Chronic Response of Waste Activated Sludge Fermentation to Titanium Dioxide Nanoparticles☆

Yinguang Chen,Hui Mu,Xiong Zheng*

Energy,Resources and Environmental Technology

Chronic Response of Waste Activated Sludge Fermentation to Titanium Dioxide Nanoparticles☆

Yinguang Chen,Hui Mu,Xiong Zheng*

State Key Laboratory of Pollution Control and Resource Reuse,School of Environmental Science and Engineering,Tongji University,Shanghai 200092,China

A R T I C L EI N F O

Article history:

Titanium dioxide nanoparticles

Waste activated sludge

Fermentation

Long-term effect

Mechanism

Due to the large-scale production and wide applications,many nanoparticles(NPs)enter wastewater treatment plantsandaccumulate inactivated sludge.It isreportedthattitaniumdioxide(TiO2)NPs showseveredamageto many model microbes.However,it is still unknown whether the long-term(e.g.,100 d)presence of TiO2NPs would affect the performance of sludge fermentation.In this study,long-term exposure experiments(105 d) were conducted to investigate the potential risk of TiO2NPs to sludge fermentation system.It is found that the presence of environmentally relevant[6 mg·(g TSS)?1]and higher[150 mg·(g TSS)?1]concentrations of TiO2NPs does not affectmethane production from sludgefermentation.The analysisoff l uorescence insituhybridization indicates that these concentrations of TiO2NPs present marginal inf l uences on abundances of bacteria and methanogenic archaea in sludge fermentation system.The viability of sludge microorganisms and activities of key enzymes related to methane production such as protease,acetate kinase,and coenzyme F420are unchanged by the long-term presence of 6 and 150 mg·(g TSS)?1of TiO2NPs.Further investigations reveal that the insolubility of NPs and the protection role of sludge extracellular polymeric substances are the main reasons for the marginal inf l uence of TiO2NPs on sludge fermentation.

?2014TheChemicalIndustry andEngineeringSocietyofChina,andChemicalIndustryPress.Allrightsreserved.

1.Introduction

Nanotechnology has been considered as one of the fastest growing sections of the‘high-tech economy’.Over the past decade,due to the unique physicochemical properties,large numbers of nanoparticles (NPs)have been manufactured and applied in our daily life,such as antibactericide coatings,biomedicine,skin creams,and toothpastes [1,2].However,some researchers have reported that the wide use of these NP-containing products inevitably releases NPs into the environment,which might pose potential risks to human health and ecosystems[3,4].

Nanoparticulate titanium dioxide(TiO2)is one of the most widely applied nanomaterials in the world and can be used in various f i elds, such as catalysts,sunscreens,cosmetics,and wastewater treatment[4]. With its increasing production and application,some studies have been conducted to examine their potential inf l uences on model organisms.For example,it is observed that TiO2NP exhibits antibacterial properties towards Bacillus subtilis and Escherichia coli[5].Compared with bulk particles,TiO2NPs may induce more damage to algae[6]. The study on the potential toxicity of NPs to human keratinocytes in vitro indicates that the long term exposure has more adverse effects thantheshorttermone[7].However,insomecases,TiO2NPisnontoxic to some aquatic microorganisms even at 20000 mg·L?1[8,9].These resultsindicatethatdifferentcelllines mightshowdissimilarresponses to TiO2NPs.

Many commercial NPs used in our daily life have been found in various environments such as wastewater[10,11].It is reported that some raw sewage contains 0.1 to nearly 3 mg Ti·L?1and these NPs f i nally enter wastewater treatment plants(WWTPs)[4].Usually,more than 95%of NPs in treatment facilities could be accumulated in activated sludge[12,13]and the titanium content in waste activated sludge (WAS)of WWTPs is in the range of 0.018-7.02 mg Ti·(g TSS)?1[10,11,13].Anaerobic fermentationisa sustainable technique for sludge and wastewater treatment,and the potential effects of NPs should be evaluated.Although a slight increase of biogas production is detected in the thermophilic anaerobic batch test during exposure to TiO2NPs [14],little work has been carried out on the effect of TiO2NPs on different microbial populations in a sludge treatment system.Since sludge fermentation is always operated continuously,it is necessary to investigate the long-term inf l uenceof TiO2NPs on sludge fermentation,which has not been documented in literature.

In this study,long-term exposure experiments are conducted to investigatethepotentialriskof TiO2NPs tosludgefermentation.Methane production is used as an indicator to assess the long-term effect of TiO2NPs to anaerobic sludge fermentation systems.After 105 d of exposure,fl uorescence in situ hybridization(FISH)is adopted to measure the abundance of bacteria and methanogenic archaea in sludge fermentation reactors.The relative viability of sludge microorganisms and activities of key enzymes related to methane production such as protease,acetate kinase(AK),and coenzyme F420are determined to analyze the chronic inf l uence of TiO2NPs.Finally,the surface integrity of sludge microorganisms and characteristics of TiO2NPs are investigated to address the potential impact of TiO2NPs on sludge fermentation.

2.Materials and Methods

2.1.Waste activated sludge

The WAS was obtained from the secondary sedimentation tank of a municipal WWTP in Shanghai,China.The sludge was concentrated by settling at 4°C for 24 h,with its main characteristics(average data and standard deviations of triplicate measurements)as follows: pH 6.7±0.2,total suspended solids(TSS)(10070±780)mg·L?1, volatile suspended solids(VSS)(7690±452)mg·L?1,total chemical oxygen demand(10710±220)mg·L?1,soluble chemical oxygen demand(90±14)mg·L?1,total protein(5685±149)mg COD·L?1, and total carbohydrate(899±530)mg COD·L?1.

2.2.Characterization of TiO2NPs

TiO2NPs(anatase)used in this study were purchased from Sigma Aldrich(USA).The stock suspension of NPs was prepared by adding 2000 mg of NPs to 1.0 L of deionized water(pH 7.0)followed by 1 h of ultrasonication(25°C,250 W,40 kHz)according to the literature [15],and transmission electron microscopy(TEM)was used to image the morphology of TiO2NPs.Due to the aggregation characteristics of NPs,sodium dodecylbenzene sulfonate(SDBS),an anionic surfactant, waschosentoimprovethedispersionofTiO2NPsinthesuspension.DynamiclightscatteringanalysisindicatedthatthesuspensionofTiO2NPs had a size distribution of 80-290 nm with an average particle size of approximately 185 nm.

2.3.Long-term exposure to TiO2NPs

In this study,the environmentally relevant concentration of TiO2NPs was chosen to be 6 mg·(g TSS)?1according to the literature[10, 16].With increasing production and wide application,more NPs will be released to the environment[17],which suggests that much higher concentration of TiO2NPs should be examined to assess the potential risks of TiO2NPs.Therefore,in this study,long-term exposure experiments were conducted to determine the potential long-term effects of 6 and 150 mg·(g TSS)?1of TiO2NPs on sludge fermentation.

Allexperimentswereconductedinfermentationreactors,eachwitha working volume of 300 ml.After the addition of WAS and TiO2NPs[0,6, and 150 mg·(g TSS)?1],all reactors were f l ushed with nitrogen gas for 10 min to remove oxygen,capped with rubber stoppers,and placed in an air-bath shaker(150 r·min?1)at(35±1)°C.Every day,20 ml of fermentationmixture was manually wasted from the reactors and the same amounts of raw sludge were supplemented,with the sludge retention time of 15 d.As sludge wasting would decrease the concentrations of TiO2NPs,inductivelycoupledplasmamassspectrometry(ICP-MS,Agilent Technologies,USA)was used to measure the content of TiO2NPs left in sludge fermentation systems before some amounts of NP suspension were added to maintain their constant concentrations.During the entire fermentation process,methane production was measured frequently.It was observed that the methane production did not change signif i cantly after 60 d of exposure.At the end of the experiment(day 105),the abundance of bacteria and methanogenic archaea,sludge viability,and activities of key enzymes related to methane production were analyzed.

2.4.Analytical methods

The methane production was measured using a gas chromatograph (Agilent Technologies,USA)equipped with a thermal conductivity detector using nitrogen as the carrier gas[18].The determinations of TSS and VSS were described in our previous publication[19].The viability of sludge microorganisms was measured using the cell counting kit-8 (CCK-8)(Dojindo)in accordance with manufacturer's instructions. The measurement of intracellular reactive oxygen species(ROS) production was according to our previous publications[20,21].The activities of protease,acetate kinase,and coenzyme F420were assayed according to the references[22,23].The lactate dehydrogenase release (LDH)was determined by using a cytotoxicity detection kit(Roche Applied Science)according to manufacturer's instructions.At the end of the exposure experiments,the mixture was centrifuged at 12,000 g for 5 min.Then,the supernatant was seeded on a 96-well plate,followed by the addition of 50 μl of substrate mix(Roche Applied Science). After 30 min of incubation at room temperature in dark,50 μl of stop solution(RocheAppliedScience)wasaddedtoeachwellandtheabsorbance was recorded at 490 nm using a microplate reader(BioTek).

After long-termexposure to TiO2NPs,f l uorescence insitu hybridization technique with 16S rRNA-targeted oligonucleotide probes was adopted tomonitortheabundanceof bacteria andarchaea inthesludge fermentation reactors.The probes of Cy-3-labeled EUB338(5′-GCTGCC TCCCGTAGGAGT-3′)and f l ourescein iso-thiocyanate(FITC)labeled ARC915(5′-GTGCTCCCCCGCCAATTCCT-3′)were used for bacteria and methanogenic archaea,respectively[24,25].The detailed procedure of FISH analysis was documented in the previous literature[21].Finally, the sections hybridized with the probes were observed with a confocal laser scanning microscope(CLSM,Leica TCS,SP2 AOBS),and the random fi elds were analyzed to determine the average abundances of sludge microorganisms.

2.5.Statistical analysis

All assays were conducted in triplicate and the results were expressed as mean±standard deviation.An analysis of variance was used to test the signif i cance of results,and p＜0.05 was considered to be statistically signif i cant.

3.Results and Discussion

3.1.Effect of TiO2NPs on methane production from sludge fermentation

Suff i cient dispersion of NPs in stock suspension plays an important roleinassessingthepotentialeffectsofNPs,becauseaggregationisacharacteristicofNPs[Fig.1(a)].WithSDBSasadispersant,Fig.1(b,c,d)shows the TiO2NPs suspensions in the absence and presence of SDBS.The presence of 4 mg·(g TSS)?1of SDBS signif i cantly improves the dispersion of TiO2NPs instock suspensionand does not affectthesludgefermentation, which is consistent with the observation of Garcia et al.[26].Thus,in this study,4 mg·(g TSS)?1of SDBS was added to all experimental systems.

The toxicity of environmental pollutants to sludge fermentation can be investigated by using the f i nal product of sludge fermentation (i.e.,methane)as a useful indicator[27].In this study,the methane productioninlong-termexposureexperimentsismeasuredtoassessthepotential effect of TiO2NPs on sludge fermentation.Fig.2 shows the variations of methane production in the absence and presence of 6 and 150 mg·(g·TSS)?1of TiO2NPs during the entire fermentation process.The methane production in all reactors increased with the fermentation time and reached stable concentrations after 60 d of fermentation.However,no signif i cant differences appeared in methane production among these reactors,indicating that longterm presence of TiO2NPs does not affect sludge fermentation.We have reported that short-term presence of TiO2NPs causes marginal effect on methane production from sludge fermentation[20].Therefore,it can be concluded that both acute exposure and chronic exposure to 6 and 150 mg·(g TSS)?1of TiO2NPs present marginal inf l uence on methane production from sludge fermentation.

Fig.1.TEM image(a)and dispersion of TiO2NP stock suspension in the absence and presence of SDBS[4 mg·(g TSS)?1]after 0.5 h(b),6 h(c),and 20 h(d)of settlement.

3.2.Effects of TiO2NPsonthe abundance,viability,and enzyme activities of sludge microorganisms

The above results indicate that the long-term presence of TiO2NPs does not have a negative effect on methane production from sludge fermentation.However,to date,the reasons for the marginal inf l uence of TiO2NPs on sludge fermentation are still unclear.It is reported that the presence of low concentration of TiO2NPs[2 mg·(g soil)?1]reduces the microbial diversity and alters the composition of soil bacterial community after 60 d of exposure[28].It is well-known that the microbial communityplaysanimportantroleinsludgefermentation,andtheabundance of bacteria and methanogenic archaea is related to the methane production from sludge fermentation.Thus,in this study,FISH analysis is conducted to investigate the potential effect of TiO2NPs on sludge microorganisms after long-term exposure,and the results are shown in Fig.3.Itwasfoundthattheabundancesofbacteria andmethanogenicarchaeainthecontrolwere51.3%and39.5%,respectively,whereasthosein the presence of 6 and 150 mg·(g TSS)?1of TiO2NPs were 50.7%versus 39.0%and 52.6%versus 38.6%,respectively.Obviously,there were no signif i cant differences in the abundances of bacteria and archaea in these sludge fermentation reactors,indicating that TiO2NPs did not affect the key sludge microorganisms'abundances.

The viability of sludge microorganisms is important for sludge fermentation.Cell viability assay is a useful tool to demonstrate the inf l uence of toxic substances on model organisms[29].Thus,after long-term exposure,the inf l uence of TiO2NPs on the viability of sludge microorganisms is determined.Although in some cases TiO2NPs could reduce the activities of biomass microorganisms[5,28,30],in this study,the relative viability of sludge does not have signif i cant differences between the absence and presence of 6 and 150 mg·(g TSS)?1of TiO2NPs.Some researchers observed that TiO2NPs did not induce signif i cant impacts on Daphnia magna and Thamnocephalus platyurus even at the concentration of 20000 mg·L?1[8].

The methane production from sludge fermentation undergoes solubilization of sludge particulate organic substances,hydrolysis,acidif i cation,and methanation.In these fermentation processes,the protease, acetate kinase,and coenzyme F420are important enzymes responsible for sludge hydrolysis,acidif i cation,and methanation,respectively [Fig.4(a)].We have reported that the methane production is inhibited by ZnO NPs,by inhibiting the activity of coenzyme F420[20,21]. Although the results have shown that methane production is not affected by TiO2NPs,it is still unknown whether the activities of these enzymes are affected by long-term exposure to TiO2NPs.Thus the chronic inf l uence of TiO2NPs on sludge fermentation is further investigated from the aspect of enzyme activity,and the results are shown in Fig.4(b).It can be seen that 6 and 150 mg·(g TSS)?1of TiO2NPs do not inf l uence the activities of protease,AK,and coenzyme F420,which is consistent with the observation that TiO2NPs do not affect the methane production from sludge fermentation.

Fig.2.Methane production from sludge fermentation in the absence and presence of 6 and 150 mg·(g TSS)?1of TiO2NPs during 105 d of fermentation.(Error bar:standard deviation of triplicate measurements).

3.3.Effects of TiO2NPs on the surface integrity of sludge microorganisms

It is reported that toxic metal ions released from the dissolution of NPs are mainlyresponsible for their acute toxicitytosome livingorganisms[21,31].For instance,comparedwithaluminum oxide(Al2O3NPs), silicon dioxide(SiO2NPs),and magnetite(Fe3O4NPs),ZnO NPs are most toxic to the growth of Arabidopsis thaliana,since only ZnO NPs releasetoxic metalions[32].Ourprevious studyalsosupportstheeffect of ZnO NP dissolution on sludge fermentation systems[20].However, no metal ions have been detected in TiO2NP suspension in this study, which may be the reason for its nontoxic effect on sludge fermentation.

It is reported thatsomeNPsare abletocauseoxidativestress and induce adverse effects on model organisms[33].High ROS production may damage the cell membrane or cytoplasmic proteins in human cells[34]and E.coli[35].Thus the ROS production caused by TiO2NPs during sludge fermentation is measured and the result is shown in Fig.5(a).No signif i cant differences in ROS production are observed between the absence and presence of 6 mg·(g TSS)?1of TiO2NPs.At the TiO2NP concentration of 150 mg·(g TSS)?1,the ROS production is increased to 126%of the control.LDH release assay further indicates that no measurable cell leakage is induced by TiO2NPs at theconcentrations of 6 and 150 mg·(g TSS)?1[Fig.5(b)].These results indicatethat thelong-term presence ofTiO2NPs hasnosignif i cant impact on the integrity of sludge microorganisms.

Fig.3.FISH images of sludge bacteria and methanogenic archaea in the absence(a1-a3)and presence of 6(b1-b3)and 150 mg·(g TSS)?1(c1-c3)of TiO2NPs.[with simultaneous hybridization with Cy-3-labeled bacteria-domain probe(EUB338)(a1,b1 and c1)and FITC-labeled archaea-domain probe(ARC915)(a2,b2 and c2),randomly viewed by CLSM and photographed at higher(×62)magnif i cation].

Fig.4.Proposed metabolic pathway(a)and activities(b)of the key enzymes related to methane production from sludge fermentation.

Extracellular polymeric substances(EPS)in sludge f l ocs are usually consideredtoprotecttheinnermicroorganismsagainsttheharshexternal environmental conditions,because EPS could establish interactionswith the polymer matrix by impeding access of these pollutants to bacterial cells[36].As seen from Fig.6,EPS fi rst risk something unpleasant with TiO2NPs in the sludge fermentation system,and the ROS production and released ions are the two key factors of NP toxicity to sludge fl ocs.It is not needed to consider the EPS complexation of toxic ions in this study,because no ions are released in this system.Fig.5(a)shows thattheROSproductionincreasesto126%ofthecontrolinthepresence of150 mg·(gTSS)?1ofTiO2NPs,whichmightdamagetheEPS.However, the EPS of activated sludge could protect the microorganisms from the toxicityof carbonnanotubes[37],and Fig.5(b)shows that such ROS production is not enough to damage the EPS,not even the protein and DNA in the cells.Therefore,the EPS protection might be the reason for the nontoxicity of TiO2NPs to microorganism in sludge.

Fig.5.Relative ROS production(a)and LDH release(cell membrane damage marker)(b)in the absence and presence of 6 and 150 mg·(g TSS)?1of TiO2NPs.(Error bar:standard deviations of triplicate measurements).

4.Conclusions

In this study,the potential effect of TiO2NPs on sludge fermentation wasinvestigated attheenvironmentallyrelevant[6 mg·(g TSS)?1]and higher[150 mg·(g TSS)?1]concentrations with long-term exposure.It is found that TiO2NPs do not affect the methane production during 105 d of sludge fermentation.Further experiments indicate that 6 and 150 mg·(g TSS)?1of TiO2NPs do not change theabundance of bacteria andmethanogenic archaeainsludgefermentationsystems.Meanwhile, the relative viability of sludge microorganisms and activities of key enzymes related to methane production such as protease,acetate kinase,and coenzyme F420are not affected by the presence 6 and 150 mg·(g TSS)?1of TiO2NPs,which is consistent with no signif i cant inf l uence on methane production.The insoluble characteristics of TiO2NPs and sludge EPS protection are the main reasons for the marginal inf l uence of TiO2NPs on sludge fermentation.

Fig.6.Diagram of possible mechanisms for the cytotoxicity of nanoparticles to sludge microorganisms.

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8 May 2013

☆SupportedbytheNationalHi-TechResearchandDevelopmentProgramofChina(863 Program)(2011AA060903),theNational NaturalScience FoundationofChina(41301558 and 51278354),and Shanghai Tongji Gao Tingyao Environmental Science&Technology Development Foundation(STGEF).

*Corresponding author.

E-mail address:xiongzheng@#edu.cn(X.Zheng).

http://dx.doi.org/10.1016/j.cjche.2014.09.007

1004-9541/?2014 The Chemical Industry and Engineering Society of China,and Chemical Industry Press.All rights reserved.

Received in revised form 28 June 2013

Accepted 29 July 2013

Available online 16 September 2014