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Extracellular biosynthesis of Cu2-xSe nanocrystallites with photocatalytic activity
Materials Research Bulletin 111 (2019) 126–132
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Extracellular biosynthesis of Cu2-xSe nanocrystallites with photocatalytic activity Shiyue Qi, Shuhui Yang, Lei Yue, Jia Wang, Xinrui Liang, Baoping Xin
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School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, 100081, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Biosynthesis Photocatalytical activity Cu2-xSe Extracellular proteins
Semiconductor Cu2-xSe nanospheres were successfully biosynthesized based on bioreduction of SeO32− into Se2by the selenite-reducing bacterium, Pantoea agglomerans. The Cu2-xSe nanospheres had excellent crystallinity with a face-centered cubic structure and an average diameter of 80 nm. Composition and oxidation states analysis using X-ray photoelectron spectroscopy and X-ray energy dispersive spectroscopy followed by optical characterization using ultraviolet-visible and Fourier transform infra-red spectroscopy confirmed that the biosynthesized Cu2-xSe nanospheres were capped by proteins. The extracellular proteins which mediated biosynthesis were visualized by excitation–emission matrix fluorescence spectroscopy. Sodium dodecyl sulfatepolyacrylamide gel electrophoretic analysis revealed that the molecular masses of proteins were about 110, 50, 38, 35 and 25 kDa. The biosynthesized Cu2−xSe nanospheres showed an excellent and stable photocatalytical activity under sunlight irradiation in the degradation of methylene blue for four cycles. This study put forward a green and toilless way to manufacture copper selenide nanoparticles using a biological process.
1. Introduction The semiconductor metal selenide nanomaterials have been drawing widespread attention, due to their unique optoelectronic properties and pervasive applications in semiconductor devices, nonlinear optical materials, photosensitive elements, photoelectric conversion devices and other fields [1–3]. At present, a variety of chemical methods have been provided to synthesize metal selenide nanomaterials [4–6]. However, chemical methods are usually strict with reaction conditions and may produce poisonous and harmful byproducts, which will raise the cost of manufacture and safety risk. It is still difficult to develop a low consumption and convenient way to synthesize plentiful metal selenide nanomaterials with controlled structures and properties. In recent years, biological synthesis of metal selenide nanomaterials has raised widespread concern because of its simple preparation process, mild reaction conditions, low cost, the controllability of the growth of microorganisms and so on, representing a green way of fabricating industrial and technological nanomaterials [7,8]. For example, several metal selenide nanomaterials including CdSe, PbSe and ZnSe have been synthesized by yeast, fungi and bacteria [9–14]. Kumar et al. made use of Fusarium oxysporum incubated at 25 ℃ to biosynthesize highly luminescent CdSe quantum dots with a mixture of CdCl2, SeCl4 and a reducing agent NaNO2, which would be of great
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importance [11]. Also, Cui et al. [9] demonstrated that multicolour CdSe quantum dots were produced in controlled synthesis from CdCl2 and Na2SeO3 by coupling yeast intracellular irrelated biochemical reactions. In the synthesis process, glutathione is necessary in the reduction of sodium selenite. In both of the studies, Se4+ is reduced to Se2− by an additional reducing agent (NaNO2) or autogenous reducing agent (glutathione), and the microorganisms just play the role of parcel agent or template package. Jacob et al. [14] manufactured PbSe quantum dots by Aspergillus terreus, a selenium and lead tolerant marine microorganism, but the role of the microorganisms was not clear yet. Bao et al. testified that the extracellular synthesis of CdTe quantum dots directly relied on the E. coli-secreted proteins [15]. Cu2-xSe is a significant p-type semiconductor exhibiting stoichiometry-dependent band gap (direct band gap of 2.2 eV) among metal selenides, which makes it appealing in diverse fields, such as solar cells, gas sensing and thermoelectric converters [16–19]. Dorfs et al. [5] reported that the Cu2-xSe nanoparticles had good plasmonic properties. Scotognella et al. [20] confirmed that the colloidal Cu2-xSe nanocrystals had an optical response to ultrafast laser pulses. It was also found that the heavily-doped Cu2-xSe nanocrystals with strong NIR absorption were qualified for in vivo biomedical imaging [21,22]. On the one hand, although the Cu2-xSe materials possess potential photocatalytic degradation capacity in the visible region due to its low band gap, there
Corresponding author. E-mail address: [email protected] (B. Xin).
https://doi.org/10.1016/j.materresbull.2018.11.014 Received 6 October 2017; Received in revised form 9 October 2018; Accepted 12 November 2018 Available online 15 November 2018 0025-5408/ © 2018 Elsevier Ltd. All rights reserved.
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flasks. The precipitates were then collected using centrifugation (1006.2 ×g, 10 min) and purified by washing five times with deionized water (20 ml) and three times with ethanol (5 ml) at room temperature, followed by heating at 105 ℃ to constant weight. The yield was 89.7% by monitoring the difference of Cu2+ concentration before and after biosynthesis using atomic absorption spectroscopy (AAS320, Shanghai Precision Instrument Company, China).
have not been any reports available regarding the photocatalytic activity of Cu2-xSe nanoparticles. On the other hand, various methods have been developed to produce Cu2-xSe nanocrystals in the past decade, such as hydrothermal method [16], low-temperature colloidal method [19], sonochemical irradiation method [23], and template method [24]. These efficient chemical approaches synthesize Cu2-xSe nanocrystallites with different morphologies, such as nanoparticles, nanoboxes, nano thin films, nanotubes and nanowires [23–26]. To our knowledge, the biological synthesis of Cu2-xSe nanocrystals and the functions of extracellular proteins in the biosynthesis have not been reported so far. In the previous work, we successfully synthesized Cu2Se nanospheres using a novel biological-chemical coupling reduction process in the presence of NaBH4 which was necessary in the synthesis of Cu2Se nanospheres via reducing Cu2+ into Cu+ that not only promoted the generation of Se2− as a catalyst but also reacted with Se2− to form Cu2Se [27]. However, the use of dangerous NaBH4 is a stigma to the biosynthesis. Herein, we report a new discovery that the Cu2-xSe nanospheres with an average particle size of 80 nm were extracellularly biosynthesized in the absence of NaBH4 using EDTA-Cu2+ and Na2SeO3 as precursors by Pantoea agglomerans (P. agglomerans), The role of extracellular proteins secreted by the bacterial cells in the biosynthesis of Cu2-xSe was preliminarily explored using Excitation–emission matrix (EEM) fluorescence spectroscopy and Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The photocatalytic activity of the synthesized Cu2-xSe nanospheres was examined using repeated photocatalytic degradation of methylene blue (MB) under sunlight irradiation.
The crystalline phase of the samples was detected by XRD (Shimadzu XD-D1) with Cu Kα radiation (λ = 1.5418 Å, 40 kV, 40 mA, scan range 2θ = 10°∼ 80°, a scan rate of 8°/ min). The morphologies and the dimensions of the nanospheres were evaluated by SEM (Hitachi S-4800, Japan) at 20 kV accelerating voltage. TEM (Hitachi H-700) images were checked at 200 kV accelerating voltage, and the crystallites were studied by high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) attached to the TEM. The atoms composition and proportion of the nanospheres were detected by EDS (Oxford) operating at 20 kV. XPS was performed using Omicron Nanotechnology, GmBH, Germany XM1000-monochromator with Al Kα radiation of 1483 eV operated at 300 W (20 mA emission current, 15 kV) with a base pressure of 5 × 10−5 mbar. Fourier transform infrared (FTIR) spectroscopy analysis was performed using an EQUINO55 spectrometer using KBr pellet in 600∼4000 cm-1 region. 200 mg KBr and 1∼2 mg sample were ground in an agate mortar for about 1∼2 min in one direction to avoid damaging the crystal structure. Then the mixtures were put into a mold to pressure to 20 MPa for 1∼2 min for FTIR analysis [28,29].
2. Materials and methods
2.6. Characterization of extracellular proteins
2.1. Materials
The types and molecular weights of the extracellular proteins involved in biosynthesis were analyzed by comparing the difference of protein profile between the experiment group containing EDTA-Cu2+ and control group without EDTA-Cu2+. After culturing for 7 days, the culture solution in both groups was centrifuged (7155.2 ×g, 10 min) to remove the cells. And then the resulting supernatants containing soluble extracellular proteins were directly characterized by using excitation-emission matrix fluorescence spectroscopy (EEM, Eclipse Fluorescence Spectrophotometer, Agilent) to uncover qualitatively the protein profile [30]. At the same time, the extracellular proteins in the resulting supernatants were precipitated with ammonium sulfate method. The protein solution was kept stirring and ammonium sulfate solid particles were slowly added into the solution until saturated (4.1 M). With the concentration of ammonium sulfate aqueous solution increasing, different solubility of proteins were precipitated completely. The process should not be too fast to ensure the different kinds of proteins to be precipitated successfully. The proteins precipitates were then put into a 3500 MVco dialysis bag to remove the ammonium sulfate, followed by dissolving in phosphate buffer. Subsequently, the purified proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the types of proteins and their molecular weights [31]. The proteins samples were loaded onto a precast 10% SDS–PAGE gel and electrophoresed at 90 V for 90 min. Gels were stained with Coomassie Brilliant Blue R-250.
2.5. Characterizations of biosynthesized Cu2-xSe nanospheres
Glucose, lactic acid, yeast extract, KNO3, MgSO4·H2O, NaCl, K2HPO4, CaCl2, NH4Cl, KH2PO4, Na2SeO3, tetrasodium EDTA (EDTANa4), 2 mol/L NaOH, ethanol, all chemical reagents were analytical grade; and in all experiments, deionised water was used. 2.2. Identification and Cultivation of the bacterium The bacterium strain was obtained from the farmland soils around Beijing, which was identified as P. agglomerans by 16S rDNA method [27]. The cells were anaerobically cultured at 32 ℃ for ca. 7 days in a growth media for the purpose of inoculum when the cell density reached OD600≈0.6. The growth media contain Lactic acid, 0.1 mol/L; Na2SeO3, 3.8 g/L; NH4Cl, 1.0 g/L; KH2PO4, 0.5 g/L; MgSO4, 0.5 g/L; CaCl2, 0.1 g/L; pH 7.2. 2.3. Preparation of the precursor EDTA-Cu chelate solution The precursor EDTA-Cu chelate solution (0.1 mol/L) was prepared via the reaction of CuSO4·5H2O (0.1 mol) and tetrasodium EDTA-Na4 (0.1 mol) in 1000 mL of deionized water at 25 ℃ for 4 h. The EDTA-Cu was applied directly without further processing. To make sure the complete chelation between EDTA and Cu2+, a mother solution containing EDTA-4Na and CuSO4 was prepared ahead of time.
2.7. Photocatalytic activity of biosynthesized Cu2-xSe nanospheres 2.4. Biosynthesis of Cu2-xSe nanospheres The photocatalytic activity of biosynthesized Cu2-xSe nanospheres was measured in a complete spectrum of sunlight illumination by exposing MB at room temperature in a 250 mL beaker on a magnetic stirrer (30 rpm, 25 ℃). Five experimental groups were established for the purpose: 50 mL MB solution at 26 mg/L + 0.1 g Cu2-xSe + sunlight (Group A); 50 mL MB solution at 26 mg/L + 0.1 g Cu2-xSe + dark (Group B); 50 mL MB solution at 26 mg/L + 0.1 g Cu2-xSe + 1.0 mL H2O2 + sunlight (Group C); 50 mL MB solution at 26 mg/L + 0.1 g Cu2-
The biosynthesis media contained glucose, 10.0 g/L; KNO3, 1.0 g/L; MgSO4·H2O, 1.0 g/L; K2HPO4, 0.5 g/L; NaCl, 0.5 g/L; Na2SeO3, 3.8 g/L; 10 mM EDTA-CuSO4; pH≈9.0. P. agglomerans inoculum (OD600≈0.6) was inoculated into the biosynthesis media in 300 mL flasks at an inoculation density of 20% (v/v), which was sealed with liquid paraffin and then incubated at 32 ℃ for 7 days. During incubation, lots of black green sediments gradually generated and deposited at the bottom of the 127
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Fig. 1. The XRD patterns (a) and EDS spectrum (b) of the biosynthesized product. The upper plot in (a) is the XRD pattern of biosynthesized product, the lower lines on the abscissa are the XRD characteristic peaks of the pure cubic phase of Cu2-xSe (JCPDS card NO.06-0680). xSe + 1.0 mL H2O2 + dark (Group D); 50 mL MB solution at 26 mg/ L + 1.0 mL H2O2 + sunlight (Group E). All the suspensions were magnetically stirred in the dark for one hour to ensure the adsorption equilibrium prior to test. 2.0 mL of the suspensions were periodically extracted and centrifuged to remove the photocatalysts (3000 rpm, 10 min). The supernatant was analyzed by using a UV–vis spectrophotometer (Perkin-Elmer Lambda 750, USA) at 664 nm to measure the remaining concentration of MB [32]. Group C was applied to further assess the repeated photocatalytic activity of the biosynthesized Cu2-xSe. When MB had been totally removed by photocatalytic degradation, a required certain volume of mother liquor containing MB for a final concentration of 26 mg/L and 1.0 mL of H2O2 were again added into the decolored solution to initiate the second photocatalytic decoloration of MB. In the same way, the third and fourth photocatalytic decoloration of MB was conducted.
Fig. 2. XPS survey spectrum of the biosynthesized Cu2-xSe nanospheres (a), Cu 2p core level spectrum (b) and Se 3d core level spectrum (c).
left represent C and O respectively, suggesting there might be proteins attaching to the surface of the Cu2-xSe crystal and certain proteins might be involved in the biosynthesis of Cu2-xSe [33]. The XPS images uncover the elemental composition and their chemical states of the biosynthesized product (Fig. 2). The full-scan map of XPS further demonstrated that the product contains both Se and Cu (Fig. 2a). The Cu 2p core-level spectrum showed that there are two intense peaks centered at 930.85 eV and 950.8 eV which respectively correspond to Cu 2p3/2 and Cu 2p1/2 of the Cu+ ion, as an evidence of the existence of Cu+ in the product. The other two weak peaks centered at 933.1 eV and 953 eV represent respectively Cu 2p3/2 and Cu 2p1/2 of the Cu2+ ion, as an evidence of the presence of Cu2+ (Fig. 2b). A single big peak in the Se 3d core-level photograph centered at 52.8 eV represents Se 3d5/2 of Se2− ion as an evidence of Se2− existence (Fig. 2c) [34]. The XPS pictures confirmed that the product contain Cu+, Cu2+ and Se2−, further demonstrating that the biosynthesized product is Cu2-xSe [35]. The results showed that Cu2-xSe crystals were formed in the presence of P. agglomerans. In contrast, the concentration of both Cu2+ and SeO32− kept almost unchanged and no any precipitate occurred
3. Results and discussion 3.1. Characterization of the product synthesized by Pantoea agglomerans Fig. 1a depicts the XRD pattern of the as-biosynthesized product. The upper plot in Fig. 1a is the XRD patterns of biosynthesized product and the lower lines on the abscissa are XRD characteristic peaks of the pure cubic phase of Cu2-xSe crystallizing in the F-43 m (216) space group (JCPDS card NO.06-0680). It was observed that the major diffraction peaks of the biosynthesized product (111, 200, 220, 311, 400, 331 and 422) matched well with the pure cubic phase of Cu2-xSe. Hence, it was concluded that the biosynthesized product is crystalline Cu2-xSe. The elemental analysis by EDS revealed that the product contained two kinds of elements, Se and Cu, with the molar ratio of Cu to Se at 1.45:1, showing the Cu2-xSe crystal had nonstoichiometric composition (Fig. 1b). The two little peaks of the EDS spectrum on the 128
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nanoparticles are solid spheres with a uniform size of ca. 80 nm (Fig. 4c, 4d). The HRTEM images taken from the near margin of a single nanosphere indicated that the nanosphere has lattice planes with a spacing of ca. 0.3468 nm, consistent with the interplanar spacing of the 400 plane of Cu2-xSe (Fig. 5a). The selected area electron diffraction (SAED) patterns displayed that the biosynthesized Cu2-xSe nanospheres are single crystalline with a face-centered cubic structure (Fig. 5b). 3.3. The optical properties of Cu2-xSe nanospheres The UV–vis absorption spectrum shows that the biosynthesized Cu2nanoparticles have a strong and wide absorption peak at 549 nm, a weak and narrow absorption peak at 283 nm and a NIR absorption peak at 992 nm (Fig. 6a). The absorption at 549 nm attributes the electronic transition of Cu2-xSe nanoparticles as a characteristic absorption peak which is closely related to its photocatalytical activity under sunlight [37], the absorption at 283 nm is due to the attached proteins [38], the NIR absorption at 992 nm belongs to a unique plasma resonance absorption [39]. The FTIR spectrum from 600 to 4000 cm−1 shows that there are vibration bands at 740, 970, 1541, 1647 and 3290 cm−1, respectively (Fig. 6b). The broad composite band around 3290 cm-1 is due to the hydrogen-bonded OeH / NeH groups. The amide I band (due primarily to the C]O stretching mode of the peptide moiety) is observed at 1647 cm−1 [40]. The strong peak at 1541 cm-1 is due to the presence of amide II band which is primarily NeH stretching modes of vibration in the amide linkage [41]. The strong absorption peak at 970 cm-1 is attributed to C–N vibrations of the peptide bond. The CeH bending mode of the aromatic residue in tryptophan is detected at 740 cm−1 [42]. All the absorption bands show the presence of amide group, carboxylic group and hydrocarbon group of proteins, demonstrating that the Cu2-xSe nanospheres are capped by proteins. xSe
Fig. 3. Schematic of the biosynthesis process of Cu2-xSe nanospheres by Pantoea agglomerans using EDTA-Cu2+ and SeO32− as precursors.
throughout the whole culture period of 7 days in the absence of cells, demonstrating the generation of Cu2-xSe crystal was due to P. agglomerans. At most cases, the red elemental selenium (Se°) is biosynthesized in the presence of SeO42−/SeO32− as a precursor by selenite-reducing microbial cultures [36]. In this study, however, only the black Cu2-xSe crystals generated driven by P. agglomerans, no any peaks representing Se° appeared in the XRD pattern and no peaks of Se° occurred in the XPS picture. It is concluded that there is a great difference in the bio-chemical mechanisms between the biosynthesis of Cu2-xSe by P. agglomerans and bio-fabrication of Se° by other selenite-reducing cells. In the biosynthesis of Cu2-xSe, a partial bio-reduction of Cu2+ into Cu+ and a complete bio-reduction of SeO32− into Se2− occurred (Fig. 3). However, a further study is required to uncover the detailed bio-chemical process involved in the biosynthesis of Cu2-xSe crystal.
3.4. Identification of the extracellular proteins adhered to the Cu2-xSe nanospheres Many bacteria are capable of secreting extracellular proteins to mediate the biosynthesis, generating biocompatible and dispersive nanospheres [28]. In our case, the components of the soluble organic substances including the extracellular proteins in the medium where
3.2. The morphology of biosynthesized Cu2-xSe nanoparticles The SEM images (Fig. 4a, b) show the biosynthesized Cu2-xSe as well dispersed nanospheres. The TEM images reveal that the Cu2-xSe
Fig. 4. SEM images (a, b) and TEM photographs ((c, d); with different magnification) of the Cu2-xSe nanoparticles fabricated by P. agglomerans. 129
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Fig. 5. HRTEM image (a) and SAED patterns (b) of Cu2-xSe nanoparticles biosynthesized by P. agglomerans.
Fig. 6. The UV–vis absorption spectrum (a) and the FTIR spectrum (b) of the Cu2-xSe nanospheres biosynthesized by P. agglomerans.
Fig. 8. SDS-PAGE profiles of extracellular proteins in the media where P. agglomerans was grown. (Lane M shows standard protein molecular weight markers with molecular weights from 15 to 180 kDa.) Lanes A and B show the profile of extracellular proteins (A) in the control medium without biosynthesis of Cu2-xSe and (B) in the biosynthesis media (the proteins attached to the Cu2xSe nanospheres as mediators are marked with red dotted lines). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Cu2-xSe was synthesized were characterized by EEM spectrometry and compared with the control medium with no addition of EDTA-Cu2+ and no generation of Cu2-xSe (Fig. 7). Using consistent excitation and emission wavelength boundaries for each EEM, horizontal and vertical lines were drawn to divide the EEM into five regions [30]. Regions I and II (peaks at shorter excitation wavelengths < 250 nm and shorter emission wavelengths < 350 nm) are related to aromatic proteins such as tyrosine, phenylalanine and tryptophan; region III (peaks at shorter excitation wavelengths < 250 nm and longer emission wavelengths > 350 nm) corresponds to fulvic acid-like materials; region IV (Peaks at
Fig. 7. EEM spectrum of the extracellular organic matter from (a) control media without biosynthesis of Cu2-xSe, (b) medium with biosynthesis of Cu2-xSe nanospheres. Different colour represents the variation in relative content of soluble organic matters, and the units are fluorescence intensity.
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Fig. 9. Experimental evaluation of the photocatalytic activity of as-biosynthesized Cu2-xSe nanospheres. (a) The performance of MB degradation photo-catalyzed by the Cu2-xSe nanospheres under different conditions; (b) Repeated photo-degradation of MB catalyzed by the Cu2-xSe nanospheres for four cycles.
the metal chalcogenides semiconductor materials are easily subjected to corrosion when photocatalytical degradation occurs, especially in the presence of H2O2 [45]. In this study, the high stability and corrosion resistance of the Cu2-xSe nanospheres is maybe due to the capping proteins. Nevertheless, more studies are needed to uncover the relationship between the capping proteins and the corrosion resistance of the Cu2-xSe nanospheres.
intermediate excitation wavelengths 250–280 nm and shorter emission wavelength < 380 nm) is relevant to a soluble microbial byproduct, e.g., including soluble amino acids; region V (Peaks at longer excitation wavelengths > 280 nm and longer emission wavelengths > 380 nm) represents humic acid-like organics which are secreted by P. agglomerans. It was observed that the area grew smaller and the colour became lighter in regions I, II and IV in the biosynthesis media than those in the control media, demonstrating certain extracellular proteins containing aromatic and soluble amino acids adhere to the Cu2-xSe nanospheres as a mediator during biosynthesis. The SDS-PAGE electrophoretogram showed there are a large number of electrophoretic bands in the control media, implying a great number of proteins secreted by the cells (Fig. 8A). However, the number of bands diminished evidently in the biosynthesis media, suggesting that quite a few of extracellular proteins adhered to the Cu2-xSe nanospheres for mediating the biosynthesis process, especially the extracellular proteins with molecular weights 110, 50, 38, 35 and 25 kDa (Fig. 8B) [15,31].
4. Conclusions Semiconductor Cu2-xSe nanospheres were successfully fabricated by P. agglomerans using EDTA-Cu2+ and SeO32− as precursors under mild conditions. The biogenic Cu2-xSe nanospheres show a relatively uniform size (ca. 80 nm), good crystallinity with a face-centered cubic structure and a wide and strong absorption centered at 549 nm. The cells secreted a great number of extracellular proteins into the medium, and some of them with molecular weights 110, 50, 38, 35 and 25 kDa adhered to the Cu2-xSe nanospheres mediating the biosynthesis process. The Cu2-xSe nanospheres can repeatedly and efficiently catalyze the photodegradation of MB driven by sunlight, and as high as 93.4% of MB degradation efficiency was still achieved even at the fourth catalytical cycle. The biosynthesized Cu2-xSe nanospheres display a good and stable photocatalytic activity under sunlight.
3.5. The photocatalytical activity of the Cu2-xSe nanospheres The photocatalytical degradation and decolorization of MB by the Cu2-xSe nanospheres under sunlight was presented in Fig. 9. The results showed that the removal of MB reached 20.3% after 8.5 h in the presence of Cu2-xSe nanospheres in the dark, displaying there was adsorption of MB by the Cu2-xSe nanospheres (Fig. 9a). The dye removal reached 24.1% in the presence of H2O2 under sunlight, indicating that a weak oxidation degradation of MB by H2O2 occurred triggered by sunlight. In the case of co-existence of Cu2-xSe nanospheres and H2O2, a MB removal of 29.2% was achieved in the absence of sunlight due to MB adsorption by the nanospheres and a slight oxidation of MB by H2O2. However, a complete removal of MB was achieved under sunlight in the presence of Cu2-xSe nanospheres after 8.5 h, showing a strong photodegradation of MB occurred catalyzed by the Cu2-xSe nanospheres which have a strong absorption at 549 nm [43] (see also Fig. 6a). And the addition of H2O2 evidently accelerated the photodegradation of MB because of the formation of superoxide radicals and hydroxyl radicals derived from the reactions between the photo-generated electrons/ holes and H2O2 [44], as a shorter period of 5.0 h was required for the complete photocatalytical degradation of MB. A repeated photocatalytical degradation of MB was carried out in the presence of both Cu2-xSe nanospheres and H2O2 (Fig. 9b). It was found that the Cu2-xSe nanospheres could repeatedly and efficiently catalyze the photodegradation of MB for four times, and as high as 93.4% of MB degradation efficiency was still achieved even at the fourth catalytical cycle, indicating that the as-biosynthesized Cu2-xSe nanospheres have high stability and corrosion resistance. In general,
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Extracellular biosynthesis of Cu2-xSe nanocrystallites with photocatalytic activity Shiyue Qi, Shuhui Yang, Lei Yue, Jia Wang, Xinrui Liang, Baoping Xin
T
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School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, 100081, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Biosynthesis Photocatalytical activity Cu2-xSe Extracellular proteins
Semiconductor Cu2-xSe nanospheres were successfully biosynthesized based on bioreduction of SeO32− into Se2by the selenite-reducing bacterium, Pantoea agglomerans. The Cu2-xSe nanospheres had excellent crystallinity with a face-centered cubic structure and an average diameter of 80 nm. Composition and oxidation states analysis using X-ray photoelectron spectroscopy and X-ray energy dispersive spectroscopy followed by optical characterization using ultraviolet-visible and Fourier transform infra-red spectroscopy confirmed that the biosynthesized Cu2-xSe nanospheres were capped by proteins. The extracellular proteins which mediated biosynthesis were visualized by excitation–emission matrix fluorescence spectroscopy. Sodium dodecyl sulfatepolyacrylamide gel electrophoretic analysis revealed that the molecular masses of proteins were about 110, 50, 38, 35 and 25 kDa. The biosynthesized Cu2−xSe nanospheres showed an excellent and stable photocatalytical activity under sunlight irradiation in the degradation of methylene blue for four cycles. This study put forward a green and toilless way to manufacture copper selenide nanoparticles using a biological process.
1. Introduction The semiconductor metal selenide nanomaterials have been drawing widespread attention, due to their unique optoelectronic properties and pervasive applications in semiconductor devices, nonlinear optical materials, photosensitive elements, photoelectric conversion devices and other fields [1–3]. At present, a variety of chemical methods have been provided to synthesize metal selenide nanomaterials [4–6]. However, chemical methods are usually strict with reaction conditions and may produce poisonous and harmful byproducts, which will raise the cost of manufacture and safety risk. It is still difficult to develop a low consumption and convenient way to synthesize plentiful metal selenide nanomaterials with controlled structures and properties. In recent years, biological synthesis of metal selenide nanomaterials has raised widespread concern because of its simple preparation process, mild reaction conditions, low cost, the controllability of the growth of microorganisms and so on, representing a green way of fabricating industrial and technological nanomaterials [7,8]. For example, several metal selenide nanomaterials including CdSe, PbSe and ZnSe have been synthesized by yeast, fungi and bacteria [9–14]. Kumar et al. made use of Fusarium oxysporum incubated at 25 ℃ to biosynthesize highly luminescent CdSe quantum dots with a mixture of CdCl2, SeCl4 and a reducing agent NaNO2, which would be of great
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importance [11]. Also, Cui et al. [9] demonstrated that multicolour CdSe quantum dots were produced in controlled synthesis from CdCl2 and Na2SeO3 by coupling yeast intracellular irrelated biochemical reactions. In the synthesis process, glutathione is necessary in the reduction of sodium selenite. In both of the studies, Se4+ is reduced to Se2− by an additional reducing agent (NaNO2) or autogenous reducing agent (glutathione), and the microorganisms just play the role of parcel agent or template package. Jacob et al. [14] manufactured PbSe quantum dots by Aspergillus terreus, a selenium and lead tolerant marine microorganism, but the role of the microorganisms was not clear yet. Bao et al. testified that the extracellular synthesis of CdTe quantum dots directly relied on the E. coli-secreted proteins [15]. Cu2-xSe is a significant p-type semiconductor exhibiting stoichiometry-dependent band gap (direct band gap of 2.2 eV) among metal selenides, which makes it appealing in diverse fields, such as solar cells, gas sensing and thermoelectric converters [16–19]. Dorfs et al. [5] reported that the Cu2-xSe nanoparticles had good plasmonic properties. Scotognella et al. [20] confirmed that the colloidal Cu2-xSe nanocrystals had an optical response to ultrafast laser pulses. It was also found that the heavily-doped Cu2-xSe nanocrystals with strong NIR absorption were qualified for in vivo biomedical imaging [21,22]. On the one hand, although the Cu2-xSe materials possess potential photocatalytic degradation capacity in the visible region due to its low band gap, there
Corresponding author. E-mail address: [email protected] (B. Xin).
https://doi.org/10.1016/j.materresbull.2018.11.014 Received 6 October 2017; Received in revised form 9 October 2018; Accepted 12 November 2018 Available online 15 November 2018 0025-5408/ © 2018 Elsevier Ltd. All rights reserved.
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flasks. The precipitates were then collected using centrifugation (1006.2 ×g, 10 min) and purified by washing five times with deionized water (20 ml) and three times with ethanol (5 ml) at room temperature, followed by heating at 105 ℃ to constant weight. The yield was 89.7% by monitoring the difference of Cu2+ concentration before and after biosynthesis using atomic absorption spectroscopy (AAS320, Shanghai Precision Instrument Company, China).
have not been any reports available regarding the photocatalytic activity of Cu2-xSe nanoparticles. On the other hand, various methods have been developed to produce Cu2-xSe nanocrystals in the past decade, such as hydrothermal method [16], low-temperature colloidal method [19], sonochemical irradiation method [23], and template method [24]. These efficient chemical approaches synthesize Cu2-xSe nanocrystallites with different morphologies, such as nanoparticles, nanoboxes, nano thin films, nanotubes and nanowires [23–26]. To our knowledge, the biological synthesis of Cu2-xSe nanocrystals and the functions of extracellular proteins in the biosynthesis have not been reported so far. In the previous work, we successfully synthesized Cu2Se nanospheres using a novel biological-chemical coupling reduction process in the presence of NaBH4 which was necessary in the synthesis of Cu2Se nanospheres via reducing Cu2+ into Cu+ that not only promoted the generation of Se2− as a catalyst but also reacted with Se2− to form Cu2Se [27]. However, the use of dangerous NaBH4 is a stigma to the biosynthesis. Herein, we report a new discovery that the Cu2-xSe nanospheres with an average particle size of 80 nm were extracellularly biosynthesized in the absence of NaBH4 using EDTA-Cu2+ and Na2SeO3 as precursors by Pantoea agglomerans (P. agglomerans), The role of extracellular proteins secreted by the bacterial cells in the biosynthesis of Cu2-xSe was preliminarily explored using Excitation–emission matrix (EEM) fluorescence spectroscopy and Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The photocatalytic activity of the synthesized Cu2-xSe nanospheres was examined using repeated photocatalytic degradation of methylene blue (MB) under sunlight irradiation.
The crystalline phase of the samples was detected by XRD (Shimadzu XD-D1) with Cu Kα radiation (λ = 1.5418 Å, 40 kV, 40 mA, scan range 2θ = 10°∼ 80°, a scan rate of 8°/ min). The morphologies and the dimensions of the nanospheres were evaluated by SEM (Hitachi S-4800, Japan) at 20 kV accelerating voltage. TEM (Hitachi H-700) images were checked at 200 kV accelerating voltage, and the crystallites were studied by high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) attached to the TEM. The atoms composition and proportion of the nanospheres were detected by EDS (Oxford) operating at 20 kV. XPS was performed using Omicron Nanotechnology, GmBH, Germany XM1000-monochromator with Al Kα radiation of 1483 eV operated at 300 W (20 mA emission current, 15 kV) with a base pressure of 5 × 10−5 mbar. Fourier transform infrared (FTIR) spectroscopy analysis was performed using an EQUINO55 spectrometer using KBr pellet in 600∼4000 cm-1 region. 200 mg KBr and 1∼2 mg sample were ground in an agate mortar for about 1∼2 min in one direction to avoid damaging the crystal structure. Then the mixtures were put into a mold to pressure to 20 MPa for 1∼2 min for FTIR analysis [28,29].
2. Materials and methods
2.6. Characterization of extracellular proteins
2.1. Materials
The types and molecular weights of the extracellular proteins involved in biosynthesis were analyzed by comparing the difference of protein profile between the experiment group containing EDTA-Cu2+ and control group without EDTA-Cu2+. After culturing for 7 days, the culture solution in both groups was centrifuged (7155.2 ×g, 10 min) to remove the cells. And then the resulting supernatants containing soluble extracellular proteins were directly characterized by using excitation-emission matrix fluorescence spectroscopy (EEM, Eclipse Fluorescence Spectrophotometer, Agilent) to uncover qualitatively the protein profile [30]. At the same time, the extracellular proteins in the resulting supernatants were precipitated with ammonium sulfate method. The protein solution was kept stirring and ammonium sulfate solid particles were slowly added into the solution until saturated (4.1 M). With the concentration of ammonium sulfate aqueous solution increasing, different solubility of proteins were precipitated completely. The process should not be too fast to ensure the different kinds of proteins to be precipitated successfully. The proteins precipitates were then put into a 3500 MVco dialysis bag to remove the ammonium sulfate, followed by dissolving in phosphate buffer. Subsequently, the purified proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the types of proteins and their molecular weights [31]. The proteins samples were loaded onto a precast 10% SDS–PAGE gel and electrophoresed at 90 V for 90 min. Gels were stained with Coomassie Brilliant Blue R-250.
2.5. Characterizations of biosynthesized Cu2-xSe nanospheres
Glucose, lactic acid, yeast extract, KNO3, MgSO4·H2O, NaCl, K2HPO4, CaCl2, NH4Cl, KH2PO4, Na2SeO3, tetrasodium EDTA (EDTANa4), 2 mol/L NaOH, ethanol, all chemical reagents were analytical grade; and in all experiments, deionised water was used. 2.2. Identification and Cultivation of the bacterium The bacterium strain was obtained from the farmland soils around Beijing, which was identified as P. agglomerans by 16S rDNA method [27]. The cells were anaerobically cultured at 32 ℃ for ca. 7 days in a growth media for the purpose of inoculum when the cell density reached OD600≈0.6. The growth media contain Lactic acid, 0.1 mol/L; Na2SeO3, 3.8 g/L; NH4Cl, 1.0 g/L; KH2PO4, 0.5 g/L; MgSO4, 0.5 g/L; CaCl2, 0.1 g/L; pH 7.2. 2.3. Preparation of the precursor EDTA-Cu chelate solution The precursor EDTA-Cu chelate solution (0.1 mol/L) was prepared via the reaction of CuSO4·5H2O (0.1 mol) and tetrasodium EDTA-Na4 (0.1 mol) in 1000 mL of deionized water at 25 ℃ for 4 h. The EDTA-Cu was applied directly without further processing. To make sure the complete chelation between EDTA and Cu2+, a mother solution containing EDTA-4Na and CuSO4 was prepared ahead of time.
2.7. Photocatalytic activity of biosynthesized Cu2-xSe nanospheres 2.4. Biosynthesis of Cu2-xSe nanospheres The photocatalytic activity of biosynthesized Cu2-xSe nanospheres was measured in a complete spectrum of sunlight illumination by exposing MB at room temperature in a 250 mL beaker on a magnetic stirrer (30 rpm, 25 ℃). Five experimental groups were established for the purpose: 50 mL MB solution at 26 mg/L + 0.1 g Cu2-xSe + sunlight (Group A); 50 mL MB solution at 26 mg/L + 0.1 g Cu2-xSe + dark (Group B); 50 mL MB solution at 26 mg/L + 0.1 g Cu2-xSe + 1.0 mL H2O2 + sunlight (Group C); 50 mL MB solution at 26 mg/L + 0.1 g Cu2-
The biosynthesis media contained glucose, 10.0 g/L; KNO3, 1.0 g/L; MgSO4·H2O, 1.0 g/L; K2HPO4, 0.5 g/L; NaCl, 0.5 g/L; Na2SeO3, 3.8 g/L; 10 mM EDTA-CuSO4; pH≈9.0. P. agglomerans inoculum (OD600≈0.6) was inoculated into the biosynthesis media in 300 mL flasks at an inoculation density of 20% (v/v), which was sealed with liquid paraffin and then incubated at 32 ℃ for 7 days. During incubation, lots of black green sediments gradually generated and deposited at the bottom of the 127
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Fig. 1. The XRD patterns (a) and EDS spectrum (b) of the biosynthesized product. The upper plot in (a) is the XRD pattern of biosynthesized product, the lower lines on the abscissa are the XRD characteristic peaks of the pure cubic phase of Cu2-xSe (JCPDS card NO.06-0680). xSe + 1.0 mL H2O2 + dark (Group D); 50 mL MB solution at 26 mg/ L + 1.0 mL H2O2 + sunlight (Group E). All the suspensions were magnetically stirred in the dark for one hour to ensure the adsorption equilibrium prior to test. 2.0 mL of the suspensions were periodically extracted and centrifuged to remove the photocatalysts (3000 rpm, 10 min). The supernatant was analyzed by using a UV–vis spectrophotometer (Perkin-Elmer Lambda 750, USA) at 664 nm to measure the remaining concentration of MB [32]. Group C was applied to further assess the repeated photocatalytic activity of the biosynthesized Cu2-xSe. When MB had been totally removed by photocatalytic degradation, a required certain volume of mother liquor containing MB for a final concentration of 26 mg/L and 1.0 mL of H2O2 were again added into the decolored solution to initiate the second photocatalytic decoloration of MB. In the same way, the third and fourth photocatalytic decoloration of MB was conducted.
Fig. 2. XPS survey spectrum of the biosynthesized Cu2-xSe nanospheres (a), Cu 2p core level spectrum (b) and Se 3d core level spectrum (c).
left represent C and O respectively, suggesting there might be proteins attaching to the surface of the Cu2-xSe crystal and certain proteins might be involved in the biosynthesis of Cu2-xSe [33]. The XPS images uncover the elemental composition and their chemical states of the biosynthesized product (Fig. 2). The full-scan map of XPS further demonstrated that the product contains both Se and Cu (Fig. 2a). The Cu 2p core-level spectrum showed that there are two intense peaks centered at 930.85 eV and 950.8 eV which respectively correspond to Cu 2p3/2 and Cu 2p1/2 of the Cu+ ion, as an evidence of the existence of Cu+ in the product. The other two weak peaks centered at 933.1 eV and 953 eV represent respectively Cu 2p3/2 and Cu 2p1/2 of the Cu2+ ion, as an evidence of the presence of Cu2+ (Fig. 2b). A single big peak in the Se 3d core-level photograph centered at 52.8 eV represents Se 3d5/2 of Se2− ion as an evidence of Se2− existence (Fig. 2c) [34]. The XPS pictures confirmed that the product contain Cu+, Cu2+ and Se2−, further demonstrating that the biosynthesized product is Cu2-xSe [35]. The results showed that Cu2-xSe crystals were formed in the presence of P. agglomerans. In contrast, the concentration of both Cu2+ and SeO32− kept almost unchanged and no any precipitate occurred
3. Results and discussion 3.1. Characterization of the product synthesized by Pantoea agglomerans Fig. 1a depicts the XRD pattern of the as-biosynthesized product. The upper plot in Fig. 1a is the XRD patterns of biosynthesized product and the lower lines on the abscissa are XRD characteristic peaks of the pure cubic phase of Cu2-xSe crystallizing in the F-43 m (216) space group (JCPDS card NO.06-0680). It was observed that the major diffraction peaks of the biosynthesized product (111, 200, 220, 311, 400, 331 and 422) matched well with the pure cubic phase of Cu2-xSe. Hence, it was concluded that the biosynthesized product is crystalline Cu2-xSe. The elemental analysis by EDS revealed that the product contained two kinds of elements, Se and Cu, with the molar ratio of Cu to Se at 1.45:1, showing the Cu2-xSe crystal had nonstoichiometric composition (Fig. 1b). The two little peaks of the EDS spectrum on the 128
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nanoparticles are solid spheres with a uniform size of ca. 80 nm (Fig. 4c, 4d). The HRTEM images taken from the near margin of a single nanosphere indicated that the nanosphere has lattice planes with a spacing of ca. 0.3468 nm, consistent with the interplanar spacing of the 400 plane of Cu2-xSe (Fig. 5a). The selected area electron diffraction (SAED) patterns displayed that the biosynthesized Cu2-xSe nanospheres are single crystalline with a face-centered cubic structure (Fig. 5b). 3.3. The optical properties of Cu2-xSe nanospheres The UV–vis absorption spectrum shows that the biosynthesized Cu2nanoparticles have a strong and wide absorption peak at 549 nm, a weak and narrow absorption peak at 283 nm and a NIR absorption peak at 992 nm (Fig. 6a). The absorption at 549 nm attributes the electronic transition of Cu2-xSe nanoparticles as a characteristic absorption peak which is closely related to its photocatalytical activity under sunlight [37], the absorption at 283 nm is due to the attached proteins [38], the NIR absorption at 992 nm belongs to a unique plasma resonance absorption [39]. The FTIR spectrum from 600 to 4000 cm−1 shows that there are vibration bands at 740, 970, 1541, 1647 and 3290 cm−1, respectively (Fig. 6b). The broad composite band around 3290 cm-1 is due to the hydrogen-bonded OeH / NeH groups. The amide I band (due primarily to the C]O stretching mode of the peptide moiety) is observed at 1647 cm−1 [40]. The strong peak at 1541 cm-1 is due to the presence of amide II band which is primarily NeH stretching modes of vibration in the amide linkage [41]. The strong absorption peak at 970 cm-1 is attributed to C–N vibrations of the peptide bond. The CeH bending mode of the aromatic residue in tryptophan is detected at 740 cm−1 [42]. All the absorption bands show the presence of amide group, carboxylic group and hydrocarbon group of proteins, demonstrating that the Cu2-xSe nanospheres are capped by proteins. xSe
Fig. 3. Schematic of the biosynthesis process of Cu2-xSe nanospheres by Pantoea agglomerans using EDTA-Cu2+ and SeO32− as precursors.
throughout the whole culture period of 7 days in the absence of cells, demonstrating the generation of Cu2-xSe crystal was due to P. agglomerans. At most cases, the red elemental selenium (Se°) is biosynthesized in the presence of SeO42−/SeO32− as a precursor by selenite-reducing microbial cultures [36]. In this study, however, only the black Cu2-xSe crystals generated driven by P. agglomerans, no any peaks representing Se° appeared in the XRD pattern and no peaks of Se° occurred in the XPS picture. It is concluded that there is a great difference in the bio-chemical mechanisms between the biosynthesis of Cu2-xSe by P. agglomerans and bio-fabrication of Se° by other selenite-reducing cells. In the biosynthesis of Cu2-xSe, a partial bio-reduction of Cu2+ into Cu+ and a complete bio-reduction of SeO32− into Se2− occurred (Fig. 3). However, a further study is required to uncover the detailed bio-chemical process involved in the biosynthesis of Cu2-xSe crystal.
3.4. Identification of the extracellular proteins adhered to the Cu2-xSe nanospheres Many bacteria are capable of secreting extracellular proteins to mediate the biosynthesis, generating biocompatible and dispersive nanospheres [28]. In our case, the components of the soluble organic substances including the extracellular proteins in the medium where
3.2. The morphology of biosynthesized Cu2-xSe nanoparticles The SEM images (Fig. 4a, b) show the biosynthesized Cu2-xSe as well dispersed nanospheres. The TEM images reveal that the Cu2-xSe
Fig. 4. SEM images (a, b) and TEM photographs ((c, d); with different magnification) of the Cu2-xSe nanoparticles fabricated by P. agglomerans. 129
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Fig. 5. HRTEM image (a) and SAED patterns (b) of Cu2-xSe nanoparticles biosynthesized by P. agglomerans.
Fig. 6. The UV–vis absorption spectrum (a) and the FTIR spectrum (b) of the Cu2-xSe nanospheres biosynthesized by P. agglomerans.
Fig. 8. SDS-PAGE profiles of extracellular proteins in the media where P. agglomerans was grown. (Lane M shows standard protein molecular weight markers with molecular weights from 15 to 180 kDa.) Lanes A and B show the profile of extracellular proteins (A) in the control medium without biosynthesis of Cu2-xSe and (B) in the biosynthesis media (the proteins attached to the Cu2xSe nanospheres as mediators are marked with red dotted lines). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Cu2-xSe was synthesized were characterized by EEM spectrometry and compared with the control medium with no addition of EDTA-Cu2+ and no generation of Cu2-xSe (Fig. 7). Using consistent excitation and emission wavelength boundaries for each EEM, horizontal and vertical lines were drawn to divide the EEM into five regions [30]. Regions I and II (peaks at shorter excitation wavelengths < 250 nm and shorter emission wavelengths < 350 nm) are related to aromatic proteins such as tyrosine, phenylalanine and tryptophan; region III (peaks at shorter excitation wavelengths < 250 nm and longer emission wavelengths > 350 nm) corresponds to fulvic acid-like materials; region IV (Peaks at
Fig. 7. EEM spectrum of the extracellular organic matter from (a) control media without biosynthesis of Cu2-xSe, (b) medium with biosynthesis of Cu2-xSe nanospheres. Different colour represents the variation in relative content of soluble organic matters, and the units are fluorescence intensity.
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Fig. 9. Experimental evaluation of the photocatalytic activity of as-biosynthesized Cu2-xSe nanospheres. (a) The performance of MB degradation photo-catalyzed by the Cu2-xSe nanospheres under different conditions; (b) Repeated photo-degradation of MB catalyzed by the Cu2-xSe nanospheres for four cycles.
the metal chalcogenides semiconductor materials are easily subjected to corrosion when photocatalytical degradation occurs, especially in the presence of H2O2 [45]. In this study, the high stability and corrosion resistance of the Cu2-xSe nanospheres is maybe due to the capping proteins. Nevertheless, more studies are needed to uncover the relationship between the capping proteins and the corrosion resistance of the Cu2-xSe nanospheres.
intermediate excitation wavelengths 250–280 nm and shorter emission wavelength < 380 nm) is relevant to a soluble microbial byproduct, e.g., including soluble amino acids; region V (Peaks at longer excitation wavelengths > 280 nm and longer emission wavelengths > 380 nm) represents humic acid-like organics which are secreted by P. agglomerans. It was observed that the area grew smaller and the colour became lighter in regions I, II and IV in the biosynthesis media than those in the control media, demonstrating certain extracellular proteins containing aromatic and soluble amino acids adhere to the Cu2-xSe nanospheres as a mediator during biosynthesis. The SDS-PAGE electrophoretogram showed there are a large number of electrophoretic bands in the control media, implying a great number of proteins secreted by the cells (Fig. 8A). However, the number of bands diminished evidently in the biosynthesis media, suggesting that quite a few of extracellular proteins adhered to the Cu2-xSe nanospheres for mediating the biosynthesis process, especially the extracellular proteins with molecular weights 110, 50, 38, 35 and 25 kDa (Fig. 8B) [15,31].
4. Conclusions Semiconductor Cu2-xSe nanospheres were successfully fabricated by P. agglomerans using EDTA-Cu2+ and SeO32− as precursors under mild conditions. The biogenic Cu2-xSe nanospheres show a relatively uniform size (ca. 80 nm), good crystallinity with a face-centered cubic structure and a wide and strong absorption centered at 549 nm. The cells secreted a great number of extracellular proteins into the medium, and some of them with molecular weights 110, 50, 38, 35 and 25 kDa adhered to the Cu2-xSe nanospheres mediating the biosynthesis process. The Cu2-xSe nanospheres can repeatedly and efficiently catalyze the photodegradation of MB driven by sunlight, and as high as 93.4% of MB degradation efficiency was still achieved even at the fourth catalytical cycle. The biosynthesized Cu2-xSe nanospheres display a good and stable photocatalytic activity under sunlight.
3.5. The photocatalytical activity of the Cu2-xSe nanospheres The photocatalytical degradation and decolorization of MB by the Cu2-xSe nanospheres under sunlight was presented in Fig. 9. The results showed that the removal of MB reached 20.3% after 8.5 h in the presence of Cu2-xSe nanospheres in the dark, displaying there was adsorption of MB by the Cu2-xSe nanospheres (Fig. 9a). The dye removal reached 24.1% in the presence of H2O2 under sunlight, indicating that a weak oxidation degradation of MB by H2O2 occurred triggered by sunlight. In the case of co-existence of Cu2-xSe nanospheres and H2O2, a MB removal of 29.2% was achieved in the absence of sunlight due to MB adsorption by the nanospheres and a slight oxidation of MB by H2O2. However, a complete removal of MB was achieved under sunlight in the presence of Cu2-xSe nanospheres after 8.5 h, showing a strong photodegradation of MB occurred catalyzed by the Cu2-xSe nanospheres which have a strong absorption at 549 nm [43] (see also Fig. 6a). And the addition of H2O2 evidently accelerated the photodegradation of MB because of the formation of superoxide radicals and hydroxyl radicals derived from the reactions between the photo-generated electrons/ holes and H2O2 [44], as a shorter period of 5.0 h was required for the complete photocatalytical degradation of MB. A repeated photocatalytical degradation of MB was carried out in the presence of both Cu2-xSe nanospheres and H2O2 (Fig. 9b). It was found that the Cu2-xSe nanospheres could repeatedly and efficiently catalyze the photodegradation of MB for four times, and as high as 93.4% of MB degradation efficiency was still achieved even at the fourth catalytical cycle, indicating that the as-biosynthesized Cu2-xSe nanospheres have high stability and corrosion resistance. In general,
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