Reduction of chalcogen oxyanions and generation of nanoprecipitates by the photosynthetic bacterium Rhodobacter capsulatus

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Journal of Hazardous Materials 269 (2014) 24–30

Contents lists available at ScienceDirect

Journal of Hazardous Materials

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

eduction of chalcogen oxyanions and generation of nanoprecipitatesy the photosynthetic bacterium Rhodobacter capsulatus

oberto Borghesea,∗, Chiara Baccolinia, Francesco Franciaa, Piera Sabatinob,aymond J. Turnerc, Davide Zannonia,∗∗

Department of Pharmacy and Biotechnology, University of Bologna, ItalyDepartment of Chemistry G. Ciamician, University of Bologna, ItalyDepartment of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

i g h l i g h t s

R. capsulatus cells produce extra-cellular chalcogens nanoprecipitateswhen lawsone is present.Lawsone acts as a redox mediatorfrom reducing equivalents to telluriteand selenite.Nanoprecipitates productiondepends on carbon source andrequires metabolically active cells.Te0 and Se0 nanoprecipitates areidentified by X-ray diffraction (XRD)spectroscopy.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 27 August 2013eceived in revised form6 November 2013ccepted 4 December 2013vailable online 25 December 2013

a b s t r a c t

The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction withthe toxic oxyanions tellurite (TeIV) and selenite (SeIV) by a highly variable level of resistance that isdependent on the growth mode making this bacterium an ideal organism for the study of the microbialinteraction with chalcogens. As we have reported in the past, while the oxyanion tellurite is taken up by R.capsulatus cells via acetate permease and it is reduced to Te0 in the cytoplasm in the form of splinter-likeblack intracellular deposits no clear mechanism was described for Se0 precipitation. Here, we present the

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eywords:elluriteeleniteanoprecipitatesawsonehodobacter capsulatus

first report on the biotransformation of tellurium and selenium oxyanions into extracellular Te and Senanoprecipitates (NPs) by anaerobic photosynthetically growing cultures of R. capsulatus as a functionof exogenously added redox-mediator lawsone, i.e. 2-hydroxy-1,4-naphthoquinone. The NPs formationwas dependent on the carbon source used for the bacterial growth and the rate of chalcogen reductionwas constant at different lawsone concentrations, in line with a catalytic role for the redox mediator.X-ray diffraction (XRD) analysis demonstrated the Te0 and Se0 nature of the nanoparticles.

∗ Corresponding author at: Via Irnerio 42, 40126 Bologna, Italy.el.: +39 0512091300; fax: +39 051242576.∗∗ Corresponding author at: Via irnerio 42, 40126 Bologna, Italy.el.: +39 0512091285; fax: +39 051242576.

E-mail addresses: [email protected] (R. Borghese),[email protected] (D. Zannoni).

304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jhazmat.2013.12.028

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Differently from bulk material, nanoparticles show peculiarphysical, chemical and electronic properties deriving from theirnano-scale dimension [1]. These physical properties derive from

their large surface to volume ratio, large surface energy, spatialconfinement and reduced imperfections. Because of their spe-cific characters, the synthesis of monodispersed nanoparticleswith different size and shape is an important goal but remains a

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hallenge in nanotechnology due to the use of toxic chemicals onhe nanoparticles surface along with non-polar solvents used in theynthesis procedure. Although these physical/chemical methodsre extensively applied, the presence of toxic chemicals limits theirpplications in clinical fields and are the subject of major concerns.wing to this, microbiological methods to generate nanoparti-les are regarded as safe, cost-effective and environment-friendlyrocesses. In the past few years, data collected from strain isola-ion, selection, optimization of nanoparticles production conditionslong with the possibility to generate genetically engineered strainsverexpressing specific reducing agents, indicate the microbialynthesis of nanoparticles as a promising field of research [2,3].

Nanoparticles produced with the chalcogens selenium andellurium show interesting optoelectronic and semiconductingroperties that grant their utilization in applications such as micro-lectronic circuits and solar cells. Microbial-based production ofe0, Te0, CdSe, ZnSe and CdTe nanoparticles has been reported inhe past [4–7].

Tellurium and selenium nanoparticles are often produced insidehe cell and this leads to the isolation of these particles fromhe bacteria requiring additional downstream processing stepsn order to release the metal spheres or rods through the usef detergents or ultrasound treatments. The possibility of induc-ng extracellular accumulation of the microbiological producedanoparticles has to be regarded as highly desirable from eas-

ness of production, better yield and purity of the materialtandpoints.

Several bacteria such as Stenotrophomonas maltophila [8],nterobacter cloacae [9], Rhodospirillum rubrum [10], Desulfovib-io desulfuricans [11], Escherichia coli [12], Pseudomonas stutzeri13] and Tetrathiobacter kashmirensis [14], have shown to reduceeO3

2− oxyanions to selenium (Se0) nanoparticles both inside andutside the cells with various physical morphologies. Similarly, tel-urite (TeO3

2−) reduction to crystal particles of elemental Te0 wereeported both inside and/or outside the cells of bacterial speciesuch as Rhodobacter capsulatus [15], Rhodobacter sphaeroides [16],seudomonas pseudoalcaligenes KF707 [17], Strain ER-Te-48 [18],ulfurospirillum barnesii [5] and Bacillus selenitireducens [4].

The photosynthetic bacterium R. capsulatus is characterized ints interaction with the toxic oxyanions tellurite and selenite by aighly variable level of resistance that is dependent on the growthode. Cells grown by aerobic respiration are very sensitive to tel-

urite with a MIC (minimal inhibitory concentration) of 0.008 mM,hereas cells grown photosynthetically in the absence of oxy-

en are highly resistant with a MIC of 0.8 mM [19]. This differentesponse holds also for selenite, albeit less marked, with a MICf 0.23 mM under aerobic conditions and 0.44 mM under photo-ynthetic growth conditions (Borghese, unpublished). As a similarrowth mode response to selenite has been seen in the closelyelated species R. rubrum [10], this behavior makes photosyntheticacteria ideal organisms for the study of the interaction betweenhalcogens and prokaryotic cells.

In R. capsulatus tellurite has been shown to induce an oxida-ive stress response and to alter the functionality of the respiratorylectron transport chain, most likely through its action on the matu-ation of cytochromes of c-type [15,20]. Similarly to other bacterialpecies, tellurite is taken up by the R. capsulatus cells and reduced inhe cytoplasm, producing black intracellular deposits. R. capsulatuss the only species, along with E. coli, for which a transport systemtilized by tellurite has been proposed. In E. coli a phosphate trans-ort system appears to be responsible for the uptake of tellurite21], while R. capsulatus cells take up the oxyanion via an acetate

ermease (ActP system) as evidenced by mutants and uptake com-etition studies [22,23]. The only transport system for selenite haseen described in E. coli, in which this chalcogen has been proposedo enter the cell through a sulphate transport system [24] and, to

us Materials 269 (2014) 24–30 25

best of our knowledge, no selenite transporter has been studied inphotosynthetic bacteria.

Recent studies have shown that redox mediators such aslawsone (2-hydroxy-1,4-naphthoquinone), AQDS (anthraquinone-2,6-disulfonate) and menadione (2-methyl-1,4-naphthoquinone)can participate in the biotransformation of azo dyes, nitroaromat-ics, polychlorinated compounds, FeIII oxides, UVI, TcVII, AsV, SeIV

and TeIV [25–27]. In particular, lawsone has been shown to medi-ate the extracellular reduction of tellurite and selenite by E. colicells with the associated accumulation of nanoprecipitates [27].Here we present the first report on the biotransformation, drivenby light-energy, of tellurium and selenium oxyanions into Te0 andSe0 nanoprecipitates (NPs) that are accumulated outside the cellsby anaerobic cultures of R. capsulatus. This result was obtainedthrough the use of exogenously added lawsone acting as redoxmediator between intracellular reducing equivalents and highlysoluble metalloid oxyanions.

2. Materials and methods

2.1. Growth conditions and nanoprecipitates preparation

R. capsulatus B100 (kindly provided by JD Wall, University ofMissouri, MO, USA) cells were grown anaerobically in the standardRCV minimal medium [28], with 30 mM malate as the carbonsource, under photosynthetic conditions. Bacterial growth wasdetermined by measuring the optical density of the cells’ suspen-sion at 660 nm. Anaerobiosis was reached upon incubation of 1Lfilled screw-capped bottles, containing 1 × 108 cells mL−1, for 18 hin the dark, to allow for the O2 consumption by bacterial respira-tion. Under these conditions the final oxygen concentration was≤20 �M, as measured by a Clark-type oxygen electrode [19]. Afterreaching anaerobiosis, K2TeO3 or Na2SeO3 and lawsone were addedat a final concentration of 1 mM, for the chalcogens, and 0.2 mM, forthe redox mediator, and the bottles were put in the light. The dif-ferent carbon sources used in the growth experiments were addedat a concentration of 30 mM each. For nanoprecipitates preparationpyruvate was routinely used as the carbon source.

NPs from both tellurium and selenium experiments were pre-pared after 48 h of incubation in the light. The cultures were firstcentrifuged at 15,300 × g for 10 min in order to collect the cells. Thesupernatant, without the cells, was then centrifuged at 22,100 × gfor 60 min and the NPs were concentrated in a tight pellet. Thematerial obtained consisted mainly of chalcogen NPs, with someresidual cells, and was suspended in a small volume of Milliporepurified water. In some experiments the suspended material wasfurther purified by filtration through a 0.22 �m pores membrane.

2.2. Biochemical analyses

Protein content of whole cells was determined by the method ofLowry et al. [29] after a 1 min incubation with 0.1 N NaOH in boil-ing water. Crystalline bovine serum albumin (Sigma) was used asthe protein standard. The quantitative determination of potassiumtellurite in liquid media was done using the reagent diethyldithio-carbamate (DDTC) (Sigma) as described by Turner et al. [30].

2.3. Electron microscopy

Transmission electron microscopy (TEM) thin section preparation.The bacterial cells pellets were first washed in 0.05 M cacodylate

buffer (pH 7.2) and then fixed for 2 h in 0.05 M cacodylate and1.5% (w/v) glutaraldehyde (pH 7.2). The same buffer was then usedfor overnight washing of the sample followed by 2 h fixation with2% (w/v) osmium tetroxide and dehydration with ethanol. Finally,

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26 R. Borghese et al. / Journal of Hazardous Materials 269 (2014) 24–30

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ig. 1. (a) TEM picture of tellurium precipitates inside the cytoplasm of a cell growollowing the addition of lawsone.

he samples were embedded in Durcopan, and thin sections pre-ared with a LKB Ultratome Nova were double-stained with uranylcetate and lead citrate [31]. TEM negative staining. A CF300 meshopper grid was first washed with deionized water. 2 �L of the cel-ular and/or NPs material, obtained as described in Section 2.1, werehen applied onto the grid, left for 3 min and dried with a filteraper. The grid was then washed with EDTA 1 mM and dried withlter paper. Finally, the material on the grid was stained with 0.05%ranyl acetate for 1 min and the excess dried up with filter paper.ll samples were examined with a Philips CM-100 transmissionlectron microscope.

.4. Spectroscopy

.4.1. Rapid flash spectroscopyAbsorbance changes induced by a xenon flash (EG&G FX201),

ischarging a 3 × 10−6 F capacitor charged to a 1.5 kV, 4 × 10−6 sulse duration at half-maximal intensity, were measured by aingle-beam spectrophotometer equipped with a double gratingono-chromator (bandwidth, 1.5 nm). Other experimental condi-

ions as in Borsetti et al. [32].

.4.2. X-ray diffraction (XRD) spectroscopy

X-ray patterns, obtained from the tellurite derived NPs after

entrifugation and air drying, and the selenite NPs after centrifu-ation, air drying and a thermal treatment by annealing for 3 ht 90 ◦C, were collected using a PANanalytical X’Pert Pro powder

0

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0.4

0.6

0.8

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1.4

1.6

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660

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ig. 2. Growth curves of R. capsulatus cultures at the lawsone concentrations specifieduspensions at 660 nm. Doubling times (dt) are shown for each growth condition. The tra

out lawsone added and (b) TEM picture of tellurium precipitates outside the cells

diffractometer, equipped with a fast X’Celerator detector using CuK� radiation generated at 40 kV and 40 mA. Samples were loaded on“zero background” sample holders 0.1 mm deep, 20 mm × 20 mmwide.

3. Results

R. capsulatus grown under photosynthetic conditions (anaer-obically in the light) in the presence of high concentrations ofTeO3

2− (0.2 mM) takes up the oxyanion and reduces it to ele-mental tellurium to form typical intracellular splinter-like deposits(Fig. 1a) [19]. Upon the addition of the redox mediator law-sone (0.2 mM), 2-hydroxy-1,4-naphthoquinone (E0′

h = −145 mV),the oxyanion tellurite (used at 1 mM) is reduced outside the cellsand it accumulates in the external medium assuming the same typ-ical splinter-like shape (Fig. 1b). The tellurium particles formed arebetween 80 and 300 nm long.

Notably, lawsone itself is toxic to cells and its effect on cell-growth gets stronger at increasing concentration. Fig. 2 shows thatcell-doubling times significantly increase, from 178 up to 548 min,in cells grown photosynthetically under anaerobic conditions in thepresence of lawsone and that they fail to reach high final densities.Owing to this, the toxic effect of this redox mediator should be

taken into account while setting the experimental conditions forcells-mediated tellurite reduction.

Rapid light-flash spectroscopy using cell-membranes frag-ments (chromatophores) from photosynthetically grown cells of

25

control

lawsone 0,0 5 mM

lawsone 0,1 mM

lawsone 0,2 mM

(min)dt

16178 ±

37283 ±

42347 ±

78 548 ±

in the figure. Growth was assessed by measuring the optical density of the cell’ces shown are representative of several separate experiments.

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R. Borghese et al. / Journal of Hazardous Materials 269 (2014) 24–30 27

Fig. 3. Multiple turnover flash on R. capsulatus membrane fragments (chro-matophores) in the presence (red traces) or absence (black traces) of 0.2 mMLawsone: (a) reaction center photooxidation; (b) total cytochrome c oxidation.Chromatophores were suspended in an open, unstirred cuvette, at 0.060 mM bac-teriochlorophyl in buffer GlyGly 50 mM, 20 mM KCl, pH 7.5 in the presence of0.005 mM Antimycin A, 0.010 mM Nigericin, 0.010 mM Valinomycin. Traces are theaverage of 4 events made by trains of 10 flashes spaced by 100 ms. (For interpreta-tt

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Table 1Extracellular reduction of tellurite in the presence of lawsone as a function of thecarbon source present in the growth medium.

Cells Carbon sourcea Lawsone TeO32− OD660

− Fructose + + 0.04+ Fructose + + 3.93+ Glucose + + 1.302+ Malate + + 0.282+ Succinate + + 0.262+ Acetate + + 0.233+ Pyruvate + + 3.160+b Pyruvate + + 0.149

a Carbon sources were added at a concentration of 30 mM.b Heat-inactivated cells (30 min at 65 ◦C).

Table 2TeO3

2− reduction rate as a function of lawsone concentration.

Lawsone concentrationa TeO32− reductionb Biomass fold

increase after 24 h

0 68 ± 9 4.350 113 ± 13 4.5

100 114 ± 15 3.1200 115 ± 14 3.1

pearance of the oxyanion from the culture, was determined in thepresence and in the absence of lawsone (Table 2). The cultures weretreated as usual (see Section 2.1) and pre-incubated in the light

ion of the references to color in text, the reader is referred to the web version ofhis article.)

. capsulatus gave strong evidence that lawsone was able tonteract, being possibly in equilibrium, with the light-induced elec-ron transport system (Fig. 3a and b). Indeed, the photosyntheticeaction center (RC) re-reduction kinetics (seen at 542 nm) onntimycin A inhibited chromatophores, which follows its photo-xidation, were accelerated by the addition of lawsone (Fig. 3a).his effect was paralleled by a faster re-reduction of the cytochrome

types (seen at 551–542 nm) oxidized by the photochemicaleaction-center (RC) activity. As shown in Fig. 3b, in the presence of.2 mM lawsone, the accumulation of oxidized c type cytochromes

nduced by multiple activation of the reaction center photochem-stry is markedly decreased, indicating that the reducing powerroduced by the photoactivity of the reaction center and trapped

n the membrane quinone pool is rapidly available for cytochrome re-reduction in the presence of lawsone. This result clearlyndicated that lawsone, 2-hydroxy-1,4-naphthoquinone, added tohotosynthetic membranes isolated from R. capsulatus, physiolog-

cally interacts (most likely at the ubiquinone-pool level) with thehoto-cyclic electron transport system.

The production of Te0 and Se0 extracellular nanoprecipitatesNPs) is dependent on the cultivation conditions, requiring anaer-bic growth, and it is linked to the carbon source added. Table 1hows how the nature of the carbon source determines theroduction of tellurium nanoparticles and that the presence of

etabolically active cells is required. The accumulation of the par-

icles was clearly seen because of the strong blackening of the cells’uspension and was measured at 660 nm by optical spectroscopy.

a mM.b nmoles/h/mg proteins.

Malate, succinate and acetate were not able to support any reduc-tion, whereas pyruvate, fructose and, to a lesser extent glucose,produced a deep blackening and a very high optical density (OD660).To be noted, when R. capsulatus cells were heat-inactivated (30 minat 65 ◦C) the production of extracellular NPs was completely abol-ished. The extracellular tellurium NPs could be separated from thecellular component by filtrating the darkened cultures through a0.22 �m pores membrane (Fig. 4). Cultures grown without lawsonedid not show the presence of extracellular precipitated materialupon filtration (not shown).

Besides lawsone, other redox mediators were also testedin trial experiments; interestingly, lawsone was the onlyquinone species found to mediate the strong reduction of tel-lurite. Menadione (2-methyl-1,4-naphthoquinone) and Juglone(5-hydroxy-1,4-naphthoquinone), characterized by having verysimilar chemical structure but different redox potentials (E0′

h =−14 mV and +50 mV, respectively) relative to lawsone (E0′

h =−145 mV), showed no redox activities in mediating tellurite reduc-tion outside the cells (not shown).

TeO32− cellular reduction, assessed by measuring the disap-

Fig. 4. TEM negative staining of tellurium nanoparticles deriving from a bacterialculture passed through a 0.22 �m filter.

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28 R. Borghese et al. / Journal of Hazardo

Fig. 5. TEM negative staining of cells incubated with selenite and lawsone showings

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elenium globular nanoparticles outside the cellular body.

or 4 h in order to obtain a greater biomass. The measurementsere started after addition of lawsone (at the indicated concentra-

ions) and tellurite (1 mM). The rate of TeO32− reduction was higher

almost doubled) in the presence of lawsone and was independentn the concentration of the redox mediator. Conversely, as seenn growth experiments (Fig. 2), lawsone showed a concentration-ependent effect on biomass increase.

Besides tellurium, the other chalcogen selenium has been testedn a vast number of studies for the production of different types ofanoparticles [5,6,33]. Therefore, our study on lawsone-dependentroduction of extracellular NPs by cells of R. capsulatus was alsoxtended to selenium. Selenite at 1 mM concentration was addedo R. capsulatus cultures, otherwise treated exactly as for the tellu-ite NPs production experiments, resulting in the accumulation ofxtracellular NPs (40–60 nm in diameter) with the globular struc-ure, similarly seen for selenium nanoparticles produced by otherrganisms (Fig. 5) [4,5,34].

The nature of both Te0 and Se0 NPs was assessed by X-rayiffraction (XRD) analysis (Fig. 6a and b). Apparently, all the diffrac-ion peaks from the tellurite-derived NPs, obtained by biochemicaleduction in aqueous phosphate solution after centrifugation andir-drying, belong to a unique crystalline phase defined as nativeellurium, Inorganic Center for Diffraction Data (ICDD) file n. 89-899 (Fig. 6a). No other crystal phases were detected, suggestinghat the tellurite phase supplied to the bacteria was quantitativelyransformed into the extracellular metalloid phase.

The XRD analysis for the selenite-derived globular NPs (Fig. 6b)hows all the main peaks belonging to Se0 file at 23.5◦, 29.7◦, 41.4◦,3.6◦, 45.4◦, 51.7◦ and 56.1◦, characteristic for the trigonal Se crys-als (ICDD file n 6-362). In addition, other crystal phases are clearlyecognizable in the diffraction pattern as belonging to sodium pyru-ate (8-687), used as a carbon source, and NaCl (5-628), besides aeak at 9.8◦ coming from the sample holder (SH).

. Discussion

During the last decade the facultative photosynthetic bacterium. capsulatus has been used as a model system for the study ofhe microbial interaction with the toxic oxyanion tellurite [3] [35].imilarly to other bacterial species, tellurite is rapidly and mas-ively taken up by anaerobic/photosynthetic cells of R. capsulatusnd reduced in the cytoplasm as black intracellular deposits [35].n contrast to E. coli, where a phosphate transport system appearso be responsible for the uptake of tellurite [21], R. capsulatus

ells take up the oxyanion via acetate permease as evidenced byutants and uptake competition studies [22,23]. In R. capsula-

us, the tellurite influx was shown to induce an oxidative andtress response and to alter the functionality of the respiratory

us Materials 269 (2014) 24–30

electron transport chain, most likely through its action on thematuration of cytochromes of c-type [15,20]. Interestingly, it hasalso been reported that the membrane-bound thiol:disulfide oxi-doreductase, DsbB, allows the transfer of reducing equivalentsfrom the membrane-embedded quinols to tellurite generating an“electron conduit” or “electron-nanowire” connecting the photo-synthetic and/or respiratory redox complexes to periplasmicallylocated metalloids [32]. This early observation encouraged us totest the possibility to use exogenously added mediators such asquinones to reduce and/or precipitate elemental Te0 both insideand outside the cells as recently shown in E. coli [27].

This report shows for the first time that in the presence of law-sone, i.e. 2-hydroxy-1,4-naphthoquinone, photosynthetic cells ofR. capsulatus catalyze the extracellular accumulation of Te0 and Se0

nanoprecipitates (NPs) in contrast to the formation of intracellulardeposits in the absence of lawsone. In a recent report [27] it wasshown that E. coli cells harvested and concentrated to high densitywere able to produce both tellurium and selenium extracellularnanoprecipitates when lawsone was added to the cell suspension.However, no cellular growth was associated to the reduction pro-cess. In this work, the extracellular production of Te0 and Se0 NPsby R. capsulatus was dependent on cells metabolic activity, withthe cells still vital and growing. Experimental work aimed to theoptimization of the production process of NPs by actively growingbacterial cultures is underway in our laboratory.

The possibility that the nanoprecipitates, accumulated intracel-lularly in the absence of lawsone, may be released by the cells andrecovered in the external environment, is unlikely as cell-free cul-ture medium obtained upon passage through a 0.22 �m filter, didnot contain NPs in the absence of lawsone added to the grow-ing culture. Notably, menadione (2-methyl-1,4-naphthoquinone)and juglone (5-hydroxy-1,4-naphthoquinone), characterized byhaving similar chemical structures but different redox potentials(E0′

h = −14 mV and +50 mV, respectively) relative to lawsone (E0′h =

−145 mV), showed no significant redox activities in mediating tel-lurite/selenite reduction and precipitation outside the cells. Thelatter result is therefore in line with the mid-point potentials atpH 7.0 of the redox-couples involved as tellurite is mainly presentin the form of HTeO3

−/TeO32− with a Eh

0′of −127 mV, a value close

to the one of lawsone but far away from that of menadione andjuglone. A similar consideration can also be applied to the redoxcouple HSeO3

−/SeO32− which is predicted to have a mid-potential

at pH 7.0 of −87 mV. This thermodynamic based reasoning stronglysupport our observation that lawsone is the only quinone, amongthose tested by us, that effectively mediates the redox-equilibriumbetween the physiological membrane quinone pool and oxyanionssuch as tellurite and/or selenite.

An interesting observation, though unexplained at the moment,was that production of these extracellular particles is linked to thecell growth carbon source as the accumulation of the telluriumparticles was not seen with malate, succinate and acetate whereaspyruvate, fructose and, to a lesser extent glucose, produced a deepblackening due to Te0 precipitates. In this respect, we have shown inthe past that the expression of acetate permease (actP gene cluster),responsible for tellurite uptake in R. capsulatus, is down-regulatedby fructose while it is up-regulated by acetate [36]. Possibly, repres-sion or induction of tellurite uptake would affect the cell viabilitywhich might, in turn, influence the cell’s delivery of reducing equiv-alents to exogenous chalcogens (this work). Further studies areclearly necessary to validate our working hypothesis and bettercorrelate the exogenous formation of metalloid nanoparticles tothe carbon source effects on the cell metabolism.

Rapid light-flash spectroscopy gave strong evidence for theinteraction of lawsone with the membrane-bound electron trans-port system. Indeed, a strong acceleration of the photocyclicelectron flow, which involves a set of redox carriers such as

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R. Borghese et al. / Journal of Hazardous Materials 269 (2014) 24–30 29

Fig. 6. (a) Continuous line: X-ray diffraction patterns of Te0 nanoparticles after centrifugation and air drying; gray bars: positions and relative intensities of the telluriumpeaks, as reported by the ICDD file n 89-4899; (b) continuous line: X-ray diffraction patterns of Se0 nanoparticles after centrifugation, air drying and annealing; gray barsi d by Id mical

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[16] M.D. Moore, S. Kaplan, Members of the family Rhodospirillaceae reduce heavy-

ndicate position and relative intensities of the trigonal selenium peaks, as reporteiamonds identify peaks of the NaCl (5-628) crystal phases coming from the bioche

biquinones (UQ-10), cytochrome bc1 complex (or complex III)nd cytochrome c2 is seen following the addition of lawsone ton vitro plasma-membrane fragments (chromatophores) isolatedrom photosynthetically grown R. capsulatus. This result (shownn Fig. 3) was interpreted to show that lawsone is able to medi-te the reduction of exogenously added metalloids through these of reducing equivalents possibly deriving from the membrane-mbedded ubiquinol pool (UQH2) generated by light energy15,20].

The rate of tellurite reduction mediated by lawsone was clearlyigher (about twofold) than the reduction with no redox mediatordded. Interestingly, the oxyanion reduction rate was independentrom the lawsone concentration in the range from 0.05 mM up to.2 mM. This suggests that under the experimental condition usedere, a high rate of electron exchange between cells and lawsoneas present. This finding will allow the optimization of the process

n order to maximize the NPs production and minimize the lawsoneoxicity so to maintain an active cellular biomass.

X-ray diffraction analysis indicated that tellurium NPs outsidehe cells represent indeed a unique crystalline phase, i.e. elementale0. When selenite (SeO3

2−) is substituted for tellurite (TeO32−),

he elemental selenium (Se0) NPs show a different shape, as com-ared to Te0, assuming a globular structure as seen by TEM analysis.

n this respect, it is important to note that a sample obtained fromreshly synthesized Se colloidal nanoparticles gave a XRD plot inhich the Se0 peaks were much less evident. In order to obtain aore ordered Se0 phase, a thermal treatment was applied to the

amples, by annealing for 3 h at 90 ◦C and letting them slowly goack to ambient temperature (see Section 2.4.2). Under these con-itions, the trigonal Se crystal phase clearly prevails over differentllotropic forms. The need for thermal treatment in order to put invidence the Se phase suggests the colloidal nature of the seleniteanoparticles and the XRD pattern of the nanospheres after anneal-

ng confirms the complete transformation of selenite into pure Se0

s the final crystalline product.

cknowledgment

We thank the University of Bologna (Grant FRA 2011-12) forupporting this work.

[

CDD file n 6-362; filled stars identify peaks of the sodium pyruvate (8-687); open treatment; open square identify the sample holder (SH).

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