Effects of metal oxide nanoparticles (TiO2, Al2O3, SiO2 and ZnO) on waste activated sludge anaerobic digestion

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<ul><li><p>2,</p><p>l Scie</p><p>anobyna</p><p>torynerer,d Zs fo</p><p> 2011 Elsevier Ltd. All rights reserved.</p><p>y havel proptricitye nanxide (receivmedicaek, 20</p><p>release. The existence of nanomaterials in WWTPs has been re-ported (Gottschalk et al., 2009; Kiser et al., 2009), and the adsorp-tion of activated sludge was reported to be the main mechanismfor nanoparticles removal in WWTPs (Ganesh et al., 2010; Kiseret al., 2009, 2010; Limbach et al., 2008). Therefore, nanoparticleswould eventually end up in sludge. Large amounts of WAS are pro-duced in municipal WWTPs, which need to be treated before being</p><p>The purpose of this study was to investigate the inuences offour metal oxide nanoparticles (TiO2, Al2O3, SiO2 and ZnO) onmethane generation during sludge anaerobic digestion and to digout the mechanisms. Firstly, the effects of three dosages (6, 30and 150 mg/g-TSS) of these four nanoparticles on methane gener-ation were studied when WAS was anaerobically digested in batchtests. Then, the mechanisms for nanoparticles affecting methanegeneration were investigated from the role of dissolved metal ionsand the changes of products and key enzymes involved each stage(solubilization, hydrolysis, acidication and methanation) ofsludge anaerobic digestion.</p><p> Corresponding author. Tel.: +86 21 65981263; fax: +86 21 65986313.</p><p>Bioresource Technology 102 (2011) 1030510311</p><p>Contents lists available at</p><p>T</p><p>elsE-mail address: yg2chen@yahoo.com (Y. Chen).With the world wide utilization of these metal oxide nanoparticles,their potential effects on environment have been investigated, butmost studies focused on the toxicity to human health, and soil andaquatic organisms (Franklin et al., 2007; Ge et al., 2011; Limbachet al., 2007).</p><p>The increasing use of nanoparticles introduces them intention-ally or unintentionally into wastewater treatment plants (WWTPs),which are the last barriers prior to nanoparticles waste (causedmainly by both daily activities and industrial use) environmental</p><p>nanoparticles on B. subtilis and E. coli increased from SiO2 to TiO2to ZnO. Nano-ZnO was observed to cause signicant toxicity tothe viability of gram negative bacterial cells (Sinha et al., 2011).It is well known that large numbers of different microorganismsparticipate in sludge anaerobic digestion due to several stages (sol-ubilization, hydrolysis, acidication and methanation) involved.Thus, it is impossible to deduce the negative effect of these metaloxide nanoparticles on sludge anaerobic digestion microorganismsaccording to the current knowledge of pure microbial species.1. Introduction</p><p>Researches about nanotechnologdue to the unique physicochemicasuch as enhanced magnetism, elecet al., 2006; Roco, 2005). Metal oxidnium dioxide (TiO2), aluminum o(SiO2) and zinc oxide (ZnO), havedue to their widespread industrial,tions (Ellsworth et al., 2000; Miziol0960-8524/$ - see front matter 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.08.100drawn much attentionerties of nanoparticles,, and optics (Maynardoparticles, such as tita-Al2O3), silicon dioxideed increasing interestsl and military applica-02; Serda et al., 2009).</p><p>discharged to the environment. WAS anaerobic digestion for meth-ane generation is a sustainable sludge treatment practice in whichboth pollution control and energy (methane) recovery can beachieved. Nevertheless the inuences of metal oxide nanoparticleson sludge anaerobic digestion have seldom been investigated.</p><p>There are some publications discussing the toxicity of metaloxide nanoparticles to pure microbes. For example, nano-Al2O3,nano-SiO2 and nano-ZnO were observed to be harmful to Bacillussubtilis, Escherichia coli and Pseudomonas uorescens (Jiang et al.,2009). Adams et al. (2006) found that the antibacterial effects ofAnaerobic digestionMethane</p><p>lysis, acidication and methanation. Also, the activities of protease, acetate kinase (AK) and coenzymeF420 were inhibited by higher dosages of nano-ZnO during WAS anaerobic digestion.Effects of metal oxide nanoparticles (TiOactivated sludge anaerobic digestion</p><p>Hui Mu, Yinguang Chen , Naidong XiaoState Key Laboratory of Pollution Control and Resources Reuse, School of Environmenta</p><p>a r t i c l e i n f o</p><p>Article history:Received 1 June 2011Received in revised form 28 July 2011Accepted 22 August 2011Available online 27 August 2011</p><p>Keywords:Metal oxide nanoparticlesWaste activated sludge</p><p>a b s t r a c t</p><p>The effect of metal oxide ndigestion was investigatedNano-TiO2, nano-Al2O3 andg-TSS) showed no inhibiincreased. The methane geg-TSS of nano-ZnO, howev150 mg/g-TSS. The releasemethane generation. It wa</p><p>Bioresource</p><p>journal homepage: www.ll rights reserved.Al2O3, SiO2 and ZnO) on waste</p><p>nce and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China</p><p>particles (nano-TiO2, nano-Al2O3, nano-SiO2 and nano-ZnO) on anaerobicfermentation experiments using waste activated sludge as the substrates.no-SiO2 in doses up to 150 milligram per gram total suspended solids (mg/effect, whereas nano-ZnO showed inhibitory effect with its dosages</p><p>ation was the same as that in the control when in the presence of 6 mg/which decreased respectively to 77.2% and 18.9% of the control at 30 andn2+ from nano-ZnO was an important reason for its inhibitory effect onund that higher dosages of nano-ZnO inhibited the steps of sludge hydro-</p><p>SciVerse ScienceDirect</p><p>echnology</p><p>evier .com/locate /bior tech</p></li><li><p>nolo2. Methods</p><p>2.1. Waste activated sludge: origin and chemical properties</p><p>The WAS used in this study was withdrawn from the secondarysedimentation tank of a municipal WWTP in Shanghai, China. Thesludge was concentrated by settling at 4 C for 24 h, and its maincharacteristics (average data and standard deviations of three tests)are as follows: pH 6.7 0.2, total suspended solids (TSS)10,070 780 mg/L, volatile suspended solids (VSS) 7700 450 mg/L, soluble chemical oxygen demand (SCOD) 90 14 mg/L, totalchemical oxygen demand (TCOD) 10,700 200 mg/L, total carbohy-drate 900 530 mg-COD/L, and total protein 5685 150 mg-COD/L.The natural concentrations of titanium, aluminum, silicon and zincin the WAS used in this study were 3.4 0.2, 14.7 0.9, 52.5 3.5and 0.8 0.2 mg/g-TSS, respectively.</p><p>2.2. Metal oxide nanoparticles and their dissolved metal ions</p><p>Nano-TiO2 (</p></li><li><p>mentation time of 18 d in the presence of 30 and 150 mg/g-TSS of</p><p>nolowas almost the same as that in WAS). Then the mixed liquid wasdivided into 13 bottles, and the heat-pretreated WAS of 30 mLwas added to each bottle with a nal sludge concentration of1000 mg/L. According to the above described nanoparticles addi-tion dosages, the suitable dosages of nano-TiO2, nano-Al2O3,nano-SiO2 and nano-ZnO were respectively added to each bottlewith a nal dosage of 6, 30 and 150 mg/L before the pH of mixedliquid was adjusted to 7.0 by adding 4 M NaOH or 4 M HCl. Afterbeing ushed with nitrogen gas to remove oxygen, all bottles werecapped with rubber stoppers, sealed and placed in an air-bathshaker (150 rpm) at 35 1 C. By analyzing the degradationefciencies of BSA and dextran, the impact of nanoparticles onsludge hydrolysis was achieved.</p><p>The same operation was conducted when the effect of nanopar-ticles on the acidication of hydrolyzed products was investigatedexcept that the 3510 mL synthetic wastewater containing 15.6 g L-glutamate (model amino acid compound) and 3.9 g glucose (modelmonosaccharide compound). The inuence of nanoparticles onacidication was obtained by measuring the concentration of gen-erated short-chain fatty acid (SCFA). The operation of nanoparticlesaffecting the methanation of acidication products was conductedwith the samemethod as described above except that the syntheticwastewater (3510 mL) containing 9.36 g sodium acetate (modelSCFA compound in this study) and the inocula was 30 mL rawWAS. By the analysis of methane generation, the effect of nanopar-ticles on methanation was obtained.</p><p>2.5. Scanning electron microscopy</p><p>The surface morphology of WAS exposed to nanoparticles wascharacterized by scanning electron microscopy (SEM) with en-ergy-dispersive X-ray (EDX) elemental analysis tted with an Ox-ford Inca 300 EDS system. Fermentation mixture of 20 mL waswithdrawn from the reactors after WAS was exposed to nanoparti-cles for 18 d, and then centrifuged at 3000 rpm for 10 min. Afterbeing washed three times with 0.1 M phosphate buffer (pH 7.4),the centrifuged pellets were xed in 0.1 M phosphate buffer (pH7.4) containing 2.5% glutaraldehyde at 4 C for 4 h. The pelletsagain were washed three times with 0.1 M phosphate buffer, andthen dehydrated in the ethanol serials (50%, 70%, 90% and 100%,15 min per step), followed by air drying.</p><p>2.6. Key enzyme activity analysis</p><p>The activity of proteasewas assayed by the folinphenolmethod(Ledoux and Lamy, 1986). Acetate kinase (AK) was the most impor-tant enzyme in acid-forming step (Fig. S2 Supplementary material).For determining its activity, fermentation mixture of 25 mL was ta-ken out of the reactors at different fermentation times and thenwashed and resuspended in 100 mM sodium phosphate buffer (pH7.4). The resuspended mixture was sonicated at 20 kHz and 4 Cfor 10 min to break down the cells structure of bacteria and thencentrifuged at 10,000 rpm and 4 C for 15 min to remove the wastedebris (Allen et al., 1964). The extracts were kept cold on ice beforethey were used for enzyme activity assay. Coenzyme F420 wasassayed by spectrophotometric study (Delafontaine et al., 1979).The specic enzyme activities of protease and coenzyme F420 weredened as the unit of enzyme activity per milligram of VSS (units/mg-VSS), whereas AK was dened as the unit of enzyme activityper milligram of protein (units/mg-protein).</p><p>2.7. Other analytical methods</p><p>H. Mu et al. / Bioresource TechGas component was measured using a gastight syringe (0.2 mLinjection volume) and a gas chromatograph (Agilent 6890N, USA)equipped with a thermal conductivity detector using nitrogen asnano-ZnO was respectively 99.5 and 24.5 mL/g-VSS, whereas in thecontrol test the methane generation was 129.1 mL/g-VSS, whichsuggested the methane inhibition rates were 22.8% and 81.1%. Inthe literature Luna-delRisco et al. (2011) studied the inuence ofparticle size of ZnO and CuO on methane generation during anaer-obic digestion of cattle manure, and found that the inhibitory ef-fects of nano-ZnO and nano-CuO were much greater than thebulk ZnO and CuO.</p><p>3.2. Inuence of dissolved metal ions from nanoparticles on methanegeneration during WAS anaerobic digestion</p><p>The toxicity of metal oxide nanoparticles is sometimes believedto be relevant to the released metal ions (Brunner et al., 2006;Franklin et al., 2007; Wong et al., 2010; Xia et al., 2008). However,the toxicity of nano-ZnO to some microorganisms (such as E. coliand P. uorescens) was found not to be caused by the releasedZn2+ but nano-ZnO (Jiang et al., 2009). Therefore, the experimentsthe carrier gas. The titanium, aluminum, silicon and zinc concen-trations in sludge were analyzed by ICP-OES (PerkinElmer Optima2100 DV, USA), and the samples were digested according to EPAMethod 3052 prior to ICP analyses. The determinations of SCFA,protein, carbohydrate, TSS and VSS were the same as describedin the previous publication (Yuan et al., 2006). The pH value wasmeasured by a pH meter. The total SCFA was calculated as thesum of measured acetic, propionic, n-butyric, iso-butyric, n-valericand iso-valeric acids. The COD (chemical oxygen demand) conver-sion factors of protein, carbohydrate and SCFA were performedaccording to Grady et al. (1999).</p><p>2.8. Statistical analysis</p><p>All assays were conducted in triplicate and the results were ex-pressed as mean standard deviation. An analysis of variance (AN-OVA) was used to test the signicance of results, and p &lt; 0.05 wasconsidered to be statistically signicant.</p><p>3. Results and discussion</p><p>3.1. Effects of nanoparticles exposure on methane generation duringWAS anaerobic digestion</p><p>A higher concentration of SDBS was observed to inuencemethane generation during WAS digestion (Jiang et al., 2007).However, the presence of 4 mg/g-TSS of SDBS (dispersing reagent)in sludge digestion experiments or 0.1 mM SDBS in syntheticwastewater tests showed no inuence on methane generation inthis study, which is consistent with Garcia et al. (2006). In the com-ing text, the control represented the reactor without nanoparticlesaddition but with a SDBS dosage of 4 mg/g-TSS (or 0.1 mM). Asshown in Fig. 1AC, the presence of 6, 30 and 150 mg/g-TSS ofnano-TiO2, nano-Al2O3 and nano-SiO2 did not signicantly affectmethane generation at any fermentation time investigated in thisstudy (p &gt; 0.05). However, when nano-ZnO was in sludge fermen-tation system, its inuence on methane generation was relevantto the dosage (Fig. 1D). No signicant difference of methane gener-ation between the control and 6 mg/g-TSS of nano-ZnO reactorswas observed at any fermentation time (p &gt; 0.05). However, theinhibitory effects of 30 and 150 mg/g-TSS of nano-ZnO on methanegeneration were signicant at fermentation time being more than10 and 5 d, respectively (p &lt; 0.05). The methane generation at fer-</p><p>gy 102 (2011) 1030510311 10307of comparisons of nano-ZnO and the corresponding released metalion (Zn2+) affecting methane generation were conducted to inves-tigate the inuence of nano-ZnO dissolution on WAS anaerobic</p></li><li><p> nolo0</p><p>20</p><p>40</p><p>60</p><p>80</p><p>100</p><p>120A</p><p>ulat</p><p>ive </p><p>met</p><p>hane</p><p> pro</p><p>duct</p><p>ion </p><p>(ml /</p><p> g-V</p><p>SS )</p><p> 0 mg/g-TSS 6 mg/g-TSS</p><p>100</p><p>120C</p><p>10308 H. Mu et al. / Bioresource Techdigestion. The experimental results showed that the presence of4.4 mg/L of Zn2+ did not induce signicant inuence on methanegeneration (p &gt; 0.05) at fermentation time of 18 d. However, themethane generation in the presence of 11.6 and 17.6 mg/L ofZn2+ was respectively 84.4% and 22.1% of the control, and themethane generation was respectively 77.2% and 18.9% of the con-trol at nano-ZnO dosage of 30 and 150 mg/g-TSS. It seems that thereleased Zn2+ was an important reason for the inhibitory effect ofnano-ZnO on WAS digestion for methane generation, which mightbe one reason for only nano-ZnO among four nanoparticles show-ing the negative inuence on WAS anaerobic digestion.</p><p>3.3. Morphology of WAS after nanoparticles addition</p><p>SEM analysis was usually applied to characterize the sludge sur-face structure after nanoparticles were added (Kiser et al., 2009).As seen in Fig. S3A1-D1 (Supplementary material), there were largenumbers of nanoparticles adsorbed on the surface of sludge whennano-TiO2, nano-Al2O3, nano-SiO2 and nano-ZnO appeared insludge digestion system. The same observations were reportedby other researchers when the behavior of nanoparticles in waste-water treatment system was studied (Kiser et al., 2010; Limbachet al., 2008). The EDX spectra further conrmed that the granulesobserved on sludge surface were nano-TiO2 (Fig. S3A2 Supplemen-tary material), nano-Al2O3 (Fig. S3B2), nano-SiO2 (Fig. S3C2) andnano-ZnO (Fig. S3D2).</p><p>Cum</p><p>0</p><p>20</p><p>40</p><p>60</p><p>80</p><p> Time (d)2 6 10 14 18</p><p>Fig. 1. Effects of different dosages (0, 6, 30 and 150 mg/g-TSS) of nano-TiO2 (A), nan...</p></li></ul>


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