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Long-term effect of ZnO nanoparticles on waste activated sludge anaerobic digestion
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Long-term effect of ZnO nanoparticles on waste activated sludge 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
article info
abstract
Article history:
The increasing use of zinc oxide nanoparticles (ZnO NPs) raises concerns about their
Received 9 May 2011
environmental impacts, but the potential effect of ZnO NPs on sludge anaerobic digestion
Received in revised form
remains unknown. In this paper, long-term exposure experiments were carried out to
10 August 2011
investigate the influence of ZnO NPs on methane production during waste activated sludge
Accepted 14 August 2011
(WAS) anaerobic digestion. The presence of 1 mg/g-TSS of ZnO NPs did not affect methane
Available online 23 August 2011
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
Keywords:
dosage dependant. Then, the mechanisms of ZnO NPs affecting sludge anaerobic digestion
Zinc oxide nanoparticles
were investigated. It was found that the toxic effect of ZnO NPs on methane production
Waste activated sludge
was mainly due to the release of Zn2þ from ZnO NPs, which may cause the inhibitory
Anaerobic digestion
effects on the hydrolysis and methanation steps of sludge anaerobic digestion. Further
Mechanisms
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
With the rapid development of nanotechnology, nanoparticles (NPs) are now widely 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 of NPs from discover source to environment receptor, and some NPs have been found in wastewater 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 of metal oxide NPs, have received increasing interest due to their widespread industrial, medical and military applications (Ellsworth et al., 2000; Miziolek,
2002; Serda et al., 2009). There are some publications discussing the toxicity of ZnO NPs on microbes. 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 composition 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
* Corresponding author. Tel.: þ86 21 65981263; fax: þ86 21 65986313. E-mail address: [email protected] (Y. Chen). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.08.022
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are produced in municipal WWTPs, which need to be treated before being discharged to the environment. It can be anticipated that most of ZnO NPs will enter into sludge treatment system. Among several sludge treatment methods anaerobic digestion for methane is a preferred one because of WAS 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 digestion and to explore the mechanisms. Furthermore, fluorescence in situ hybridization (FISH) technique with 16S rRNAtargeted 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 ZnO NPs was 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 ZnO NPs was approximately 140 20 nm on 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. The sludge was concentrated by settling at 4 C for 24 h, and its main characteristics (average data plus standard deviations of triplicate tests) are as follows: pH 6.7 0.2, total
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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 220 mg/L, total carbohydrate 899 530 mg-COD/L, total protein 5685 149 mg-COD/L, and total zinc 0.8 0.2 mg/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 withdrawn and centrifuged at 12000 rpm for 30 min, and the supernatant was collected, and filtered through 0.22 mm mixed cellulose ester membrane (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 and their released Zn2þ on WAS anaerobic digestion for methane production Three dosages (1, 30 and 150 mg/g-TSS) of ZnO NPs were 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 scientists suggested that although lower nanomaterial content (50 mg C60/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 reference (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 SDBS was used as the dispersing reagent in this study, two controls, one with only sludge, and another one with 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 105 d, the daily methane production did not 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 according to our previous publication (Zhao et al., 2010).
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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 involved in 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 longterm effects of ZnO NPs on the hydrolysis of sludge solubilized 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 consisted 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 1200 mL synthetic wastewater, the mixture 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 4 M NaOH or 4 M HCl. 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 longterm 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 products 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 acid was the main short-chain fatty acid (SCFA) of sludge acidification product (Yuan et al., 2006) and the preferred substrate for methane production (Fig. 1), the longterm effect of ZnO NPs on methanation of the acidification product was conducted with 300 mL synthetic wastewater (see the above description) containing 0.72 g sodium acetate (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 concentration 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.
Particulate organic matters of waste active sludge
Solubilization
Solubilization
Soluble polysaccharide
Soluble protein protease
Hydrolysis
Amino acids
Monosaccharide
Hydrolysis
Pyruvic acid Acidification
Acetyl-CoA AK
Propionic acid
Acetic acid
Methanation F420
Methane
Butyric acid Carbon dioxide
Hydrogen
F420
Fig. 1 e Proposed metabolic pathway for methane production from WAS anaerobic digestion. Only the key enzymes assayed in this study are labeled.
Relativ e m eth an e p ro d uction ( % o f co ntro l)
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*
100
* 80
60
* 40
* 20
0 1
30
150
1.2
ZnO NPs (mg/g-TSS)
11.6
17.6
Zn2+ (mg/L)
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.
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 represents the reactor without ZnO NPs addition but with an SDBS dosage of 4 mg/g-TSS in sludge digestion experiments or 0.1 mM in synthetic wastewater 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 ZnO NPs was 30 mg/gTSS, 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-
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TSS. Apparently, higher concentrations (30 and 150 mg/g-TSS) of ZnO NPs were capable of inhibiting the methane production. 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). Nevertheless, 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 the microbial community and each step involved in anaerobic digestion have never been documented, which will be investigated in detail in the following text to understand the mechanisms of ZnO NPs affecting methane production during WAS anaerobic digestion.
3.2. Effects of ZnO nanoparticles on sludge surface and Zn2þ 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 observations were 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 150 mg/g-TSS, respectively, the corresponding released Zn2þ concentrations were 1.2, 11.6 and 17.6 mg/L (Fig. S2, Supplementary Information). The longterm 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 bioconverted to sulfide by sulfate reducing bacterial under anaerobic conditions, and then the sulfide reacted with Zn2þ and thus reduced the toxicity of Zn2þ. However, the methane 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
Fig. 3 e Scanning electron micrographs imaging of sludge long-term exposed to 0 mg/g-TSS (A), 1 mg/g-TSS (B), 30 mg/g-TSS (C), and 150 mg/g-TSS (D) of ZnO NPs during WAS anaerobic digestion.
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160
*
120
120
* 80
80
*
40
40
0
Relativ e activ ity o f en zy m e ( % o f co nt r o l)
160
200
*
ROS production Biomass viability
Relative biomass viability (% of control)
Relative ROS production (% of control)
200
1 mg/g-TSS
30 mg/g-TSS
150 mg/g-TSS
*
100
* 80
*
60
40
20
0 1
30
150
0 Protease
ZnO NPs (mg/g-TSS)
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.
A
Protein
1500
40
20
0 80
Degradation efficiency (%)
Individual SCFA concentration (mg-COD / L)
60
B
D
200
*
*
40
20
0
61
30
150
Innoculum sludge long-term exposed to different dosages of ZnO NPs (mg/g-TSS)
Propionic n-Butyric
200
Dextran
60
0
Acetic iso-Butyric
400
0
BSA
C
1400
Cumulative methane production (mL / g-COD )
Concentrations (mg-COD/L)
80
F 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.
Polysaccharide
100
AK
*
150
100
* 50
0
0
16
30
150
Innoculum sludge long-term exposed to different dosages of ZnO NPs (mg/g-TSS)
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: the methanation 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.
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from Fig. 2 that the impact of ZnO NPs on methane production mainly resulted from the dissolved Zn2þ. 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 microbes was also observed to come from the released Zn2þ, but those studies focused on cell growth instead of microbial function (Franklin et al., 2007; Wong et al., 2010; Xia et al., 2008).
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The ROS induced by ZnO NPs was 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,
Fig. 7 e Fluorescence in situ hybridization of sectios of biomass long-term (more than 105 d) cultured respectively in the absence of ZnO NPs (A1-A3) and in the presence of 1 mg/gTSS (B1-B3), 30 mg/g-TSS (C1-C3) and 150 mg/g-TSS of ZnO NPs (D1-D3) viewed by CLSM and photographed at higher (362) magnification. The sections were simultaneously hybridized with Cy-3-labeled bacterial-domain probe (EUB338) (red) and FITC-labeled archaeal-domain probe (ARC915) (green). Overlay of ARC915 (green) and EUB338 (red) are shown in A3, B3, C3 and D3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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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 production, 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.
the bio-conversion step of acetic acid to methane. 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 the methanation step was the most serious one among the four steps.
3.3. Effects of ZnO nanoparticles on each step involved in methane production
3.4.
Protein and carbohydrate, the main constituents of WAS (accounting for 61.5% of sludge TCOD), are usually in particulate state. The batch experiments were conducted to investigate 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 carbohydrate 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 dependent. 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 concentrations 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 ZnO NPs on the methanation step, the data in Fig. 5D indicated that there was no significant difference 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
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 ZnO NPs dosages ( p > 0.05). To the protease, the dosage of 150 mg/g-TSS of ZnO NPs 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 Archaea were 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 ZnO NPs 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 1 mg/g-TSS. Nevertheless, due to large numbers of Zn2þ release the higher dosages (30 and 150 mg/g-TSS) of ZnO NPs
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inhibited the production of methane. By investigating the four stages involved in sludge anaerobic digestion, i.e., solubilization, hydrolysis, acidification and methanation, it was found that the activities of protease and coenzyme F420 were negatively influenced by higher dosages of ZnO NPs, which suggested that only the steps of hydrolysis and methanation were 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.
references
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Ge, Y., Schimel, J.P., Holden, P.A., 2011. Evidence for negative effects of TiO2 and ZnO nanoparticles on soil bacterial communities. Environmental Science and Technology 45 (4), 1659e1664. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for different regions. Environmental Science and Technology 43 (24), 9216e9222. Grady, C.P.L., Daigger, G.T., Lim, H.C., 1999. Biological Wastewater Treatment. Marcel Dekker Inc., New York. Jiang, W., Mashayekhi, H., Xing, B., 2009. Bacterial toxicity comparison between nano- and micro-scaled oxide particles. Environmental Pollution 157 (5), 1619e1625. Joshi, P., Chakraborti, S., Chakrabarti, P., Haranath, D., Shanker, V., Ansari, Z.A., Singh, S.P., Gupta, V., 2009. Role of surface adsorbed anionic species in antibacterial activity of ZnO quantum dots against Escherichia coli. Journal of Nanoscience and Nanotechnology 9 (11), 6427e6433. Keller, A.A., Wang, H.T., Zhou, D.X., Lenihan, H.S., Cherr, G., Cardinale, B.J., Miller, R., Ji, Z.X., 2010. Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environmental Science and Technology 44 (6), 1962e1967. Kiser, M.A., Ryu, H., Jang, H.Y., Hristovski, K., Westerhoff, P., 2010. Biosorption of nanoparticles to heterotrophic wastewater biomass. Water Research 44 (14), 4105e4114. Kiser, M.A., Westerhoff, P., Benn, T., Wang, Y., Perez-Rivera, J., Hristovski, K., 2009. Titanium nanomaterial removal and release from wastewater treatment plants. Environmental Science and Technology 43 (17), 6757e6763. Li, M., Zhu, L., Lin, D., 2011. Toxicity of ZnO nanoparticles to Escherichia coli: mechanism and the influence of medium components. Environmental Science and Technology 45 (5), 1977e1983. Limbach, L.K., Bereiter, R., Mueller, E., Krebs, R., Gaelli, R., Stark, W.J., 2008. Removal of oxide nanoparticles in a model wastewater treatment plant: influence of agglomeration and surfactants on clearing efficiency. Environmental Science and Technology 42 (15), 5828e5833. Liu, G.Q., Wang, D.M., Wang, J.M., Mendoza, C., 2011. Effect of ZnO particles on activated sludge: Role of particle dissolution. Science of the Total Environment 409 (14), 2852e2857. Maynard, A.D., Aitken, R.J., Butz, T., Colvin, V., Donaldson, K., Oberdorster, G., Philbert, M.A., Ryan, J., Seaton, A., Stone, V., Tinkle, S.S., Tran, L., Walker, N.J., Warheit, D.B., 2006. Safe handling of nanotechnology. Nature 444 (7117), 267e269. Miziolek, A., 2002. Nanoenergetics: an emerging technology area of national importance. AMPTIAC Quarterly 6 (1), 43e48. Murphy, M.P., 2009. How mitochondria produce reactive oxygen species. Biochemical Journal 417, 1e13. Nyberg, L., Turco, R.F., Nies, L., 2008. Assessing the impact of nanomaterials on anaerobic microbial communities. Environmental Science and Technology 42 (6), 1938e1943. Oh, S.E., Van Ginkel, S., Logan, B.E., 2003. The relative effectiveness of pH control and heat treatment for enhancing biohydrogen gas production. Environmental Science and Technology 37 (22), 5186e5190. Roco, M.C., 2005. The emergence and policy implications of converging new technologies integrated from the nanoscale. Journal of Nanoparticle Research 7 (2e3), 129e143. Serda, R.E., Ferrati, S., Godin, B., Tasciotti, E., Liu, X.W., Ferrari, M., 2009. Mitotic trafficking of silicon microparticles. Nanoscale 1 (2), 250e259. Sharma, V., Shukla, R.K., Saxena, N., Parmar, D., Das, M., Dhawan, A., 2009. DNA damaging potential of zinc oxide nanoparticles in human epidermal cells. Toxicology Letters 185 (3), 211e218. Simon-Deckers, A., Loo, S., Mayne-L’Hermite, M., HerlinBoime, N., Menguy, N., Reynaud, C., Gouget, B., Carriere, M.,
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Long-term effect of ZnO nanoparticles on waste activated sludge 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
article info
abstract
Article history:
The increasing use of zinc oxide nanoparticles (ZnO NPs) raises concerns about their
Received 9 May 2011
environmental impacts, but the potential effect of ZnO NPs on sludge anaerobic digestion
Received in revised form
remains unknown. In this paper, long-term exposure experiments were carried out to
10 August 2011
investigate the influence of ZnO NPs on methane production during waste activated sludge
Accepted 14 August 2011
(WAS) anaerobic digestion. The presence of 1 mg/g-TSS of ZnO NPs did not affect methane
Available online 23 August 2011
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
Keywords:
dosage dependant. Then, the mechanisms of ZnO NPs affecting sludge anaerobic digestion
Zinc oxide nanoparticles
were investigated. It was found that the toxic effect of ZnO NPs on methane production
Waste activated sludge
was mainly due to the release of Zn2þ from ZnO NPs, which may cause the inhibitory
Anaerobic digestion
effects on the hydrolysis and methanation steps of sludge anaerobic digestion. Further
Mechanisms
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
With the rapid development of nanotechnology, nanoparticles (NPs) are now widely 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 of NPs from discover source to environment receptor, and some NPs have been found in wastewater 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 of metal oxide NPs, have received increasing interest due to their widespread industrial, medical and military applications (Ellsworth et al., 2000; Miziolek,
2002; Serda et al., 2009). There are some publications discussing the toxicity of ZnO NPs on microbes. 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 composition 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
* Corresponding author. Tel.: þ86 21 65981263; fax: þ86 21 65986313. E-mail address: [email protected] (Y. Chen). 0043-1354/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2011.08.022
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2 e5 6 2 0
are produced in municipal WWTPs, which need to be treated before being discharged to the environment. It can be anticipated that most of ZnO NPs will enter into sludge treatment system. Among several sludge treatment methods anaerobic digestion for methane is a preferred one because of WAS 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 digestion and to explore the mechanisms. Furthermore, fluorescence in situ hybridization (FISH) technique with 16S rRNAtargeted 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 ZnO NPs was 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 ZnO NPs was approximately 140 20 nm on 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. The sludge was concentrated by settling at 4 C for 24 h, and its main characteristics (average data plus standard deviations of triplicate tests) are as follows: pH 6.7 0.2, total
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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 220 mg/L, total carbohydrate 899 530 mg-COD/L, total protein 5685 149 mg-COD/L, and total zinc 0.8 0.2 mg/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 withdrawn and centrifuged at 12000 rpm for 30 min, and the supernatant was collected, and filtered through 0.22 mm mixed cellulose ester membrane (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 and their released Zn2þ on WAS anaerobic digestion for methane production Three dosages (1, 30 and 150 mg/g-TSS) of ZnO NPs were 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 scientists suggested that although lower nanomaterial content (50 mg C60/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 reference (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 SDBS was used as the dispersing reagent in this study, two controls, one with only sludge, and another one with 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 105 d, the daily methane production did not 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 according to our previous publication (Zhao et al., 2010).
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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 involved in 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 longterm effects of ZnO NPs on the hydrolysis of sludge solubilized 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 consisted 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 1200 mL synthetic wastewater, the mixture 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 4 M NaOH or 4 M HCl. 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 longterm 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 products 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 acid was the main short-chain fatty acid (SCFA) of sludge acidification product (Yuan et al., 2006) and the preferred substrate for methane production (Fig. 1), the longterm effect of ZnO NPs on methanation of the acidification product was conducted with 300 mL synthetic wastewater (see the above description) containing 0.72 g sodium acetate (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 concentration 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.
Particulate organic matters of waste active sludge
Solubilization
Solubilization
Soluble polysaccharide
Soluble protein protease
Hydrolysis
Amino acids
Monosaccharide
Hydrolysis
Pyruvic acid Acidification
Acetyl-CoA AK
Propionic acid
Acetic acid
Methanation F420
Methane
Butyric acid Carbon dioxide
Hydrogen
F420
Fig. 1 e Proposed metabolic pathway for methane production from WAS anaerobic digestion. Only the key enzymes assayed in this study are labeled.
Relativ e m eth an e p ro d uction ( % o f co ntro l)
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*
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* 80
60
* 40
* 20
0 1
30
150
1.2
ZnO NPs (mg/g-TSS)
11.6
17.6
Zn2+ (mg/L)
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.
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 represents the reactor without ZnO NPs addition but with an SDBS dosage of 4 mg/g-TSS in sludge digestion experiments or 0.1 mM in synthetic wastewater 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 ZnO NPs was 30 mg/gTSS, 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-
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TSS. Apparently, higher concentrations (30 and 150 mg/g-TSS) of ZnO NPs were capable of inhibiting the methane production. 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). Nevertheless, 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 the microbial community and each step involved in anaerobic digestion have never been documented, which will be investigated in detail in the following text to understand the mechanisms of ZnO NPs affecting methane production during WAS anaerobic digestion.
3.2. Effects of ZnO nanoparticles on sludge surface and Zn2þ 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 observations were 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 150 mg/g-TSS, respectively, the corresponding released Zn2þ concentrations were 1.2, 11.6 and 17.6 mg/L (Fig. S2, Supplementary Information). The longterm 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 bioconverted to sulfide by sulfate reducing bacterial under anaerobic conditions, and then the sulfide reacted with Zn2þ and thus reduced the toxicity of Zn2þ. However, the methane 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
Fig. 3 e Scanning electron micrographs imaging of sludge long-term exposed to 0 mg/g-TSS (A), 1 mg/g-TSS (B), 30 mg/g-TSS (C), and 150 mg/g-TSS (D) of ZnO NPs during WAS anaerobic digestion.
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Relativ e activ ity o f en zy m e ( % o f co nt r o l)
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*
ROS production Biomass viability
Relative biomass viability (% of control)
Relative ROS production (% of control)
200
1 mg/g-TSS
30 mg/g-TSS
150 mg/g-TSS
*
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* 80
*
60
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30
150
0 Protease
ZnO NPs (mg/g-TSS)
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.
A
Protein
1500
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0 80
Degradation efficiency (%)
Individual SCFA concentration (mg-COD / L)
60
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*
*
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Innoculum sludge long-term exposed to different dosages of ZnO NPs (mg/g-TSS)
Propionic n-Butyric
200
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400
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BSA
C
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Cumulative methane production (mL / g-COD )
Concentrations (mg-COD/L)
80
F 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.
Polysaccharide
100
AK
*
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* 50
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0
16
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Innoculum sludge long-term exposed to different dosages of ZnO NPs (mg/g-TSS)
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: the methanation 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.
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2 e5 6 2 0
from Fig. 2 that the impact of ZnO NPs on methane production mainly resulted from the dissolved Zn2þ. 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 microbes was also observed to come from the released Zn2þ, but those studies focused on cell growth instead of microbial function (Franklin et al., 2007; Wong et al., 2010; Xia et al., 2008).
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The ROS induced by ZnO NPs was 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,
Fig. 7 e Fluorescence in situ hybridization of sectios of biomass long-term (more than 105 d) cultured respectively in the absence of ZnO NPs (A1-A3) and in the presence of 1 mg/gTSS (B1-B3), 30 mg/g-TSS (C1-C3) and 150 mg/g-TSS of ZnO NPs (D1-D3) viewed by CLSM and photographed at higher (362) magnification. The sections were simultaneously hybridized with Cy-3-labeled bacterial-domain probe (EUB338) (red) and FITC-labeled archaeal-domain probe (ARC915) (green). Overlay of ARC915 (green) and EUB338 (red) are shown in A3, B3, C3 and D3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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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 production, 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.
the bio-conversion step of acetic acid to methane. 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 the methanation step was the most serious one among the four steps.
3.3. Effects of ZnO nanoparticles on each step involved in methane production
3.4.
Protein and carbohydrate, the main constituents of WAS (accounting for 61.5% of sludge TCOD), are usually in particulate state. The batch experiments were conducted to investigate 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 carbohydrate 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 dependent. 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 concentrations 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 ZnO NPs on the methanation step, the data in Fig. 5D indicated that there was no significant difference 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
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 ZnO NPs dosages ( p > 0.05). To the protease, the dosage of 150 mg/g-TSS of ZnO NPs 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 Archaea were 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 ZnO NPs 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 1 mg/g-TSS. Nevertheless, due to large numbers of Zn2þ release the higher dosages (30 and 150 mg/g-TSS) of ZnO NPs
w a t e r r e s e a r c h 4 5 ( 2 0 1 1 ) 5 6 1 2 e5 6 2 0
inhibited the production of methane. By investigating the four stages involved in sludge anaerobic digestion, i.e., solubilization, hydrolysis, acidification and methanation, it was found that the activities of protease and coenzyme F420 were negatively influenced by higher dosages of ZnO NPs, which suggested that only the steps of hydrolysis and methanation were 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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