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Journal of Hazardous Materials 283 (2015) 80–88

Contents lists available at ScienceDirect

Journal of Hazardous Materials

jo ur nal ho me p ag e: www.elsev ier .com/ locate / jhazmat

haracterizing reactive oxygen generation and bacterial inactivationy a zerovalent iron-fullerene nano-composite device at neutral pHnder UV-A illumination

sra Erdima,b, Appala Raju Badireddya,c, Mark R. Wiesnera,c,∗

Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708, USAEnvironmental Engineering Department, Marmara University, Istanbul 34469, TurkeyDepartment of Civil and Environmental Engineering, Duke University, Durham, NC 27708, USA

i g h l i g h t s

We synthesized a novel ZVI/nC60 nano-composite device for multi-ROS generation.O2

• − (UV-A independent) and 1O2 (UV-A dependent) are generated at neutral pH.At low Fe concentration, ZVI/nC60 device is a better ROS generator than ZVI alone.C60 mediates electron transfer from ZVI surface to dissolved O2 to produce O2

• −.Bacteria are rapidly inactivated by O2

• − even at low ZVI/nC60 ratio.

r t i c l e i n f o

rticle history:eceived 18 February 2014eceived in revised form 16 July 2014ccepted 13 August 2014vailable online 16 September 2014

eywords:ano-scale zerovalent ironullerene

a b s t r a c t

A nano-composite device composed of nano-scale zerovalent iron (ZVI) and C60 fullerene aggregates(ZVI/nC60) was produced via a rapid nucleation method. The device was conceived to deliver reactiveoxygen species (ROS) generated by photosensitization and/or electron transfer to targeted contaminants,including waterborne pathogens under neutral pH conditions. Certain variations of the nano-compositewere fabricated differing in the amounts of (1) ZVI (0.1 mM and 2 mM) but not nC60 (2.5 mg-C/L), and (2)nC60 (0–25 mg-C/L) but not ZVI (0.1 mM). The generation of ROS by the ZVI/nC60 nano-composites andZVI nanoparticles was quantified using organic probe compounds. 0.1 mM ZVI/2.5 mg-C/L C60 generated3.74-fold higher O2

•− concentration and also resulted in an additional 2-log inactivation of Pseudomonas

ano-compositeeactive oxygen specieshotosensitization

aeruginosa when compared to 0.1 mM ZVI (3-log inactivation). 2 mM ZVI/2.5 mg-C/L nC60 showed neg-ligible improvement over 2 mM ZVI in terms of O2

•− generation or inactivation. Further, incrementalamounts of nC60 in the range of 0–25 mg-C/L in 0.1 mM ZVI/nC60 led to increased O2

•− concentration,independent of UV-A. This study demonstrates that ZVI/nC60 device delivers (1) enhanced O2

•− with nC60

as a mediator for electron transfer, and (2) 1O2 (only under UV-A illumination) at neutral pH conditions.

. Introduction

Previous investigators have observed that the fullerene C60 isn efficient electron shuttle with the capability to accept up to

ix electrons from a donor molecule [1,2]. The photochemical andhotophysical properties of C60 have been well studied and it haseen shown that the C60 when subjected to photosensitization

∗ Corresponding author at: Department of Civil and Environmental Engineering,uke University, Durham, NC 27708, USA. Tel.: +1 919 660 5292;

ax: +1 919 660 5219.E-mail address: wiesner@duke.edu (M.R. Wiesner).

ttp://dx.doi.org/10.1016/j.jhazmat.2014.08.049304-3894/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

transforms to an excited state (1C60* and 3C60*), which may pro-duce 1O2 via energy transfer or O2

•− via electron transfer to oxygen[3–6]. The O2

•− generation occurs through the formation of C60•−

by photoexcited C60 in the presence of an electron donor [7]. Zero-valent iron nanoparticles (ZVI NPs) have been observed to induceboth reductive and oxidative reactions [8]. In the absence of oxygen,some contaminants are degraded by ZVI through direct electrontransfer reactions (e.g., reductive dehalogenation of chlorinatedsolvents) [9–13], whereas under oxygenated conditions a variety of

recalcitrant contaminants (e.g., pesticides, dyes, and aromatic com-pounds) may be oxidized by reactive oxygen species (ROS) that areby-products of ZVI NP oxidation [14–19]. Also, several studies havereported on the antimicrobial activity of iron-based nanoparticles

ardous Materials 283 (2015) 80–88 81

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E. Erdim et al. / Journal of Haz

gainst Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseu-omonas fluorescens [20–22], and bacteriophage [23]. Under acidiconditions, oxidation of ZVI produces Fenton reagent (Fe2+ + H2O2)n situ that is well known for generating hydroxyl radicals (•OH). Atear-neutral pH, in the absence of stabilizing ligands (e.g., EDTA)xidation of ferrous ion (generated from ZVI) produces superox-de radicals (O2

•−) [24]. Under ambient aqueous conditions ZVIxidation has been shown to produce ROS [25–27], however, thisrocess is severely limited by other competing reactions, whichesults in loss of reactive iron species (ferrous and ferric ions) in theorm of iron corrosion products, such as iron oxides and hydrox-des [16,28]. These competing reactions in oxygenated systemsapidly reduce the efficiency of the ZVI NPs through passivationf ZVI surfaces by corrosion products and subsequent decrease inhe electron transfer rate. Recently, it has been suggested that in ZVIystems the efficiency of electron transfer processes and the yield ofOS production could be enhanced using ligands (e.g., ethylenedi-minetetraacetate (EDTA)), electron shuttles (e.g., polyoxometalatePOM; a metal–oxygen anion) or natural organic matter (NOM))14,29–32]. The ZVI/EDTA/O2 system necessitates a continuousupply of EDTA and results in a partial quenching of the ROS byDTA itself. At pH 2 and 7, ZVI/POM/O2 has been shown to enhanceOH generation, ascribed to the capability of POM to shuttle elec-rons and form complexes with Fe ions. But, the application of POMor real-world treatment systems may not be cost-effective andOM regenerative ability after treatment is unclear. In ZVI/NOM/O2ystem, NOM have been shown to be an effective mediator for elec-ron transfer under acidic conditions (pH 2.5), which is interestingor the understanding of NOM role during ZVI oxidation mecha-isms but this system may not be of practical value for treatmentpplications. However, both ZVI/POM/O2 and ZVI/NOM/O2 systemsave revealed the importance of presence of an electron mediatornd its coordinative nature with iron, which not only facilitatedransfer of electrons from ZVI surface to O2 but also likely min-mized iron precipitation through co-ordination. The precedingxamples, ZVI/electron-mediator/O2 systems, have only exploredOS generation capability of ZVI in the absence of light (i.e., “dark”eaction). In the current study nC60, which is known for its stabletructure and resistance to oxidation, was employed as a photo-ensitizer and electron-mediator to elucidate the ROS generationechanism both in the presence and absence of UV-A at near-

eutral pH condition.Based on the photophysical and photochemical properties of

60 and electron-donating nature of ZVI, we hypothesize thathotosensitization of ZVI/nC60 nanocomposite enhances the ROSO2

•− and 1O2) generation at near-neutral pH, which is a com-only desired condition in treatment systems. The objective of

he present study was to evaluate the electron-mediating capac-ty of nC60 in ZVI/nC60 nano-composite under ultraviolet (UV)-Allumination for enhancing ROS generation by ZVI at pH 7.8. UV-A

as employed for photosensitization and possibly to drive photo-enton-like reaction at near-neutral pH. In the present work, weescribe a nanoparticulate device to produce ROS (O2

•− and 1O2)hat employs C60 fullerene aggregates (nC60) as photosensitizer andlectron mediator in a composite form with ZVI, which serves as anlectron donor (see Fig. 1). In the presence or absence of UV-A, weompare the ROS generation and the efficacies of bacterial inacti-ation by these ZVI/nC60 devices with that of ZVI NPs in oxygenatedystem at pH 7.8.

. Materials and methods

We synthesized and characterized ZVI/nC60 nanoparticulateevices along with ZVI NPs and compared their ROS generationapacities in the presence or absence of UV-A. ROS (O2

•− and 1O2)

Fig. 1. Schematic representation of reactive oxygen species (ROS) generation byZVI/nC60 device under UV-A illumination at ambient conditions.

measurements were performed using probe compounds and theeffect of the ZVI/nC60 nano-composites and ZVI NPs on the viabilityof Pseudomonas aeruginosa was assessed.

2.1. Chemicals

Fullerene powder (C60, 99.9%) was purchased from MaterialsElectronics Research Corporation (Tucson, AZ). Ferrous chloride(FeCl2·4H2O), sodium borohydride (NaBH4), 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT),superoxide dismutase (SOD), and furfuryl alcohol (FFA) werereagent grade and purchased from Sigma–Aldrich Co., USA. Glass-ware was cleaned by soaking in diluted nitric acid (5%) and rinsedwith ultrapure water with resistivity > 18 M� cm (NANOpure,Barnstead, Dubuque, IA). 15 mM phosphate buffered-saline (PBS)was prepared by 10-fold dilution of 1× PBS in ultrapure water.

2.2. Preparation of fullerene nanoparticle (nC60) suspensions

A stock suspension of colloidal aggregates of C60 (referred to asnC60 where, an average aggregate is composed of “n” C60 molecules)was prepared using a previously described procedure [33,34]. Inbrief, 100 mg of fullerene powder in 200 mL of ultrapure water wasultra-sonicated (Misonix, QSonica, S-4000, Newtown, CT) for 9 hwith a sequence of 5 min pulse-on/15 min pulse-off. The beakerwas kept in an ice bath to dissipate heat and was covered with alu-minum foil to avoid light as well as any contaminant interference.The resultant suspension was filtered through a 0.22 �m PVDF fil-ter. The total carbon (TC) concentration of the filtrate was measuredto be 38 mg-C/L by total organic carbon analyzer (TOC-5050A, Shi-madzu, Japan). The number-average diameter of nC60 in ultrapurewater was 13 nm.

2.3. Synthesis of ZVI and ZVI/nC60 nanoparticles

ZVI nanoparticles were synthesized immediately before use byaqueous-phase reduction of ferrous chloride with sodium borohy-dride according to the following chemical reaction [35]:

Fe2+ + 2BH−4 + 6H2O → Fe0 + 2B(OH)3 + 7H2 (1)

Specifically, 10 mM FeCl2·4H2O was reduced by drop-wise(1–2 drops/s) addition of ice-cold 100 mM NaBH4 at a molar ratioof 1:2.5 in N2-sparged water. Excess NaBH4 was used to ensurecomplete reduction of Fe(II) to zero-valent iron in the solution.This reaction resulted in the formation of a black-colored suspen-sion of ZVI NPs along with hydrogen, which was allowed to escape.Similarly, ZVI/nC60 nano-composites were prepared by reducing amixture of FeCl2·4H2O and nC60 with excess NaBH4. Suspensionsof ZVI and ZVI/nC60 were synthesized and stored in an anaerobicchamber (Coy Lab Products, Michigan) until further analysis andexperimentation. Two different levels of ZVI in the nano-composite

devices were achieved by varying the ferrous chloride initiallyintroduced to form the ZVI/nC60 devices at concentrations of either0.1 mM or 2 mM while keeping the nC60 at a value of 2.5 mg-C/L.An additional set of 0.1 mM ZVI/nC60 nano-composite devices were

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2 E. Erdim et al. / Journal of Haz

abricated by incrementally varying nC60 concentration such as 0,.25, 2.5, and 25 mg-C/L.

.4. Characterization

The dynamic light scattering (DLS) (ALV CGS-3, Langen,ermany) technique (� = 633.3 nm, scattering angle of 90◦) wassed to measure the number-weighted average hydrodynamic sizef the ZVI and ZVI/nC60. The morphology characterization waserformed using transmission electron microscopy (TEM). TEMark-field images were also collected for the nanoparticles to dis-ern the location of nC60 in the nano-composite samples. X-rayhotoelectron spectroscopy (XPS) (Kratos Axis ULTRA, Shimadzu)as used to determine the composition and oxidation state of iron

n the ZVI and ZVI/nC60. Survey and high-resolution scans of eachanoparticle powder (obtained by drying the droplets of nanopar-icle suspensions) deposited on double-sticky copper tape to ahickness greater than 500 �m (scan area: 300 �m × 700 �m) wereollected. The survey spectra were acquired using pass energy of60 eV (step size 1 eV) and high-resolution scans were collected atass energy of 20 eV (step size 0.1 eV). The binding energies werealibrated using C 1s peak (284.6 eV).

.5. Experimental setup

The glass reaction beakers (O.D. = 50 mm and height = 35 mm)rimarily consisted of the following nanoparticle suspensions: (1).1 mM ZVI, (2) 2 mM ZVI, (3) 0.1 mM ZVI/2.5 mg-C/L nC60 compos-

te and (4) 2 mM ZVI/2.5 mg-C/L nC60 composite. These suspensionsere exposed to ultraviolet-A (315-400 nm) light source (Philips

LD 2X 15W/08) in an EMS UV/Cryo chamber (Hatfield, PA). Similaret of experiments were also performed in the absence of UV-A. Theotal irradiance of the UV lamps was approximately 2.0 mW/cm2 athe surface of the suspensions as measured by a radiometer. For theuration of the experiment (60 min) the suspensions were shakent 120 rpm on an orbital shaker placed in the chamber. The tem-erature in the UV/Cryo chamber was kept at around 25 ◦C using aooling jacket that was connected to a cooling chamber. Samplesere periodically withdrawn with a syringe during 60 min and fil-

ered through a 0.22 �m nylon filter (Millipore), prior to analysisf the probe compounds. The filtration of samples minimized theight shielding and potential clogging effects arising from the pres-nce of nanoparticulates in the reaction system during UV–vis andigh performance liquid chromatograph (HPLC) analysis, respec-ively. Under similar experimental conditions described above the.1 mM ZVI/(0–25 mg-C/L) nC60 nano-composites were tested foruperoxide generation in the presence and absence of UV-A. Theeactive oxygen species (ROS) generation by ZVI and ZVI/nC60 sus-ensions in the presence and absence of UV-A was characterizedsing protocols described below.

Superoxide radical (O2•−) production was quantified by

,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-arboxanilide (XTT) reduction method. The reduction of XTT by2

•− results in the formation of XTT-formazan, which has a uniquebsorbance at 470 nm (ε470 nm = 21,600 M−1 cm−1) measured byV–vis spectrophotometer (Cary). The increase in absorbanceas used to quantify the relative amount of superoxide present

36]. The concentration of O2•− was determined by comparing

TT reduction with and without a quencher for O2•−, superoxide

ismutase (SOD) (30 U/mL SOD), which allowed accounting forTT reduction by sources unrelated to O2

•−. Each nanoparticle

uspension was exposed to 300 �M or 1 mM XTT (depending onhe ZVI concentration) for 60 min in the presence and absence ofV-A. These samples were buffered with 15 mM PBS to maintainH at 7.8 since the XTT assay works best at this pH.

s Materials 283 (2015) 80–88

Singlet oxygen (1O2) concentration was measured using furfurylalcohol (FFA) as an indicator (k(FFA + 1O2) = 1.2 × 108 M−1 s−1) [37].To maintain loss of FFA as first-order and to prevent suppression of[1O2], it is necessary to limit [FFA] < 200 �M [37]. Reaction solutionscontaining 0.1 mM FFA were buffered at pH 7.8 with PBS. The reac-tion of 1O2 and FFA is independent of pH between 5 and 12 [38]. Theresidual FFA concentration during the course of 60 min was mea-sured using a HPLC (ProStar, Varian, PaloAlto, CA) equipped witha reverse phase C-18 column (5 �m, 150 × 4, 6 mm, RESTEK, Belle-fonte, PA) and a photodiode-array detector. The isocratic elutionwas 40% HPLC-grade acetonitrile and 60% 20 mM KH2PO4 (pH 2.5)at a flow rate of 1 mL/min. FFA was quantified at 279 nm.

2.6. Bacterial inactivation

The efficacy of bacterial inactivation in ZVI and ZVI/nC60 sus-pensions was tested using P. aeruginosa. This bacterium was usedas a model bacterium since it is commonly found in ultrapure watersystems, humidifiers, freshwaters, marine waters, and recreationalwaters [39]. These bacteria were cultured in Luria–Bertani (LB)broth and stock suspensions free of bacterial debris and secretionswere prepared in 15 mM PBS (pH 7.8). Bacteria were inoculated inLB and grown while shaking on an orbital shaker at 37 ◦C overnight(18 h). These overnight cultures were centrifuged, washed, andresuspended in 15 mM PBS twice to prepare stock bacteria free ofcell debris and secretions. The stock concentration of the washedbacteria was 2 × 109 colony forming units (CFU)/mL. The numberof cells was determined using spread-plate method, in which P.aeruginosa were spread on LB agar and incubated at 37 ◦C for 18 hand then the number of the colonies was counted. P. aeruginosainactivation was determined by comparing the control bacteria (noUV-A or nanoparticles) to the bacteria exposed to UV-A alone aswell as various nanoparticle suspensions for 5 min in 15 mM PBS(pH 7.8). The inactivation was reported as log (Nt/N0), where N0and Nt are the number of bacteria (CFU/mL) at time 0 and t min-utes. The initial number of bacteria (N0) in the reaction vials was2 × 107 CFU/mL.

3. Results and discussion

3.1. Characterization of ZVI and ZVI/nC60 nanoparticles

Suspensions of the ZVI and ZVI/nC60 nanoparticles had number-average hydrodynamic diameters of ∼33 nm (0.1 mM ZVI), ∼33 nm(0.1 mM ZVI/2.5 mg-C/L nC60), ∼55 nm (2 mM ZVI), and ∼111 nm(2 mM ZVI/2.5 mg-C/L nC60) as measured by DLS (Fig. 2a–d).Nanoparticles were observed to be nearly spherical as observed byTEM (Fig. 2e–h). DLS measurements suggest that the concentrationof nC60 in the range 0–2.5 mg-C/L may have negligible affect onaverage diameter of 0.1 mM ZVI/nC60 nano-composite (∼33 nm),but further increase in nC60 concentration (e.g., 25 mg-C/L) resultsin change in diameter to ∼52 nm. Similarly, increasing Fe concen-tration to 2 mM while keeping nC60 concentration constant at 0or 2.5-mg-C/L also results in change in diameter to ∼55 nm or∼111 nm, respectively. Further, TEM images indicate that nC60 maylikely have a role in influencing the morphology of the ZVI/nC60nano-composite, which in turn proved to have implications on sta-bility and reactivity of such nano-structures in aqueous systems.

TEM dark-field images of 2 mM ZVI/nC60 showed the map ofmetallic iron crystallites (bright lattice-like spots) within the nano-composites (Fig. 3a). These observations of iron crystallites within

larger ZVI nanoparticles are consistent with the previous reportson characteristics of iron nanoparticles [40]. XPS survey scans ofthe ZVI/nC60 composites showed a C 1s peak arising from nC60,and signals corresponding to iron Fe 2p (Fig. 3b). The iron signals

E. Erdim et al. / Journal of Hazardous Materials 283 (2015) 80–88 83

F imageZ 0 conc∼

(itroi

ig. 2. The dynamic light scattering (a–d) and the transmission electron microscopyVI (a, e), 0.1 mM ZVI/nC60 (b, f), 2 mM ZVI (c, g), and 2 mM ZVI/nC60 (d, h). The nC6

33 nm (b), ∼55 nm (c), and ∼110 nm (d).

Fe 2p) related zero-valent iron (Fe0) and iron oxides are shownn Fig. 3c. The peaks at 711.2 eV, 719 eV, and 724 eV are attributed

o binding energies of Fe 2p3/2, shake-up satellites 2p3/2 and 2p1/2,espectively [41]. The peak centered at 711.2 eV corresponds to ironxide. The shoulder that appears at 712.3 eV is very close to bind-ng energy values reported earlier for iron oxohydroxide (FeOOH)

s (e–h) of ZVI nanoparticles and ZVI/nC60 nano-composites are shown here. 0.1 mMentration was 2.5 mg-C/L. The average diameter measured by DLS are: ∼33 nm (a),

[42]. The feature at 706.6 eV is close to the value reported for Fein its zero oxidation state [40–42]. This feature originates from

the core region of the nanoparticles. The signal of C 1s (Fig. 3d)in 2 mM ZVI/nC60 samples matched with the C 1s signal of purenC60 nanoparticles samples [43], further confirming the presenceof nC60 within ZVI/nC60 nano-composites.

84 E. Erdim et al. / Journal of Hazardous Materials 283 (2015) 80–88

F -ray pC zeron

bis(0ZlumFo(ofrOhZcoo

3Z

i

pH 7.8,

ig. 3. The dark-field transmission electron micrograph of 2 mM ZVI/nC60 (a), the X 1s and Fe 2p (b), the XPS high-resolution scan of Fe 2p peak showing signal fromC60 in 2 mM ZVI/nC60 (d).

Upon exposure to dissolved O2 in aqueous suspension, iron inoth the 2 mM ZVI and 2 mM ZVI/nC60 was rapidly oxidized result-

ng in transformation of spherical nanoparticles into “needle”-liketructures, a characteristic of iron-oxides and -oxohydroxides [44]Fig. 4a). However, some differences in oxidation behavior of.1 mM ZVI and 0.1 mM ZVI/nC60 were noted. For example, 0.1 mMVI was oxidized similarly to the 2 mM ZVI resulting in needle-ike structures, while the 0.1 mM ZVI/nC60 consistently showedniformly distributed spherical ZVI NPs interspersed among theixture of iron oxidation products and nC60 structures (Fig. 4b).

urther, the XPS analysis of the mixture confirmed the presencef residual zero-valent iron in ZVI/nC60 even at the end of 60 minFig. 4c). The presence of the three Fe 2p signals similar to thosebserved in Fig. 3c can also be seen in Fig. 4c. A possible explanationor these observations is that nC60 in the ZVI/nC60 nano-compositeapidly mediated electron transfer from ZVI surface to dissolved2 before dissolved iron ions could combine with O2 to form ironydroxides and oxides. Visual examination of vials of ZVI andVI/nC60 nano-composite suspensions for a period of four days indi-ated a significant qualitative difference in the oxidation behaviorf ZVI (dark vs. yellow color), which is attributed to the presencer absence of nC60 in the nano-composites (Fig. 4d).

.2. Effect of fullerene on superoxide radical (O2•−) generation by

VI in ZVI/nC60 nano-composite

The results of O2•− generation by aqueous suspensions of var-

ous ZVI and ZVI/nC60 in the presence and absence of UV-A are

hotoelectron spectroscopy (XPS) survey-scan of 2 mM ZVI/nC60 showing signals of-valent iron (Fe0) at 706.6 eV (c), and XPS high-resolution scan of C 1s signal from

shown in Fig. 5. The data were fitted using B-spline function in Ori-gin 8.6 (OriginLab: Data Analysis and Graphing software). Controlexperiments showed that in the absence of the ZVI, nC60 nanopar-ticles did not generate O2

•− (or •OH) at pH 7.8, which is consistentwith the results from previous reports [33,45–47]. At pH 7.8, theO2

•− generation was almost instantaneous, which reached a maxi-mal concentration within 5 and 15 min (Fig. 5a and b), respectively,in both 0.1 mM and 2 mM ZVI (and ZVI/nC60) suspensions inde-pendent of presence or absence of UV-A. A 3.74-fold increasein O2

•− concentration was achieved with 0.1 mM ZVI/2.5 mg-C/LnC60 whereas no such improvement was observed with 2 mMZVI/2.5 mg-C/L nC60 nano-composites when compared to 0.1 mMZVI and 2 mM ZVI NPs, respectively. This result suggests that theratio of ZVI to nC60 concentration in the nano-composite likelydetermines the electron-transferring activity of nC60, which is effi-cient when Fe concentration is low relative to nC60, and rules out thepossibility of the photosensitization pathway for O2

•− generation.The effect of nC60 concentration in the 0.1 mM ZVI/nC60 nano-composite on O2

•− generation is evident in Fig. 6, which showsa proportionate improvement in O2

•− generation with increas-ing nC60 concentration. In summary, Figs. 5a and 6 suggest thatO2

•− generation likely occurs via electron transfer pathway medi-ated by nC60 from ZVI to O2 through the following process at

C60. . .Fe0 → C2•−60 . . .Fe2+ (2)

C2•−60 · · ·Fe2+ + 2O

•−2 (3)

E. Erdim et al. / Journal of Hazardous Materials 283 (2015) 80–88 85

F I/nC60

p he blad own c

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C

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Fo(

ig. 4. The transmission electron micrographs of 2 mM ZVI/nC60 (a) and 0.1 mM ZVresence of zero-valent iron (Fe0) through the signal at 706.6 eV in the sample (c). Tays of storage at ambient conditions compared with the 0.1 mM ZVI (yellowish-br

60· · ·Fe2+ → C•−60 · · ·Fe3+ (4)

•−60 · · ·Fe3+ + O2 → C60· · ·Fe3+ + O

•−2 (5)

According to the reduction potential of Fe2+/Fe0 (EH0 = −0.447

NHE), Fe3+/Fe2+ (EH0 = +0.771 VNHE), C60/C60

•− (EH0 = −0.178 VNHE)

nd, O2/O2•− (EH

0 = −0.160 VNHE), the electron transfer from ZVI

o nC60 to O2 is thermodynamically favored, and in the ZVI-nC60omposite form it appears to be an enhanced transfer to O2 whenompared to ZVI alone (Fig. 5a). Further, the association of Fe2+

nd/or Fe3+ with nC60 would likely diminish the direct interaction

ig. 5. Superoxide radical generation by 0.1 mM ZVI (a), and 2 mM ZVI (b), in the presencf the suspensions was maintained at 7.8 using 15 mM phosphate buffered-saline (PBS).and ZVI/nC60) suspensions.

(b) exposed to UV-A at the end of 60 min. The high-resolution XPS scan shows theck color (d) of the suspension of 0.1 mM ZVI/nC60 suggested little oxidation after 4olored suspension) (d).

of Fe ions with H2O/O2, and consequently may abate rapid passiv-ation of ZVI surface.

3.3. Effect of ZVI on singlet oxygen (1O2) generation by fullerenein ZVI/nC60 nano-composite

The results of 1O2 generation in aqueous suspensions of 2.5 mg-C/L nC60, 0.1 mM ZVI and 0.1 mM ZVI/2.5 mg-C/L nC60 in thepresence of UV-A are shown in Fig. 7. Under UV-A illumination,0.1 mM ZVI alone did not generate any 1O2 whereas 2.5 mg-C/L

e and absence of 2.5 mg/L nC60, upon exposure to UV-A and dark (no UV-A). The pH The initial XTT concentration was 300 �M and 1000 �M in 0.1 mM and 2 mM ZVI

86 E. Erdim et al. / Journal of Hazardous Materials 283 (2015) 80–88

Fig. 6. The effect of nC60 concentration on superoxide radical generation by theZ1p

npstadnsit[tAtrcti

3

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Feaw

Fig. 8. The inactivation of P. aeruginosa by ZVI and ZVI/nC60 in the presence andabsence of UV-A are shown here. The initial concentration (N0) of the bacteria was2 × 107 CFU/mL. The concentration of nC60 was 2.5 mg-C/L. All experiments were

VI/nC60 nano-composite. The pH of the suspensions was maintained at 7.8 using5 mM phosphate buffered-saline (PBS). The initial XTT concentration in the sus-ensions was 300 �M.

C60 alone generated 1O2, which was shown as a first-order decom-osition of FFA (Fig. 7). Further, 1O2 was also generated by UV-Aensitized 0.1 mM ZVI/nC60 at a rate 2-fold lower when comparedo 2.5 mg-C/L nC60 alone. This decrease in 1O2 generation rate wasttributed to the composite form of nC60 with ZVI in ZVI/nC60evice, wherein the UV-A excitation of C60 in ZVI/nC60 was sig-ificantly suppressed when compared to free nC60 (no ZVI). Recenttudies have also shown that tight aggregates of nC60 either withtself (or with ZVI as in this study), results in self-quenching ofriplet-triplet excitation of C60 and lowering the 1O2 quantum yield48]. These results in Fig. 7 indicate that nC60 generates 1O2 via pho-osensitization pathway through transfer energy to dissolved O2.lthough 1O2 concentration in ZVI/nC60/O2 is low, when compared

o O2•−, it may still be available for realizing 1O2-specific oxidation

eactions [49]. Additionally, it may be possible to enhance the 1O2oncentration in the suspensions by increasing the nC60 concentra-ion in ZVI/nC60 device (i.e. changing Fe-to-C60 ratio), which wouldncrease the availability of both 1O2 as well as O2

•− (Figs. 5a and 6).

.4. Effect of ZVI concentration on bacterial inactivation

The results of P. aeruginosa inactivation after 5 min of expo-ure to various aqueous suspensions of ZVI and ZVI/nC60 (at pH.8) in the presence and absence of UV-A are shown in Fig. 8.

ig. 7. Singlet oxygen generation under UV-A by 2.5 mg/L aqu-nC60 in the pres-nce and absence of 0.1 mM ZVI and by ZVI only. Singlet oxygen generation wasssessed by furfuryl alcohol degradation. The initial furfuryl alcohol concentrationas 200 �M.

done in triplicates and the mean log (Nt /N0) and standard deviation are reportedhere. L and D in the legend refer to “Light (UV-A)” and “Dark (no UV-A)” conditions,respectively.

When exposed to UV-A alone or 2.5 mg-C/L nC60 alone (light anddark) the bacteria were negligibly inactivated, which suggests thatUV-A or 1O2 from UV-A sensitized 2.5 mg-C/L nC60 had no damag-ing effect on bacteria during the 5 min of treatment. On contrary,various levels of bacterial inactivation were observed in ZVI andZVI/nC60 suspensions due to differences in O2

•− concentrations(Fig. 5a and b). For example, independent of UV-A, 3-log and 5-loginactivation was achieved with 0.1 mM ZVI and 0.1 mM ZVI/2.5 mg-C/L nC60, respectively, whereas 6-log inactivation was observed inboth 2 mM ZVI and 2 mM ZVI/2.5 mg-C/L nC60, independent of UV-A. These results indicate that with the aid of a small amount ofnC60 (an electron transferring agent) and 20-fold lower ZVI rel-ative to 2 mM ZVI concentration it is possible to obtain greaterthan 4-log inactivation within 5 min of treatment. This shouldensure disinfection of pathogens in both surface and ground waterssince according to USA Environmental Protection Agency’s sur-face water treatment rule if 3-log and 4-log removal/inactivationof Giardia lamblia and viruses, respectively, were achieved thenall other pathogens in water could be controlled. TEM examina-tion of the samples revealed that P. aeruginosa suffered sufficientcell wall damage and lysis (Fig. 9a and b), which is consistentwith the previous Escherichia coli-ZVI study wherein the oxidiz-ing species were reactive oxygen and/or ferryl ion (in the presenceof a stabilizing agent such as EDTA) [21,50]. In addition to thepresence of damaged bacteria the TEM images also showed thepresence of spherical ZVI/nC60 nano-composites at the end of 5 minof treatment. This suggests that at ambient aqueous conditions,unlike ZVI, the transformation of ZVI/nC60 nano-composites intocorresponding oxides and hydroxides may not be rapid other-wise these spherical nano-composites should resemble needle-likestructures, which are characteristic of oxidation precipitates. Thisclaim is further supported by the results shown in Figs. 5a and 6,which shows enhanced O2

•− generation and improved reactivity ofZVI/nC60 compared to ZVI under the experimental conditions of thisstudy. The spherical nanoparticle appearance was still observed for0.1 mM ZVI/2.5 mg-C/L nC60 nano-composites (Fig. 4b) but not for

2 mM ZVI/2.5 mg-C/L nC60, which resembled needle-like structuresat the end of 60 min of treatment (Fig. 4a). These morphologicaldifferences before and after oxidation reactions of ZVI/nC60 nano-composites were attributed to the presence of nC60.

E. Erdim et al. / Journal of Hazardous Materials 283 (2015) 80–88 87

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Fig. 9. Transmission electron micrographs of P. aeruginosa after 5 min

. Conclusions

The findings of this study demonstrate that 0.1 mM ZVI/2.5 mg-/L nC60 nano-composite is a much better O2

•− generator andn efficient disinfection device than 0.1 mM ZVI alone. Enhanced2

•− generation was observed only when nC60 was present atomparable or higher amounts than that of ZVI in the ZVI/nC60ano-composite otherwise no improvement was noted comparedo ZVI alone. UV-A played no role in O2

•− generation, despite theresence of nC60 and an electron donor such as ZVI. On the contrary,he presence of UV-A and nC60 were responsible for 1O2 generationhereas ZVI in the ZVI/nC60 nano-composite form had a slightly

uppressing effect on 1O2 generation by the device. ZVI/2.5 mg-C/LC60 at 20-fold lower concentration relative to 2 mM ZVI resulted in-log inactivation of P. aeruginosa, which was only 1-log lower thanhose obtained with 2 mM ZVI or 2 mM ZVI/2.5 mg-C/L nC60 nano-omposite (6-log inactivation). The electron-transferring activity,ot photosensitization, of nC60 in ZVI/nC60 nano-composite is theominant mechanism for enhanced O2

•− generation, which is con-rary to well known photosensitization pathway of nC60 in the pres-nce of an electron donor such as ZVI. However, 1O2 is generatednly through photosensitization of nC60 in ZVI/nC60 nanocompos-te. Further, by modulating nC60 concentration in ZVI/nC60, O2

•−

nd 1O2 could be generated to realize oxidative reactions at neu-ral pH conditions. The amount of nC60 in ZVI/nC60 determineshe morphology of the nano-particulates, which is very impor-ant for ROS generation. The details on exact association of nC60ith ZVI surface and Fe ions during the oxidation reaction are stillnclear, but the presence of nC60 in the ZVI/nC60 nano-compositesppears to enhance the stability and reactivity of the nanopartic-late device. Further studies are needed to determine the optimalatios of ZVI and nC60 for maximal ROS generation under pH con-itions that are relevant for treatment systems. These ZVI/nC60evices open up opportunities to harvest photochemical and oxida-ive/reductive reactions simultaneously and shows potential foreveloping systems that could generate multiple ROS species orlectron transfer under environmental conditions for applicationsn engineered water treatment and environmental remediation.

cknowledgements

This work was funded through the The Scientific and Tech-

ological Research Council of Turkey (TUBITAK), Center for thenvironmental Implications of NanoTechnology (CEINT) by theSF and the EPA under NSF Cooperative Agreement Number EF-830093, and the NSF Partnership in International Research and

[

osure to 0.1 mM ZVI/nC60 (a) and 2 mM ZVI/nC60 (b) are shown here.

Education (PIRE) program. Any opinions, findings, conclusions orrecommendations expressed in this material are those of theauthor(s) and do not necessarily reflect the views of the TUBITAK orthe NSF or the EPA. This work has not been subjected to EPA reviewand no official endorsement should be inferred.

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