Journal of Hazardous Materials 269 (2014) 24–30

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Reduction of chalcogen oxyanions and generation of nanoprecipitates by the photosynthetic bacterium Rhodobacter capsulatus Roberto Borghese a,∗ , Chiara Baccolini a , Francesco Francia a , Piera Sabatino b , Raymond J. Turner c , Davide Zannoni a,∗∗ a

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

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• R. capsulatus cells produce extracellular chalcogens nanoprecipitates when lawsone is present. • Lawsone acts as a redox mediator from reducing equivalents to tellurite and selenite. • Nanoprecipitates production depends on carbon source and requires metabolically active cells. • Te0 and Se0 nanoprecipitates are identified by X-ray diffraction (XRD) spectroscopy.

a r t i c l e

i n f o

Article history: Received 27 August 2013 Received in revised form 26 November 2013 Accepted 4 December 2013 Available online 25 December 2013 Keywords: Tellurite Selenite Nanoprecipitates Lawsone Rhodobacter capsulatus

a b s t r a c t The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction with the toxic oxyanions tellurite (TeIV ) and selenite (SeIV ) by a highly variable level of resistance that is dependent on the growth mode making this bacterium an ideal organism for the study of the microbial interaction 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-like black intracellular deposits no clear mechanism was described for Se0 precipitation. Here, we present the first report on the biotransformation of tellurium and selenium oxyanions into extracellular Te0 and Se0 nanoprecipitates (NPs) by anaerobic photosynthetically growing cultures of R. capsulatus as a function of exogenously added redox-mediator lawsone, i.e. 2-hydroxy-1,4-naphthoquinone. The NPs formation was dependent on the carbon source used for the bacterial growth and the rate of chalcogen reduction was 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. © 2013 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: Via Irnerio 42, 40126 Bologna, Italy. Tel.: +39 0512091300; fax: +39 051242576. ∗∗ Corresponding author at: Via irnerio 42, 40126 Bologna, Italy. Tel.: +39 0512091285; fax: +39 051242576. E-mail addresses: [email protected] (R. Borghese), [email protected] (D. Zannoni). 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.12.028

Differently from bulk material, nanoparticles show peculiar physical, chemical and electronic properties deriving from their nano-scale dimension [1]. These physical properties derive from their large surface to volume ratio, large surface energy, spatial confinement and reduced imperfections. Because of their specific characters, the synthesis of monodispersed nanoparticles with different size and shape is an important goal but remains a

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

challenge in nanotechnology due to the use of toxic chemicals on the nanoparticles surface along with non-polar solvents used in the synthesis procedure. Although these physical/chemical methods are extensively applied, the presence of toxic chemicals limits their applications in clinical fields and are the subject of major concerns. Owing to this, microbiological methods to generate nanoparticles are regarded as safe, cost-effective and environment-friendly processes. In the past few years, data collected from strain isolation, selection, optimization of nanoparticles production conditions along with the possibility to generate genetically engineered strains overexpressing specific reducing agents, indicate the microbial synthesis of nanoparticles as a promising field of research [2,3]. Nanoparticles produced with the chalcogens selenium and tellurium show interesting optoelectronic and semiconducting properties that grant their utilization in applications such as microelectronic circuits and solar cells. Microbial-based production of Se0 , Te0 , CdSe, ZnSe and CdTe nanoparticles has been reported in the past [4–7]. Tellurium and selenium nanoparticles are often produced inside the cell and this leads to the isolation of these particles from the bacteria requiring additional downstream processing steps in order to release the metal spheres or rods through the use of detergents or ultrasound treatments. The possibility of inducing extracellular accumulation of the microbiological produced nanoparticles has to be regarded as highly desirable from easiness of production, better yield and purity of the material standpoints. Several bacteria such as Stenotrophomonas maltophila [8], Enterobacter cloacae [9], Rhodospirillum rubrum [10], Desulfovibrio desulfuricans [11], Escherichia coli [12], Pseudomonas stutzeri [13] and Tetrathiobacter kashmirensis [14], have shown to reduce SeO3 2− oxyanions to selenium (Se0 ) nanoparticles both inside and outside the cells with various physical morphologies. Similarly, tellurite (TeO3 2− ) reduction to crystal particles of elemental Te0 were reported both inside and/or outside the cells of bacterial species such as Rhodobacter capsulatus [15], Rhodobacter sphaeroides [16], Pseudomonas pseudoalcaligenes KF707 [17], Strain ER-Te-48 [18], Sulfurospirillum barnesii [5] and Bacillus selenitireducens [4]. The photosynthetic bacterium R. capsulatus is characterized in its interaction with the toxic oxyanions tellurite and selenite by a highly variable level of resistance that is dependent on the growth mode. Cells grown by aerobic respiration are very sensitive to tellurite with a MIC (minimal inhibitory concentration) of 0.008 mM, whereas cells grown photosynthetically in the absence of oxygen are highly resistant with a MIC of 0.8 mM [19]. This different response holds also for selenite, albeit less marked, with a MIC of 0.23 mM under aerobic conditions and 0.44 mM under photosynthetic growth conditions (Borghese, unpublished). As a similar growth mode response to selenite has been seen in the closely related species R. rubrum [10], this behavior makes photosynthetic bacteria ideal organisms for the study of the interaction between chalcogens and prokaryotic cells. In R. capsulatus tellurite has been shown to induce an oxidative stress response and to alter the functionality of the respiratory electron transport chain, most likely through its action on the maturation of cytochromes of c-type [15,20]. Similarly to other bacterial species, tellurite is taken up by the R. capsulatus cells and reduced in the cytoplasm, producing black intracellular deposits. R. capsulatus is the only species, along with E. coli, for which a transport system utilized by tellurite has been proposed. In E. coli a phosphate transport system appears to be responsible for the uptake of tellurite [21], while R. capsulatus cells take up the oxyanion via an acetate permease (ActP system) as evidenced by mutants and uptake competition studies [22,23]. The only transport system for selenite has been described in E. coli, in which this chalcogen has been proposed to enter the cell through a sulphate transport system [24] and, to

25

best of our knowledge, no selenite transporter has been studied in photosynthetic bacteria. Recent studies have shown that redox mediators such as lawsone (2-hydroxy-1,4-naphthoquinone), AQDS (anthraquinone2,6-disulfonate) and menadione (2-methyl-1,4-naphthoquinone) can participate in the biotransformation of azo dyes, nitroaromatics, polychlorinated compounds, FeIII oxides, UVI , TcVII , AsV , SeIV and TeIV [25–27]. In particular, lawsone has been shown to mediate the extracellular reduction of tellurite and selenite by E. coli cells with the associated accumulation of nanoprecipitates [27]. Here we present the first report on the biotransformation, driven by light-energy, of tellurium and selenium oxyanions into Te0 and Se0 nanoprecipitates (NPs) that are accumulated outside the cells by anaerobic cultures of R. capsulatus. This result was obtained through the use of exogenously added lawsone acting as redox mediator between intracellular reducing equivalents and highly soluble metalloid oxyanions. 2. Materials and methods 2.1. Growth conditions and nanoprecipitates preparation R. capsulatus B100 (kindly provided by JD Wall, University of Missouri, MO, USA) cells were grown anaerobically in the standard RCV minimal medium [28], with 30 mM malate as the carbon source, under photosynthetic conditions. Bacterial growth was determined by measuring the optical density of the cells’ suspension at 660 nm. Anaerobiosis was reached upon incubation of 1L filled screw-capped bottles, containing 1 × 108 cells mL−1 , for 18 h in the dark, to allow for the O2 consumption by bacterial respiration. Under these conditions the final oxygen concentration was ≤20 ␮M, as measured by a Clark-type oxygen electrode [19]. After reaching anaerobiosis, K2 TeO3 or Na2 SeO3 and lawsone were added at a final concentration of 1 mM, for the chalcogens, and 0.2 mM, for the redox mediator, and the bottles were put in the light. The different carbon sources used in the growth experiments were added at a concentration of 30 mM each. For nanoprecipitates preparation pyruvate was routinely used as the carbon source. NPs from both tellurium and selenium experiments were prepared after 48 h of incubation in the light. The cultures were first centrifuged at 15,300 × g for 10 min in order to collect the cells. The supernatant, without the cells, was then centrifuged at 22,100 × g for 60 min and the NPs were concentrated in a tight pellet. The material obtained consisted mainly of chalcogen NPs, with some residual cells, and was suspended in a small volume of Millipore purified water. In some experiments the suspended material was further purified by filtration through a 0.22 ␮m pores membrane. 2.2. Biochemical analyses Protein content of whole cells was determined by the method of Lowry et al. [29] after a 1 min incubation with 0.1 N NaOH in boiling water. Crystalline bovine serum albumin (Sigma) was used as the protein standard. The quantitative determination of potassium tellurite in liquid media was done using the reagent diethyldithiocarbamate (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 and 1.5% (w/v) glutaraldehyde (pH 7.2). The same buffer was then used for overnight washing of the sample followed by 2 h fixation with 2% (w/v) osmium tetroxide and dehydration with ethanol. Finally,

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Fig. 1. (a) TEM picture of tellurium precipitates inside the cytoplasm of a cell grown without lawsone added and (b) TEM picture of tellurium precipitates outside the cells following the addition of lawsone.

the samples were embedded in Durcopan, and thin sections prepared with a LKB Ultratome Nova were double-stained with uranyl acetate and lead citrate [31]. TEM negative staining. A CF300 mesh copper grid was first washed with deionized water. 2 ␮L of the cellular and/or NPs material, obtained as described in Section 2.1, were then applied onto the grid, left for 3 min and dried with a filter paper. The grid was then washed with EDTA 1 mM and dried with filter paper. Finally, the material on the grid was stained with 0.05% uranyl acetate for 1 min and the excess dried up with filter paper. All samples were examined with a Philips CM-100 transmission electron microscope.

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

3. Results R. capsulatus grown under photosynthetic conditions (anaerobically in the light) in the presence of high concentrations of TeO3 2− (0.2 mM) takes up the oxyanion and reduces it to elemental 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 (Eh0 = −145 mV), the oxyanion tellurite (used at 1 mM) is reduced outside the cells and it accumulates in the external medium assuming the same typical splinter-like shape (Fig. 1b). The tellurium particles formed are between 80 and 300 nm long. Notably, lawsone itself is toxic to cells and its effect on cellgrowth gets stronger at increasing concentration. Fig. 2 shows that cell-doubling times significantly increase, from 178 up to 548 min, in cells grown photosynthetically under anaerobic conditions in the presence 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 for cells-mediated tellurite reduction. Rapid light-flash spectroscopy using cell-membranes fragments (chromatophores) from photosynthetically grown cells of

2.4. Spectroscopy 2.4.1. Rapid flash spectroscopy Absorbance changes induced by a xenon flash (EG&G FX201), discharging a 3 × 10−6 F capacitor charged to a 1.5 kV, 4 × 10−6 s pulse duration at half-maximal intensity, were measured by a single-beam spectrophotometer equipped with a double grating mono-chromator (bandwidth, 1.5 nm). Other experimental conditions as in Borsetti et al. [32]. 2.4.2. X-ray diffraction (XRD) spectroscopy X-ray patterns, obtained from the tellurite derived NPs after centrifugation and air drying, and the selenite NPs after centrifugation, air drying and a thermal treatment by annealing for 3 h at 90 ◦ C, were collected using a PANanalytical X’Pert Pro powder 1.6

1.4

1.2

OD 660 nm

dt (min) 1

control

178 ± 16

0.8

lawsone 0,05 mM 283 ± 37

0.6

lawsone 0,1 mM

347 ± 42

lawsone 0,2 mM

548 ± 78

0.4

0.2

0 0

5

10

15

20

25

Time (h) Fig. 2. Growth curves of R. capsulatus cultures at the lawsone concentrations specified in the figure. Growth was assessed by measuring the optical density of the cell’ suspensions at 660 nm. Doubling times (dt) are shown for each growth condition. The traces shown are representative of several separate experiments.

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

Carbon sourcea

Lawsone

TeO3 2−

OD660

− + + + + + + +b

Fructose Fructose Glucose Malate Succinate Acetate Pyruvate Pyruvate

+ + + + + + + +

+ + + + + + + +

0.04 3.93 1.302 0.282 0.262 0.233 3.160 0.149

a b

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

Table 2 TeO3 2− reduction rate as a function of lawsone concentration. Lawsone concentrationa

TeO3 2− reductionb

0 50 100 200

68 113 114 115

a b

Fig. 3. Multiple turnover flash on R. capsulatus membrane fragments (chromatophores) in the presence (red traces) or absence (black traces) of 0.2 mM Lawsone: (a) reaction center photooxidation; (b) total cytochrome c oxidation. Chromatophores were suspended in an open, unstirred cuvette, at 0.060 mM bacteriochlorophyl in buffer GlyGly 50 mM, 20 mM KCl, pH 7.5 in the presence of 0.005 mM Antimycin A, 0.010 mM Nigericin, 0.010 mM Valinomycin. Traces are the average of 4 events made by trains of 10 flashes spaced by 100 ms. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)

R. capsulatus gave strong evidence that lawsone was able to interact, being possibly in equilibrium, with the light-induced electron transport system (Fig. 3a and b). Indeed, the photosynthetic reaction center (RC) re-reduction kinetics (seen at 542 nm) on antimycin A inhibited chromatophores, which follows its photooxidation, were accelerated by the addition of lawsone (Fig. 3a). This effect was paralleled by a faster re-reduction of the cytochrome c types (seen at 551–542 nm) oxidized by the photochemical reaction-center (RC) activity. As shown in Fig. 3b, in the presence of 0.2 mM lawsone, the accumulation of oxidized c type cytochromes induced by multiple activation of the reaction center photochemistry is markedly decreased, indicating that the reducing power produced by the photoactivity of the reaction center and trapped in the membrane quinone pool is rapidly available for cytochrome c re-reduction in the presence of lawsone. This result clearly indicated that lawsone, 2-hydroxy-1,4-naphthoquinone, added to photosynthetic membranes isolated from R. capsulatus, physiologically interacts (most likely at the ubiquinone-pool level) with the photo-cyclic electron transport system. The production of Te0 and Se0 extracellular nanoprecipitates (NPs) is dependent on the cultivation conditions, requiring anaerobic growth, and it is linked to the carbon source added. Table 1 shows how the nature of the carbon source determines the production of tellurium nanoparticles and that the presence of metabolically active cells is required. The accumulation of the particles was clearly seen because of the strong blackening of the cells’ suspension and was measured at 660 nm by optical spectroscopy.

± ± ± ±

9 13 15 14

Biomass fold increase after 24 h 4.3 4.5 3.1 3.1

mM. nmoles/h/mg proteins.

Malate, succinate and acetate were not able to support any reduction, 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 min at 65 ◦ C) the production of extracellular NPs was completely abolished. The extracellular tellurium NPs could be separated from the cellular component by filtrating the darkened cultures through a 0.22 ␮m pores membrane (Fig. 4). Cultures grown without lawsone did not show the presence of extracellular precipitated material upon filtration (not shown). Besides lawsone, other redox mediators were also tested in trial experiments; interestingly, lawsone was the only quinone species found to mediate the strong reduction of tellurite. Menadione (2-methyl-1,4-naphthoquinone) and Juglone (5-hydroxy-1,4-naphthoquinone), characterized by having very  similar chemical structure but different redox potentials (Eh0 = 

−14 mV and +50 mV, respectively) relative to lawsone (Eh0 = −145 mV), showed no redox activities in mediating tellurite reduction outside the cells (not shown). TeO3 2− cellular reduction, assessed by measuring the disappearance of the oxyanion from the culture, was determined in the presence and in the absence of lawsone (Table 2). The cultures were treated as usual (see Section 2.1) and pre-incubated in the light

Fig. 4. TEM negative staining of tellurium nanoparticles deriving from a bacterial culture passed through a 0.22 ␮m filter.

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Fig. 5. TEM negative staining of cells incubated with selenite and lawsone showing selenium globular nanoparticles outside the cellular body.

for 4 h in order to obtain a greater biomass. The measurements were started after addition of lawsone (at the indicated concentrations) and tellurite (1 mM). The rate of TeO3 2− reduction was higher (almost doubled) in the presence of lawsone and was independent on the concentration of the redox mediator. Conversely, as seen in growth experiments (Fig. 2), lawsone showed a concentrationdependent effect on biomass increase. Besides tellurium, the other chalcogen selenium has been tested in a vast number of studies for the production of different types of nanoparticles [5,6,33]. Therefore, our study on lawsone-dependent production of extracellular NPs by cells of R. capsulatus was also extended to selenium. Selenite at 1 mM concentration was added to R. capsulatus cultures, otherwise treated exactly as for the tellurite NPs production experiments, resulting in the accumulation of extracellular NPs (40–60 nm in diameter) with the globular structure, similarly seen for selenium nanoparticles produced by other organisms (Fig. 5) [4,5,34]. The nature of both Te0 and Se0 NPs was assessed by X-ray diffraction (XRD) analysis (Fig. 6a and b). Apparently, all the diffraction peaks from the tellurite-derived NPs, obtained by biochemical reduction in aqueous phosphate solution after centrifugation and air-drying, belong to a unique crystalline phase defined as native tellurium, Inorganic Center for Diffraction Data (ICDD) file n. 894899 (Fig. 6a). No other crystal phases were detected, suggesting that the tellurite phase supplied to the bacteria was quantitatively transformed into the extracellular metalloid phase. The XRD analysis for the selenite-derived globular NPs (Fig. 6b) shows all the main peaks belonging to Se0 file at 23.5◦ , 29.7◦ , 41.4◦ , 43.6◦ , 45.4◦ , 51.7◦ and 56.1◦ , characteristic for the trigonal Se crystals (ICDD file n 6-362). In addition, other crystal phases are clearly recognizable in the diffraction pattern as belonging to sodium pyruvate (8-687), used as a carbon source, and NaCl (5-628), besides a peak at 9.8◦ coming from the sample holder (SH). 4. Discussion During the last decade the facultative photosynthetic bacterium R. capsulatus has been used as a model system for the study of the microbial interaction with the toxic oxyanion tellurite [3] [35]. Similarly to other bacterial species, tellurite is rapidly and massively taken up by anaerobic/photosynthetic cells of R. capsulatus and reduced in the cytoplasm as black intracellular deposits [35]. In contrast to E. coli, where a phosphate transport system appears to be responsible for the uptake of tellurite [21], R. capsulatus cells take up the oxyanion via acetate permease as evidenced by mutants and uptake competition studies [22,23]. In R. capsulatus, the tellurite influx was shown to induce an oxidative and stress response and to alter the functionality of the respiratory

electron transport chain, most likely through its action on the maturation of cytochromes of c-type [15,20]. Interestingly, it has also been reported that the membrane-bound thiol:disulfide oxidoreductase, DsbB, allows the transfer of reducing equivalents from the membrane-embedded quinols to tellurite generating an “electron conduit” or “electron-nanowire” connecting the photosynthetic and/or respiratory redox complexes to periplasmically located metalloids [32]. This early observation encouraged us to test the possibility to use exogenously added mediators such as quinones to reduce and/or precipitate elemental Te0 both inside and outside the cells as recently shown in E. coli [27]. This report shows for the first time that in the presence of lawsone, i.e. 2-hydroxy-1,4-naphthoquinone, photosynthetic cells of R. capsulatus catalyze the extracellular accumulation of Te0 and Se0 nanoprecipitates (NPs) in contrast to the formation of intracellular deposits in the absence of lawsone. In a recent report [27] it was shown that E. coli cells harvested and concentrated to high density were able to produce both tellurium and selenium extracellular nanoprecipitates when lawsone was added to the cell suspension. However, no cellular growth was associated to the reduction process. In this work, the extracellular production of Te0 and Se0 NPs by R. capsulatus was dependent on cells metabolic activity, with the cells still vital and growing. Experimental work aimed to the optimization of the production process of NPs by actively growing bacterial cultures is underway in our laboratory. The possibility that the nanoprecipitates, accumulated intracellularly in the absence of lawsone, may be released by the cells and recovered in the external environment, is unlikely as cell-free culture medium obtained upon passage through a 0.22 ␮m filter, did not contain NPs in the absence of lawsone added to the growing culture. Notably, menadione (2-methyl-1,4-naphthoquinone) and juglone (5-hydroxy-1,4-naphthoquinone), characterized by having similar chemical structures but different redox potentials   (Eh0 = −14 mV and +50 mV, respectively) relative to lawsone (Eh0 = −145 mV), showed no significant redox activities in mediating tellurite/selenite reduction and precipitation outside the cells. The latter result is therefore in line with the mid-point potentials at pH 7.0 of the redox-couples involved as tellurite is mainly present  in the form of HTeO3 − /TeO3 2− with a Eh 0 of −127 mV, a value close to the one of lawsone but far away from that of menadione and juglone. A similar consideration can also be applied to the redox couple HSeO3 − /SeO3 2− which is predicted to have a mid-potential at pH 7.0 of −87 mV. This thermodynamic based reasoning strongly support our observation that lawsone is the only quinone, among those tested by us, that effectively mediates the redox-equilibrium between the physiological membrane quinone pool and oxyanions such as tellurite and/or selenite. An interesting observation, though unexplained at the moment, was that production of these extracellular particles is linked to the cell growth carbon source as the accumulation of the tellurium particles was not seen with malate, succinate and acetate whereas pyruvate, fructose and, to a lesser extent glucose, produced a deep blackening due to Te0 precipitates. In this respect, we have shown in the past that the expression of acetate permease (actP gene cluster), responsible for tellurite uptake in R. capsulatus, is down-regulated by fructose while it is up-regulated by acetate [36]. Possibly, repression or induction of tellurite uptake would affect the cell viability which might, in turn, influence the cell’s delivery of reducing equivalents to exogenous chalcogens (this work). Further studies are clearly necessary to validate our working hypothesis and better correlate the exogenous formation of metalloid nanoparticles to the carbon source effects on the cell metabolism. Rapid light-flash spectroscopy gave strong evidence for the interaction of lawsone with the membrane-bound electron transport system. Indeed, a strong acceleration of the photocyclic electron flow, which involves a set of redox carriers such as

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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 tellurium peaks, 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 bars indicate position and relative intensities of the trigonal selenium peaks, as reported by ICDD file n 6-362; filled stars identify peaks of the sodium pyruvate (8-687); open diamonds identify peaks of the NaCl (5-628) crystal phases coming from the biochemical treatment; open square identify the sample holder (SH).

ubiquinones (UQ-10), cytochrome bc1 complex (or complex III) and cytochrome c2 is seen following the addition of lawsone to in vitro plasma-membrane fragments (chromatophores) isolated from photosynthetically grown R. capsulatus. This result (shown in Fig. 3) was interpreted to show that lawsone is able to mediate the reduction of exogenously added metalloids through the use of reducing equivalents possibly deriving from the membraneembedded ubiquinol pool (UQH2 ) generated by light energy [15,20]. The rate of tellurite reduction mediated by lawsone was clearly higher (about twofold) than the reduction with no redox mediator added. Interestingly, the oxyanion reduction rate was independent from the lawsone concentration in the range from 0.05 mM up to 0.2 mM. This suggests that under the experimental condition used here, a high rate of electron exchange between cells and lawsone was present. This finding will allow the optimization of the process in order to maximize the NPs production and minimize the lawsone toxicity so to maintain an active cellular biomass. X-ray diffraction analysis indicated that tellurium NPs outside the cells represent indeed a unique crystalline phase, i.e. elemental Te0 . When selenite (SeO3 2− ) is substituted for tellurite (TeO3 2− ), the elemental selenium (Se0 ) NPs show a different shape, as compared to Te0 , assuming a globular structure as seen by TEM analysis. In this respect, it is important to note that a sample obtained from freshly synthesized Se colloidal nanoparticles gave a XRD plot in which the Se0 peaks were much less evident. In order to obtain a more ordered Se0 phase, a thermal treatment was applied to the samples, by annealing for 3 h at 90 ◦ C and letting them slowly go back to ambient temperature (see Section 2.4.2). Under these conditions, the trigonal Se crystal phase clearly prevails over different allotropic forms. The need for thermal treatment in order to put in evidence the Se phase suggests the colloidal nature of the selenite nanoparticles and the XRD pattern of the nanospheres after annealing confirms the complete transformation of selenite into pure Se0 as the final crystalline product.

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

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

The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction with the toxic oxyanions tellurite (Te(IV)) and se...
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