and 75.1% of inhibition

respectively, which showed that the impact of ZnO NPs on methane production was

dosage dependant. Then, the mechanisms of ZnO NPs affecting sludge anaerobic digestion

were investigated. It was found that the toxic effect of ZnO NPs on methane production

was mainly due to the release of Zn2þ from ZnO NPs, which may cause the inhibitory

effects on the hydrolysis and methanation steps of sludge anaerobic digestion. Further

investigations with enzyme and fluorescence in situ hybridization (FISH) assays indicated

that higher concentration of ZnO NPs decreased the activities of protease and coenzyme

F420, and the abundance of methanogenesis Archaea.

ª 2011 Elsevier Ltd. All rights reserved.

1. Introduction 2002; Serda et al., 2009). There are some publications discus-

With the rapid development of nanotechnology, nanoparticles

(NPs) are nowwidely used in some industrial products, such as

antibactericide coatings, catalysts, biomedicine, skin creams

and toothpastes because of their unique physicochemical

properties of enhanced magnetic, electrical, optical, and etc

(Maynard et al., 2006; Roco, 2005). It is inevitable for the release

ofNPs fromdiscover source toenvironment receptor, and some

NPs have been found inwastewater treatment plants (WWTPs)

and waste sludge (Brar et al., 2010). It is therefore necessary to

evaluate their impacts on the environment.

Zinc oxide (ZnO)NPs, one ofmetal oxideNPs, have received

increasing interest due to their widespread industrial, medical

and military applications (Ellsworth et al., 2000; Miziolek,

3; fax: þ86 21 65986313.m (Y. Chen).ier Ltd. All rights reserve

sing the toxicity of ZnO NPs onmicrobes. For example, Adams

et al. (2006) reported that 500 mg/L of ZnO NPs significantly

inhibited the growth of Bacillus subtilis up to 90%, but only

induced 38% of the growth inhibition of Escherichia coli,

meaning the different toxicity of ZnO NPs on different species

of bacteria. Previous study also showed that ZnO NPs reduced

the microbial biomass, and altered the diversity and compo-

sition of soil bacterial community (Ge et al., 2011).

The release of ZnO NPs to WWTPs, which are usually

operated with an activated sludge process, has been reported

recently (Gottschalk et al., 2009). The released ZnO NPs were

observed to be removed by activated sludge via adsorption,

aggregation and settling in WWTPs (Kiser et al., 2010, 2009;

Kiser et al., 2010; Limbach et al., 2008). Large amounts of WAS

d.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2e5 6 2 0 5613

are produced in municipal WWTPs, which need to be treated

before being discharged to the environment. It can be antici-

pated that most of ZnO NPs will enter into sludge treatment

system. Among several sludge treatment methods anaerobic

digestion formethane is a preferred one because ofWAS being

reused and the energy being recovered. Nevertheless, the

toxicity of ZnO NPs to sludge anaerobic digestion has seldom

been investigated.

Some studies addressed that the toxicity of ZnO NPs came

from the released zinc ions (Zn2þ) (Franklin et al., 2007; Wong

et al., 2010; Xia et al., 2008), but others found that the toxicity

of ZnO NPs to some microorganisms (such as E. coli and

Pseudomonas fluorescens) was not caused by the released Zn2þ

but ZnO NPs themselves (Jiang et al., 2009). Thus, the role of

released Zn2þ from ZnO NPs on sludge anaerobic digestion

should be taken into account. Moreover, oxidative stress

induced by ZnO NPs was reported to cause the loss of cell

viability, and the increase of intracellular reactive oxygen

species (ROS) was found to be toxic to cytoplasmic lipids,

proteins and other intermediates in cells (Sharma et al., 2009;

Xia et al., 2008). This study was to evaluate the impact of ZnO

NPs on methane production during sludge anaerobic diges-

tion and to explore the mechanisms. Furthermore, fluores-

cence in situ hybridization (FISH) technique with 16S rRNA-

targeted oligonucleotide probes was employed to monitor

the quantity change of bacteria and Archaea community after

WAS anaerobic digestion system long-term exposed to ZnO

NPs.

2. Materials and methods

2.1. Nanoparticles and waste activated sludge

ZnO NPs were purchased from Sigma Aldrich (St. Louis, MO).

The X-ray diffraction (XRD) pattern of ZnO NPs was measured

using a Rigaku D/Max-RB (Rigaku, Japan) diffractometer

equipped with a rotating anode and a Cu Ka radiation source

and shown in Fig. S1 (Supplementary Information). In this

study, stock dispersion of ZnONPswas produced by adding 2 g

ZnO NPs to 1.0 L distilled water (pH 7.0) containing 0.1 mM

sodium dodecylbenzene sulfonate (SDBS) (Sigma Aldrich, St.

Louis, MO) to enhance the stability of nano-suspension

because the particles almost immediately aggregated in

surrounding medium (Adams et al., 2006; Franklin et al., 2007;

Ganesh et al., 2010; Keller et al., 2010; Simon-Deckers et al.,

2009; Xia et al., 2008). The stock dispersion was sonicated

(25 �C, 250 W, 40 kHz) for 1 h to break aggregates before being

diluted to the exposure concentrations. Analysis of the

suspension by dynamic light scattering (DLS) (Franklin et al.,

2007; Simon-Deckers et al., 2009) using a Malvern Autosizer

4700 (Malvern Instruments, UK) indicated that the average

particle size of ZnONPswas approximately 140� 20 nmon the

basis of the number distribution with more than five separate

measurements per sample.

The WAS used in this study was withdrawn from the

secondary sedimentation tank of a municipal WWTP in

Shanghai,China.Thesludgewasconcentratedbysettlingat4 �Cfor24h,and itsmaincharacteristics (averagedataplusstandard

deviations of triplicate tests) are as follows: pH 6.7 � 0.2, total

suspended solids (TSS) 10070 � 780 mg/L, volatile suspended

solids (VSS) 7690� 452 mg/L, soluble chemical oxygen demand

(SCOD) 90 � 5 mg/L, total chemical oxygen demand (TCOD)

10710� 220mg/L, total carbohydrate 899� 530mg-COD/L, total

protein 5685� 149mg-COD/L, and total zinc 0.8� 0.2mg/g-TSS.

2.2. Determination of ZnO nanoparticles dissolution

In order to measure the concentration of released Zn2þ from

ZnO NPs, three concentrations of ZnO NPs in 0.1 mM SDBS

solutions were prepared with the stock dispersion, and the

mixtures were maintained in an air-batch shaker (150 rpm) at

35 � 1 �C for 48 h. At different time, the samples were with-

drawn and centrifuged at 12000 rpm for 30 min, and the

supernatantwas collected, and filtered through 0.22 mmmixed

cellulose estermembrane (Jiang et al., 2009; Li et al., 2011). The

released zinc was determined by inductively coupled plasma

optical emission spectrometry (ICP-OES, PerkinElmer Optima

2100 DV, USA) after acidified with 4% ultrahigh purity HNO3.

2.3. Experiments of effects of ZnO nanoparticles andtheir released Zn2þ on WAS anaerobic digestion for methaneproduction

Three dosages (1, 30 and 150mg/g-TSS) of ZnONPswere used to

investigate the impact of ZnO NPs on WAS digestion in this

paper. The dosage of 1 mg/g-TSS of ZnO NPs was chosen to be

the environmentally relevant concentration according to the

literature (EPA, 2009; Gottschalk et al., 2009). Also some scien-

tists suggested that although lower nanomaterial content

(50mgC60/g-TSS in their study) showed almost no influence on

anaerobic community, a much higher nanomaterial dosage

should be investigated before the final conclusion regarding the

toxicity of nanomaterial was reached (Nyberg et al., 2008).

Moreover, since the environmental release of NPs might be

increased due to their large-scale production, the potential

effects of higher concentrations (30 and 150 mg/g-TSS) of ZnO

NPs were also investigated in this study according to the refer-

ence (Adams et al., 2006). The influence of ZnO NPs long-term

exposure on methane production was conducted in series of

serum bottles (500 mL each), with a sludge volume of 300 mL

each. As SDBSwas used as the dispersing reagent in this study,

twocontrols, onewithonly sludge, andanother onewith sludge

plus 4 mg/g-TSS of SDBS, were conducted to investigate

whether methane production was affected by SDBS addition.

After flushed with nitrogen gas for 5 min to remove oxygen, all

bottles were capped with rubber stoppers, sealed and placed in

an air-bath shaker (150 rpm) at 35 � 1 �C. Every day, 15 mL

fermentation mixture was manually withdrawn from each

serum bottle and the same amounts of raw sludge, SDBS and

ZnO NPs were supplemented, which resulted in a hydrolytic

retention time (HRT) or sludge retention time (SRT) of 20 d. The

sampling was operated in a glove box. After the reactors were

operated for 105d, thedailymethaneproductiondidnot change

significantly with time, and then the analyses of methane

production, enzyme activity, biomass viability and microbial

community were conducted. The total gas volume was

measured by releasing the pressure in the bottles using a glass

syringe (100 mL) to equilibrate with the room pressure accord-

ing to our previous publication (Zhao et al., 2010).

Methane

Amino acids

Aceticacid

Butyricacid

Pyruvic acid

Propionicacid

Acetyl-CoA

HydrolysisHydrolysis

Soluble polysaccharide

Monosaccharide

HydrogenCarbondioxide

Acidification

Methanation

Soluble protein

protease

AK

F420

Particulate organic matters of

waste active sludge SolubilizationSolubilization

F420Methane

Amino acids

Aceticacid

Butyricacid

Pyruvic acid

Propionicacid

Acetyl-CoA

HydrolysisHydrolysis

Soluble polysaccharide

Monosaccharide

HydrogenCarbondioxide

Acidification

Methanation

Soluble protein

protease

AK

F420

Particulate organic matters of

waste active sludge SolubilizationSolubilization

F420

Fig. 1 e Proposed metabolic pathway for methane

production from WAS anaerobic digestion. Only the key

enzymes assayed in this study are labeled.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2e5 6 2 05614

Stock solution (50 mg/L) of ZnCl2 (Sigma-Aldrich) was

prepared in 0.1 mM SDBS solution (pH 7.0). The long-term

experiments of released Zn2þ from ZnO NPs affecting WAS

digestion were conducted with the same method described

above except that the ZnCl2 was used to replace ZnO NPs, and

the total amount of Zn2þ added to the serum bottles was 1.2,

11.6, and 17.6 mg/L, respectively.

2.4. Effects of ZnO nanoparticles on each step involvedin methane production

It is well known that sludge anaerobic digestion usually

undergoes solubilization of sludge particulate organic-carbon,

hydrolysis, acidification and methanation. The experiments

of long-term effects of ZnO NPs on these four stages were

conducted with the inoculum seeds from four long-term

operated reactors with ZnO NPs dosage of 0, 1, 30 and

150 mg/g-TSS, respectively. The experiments of long-term

effects of ZnO NPs on sludge particulate organic maters

solubilization were conducted as follows. WAS of 300 mL and

30 mL inocula were added to each serum bottle. After flushed

with nitrogen gas for 5 min to remove oxygen, all bottles were

capped with rubber stoppers, sealed and placed in an air-bath

shaker (150 rpm) at 35 � 1 �C. The concentrations of soluble

protein and carbohydrate were measured after fermentation

for 2 d.

As soluble protein and polysaccharide were the main

sludge solubilized products, in order to investigate the long-

term effects of ZnO NPs on the hydrolysis of sludge solubi-

lized products, the following batch tests with synthetic

wastewater containing bovine serum albumin (BSA, average

molecular weight Mw 67000, model protein compound used in

this study) and dextran (Mww23800, model polysaccharide

compound) were conducted. The synthetic wastewater con-

sisted of (mg/L of distilled water) 1000 KH2PO4, 400 CaCl2, 600

MgCl2$6H2O, 100 FeCl3, 0.5 ZnSO4$7H2O, 0.5 CuSO4$5H2O, 0.5

CoCl2$6H2O, 0.5 MnCl2$4H2O, 1 NiCl2$6H2O and 34.8 SDBS.

After 4.8 g BSA and 1.2 g dextran (the mass ratio of protein to

carbohydrate was almost the same as that in WAS) were

dissolved in 1200mL synthetic wastewater, themixture liquid

was divided equally into 4 bottles, and then 30 mL inocula,

which was heat-pretreated at 102 �C for 30 min to kill

methanogens (Oh et al., 2003), was added before the pH in

each bottle was adjusted to 7.0 by adding 4MNaOH or 4MHCl.

After flushed with nitrogen gas to remove oxygen, all bottles

were capped with rubber stoppers, sealed and placed in an

air-bath shaker (150 rpm) at 35 � 1 �C. By analyzing

the degradation efficiencies of protein and dextran, the long-

term effects of ZnO NPs on sludge hydrolysis were obtained.

The same operations were conducted when the long-term

effects of ZnO NPs on the acidification of hydrolyzed prod-

ucts were investigated except that the synthetic wastewater

containing 4.8 g L-glutamate (model amino acid compound)

and 1.2 g glucose (model monosaccharide compound).

As acetic acidwas themain short-chain fatty acid (SCFA) of

sludge acidification product (Yuan et al., 2006) and the

preferred substrate for methane production (Fig. 1), the long-

term effect of ZnO NPs on methanation of the acidification

productwas conductedwith 300mL syntheticwastewater (see

theabovedescription) containing 0.72 g sodiumacetate (model

SCFA compound) in each serum bottle. All other operations

were the same as described above. By the analysis of methane

production, the long-term effect of ZnO NPs on methanation

was obtained.

2.5. Analytical methods

Gas component was measured via a gas chromatograph

(Agilent 6890N, USA) equipped with a thermal conductivity

detector using nitrogen as the carrier gas. The zinc concen-

tration was analyzed by ICP-OES (PerkinElmer Optima 2100

DV, USA). To measure the zinc content in sludge, the sample

was digested according to EPA Method 200.2 prior to ICP

analyses. The pH value was measured by a pH meter. The

determinations of SCFA, protein, carbohydrate, TSS and VSS

were the same as those described in the previous publication

(Yuan, et al., 2006). The total SCFA was calculated as the sum

of measured acetic, propionic, n-butyric, iso-butyric, n-valeric

and iso-valeric acids. The COD (chemical oxygen demand)

conversion factors of protein, carbohydrate and SCFA were

performed according to Grady et al. (1999). The detailed

analytical procedures of scanning electron microscopy (SEM),

intracellular ROS, Cell counting kit-8 (CCK-8), FISH, protease,

acetate kinase (AK) and coenzyme F420 activities are presented

in Supplementary Information.

2.6. Statistical analysis

All assays were conducted in triplicate and the results were

expressed as mean � standard deviation. An analysis of

variance (ANOVA) was used to test the significance of results

and p < 0.05 was considered to be statistically significant.

Zn2+ (mg/L)ZnO NPs (mg/g-TSS)

0

20

40

60

80

100

1 30 150 1.2 11.6 17.6

)lortnocf

o%(

noitcudorp

enahtem

evitale

R

*

*

*

*

Fig. 2 e Effects of different dosages of ZnO NPs (1, 30 and

150 mg/g-TSS) and the corresponding released Zn2D (1.2,

11.6 and 17.6 mg/L) on methane production during WAS

digestion. Asterisks indicate statistical differences

( p < 0.05) from the control. Error bars represent standard

deviations of triplicate tests.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2e5 6 2 0 5615

3. Results and discussions

3.1. Effect of ZnO nanoparticles on methane production

In this study, the addition of dispersing reagent (SDBS) at

a dosage of 4 mg/g-TSS in sludge digestion experiments or

0.1 mM in synthetic wastewater tests was not observed to

affect the methane production. This observation is consistent

with Garcia et al. (2006). In the coming text, the control repre-

sents the reactor without ZnO NPs addition but with an SDBS

dosage of 4 mg/g-TSS in sludge digestion experiments or

0.1mM in syntheticwastewater tests. As shown in Fig. 2, when

ZnO NPs were added to sludge fermentation system, their

influence on methane production was relevant to the dosage.

At a lower ZnO NPs dosage (1 mg/g-TSS), no inhibitory

effect was observed (Fobserved ¼ 0.05, Fsignificance ¼ 7.71,

P (0.05)¼ 0.83> 0.05).When the dosage of ZnONPswas 30mg/g-

TSS, however, the average methane production decreased to

81.7% of the control, which was further decreased to 24.9% of

the control as the dosage of ZnO NPs increased to 150 mg/g-

Fig. 3 e Scanning electronmicrographs imaging of sludge long-te

(C), and 150 mg/g-TSS (D) of ZnO NPs during WAS anaerobic di

TSS. Apparently, higher concentrations (30 and 150mg/g-TSS)

of ZnONPswere capable of inhibiting themethaneproduction.

Some nanomaterials, such as fullerene, Au, Ag and Fe3O4,

were reported to give marginal influence on anaerobic

community (Barrena et al., 2009; Nyberg et al., 2008). Never-

theless, the Gram-positive B. subtilis was observed to be more

sensitive to ZnO NPs than Gram-negative E. coli. (Adams et al.,

2006), and ZnO NPs were found to negatively affect the soil

bacterial community (Ge et al., 2011). It seems that it is difficult

to figure out the toxicity of ZnO NPs on microorganism

involved in WAS digestion according to the current ZnO NPs

toxicology information as various species of bacteria are in

sludge anaerobic digestion system, and WAS anaerobic

digestion for methane production usually includes sludge

solubilization, hydrolysis, acidification and methanation

(Fig. 1). According to our knowledge, the effects of ZnO NPs on

themicrobial community and each step involved in anaerobic

digestion have never been documented, which will be inves-

tigated in detail in the following text to understand the

mechanisms of ZnONPs affectingmethane production during

WAS anaerobic digestion.

3.2. Effects of ZnO nanoparticles on sludge surface andZn2þ release as well as ROS change

The SEM analysis has been applied in literature to investigate

the adsorption of NPs to sludge (Kiser et al., 2009). As seen in

Fig. 3, there were large numbers of ZnO NPs on the surface of

sludge after long-term exposed to ZnO NPs. The same obser-

vationswere reported by other researchers when the behavior

of NPs in wastewater treatment system was studied (Kiser

et al., 2010; Limbach et al., 2008).

At ZnO NPs dosages of 1, 30 and 150mg/g-TSS, respectively,

the corresponding released Zn2þ concentrations were 1.2, 11.6

and 17.6 mg/L (Fig. S2, Supplementary Information). The long-

term impact of released Zn2þ on methane production during

WAS anaerobic digestion is shown in Fig. 2. The presence of

1.2 mg/L of Zn2þ did not give any significant impact on the

methane production ( p> 0.05). It might be that some chemical

compounds, such as sulfate (11.5 mg/L) in sludge, was bio-

converted to sulfide by sulfate reducing bacterial under anaer-

obic conditions, and then the sulfide reactedwithZn2þ and thus

reduced the toxicity of Zn2þ. However, themethane production

was 90.6% of the control at a Zn2þ concentration of 11.6 mg/L.

When the Zn2þ was 17.6 mg/L, a much lower methane

production (36.2% of the control) was observed. It can be seen

rm exposed to 0mg/g-TSS (A), 1 mg/g-TSS (B), 30 mg/g-TSS

gestion.

1 30 1500

40

80

120

160

200

0

40

80

120

160

200

*

*

*

*

)lortnoc fo %( ytilibaiv ssa

moib evitaleR

)lortnoc fo %( noitcudorp S

OR evitale

R

ZnO NPs (mg/g-TSS)

ROS production Biomass viability

Fig. 4 e Effects of different dosages of ZnO NPs (1, 30 and

150 mg/g-TSS) on the intracellular ROS production and

biomass viability. Asterisks indicate statistical differences

( p < 0.05) from the control. Error bars represent standard

deviations of triplicate tests.

Acetic Propionic iso-Butyric n-Butyric

0

20

40

60

80

100

) L / D

O

C

- g

m

( s n o i t a r t n e c n o

C

Protein Polysaccharide

*

0 6 30 150 0

20

40

60

80

1

*

BSA Dextran

) %

(

y c n e i c i f f e n o i t a d a r g e

D

Innoculum sludge long-term exposed to different dosages of ZnO NPs (mg/g-TSS)

0 6 30 150 0

50

100

150

200 *

*

n o i t c u d o r p e n a h t e

m

e v i t a l u m

u

C

) D

O

C

- g /

L m

(

Innoculum sludge long-term exposed to different dosages of ZnO NPs (mg/g-TSS)

0

200

400

1400

1500

1

n o i t a r t n e c n o c A F

C

S l a u d i v i d n I ) L /

D

O

C

- g m

(

A C

DB

Fig. 5 e Effects of ZnO NPs on each step of sludge anaerobic digestion. A: the concentrations of soluble protein and

carbohydrate during the initial 2 d; B: the degradation of solubilized products (BSA and dextran) with time of 4 d; C: the

concentrations of acidification products (individual SCFA) with time of 4 d; D: themethanation products (methane) at time of

14 d. Asterisks indicate statistical differences ( p < 0.05) from the control. Error bars represent standard deviations of

triplicate tests.

0

20

40

60

80

100

Protease AK F

)lortnocfo

%(e

myznefo

ytivitcaevitale

R

1 mg/g-TSS 30 mg/g-TSS 150 mg/g-TSS

*

**

420

Fig. 6 e Comparisons of the activities of protease, AK and

coenzyme F420 in the long-term operated reactors exposed

to different dosages of ZnO NPs (1, 30 and 150 mg/g-TSS).

Asterisks indicate statistical differences ( p < 0.05) from

the control reactor. Error bars represent standard

deviations of triplicate tests.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2e5 6 2 05616

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2e5 6 2 0 5617

from Fig. 2 that the impact of ZnO NPs onmethane production

mainly resulted fromthedissolvedZn2þ. In a recent publication

Liu et al. (2011) also reported that the released Zn2þ from ZnO

NPs played an important role on the adverse effect of ZnO NPs

on the performance of biological wastewater treatment

process. In the literature the toxicity of ZnO NPs to some

microbeswasalsoobserved tocomefromthe releasedZn2þ, butthose studies focused on cell growth instead of microbial

function (Franklin et al., 2007;Wong et al., 2010; Xia et al., 2008).

Fig. 7 e Fluorescence in situ hybridization of sectios of biomass

absence of ZnO NPs (A1-A3) and in the presence of 1 mg/gTSS (

(D1-D3) viewed by CLSM and photographed at higher (362) mag

with Cy-3-labeled bacterial-domain probe (EUB338) (red) and FIT

of ARC915 (green) and EUB338 (red) are shown in A3, B3, C3 an

figure legend, the reader is referred to the web version of this a

The ROS induced by ZnONPswas reported to be one reason

for their toxicity, which caused the loss of cell viability (Xia

et al., 2008). ZnO NPs were regarded as an exogenous source

of ROS for cells or organisms in some previous reports (Joshi et

al., 2009; Xia et al., 2008). As seen in Fig. 4, an increase of the

intracellular ROS production was observed with the increase

of ZnO NPs. Usually, ROS, including superoxide (O2�-),

hydrogen peroxide (H2O2), and the hydroxyl radical (OH�), are

produced in the presence of oxygen (Murphy, 2009). However,

long-term (more than 105 d) cultured respectively in the

B1-B3), 30 mg/g-TSS (C1-C3) and 150 mg/g-TSS of ZnO NPs

nification. The sections were simultaneously hybridized

C-labeled archaeal-domain probe (ARC915) (green). Overlay

d D3. (For interpretation of the references to colour in this

rticle.)

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2e5 6 2 05618

it has been reported that H2O2 can also be produced under

anaerobic conditions (Degli-Esposti and McLennan, 1998). The

increase of ROS in the sludge exposed to higher dosages of

ZnO NPs was a likely reason for their adverse effect on sludge

anaerobic digestion. It can be seen from Fig. 4 that the result of

biomass viability assay was consistent with the ROS produc-

tion, which decreased from 97.3% of the control ( p > 0.05) to

88.7% of the control when ZnO NPs increased from 1 to 30 mg/

g-TSS. At ZnO NPs dosage of 150 mg/g-TSS, the relative

biomass viability further decreased to 62.4% of the control.

3.3. Effects of ZnO nanoparticles on each step involvedin methane production

Protein and carbohydrate, the main constituents of WAS

(accounting for 61.5% of sludge TCOD), are usually in partic-

ulate state. The batch experiments were conducted to inves-

tigate the long-term effects of ZnO NPs on sludge particulate

protein and carbohydrate solubilization. The effects of ZnO

NPs on solubilization of sludge particulate organic matters

were expressed by the changes of soluble protein and carbo-

hydrate production in this study. As seen from Fig. 5A, there

were no significant differences in the concentrations of

soluble protein and carbohydrate after the initial 2 d

fermentation ( p > 0.05). It might be that the solubilization of

sludge particulate organic matters was not a microbial

process, which resulted in the influences of ZnO NPs on the

concentrations of soluble protein and carbohydrate not being

observed.

The long-term effects of three dosages of ZnO NPs on the

hydrolysis of sludge solubilized products (soluble protein and

carbohydrate) with time of 4 d are shown in Fig. 5B. The

degradation of dextran (model carbohydrate mater) in the

control reactor was almost the same as those in other three

ZnO NPs reactors. Nevertheless, the influence of ZnO NPs on

the degradation of BSA (model protein) was dosage depen-

dent. At dosages of 1 and 30 mg/g-TSS, the influences of ZnO

NPs were insignificant ( p > 0.05), but the degradation of BSA

at 150 mg/g-TSS of ZnO NPs was lower than that in the

control (58.5% versus 65.1%). It might be one reason for the

decreased methane production exposed to higher concen-

trations of ZnO NPs.

Fig. 5C illustrates the long-term effects of different

concentrations of ZnO NPs on the acidification of main

hydrolyzed products (amino acid and monosaccharide) to

SCFA during the initial 4 d. The influences of ZnO NPs on the

composition of SCFA were insignificant (see Table S1 for

statistical analysis, Supplementary Information). The total

SCFA concentrations, which calculated from Fig. 5C, were

2078 � 80, 2057 � 80, 2045 � 69 and 2050 � 50 mg-COD/L in the

reactors of control, and 1, 30 and 150 mg/g-TSS of ZnO NPs,

respectively. Obviously, the acidification step involved in

sludge digestion was not affected by ZnO NPs.

As to the influence of ZnONPs on themethanation step, the

data in Fig. 5D indicated that there was no significant differ-

ence in the cumulative methane production between the

control and the 1 mg/g-TSS of ZnO NPs reactors at time of 14 d

( p > 0.05). However, the methane productions were 83.0% and

28.1% of the control at 30 and 150 mg/g-TSS of ZnO NPs,

respectively, suggesting that ZnO NPs significantly inhibited

the bio-conversion step of acetic acid tomethane. At other time

the same observations could be made (Fig. S3, Supplementary

Information). By comparing the data in Fig. 5C and D, it

seems that methanogens are more sensitive to the toxicity of

ZnO NPs than acidogens. In the literature, some researchers

reported that acidogens were more resistant to metal toxicity

than methanogens (Zayed and Winter, 2000). In addition, the

data in Fig. 5AeD suggested that the negative influence of ZnO

NPs on themethanation step was the most serious one among

the four steps.

3.4. Determination of key enzyme activity

Further investigation showed that ZnO NPs influenced the

activities of enzymes relevant to sludge anaerobic digestion.

Although large numbers of enzymes took part in methane

production during sludge anaerobic digestion, in this study

only three enzymes responsible respectively for sludge

hydrolysis (i.e., protease), acidification (AK) and methanation

(coenzyme F420) (Fig. 1) were assayed as examples. The relative

activities of these enzymes in the long-term operated reactors

are demonstrated in Fig. 6. The AK activity did not change

significantly with ZnONPs dosages ( p> 0.05). To the protease,

the dosage of 150mg/g-TSS of ZnONPs remarkably reduced its

activity. The coenzyme F420 activity, however, was ZnO NPs

dosage dependent, which was respectively 99.3%, 89.8% and

66.2% of the control at ZnO NPs of 1, 30 and 150 mg/g-TSS.

Apparently, not only the hydrolysis of soluble protein but the

transformation activity of electron donors of the redox-driven

proton translocation in methanogenic Archaea (expressed by

coenzyme F420 (Deppenmeier, 2002)) was significantly

restrained by higher concentrations of ZnO NPs. All these

consisted well with the above observed synthetic wastewater

experimental results.

3.5. FISH analysis results

For the purpose of investigating the influence of ZnO NPs on

the abundance of bacteria and Archaea, the FISH assay was

further conducted (Fig. 7), and the results were analyzed with

image analysis system (Image-Pro Plus, V6.0). It was found

that there were 39.5% of Archaea and 52.6% of bacteria in the

control reactor. In ZnO NPs reactors, the Archaeawere 38.6% (1

mg/g-TSS), 27.1% (30 mg/g-TSS), and 3.5% (150 mg/g-TSS), and

the corresponding bacteria were 51.3%, 60.8% and 87.4%,

respectively. The ratios of Archaea to bacteria were 0.8:1, 0.9:1,

0.4:1 and 0.04:1, respectively, in the reactors of control, and 1,

30 and 150 mg/g-TSS of ZnO NPs, respectively. Obviously, the

more ZnONPs appeared in sludge anaerobic digestion system,

the less Archaea remained, which was consistent with the

observed methane production when WAS was long-term

exposed to different dosages of ZnO NPs.

4. Conclusions

The above studies indicated that the methane production

during sludge anaerobic digestion was not affected by ZnO

NPs of 1mg/g-TSS. Nevertheless, due to large numbers of Zn2þ

release the higher dosages (30 and 150 mg/g-TSS) of ZnO NPs

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2e5 6 2 0 5619

inhibited the production of methane. By investigating the four

stages involved in sludge anaerobic digestion, i.e., solubiliza-

tion, hydrolysis, acidification and methanation, it was found

that the activities of protease and coenzyme F420 were nega-

tively influenced by higher dosages of ZnO NPs, which sug-

gested that only the steps of hydrolysis andmethanationwere

inhibited. The molecular biology studies indicated that

a lower abundance of methanogenesis Archaea was observed

at higher ZnO NPs dosages exposure.

Acknowledgements

This work was financially supported by the Foundation of

State Key Laboratory of Pollution Control and Resource Reuse

(PCRRK09002).

Appendix. Supplementary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.watres.2011.08.022.

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