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Biogenic synthesis of gold nanoparticles by yeast Magnusiomyces ingens LH-F1 for catalytic reduction of nitrophenols
Colloids and Surfaces A: Physicochem. Eng. Aspects 497 (2016) 280–285
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Biogenic synthesis of gold nanoparticles by yeast Magnusiomyces ingens LH-F1 for catalytic reduction of nitrophenols Xuwang Zhang, Yuanyuan Qu ∗ , Wenli Shen, Jingwei Wang, Huijie Li, Zhaojing Zhang, Shuzhen Li, Jiti Zhou State Key Laboratory of Fine Chemicals, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China
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
• This is the first report on AuNPs biosynthesis by Magnusiomyces ingens. • Various shaped AuNPs were synthesized by strain LH-F1. • Some biomolecules were found to be absorbed on the surface of AuNPs. • AuNPs showed excellent catalytic activities for the reduction of nitrophenols.
a r t i c l e
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Article history: Received 29 October 2015 Received in revised form 17 February 2016 Accepted 24 February 2016 Keywords: Gold nanoparticles Magnusiomyces ingens Nitrophenols Biosynthesis
a b s t r a c t In the present study, the biogenic synthesis of gold nanoparticles (AuNPs) was achieved using the yeast cells of Magnusiomyces ingens LH-F1. Based on UV–vis spectral analysis, 2.2 mg/mL biomass (OD600 = 2.0) and 1.0 mM HAuCl4 were preferable for AuNPs synthesis by strain LH-F1. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed that various shaped nanoparticles were obtained, including sphere, triangle and hexagon. Based on the analyses of Fourier transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), some biomolecules were absorbed on the surface of AuNPs, which could be involved in the formation of AuNPs. The as-synthesized AuNPs exhibited excellent catalytic activities for the reduction of nitrophenols (i.e. 4-nitrophenol, 3-nitrophenol and 2-nitrophenol) to aminophenols in the presence NaBH4 . This is the first report on AuNPs biosynthesis by Magnusiomyces ingens, which may serve as an efficient candidate for green synthesis of metal nanoparticles. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Gold nanoparticles (AuNPs) have attracted enormous attention in recent decades due to their unique photophysical, electronic and
∗ Corresponding author. E-mail address: [email protected] (Y. Qu). http://dx.doi.org/10.1016/j.colsurfa.2016.02.033 0927-7757/© 2016 Elsevier B.V. All rights reserved.
catalytic properties. They have broad applications in the fields of electronics, environmental sensing, biomedicine and fine chemical synthesis [1,2]. Various physical and chemical methods have been successfully used to produce AuNPs, but most of them are either of high-cost or involved with the usage of hazardous chemicals [3,4]. The biological methods are considered to be the green and ecofriendly alternatives for the synthesis of nanoparticles owing to their nature of rich diversity, cost-effective, and mild condition [5].
X. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 497 (2016) 280–285
Biological systems, such as bacteria, fungi and plants, have been shown to possess the ability of producing AuNPs via intra- and/or extra-cellular ways [4]. Among them, fungi are gaining more interests for the green synthesis of nanoparticles due to their practical advantages of easy handling, large amounts of secreted proteins and high yields of biomass, which make them more promising for industrial AuNPs production [3,6]. Up to now, a few fungal species have been reported for biogenic synthesis of AuNPs, and these nanoparticles are found to be multi-shaped, e.g. sphere, triangle, hexagon, cube and rod [3,4]. Some proteins or biomolecules secreted by fungi may act as reducing, capping and stabilizing agents responsible for the synthesis of AuNPs, while NAD(P)Hdependent reductases are considered to be the most common type [3,4,7]. Yeasts such as Candida albicans [8], Hansenula anomala [9], Pichia jadinii [10] and Yarrowia lipolytica [11] have also been reported to produce AuNPs, which have a good potential for the bulk production of nanoparticles [4]. However, other yeast species for AuNPs synthesis are less investigated. The reduction of nitrophenols to aminophenols in the presence of NaBH4 has been generally reported to examine the catalytic behaviors of AuNPs [12,13] Nitrophenols, like 4-nitrophenol (4-NP), are widely used in the manufacture of dyes, pharmaceuticals and pesticides, and they pose a great threat to human and the environment due to the carcinogenic and mutagenic properties [14]. Previous studies have already indicated that AuNPs can efficiently catalyze the hydrogenation of nitroarene to anilinem at ambient temperature [2,12]. The reduction product of nitrophenols, like 4-aminophenol (4-AP), have a broad application in various industries, including photographic developer, corrosion inhibitor, drying agent and the manufacture of analgesic and antipyretic drugs [15]. The catalytic activity of AuNPs is generally related to the particle size, and the functional groups on the surface of AuNPs may also affect the catalytic behavior [15]. Therefore, developing a facile and green approach to synthesize AuNPs with high catalytic activities for nitrophenols reduction is of environmental and industrial importance. Magnusiomyces ingens (synonym of Dipodascus ingens) is a nonconventional yeast, but the green synthesis of metal nanoparticles by Magnusiomyces ingens has been rarely described. In previous study, a yeast strain Magnusiomyces ingens LH-F1 was isolated from sea mud, which exhibited a good capability in decolorizing various azo dyes under aerobic condition [16]. Herein, the biogenic synthesis of AuNPs from HAuCl4 was investigated using the yeast cells of Magnusiomyces ingens LH-F1. The effects of reaction conditions on AuNPs synthesis were investigated, and the resulting nanoparticles were characterized by UV–vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the catalytic activities of the as-synthesized AuNPs were also determined for the reduction of nitrophenols to aminophenols. To the best of our knowledge, this is the first study regarding the biogenic synthesis of AuNPs by yeast Magnusiomyces ingens.
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Fig. 1. Time evolution of UV–vis spectra during the formation of AuNPs by the cells of Magnusiomyces ingens LH-F1. The insert shows the intensity change of SPR peak during the formation of AuNPs. Strain LH-F1 cells (OD600 2.0) were incubated with 1.0 mM HAuCl4 at 30 ◦ C for 24 h under continuous shaking.
3-aminophenol (3-AP), 2-aminophenol (2-AP). All other chemicals were of analytical grade. The yeast strain, Magnusiomyces ingens LH-F1 (CGMCC No. 10367), was isolated previously from the sea mud of a harbor industrial zone in Dalian, China, which was routinely cultivated in the culture medium containing KH2 PO4 1.0 g/L, (NH4 )2 SO4 1.0 g/L, MgSO4 ·7H2 O 0.5 g/L and glucose 4.0 g/L [16]. 2.2. Biosynthesis of AuNPs by yeast Magnusiomyces ingens LH-F1 Strain LH-F1 was grown aerobically at 30 ◦ C in the culture medium until it reached the late log-phase. Cells were harvested by centrifugation (10,000g for 10 min at 4 ◦ C), washed twice with double distilled water (ddH2 O), and stored at −80 ◦ C prior to use. Then, cells were re-suspended in the same ddH2 O to the optical density at 600 nm (OD600 ) of 2.0 (corresponding to the dry cell weight of 2.2 mg/mL). For AuNPs synthesis, the HAuCl4 stock solution (50 mM) was added to the cell suspension with a final concentration of 1.0 mM, and the reaction mixture was incubated at 30 ◦ C for 24 h under continuous shaking. Subsequently, the mixture was centrifuged at 3000g for 5 min, and the supernatant was collected, which was then filtered through 0.45 m syringe Millipore filters to remove the cell debris before further characterization. Effects of different parameters on AuNPs synthesis by strain LHF1 were investigated, including the concentrations of biomass and initial gold ion. To check the influences of biomass concentration, strain LH-F1 cells were re-suspended to OD600 1.0, 1.5, 2.0, 2.5 and 3.0, which were then mixed with 1.0 mM HAuCl4 . As for the effects of initial gold ion, strain LH-F1 cells (OD600 2.0) were mixed with 0.1, 0.5, 1.0, 2.0 and 5.0 mM HAuCl4 . The mixtures were incubated at 30 ◦ C for 24 h under continuous shaking, and UV–vis spectra were monitored to evaluate the formation of AuNPs. 2.3. Characterization of AuNPs
2. Experimental 2.1. Materials Hydrogen tetrachloroaurate (III) hydrate (HAuCl4 ·3H2 O) was purchased from J&K Scientific Ltd. (China). Nitrophenols and aminophenols were obtained from Sinopharm Chemical Regent Beijing Co., Ltd. (China), including 4-nitrophenol (4-NP), 3nitrophenol (3-NP), 2-nitrophenol (2-NP), 4-aminophenol (4-AP),
Spectral analysis of AuNPs was performed using a UV–vis spectrophotometer (Metash UV-9000, China). Gold concentrations were determined using inductively coupled plasma optical emission spectrometer (ICP-OES, Perkin-Elmer Optima 2000 DV, USA). SEM (Hitachi SU8020, Japan) and TEM (FEI Tecnai G220 S-Twin, USA) were used to evaluate morphology of AuNPs. DLS measurement was carried out by Zetasizer Nano ZS (Marvin Instruments, UK) to analyze the average particle size of AuNPs. FTIR spectra of AuNPs and strain LH-F1 cells were obtained using a Shimadzu
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IRPrestige-21 FTIR spectrophotometer (Japan) with the wavelength ranging from 750 to 4000 cm−1 . 2.4. SDS-PAGE analysis of AuNPs To investigate the possible proteins involved in AuNPs synthesis by strain LH-F1, the SDS-PAGE analysis were performed according to the methods by Das et al. [17] with some modifications. Briefly, after AuNPs synthesis by strain LH-F1, the unbound proteins on AuNPs surface were separated by centrifugation at 12,000g for 30 min. The supernatant containing the unbound proteins was collected and electrophoresed by SDS-PAGE. The pellet containing the nanoparticle-bound proteins was washed twice with ddH2 O, re-suspended in ddH2 O, and then subjected to SDS-PAGE. The SDSPAGE was performed with 12% acrylamide gels as described by Laemmli [18]. To obtain the cell-free protein extract of strain LH-F1, the frozen cells were re-suspended in phosphate sodium buffer (PBS, 50 mM, pH 7.0) and lysed by freezing and thawing followed by sonication at 4 ◦ C for 30 min (Ultrasonic Processor CPX 750, USA). After centrifugation at 15,000g for 30 min (4 ◦ C), the supernatant was filtered through 0.45 m syringe Millipore filters and used as the cellfree protein extract. Bradford assays were performed to determine the concentrations of protein in the cell-free extract. The protein extract (0.2 mg/mL) was then incubated with 1.0 mM HAuCl4 and 100 M NADH at 30 ◦ C for 24 h under continuous shaking. The formation of AuNPs was examined by UV–vis spectrophotometer. The experiment was also carried out under identical conditions excepting the addition of NADH.
amounts of strain LH-F1 cells (OD600 1.0–3.0) with 1.0 mM HAuCl4 . As shown in Fig. S2, the SPR peak of AuNPs was observed in the range of 539–565 nm with different concentrations of biomass, suggesting the sizes and shapes of the as-synthesized nanoparticles could be different [20]. Meanwhile, the intensity of SPR peaks increased with biomass concentration, reaching maximum at OD600 2.0, but then decreased when further increasing the biomass concentration (OD600 > 2.0). The effects of initial gold ion concentration (0.1–5.0 mM) on AuNPs synthesis were also investigated. Results indicated that almost no SPR peaks were observed when incubating strain LH-F1 cells with 0.1 or 5.0 mM HAuCl4 (Fig. S3). Furthermore, the AuNPs synthesized by strain LH-F1 had a higher SPR intensity when using 1.0 mM HAuCl4 as the substrate. Thus, the biomass concentration of OD600 2.0 (corresponding to the dry cell weight of 2.2 mg/mL) and HAuCl4 concentration of 1.0 mM were chosen for AuNPs synthesis in the further studies. In previous studies, only a few kinds of yeasts were shown to have a good potential in AuNPs synthesis, such as Candida albicans [8], Hansenula anomala [9], Pichia jadinii [10] and Yarrowia lipolytica [11]. Little information was available concerning the AuNPs synthesis by Magnusiomyces ingens strains. Magnusiomyces ingens LH-F1 was reported to be capable of aerobic decolorization and degradation of various azo dyes [16]. Herein, strain LH-F1 showed an excellent activity for AuNPs synthesis, possessing a promising application in the green synthesis of noble metal nanoparticles.
2.5. Catalytic activity of AuNPs The catalytic activity of AuNPs was measured using nitrophenols as the substrates. In a typical assay, 150 L of 2 mM stock solution of nitrophenols were mixed with 1 mL of 30 mM freshly prepared NaBH4 solution and 1.75 mL ddH2 O. To initiate the reaction, 100 L of biogenic AuNPs (2.6 × 10−5 mmol, which was determined by ICP-OES) was added, and the evolution of the UV–vis spectra was recorded. The apparent rate constants (kapp ) of catalytic reaction were determined through measuring the decrease in absorbance at 400, 396 and 415 nm for 4-NP, 3-NP and 2-NP, respectively, over an appropriate time period. Control experiments were performed under identical conditions without AuNPs or using strain LH-F1 cells in place of AuNPs. All the reactions were carried out in triplicate at ambient temperature. 3. Results and discussion 3.1. Biosynthesis of AuNPs by Magnusiomyces ingens LH-F1 The biosynthesis of AuNPs was carried out using the yeast cells of Magnusiomyces ingens LH-F1. As shown in Fig. S1, the color of the reaction supernatant changed from yellow to light purple in 4 h, and then turned to deep purple after 6 h, indicating the rapid synthesis of AuNPs by Magnusiomyces ingens LH-F1. According to the UV–vis spectra (Fig. 1), no significant peaks were observed within 2 h when incubating strain LH-F1 cells with HAuCl4 . Then, a distinct surface plasmon resonance (SPR) peak at ∼540 nm appeared after 4 h of incubation, and the SPR peak intensity increased rapidly within 8 h, followed by steadily increasing to saturation in 24 h. It could be noted that the SPR peak (540 ± 2 nm) had no significant shift during the process of AuNPs synthesis, suggesting the presence of some capping agents secreted by strain LH-F1 that might prevent the agglomeration of AuNPs [19,20]. To investigate the effects of biomass concentration on the biosynthesis of AuNPs, assays were performed using different
3.2. Characterization of AuNPs biosynthesized by Magnusiomyces ingens LH-F1 The morphology of AuNPs synthesized by strain LH-F1 was examined by SEM and TEM (Fig. 2). The results showed that a mixture of sphere, plates (triangle, hexagon, pentagon), and irregular shaped nanoparticles were obtained. The plate-shaped nanoparticles, with the edge length ranging from 16 to 420 nm based on TEM image analysis, seemed to be larger than the spherical-like particles, whose diameter was in the range of 10–80 nm. The average particles size of AuNPs was also calculated and found to be 80.1 ± 9.8 nm. However, DLS data indicated that the average size of as-synthesized AuNPs was 137.8 ± 4.6 nm (Fig. S4), which was larger than that observed in TEM. Previous studies indicated the sizes of nanoparticles recorded by DLS were generally larger in comparison to TEM measurements, probably due to the interference during DLS measurements resulting from the overlapping of nanoparticles or the enveloping of bioorganic compounds on the core of AuNPs [7,12] FTIR analysis was used to characterize the strain LH-F1 and its biogenic synthesized AuNPs. As shown in Fig. 3, the IR spectrum of AuNPs closely resembled the one of strain LH-F1 cells, suggesting the AuNPs might combine with some biomolecules from the yeast strain during the synthetic process. The intense broad band at ∼3400 cm−1 should be attributed to the stretching vibrations of hydroxide (-OH) or amide (-NH) groups [10]. The bands at ∼1650 and ∼1540 cm−1 might correspond to the amide I and amide II, respectively [19]. The two bands observed at ∼1730 and ∼1400 cm−1 could be assigned to the carboxyl groups [21,22]. The bands at ∼2900 and ∼1040 cm−1 were probably corresponding to C H stretching and C N stretching, respectively [19,23]. These results suggested that some proteins containing amide and carboxyl groups might be adsorbed on the surface of AuNPs, which could be involved in the reduction of Au3+ and the stabilization of AuNPs [19,21].
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Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of strain LH-F1 proteins responsible for gold biomineraliazaition. Lane M, standard protein molecular weight marker; lane 1, unbound proteins after AuNPs biosynthesis; lane 2, bound proteins absorbed on AuNPs. Arrows indicate ∼42 and ∼50 kDa proteins.
Fig. 2. Scanning electron microscopy (SEM) (a) and transmission electron microscopy (TEM) (b) images of AuNPs synthesized by strain LH-F1 cells.
Fig. 3. Fourier transform infrared spectra (FTIR) of strain LH-F1 and the biogenic AuNPs.
3.3. SDS-PAGE analysis of AuNPs biosynthesized by Magnusiomyces ingens LH-F1 To investigate the possible proteins associated with the synthesis of AuNPs by strain LH-F1, SDS-PAGE analysis was performed.
After AuNPs biosynthesis using the yeast cells of strain LH-F1, the unbound and bound proteins were separated by centrifugation (12000g for 30 min). The supernatant containing the unbound proteins was then subjected to SDS-PAGE, while the pellet containing the bound proteins was washed twice with ddH2 O before SDS-PAGE analysis to get rid of free proteins that were not capping the AuNPs. The profile of unbound proteins exhibited two prominent bands with molecular weight of ∼42 and ∼50 kDa (Fig. 4, lane 1), while the profile of the bound proteins showed a number of bands (Fig. 4, lane 2), which further confirmed the adsorption of proteins on the surface of AuNPs. Previous study by Das et al. has shown that two unbound proteins with molecular weight of ∼42 and ∼45 kDa were observed by SDS-PAGE, which could act as reducing agents for the synthesis of AuNPs by Rhizopous oryzae, while the ∼80 kDa bound protein could act as capping agent for the stabilization of AuNPs [17]. Furthermore, Malhotra et al. found that some low molecular weight proteins (∼36.9, ∼17.6 and ∼14.9 kDa) could be involved in the biosynthesis of AuNPs and AgNPs by Stenotrophomonas spp. [7], while Reddy et al. suggested that proteins with molecular weight between 25 and 66 kDa released by Bacillus Subtilis could be responsible for Au3+ ions reduction [24]. Thus, the two unbound proteins (∼42 and ∼50 kDa) were also probably involved in formation of AuNPs by strain LH-F1, which might play a key role in the reduction process of Au3+ ions. In the meanwhile, since a variety of proteins were detected on the surface of AuNPs, there should be plenty of biomolecules secreted by strain LH-F1 that could act as the capping agents, especially the proteins with molecular weights below 30 kDa. Although various proteins could be involved in the biosynthesis of AuNPs by fungal or bacterial strains, the NAD(P)H-dependent reductases were suggested to be the common type of functional enzymes responsible for the reduction process of Au3+ ions [3,4,25]. The NADH-dependent reductases in the protein extract of Fusarium oxysporum were found to be involved in the reduction of Au3+
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Fig. 5. Time-dependent UV–vis spectra during the catalytic reduction of nitrophenols to aminophenols by NaBH4 using AuNPs as the catalysts. (a) 4-nitrophenol (4-NP); (b) 3-nitrophenol (3-NP); (c) 2-nitrophenol (2-NP).
ions to Au0 [26], and the NADPH-dependent enzyme from the cellfree extract of Sclerotium rolfsii had the ability to synthesize AuNPs at ambient temperature [23]. Since strain LH-F1 could efficiently decolorize various azo dyes through biodegradation process, the azoreductases, typical kinds of NAD(P)H-dependent enzymes, were suggested to be responsible for the reduction of azo dyes [16], which might be involved in AuNPs biosynthesis. Further investigation was carried out using the cell-free protein extract of strain LH-F1 for the synthesis of AuNPs. The purple product with an intense SPR peak at ∼540 nm was obtained when incubating the cell-free extract (0.2 mg/mL) with 1.0 mM HAuCl4 in the presence of NADH (100 M) (Fig. S5), suggesting the formation of AuNPs. However, similar result was also observed even without NADH (Fig. S5), indicating that some NAD(P)H-independent enzymes or other biomolecules could be also secreted by strain LH-F1 and played important roles in AuNPs biosynthesis. Chauhan et al. found that the glutathione-like compound, phytochelatin, synthesized by Candida albicans could synthesize AuNPs from Au3+ [27], while Apte et al. found that the melanin isolated from Yarrowia lipolytica NCIM 3590 could be used as a reducing and capping agent for the synthesis of AuNPs [28]. However, the specific biomolecules involved in AuNPs synthesis by strain LH-F1 were unable to be identified. According to the results shown above, the mechanism of AuNPs synthesis by Magnusiomyces ingens LH-F1 could be proposed. Au3+ ions were firstly reduced to Au0 by some specific biomolecules in the cell surface or cytoplasm of strain LH-F1. The small Au0 nanocrystals could be capped and stabilized by the biomolecules, leading to the formation of dispersed AuNPs. In the meanwhile, the as-synthesized AuNPs were released to the outside of cells, thus the nanoparticles could be easily separated from the cells through centrifugation.
3.4. Catalytic activity of AuNPs for the reduction of nitrophenols The catalytic activity of AuNPs synthesized by strain LH-F1 was quantitatively determined for the reduction of nitrophenols, i.e. 4NP, 3-NP and 2-NP, with excess amount of NaBH4 . For 4-NP (Fig. 5a), when mixing with NaBH4 , the absorption peak underwent a red shift from 317 nm to 400 nm, and the color of the solution changed from light yellow to deep yellow correspondingly, which could be resulted from the formation of 4-nitrophenolate ion in the alkaline condition. Then, after the addition of 100 L nanoparticles with an Au content of 2.6 × 10−5 mmol measured by ICP-OES, the absorption intensity of 4-NP at 400 nm decreased immediately with a new absorption peak appeared at ∼300 nm, indicating the formation of the reduction product, 4-AP [19]. 4-NP could be completely reduced within 3 min, and the concomitant yellow color of the solution faded and turned light purple due to the presence of AuNPs (Fig. 5a). According to the first-order rate model [29,30], the apparent kinetic rate constant of 4-NP (kapp-4NP ) was calculated to be 32.5 × 10−3 s−1 . Similarly, the absorption peaks of 3-NP and 2-NP shifted from 329 and 349 nm to 396 and 415 nm, respectively, when NaBH4 was added, both of which decreased rapidly after the addition of AuNPs in company with the appearance of new peaks at ∼290 and ∼291 nm due to the formation of 3-AP and 2-AP, respectively (Fig. 5b and c). The apparent kinetic rate constants of 3-NP (kapp-3NP ) and 2-NP (kapp-2NP ) were also determined as 13.2 × 10−3 and 13.8 × 10−3 s−1 , respectively. It is well-recognized that the size of nanoparticles may play an important role in catalytic reduction, and smaller particles generally exhibit higher activities [15,31]. Compared with previous studies concerning the AuNPs catalysts of 4-NP reduction (Table S1), the AuNPs synthesized by strain LH-F1 with relatively large particle size seemed to exhibit a much better catalytic activity.
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For instance, AuNPs synthesized by the stem extract of Breynia rhamnoides (5% vol fraction) with an average sizes of ∼25 nm could catalyze the reduction of 4-NP at a rate constant of 9.2 × 10−3 s−1 [19], while the AuNPs synthesized by Escherichia coli K12 cells with the particle size around 50 nm could be used as a heterogeneous catalyst in 4-NP reduction with a rate constant of 0.2 × 10−3 s−1 [32]. Due to the “naked” AuNPs might not act as catalysts in some reactions, an appropriate stabilizer could not only help to stabilize the AuNPs but also improve the catalytic properties [15]. Various ligands have been introduced into AuNPs synthesis as function of stabilizer, including polymers [33], dendrimer [13], carbon nanotubes [34], silica nanotubes [35] and natural extracts [19,32], and the as-synthesized AuNPs exhibited a good catalytic activity for 4-NP reduction [15]. In the present study, biomolecules absorbed on the surface of AuNPs might act as organic ligands, which could improve the binding or reducing of substrates on nanoparticles, leading to excellent catalytic activities for nitrophenols reduction under mild conditions. Nevertheless, further investigation on biosynthesis of AuNPs should be performed to control the size, shape and stability of nanoparticles in order to improve their catalytic activities. 4. Conclusions A simple and eco-friendly method for the biogenic synthesis of AuNPs by the yeast cells of Magnusiomyces ingens LH-F1 was developed for the first time. The conditions for the synthesis of AuNPs were optimized and the nanoparticles were characterized. Some biomolecules were probably absorbed on the surface of AuNPs, which could play an important role in AuNPs synthesis. These biomolecules also enhanced the catalytic activity of as-synthesized AuNPs, which could serve as efficient catalysts for the reduction of nitrophenols. The present study should provide a novel candidate for green synthesis of noble metal nanoparticles. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21176040 and 51508068), the Program for New Century Excellent Talents in University (No. NCET-13-0077), and the Fundamental Research Funds for the Central Universities (No. DUT14YQ107). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2016.02. 033. References [1] E.C. Dreaden, A.M. Alkilany, X. Huang, C.J. Murphy, M.A. El-Sayed, The golden age: gold nanoparticles for biomedicine, Chem. Soc. Rev. 41 (2012) 2740–2779. [2] Y. Zhang, X. Cui, F. Shi, Y. Deng, Nano-gold catalysis in fine chemical synthesis, Chem. Rev. 112 (2012) 2467–2505. [3] M. Kitching, M. Ramani, E. Marsili, Fungal biosynthesis of gold nanoparticles: mechanism and scale up, Microb. Biotechnol. 8 (2015) 904–917. [4] U. Shedbalkar, R. Singh, S. Wadhwani, S. Gaidhani, B.A. Chopade, Microbial synthesis of gold nanoparticles: current status and future prospects, Adv. Colloid Interface Sci. 209 (2014) 40–48. [5] O.V. Kharissova, H.V.R. Dias, B.I. Kharisov, B.O. Pérez, V.M. Pérez, The greener synthesis of nanoparticles, Trends Biotechnol. 31 (2013) 240–248. [6] G.S. Dhillon, S.K. Brar, S. Kaur, M. Verma, Green approach for nanoparticle biosynthesis by fungi: current trends and applications, Crit. Rev. Biotechnol. 32 (2012) 49–73. [7] A. Malhotra, K. Dolma, N. Kaur, Y.S. Rathore, Ashish, S. Mayilraj, A.R. Choudhury, Biosynthesis of gold and silver nanoparticles using a novel marine strain of Stenotrophomonas, Bioresour. Technol. 142 (2013) 727–731.
285
[8] T. Ahmad, I.A. Wani, N. Manzoor, J. Ahmed, A.M. Asiri, Biosynthesis, structural characterization and antimicrobial activity of gold and silver nanoparticles, Colloids Surf. B Biointerfaces 107 (2013) 227–234. [9] K. Sathish Kumar, R. Amutha, P. Arumugam, S. Berchmans, Synthesis of gold nanoparticles: an ecofriendly approach using Hansenula anomala, ACS Appl. Mater. Interfaces 3 (2011) 1418–1425. [10] M. Gericke, A. Pinches, Microbial production of gold nanoparticles, Gold Bull. 39 (2006) 22–28. [11] P.S. Pimprikar, S.S. Joshi, A.R. Kumar, S.S. Zinjarde, S.K. Kulkarni, Influence of biomass and gold salt concentration on nanoparticle synthesis by the tropical marine yeast Yarrowia lipolytica NCIM 3589, Colloids Surf. B Biointerfaces 74 (2009) 309–316. [12] A. Mishra, M. Kumari, S. Pandey, V. Chaudhry, K.C. Gupta, C.S. Nautiyal, Biocatalytic and antimicrobial activities of gold nanoparticles synthesized by Trichoderma sp, Bioresour. Technol. 166 (2014) 235–242. [13] A. Kannan, P. Rajakumar, Synthesis and catalytic application of glycodendrimers decorated with gold nanoparticles—reduction of 4-nitrophenol, RSC Adv. 5 (2015) 46908–46915. [14] P.K. Arora, A. Srivastava, V.P. Singh, Bacterial degradation of nitrophenols and their derivatives, J. Hazard. Mater. 266 (2014) 42–59. [15] P.X. Zhao, X.W. Feng, D.S. Huang, G.Y. Yang, D. Astruc, Basic concepts and recent advances in nitrophenol reduction by gold- and other transition metal nanoparticles, Coord. Chem. Rev. 287 (2015) 114–136. [16] L. Tan, H. Li, S.X. Ning, B.W. Xu, Aerobic decolorization and degradation of azo dyes by suspended growing cells and immobilized cells of a newly isolated yeast Magnusiomyces ingens LH-F1, Bioresour. Technol. 158 (2014) 321–328. [17] S.K. Das, J. Liang, M. Schmidt, F. Laffir, E. Marsili, Biomineralization mechanism of gold by zygomycete fungi Rhizopous oryzae, ACS Nano 6 (2012) 6165–6173. [18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [19] A. Gangula, R. Podila, M. Ramakrishna, L. Karanam, C. Janardhana, A.M. Rao, Catalytic reduction of 4-nitrophenol using biogenic gold and silver nanoparticles derived from Breynia rhamnoides, Langmuir 27 (2011) 15268–15274. [20] S.K. Das, A.R. Das, A.K. Guha, Microbial synthesis of multishaped gold nanostructures, Small 6 (2010) 1012–1021. [21] J.Y. Song, H.K. Jang, B.S. Kim, Biological synthesis of gold nanoparticles using Magnolia kobus and Diopyros kaki leaf extracts, Process Biochem. 44 (2009) 1133–1138. [22] S.K. Das, M.M.R. Khan, A.K. Guha, N. Naskar, Bio-inspired fabrication of silver nanoparticles on nanostructured silica: characterization and application as a highly efficient hydrogenation catalyst, Green Chem. 15 (2013) 2548–2557. [23] K.B. Narayanan, N. Sakthivel, Facile green synthesis of gold nanostructures by NADPH-dependent enzyme from the extract of Sclerotium rolfsii, Colloids Surf. A Physicochem. Eng. Asp. 380 (2011) 156–161. [24] A.S. Reddy, C.Y. Chen, C.C. Chen, J.S. Jean, H.R. Chen, M.J. Tseng, C.W. Fan, J.C. Wang, Biological synthesis of gold and silver nanoparticles mediated by the bacteria Bacillus subtilis, J. Nanosci. Nanotechnol. 10 (2010) 6567–6574. [25] N. Durán, P.D. Marcato, M. Durán, A. Yadav, A. Gade, M. Rai, Mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides bacteria, fungi, and plants, Appl. Microbiol. Biotechnol. 90 (2011) 1609–1624. [26] P. Mukherjee, S. Senapati, D. Mandal, A. Ahmad, M.I. Khan, R. Kumar, M. Sastry, Extracellular synthesis of gold nanoparticles by the fungus Fusarium oxysporum, ChemBioChem 3 (2002) 461–463. [27] A. Chauhan, S. Zubair, S. Tufail, A. Sherwani, M. Sajid, S.C. Raman, A. Azam, M. Owais, Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer, Int. J. Nanomed. 6 (2011) 2305–2319. [28] M. Apte, G. Girme, A. Bankar, A. Ravikumar, S. Zinjarde, 3,4-Dihydroxy-l-phenylalanine-derived melanin from Yarrowia lipolytica mediates the synthesis of silver and gold nanostructures, J. Nanobiotechnol. 11 (2013) 2. [29] S. Wunder, F. Polzer, Y. Lu, Y. Mei, M. Ballauff, Kinetic analysis of catalytic reduction of 4-nitrophenol by metallic nanoparticles immobilized in spherical polyelectrolyte brushes, J. Phys. Chem. C 114 (2010) 8814–8820. [30] S. Gu, S. Wunder, Y. Lu, M. Ballauff, Kinetic analysis of the catalytic reduction of 4-nitrophenol by metallic nanoparticles, J. Phys. Chem. C 118 (2014) 18618–18625. [31] S. Panigrahi, S. Basu, S. Praharaj, S. Pande, S. Jana, A. Pal, S.K. Gosh, T. Pal, Synthesis and size-selective catalysis by supported gold nanoparticles: study on heterogeneous and homogeneous catalytic process, J. Phys. Chem. C 111 (2007) 4596–4605. [32] S.K. Srivastava, R. Yamada, C. Ogino, A. Kondo, Biogenic synthesis and characterization of gold nanoparticles by Escherichia coli K12 and its heterogeneous catalysis in degradation of 4-nitrophenol, Nanoscale Res. Lett. 8 (2013) 70. [33] K. Kuroda, T. Ishida, M. Haruta, Reduction of 4-nitrophenol to 4-aminophenol over Au nanoparticles deposited on PMMA, J. Mol. Catal. A Chem. 298 (2009) 7–11. [34] H. Li, J.K. Jo, L. Zhang, C.-S. Ha, H. Suh, I. Kim, A general and efficient route to fabricate carbon nanotube-metal nanoparticles and carbon nanotube-inorganic oxides hybrids, Adv. Funct. Mater. 20 (2010) 3864–3873. [35] Z. Zhang, C. Shao, P. Zou, P. Zhang, M. Zhang, J. Mu, Z. Guo, X. Li, C. Wang, Y. Liu, In situ assembly of well-dispersed gold nanoparticles on electrospun silica nanotubes for catalytic reduction of 4-nitrophenol, Chem. Commun. 47 (2011) 3906–3908.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Biogenic synthesis of gold nanoparticles by yeast Magnusiomyces ingens LH-F1 for catalytic reduction of nitrophenols Xuwang Zhang, Yuanyuan Qu ∗ , Wenli Shen, Jingwei Wang, Huijie Li, Zhaojing Zhang, Shuzhen Li, Jiti Zhou State Key Laboratory of Fine Chemicals, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China
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
• This is the first report on AuNPs biosynthesis by Magnusiomyces ingens. • Various shaped AuNPs were synthesized by strain LH-F1. • Some biomolecules were found to be absorbed on the surface of AuNPs. • AuNPs showed excellent catalytic activities for the reduction of nitrophenols.
a r t i c l e
i n f o
Article history: Received 29 October 2015 Received in revised form 17 February 2016 Accepted 24 February 2016 Keywords: Gold nanoparticles Magnusiomyces ingens Nitrophenols Biosynthesis
a b s t r a c t In the present study, the biogenic synthesis of gold nanoparticles (AuNPs) was achieved using the yeast cells of Magnusiomyces ingens LH-F1. Based on UV–vis spectral analysis, 2.2 mg/mL biomass (OD600 = 2.0) and 1.0 mM HAuCl4 were preferable for AuNPs synthesis by strain LH-F1. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed that various shaped nanoparticles were obtained, including sphere, triangle and hexagon. Based on the analyses of Fourier transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), some biomolecules were absorbed on the surface of AuNPs, which could be involved in the formation of AuNPs. The as-synthesized AuNPs exhibited excellent catalytic activities for the reduction of nitrophenols (i.e. 4-nitrophenol, 3-nitrophenol and 2-nitrophenol) to aminophenols in the presence NaBH4 . This is the first report on AuNPs biosynthesis by Magnusiomyces ingens, which may serve as an efficient candidate for green synthesis of metal nanoparticles. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Gold nanoparticles (AuNPs) have attracted enormous attention in recent decades due to their unique photophysical, electronic and
∗ Corresponding author. E-mail address: [email protected] (Y. Qu). http://dx.doi.org/10.1016/j.colsurfa.2016.02.033 0927-7757/© 2016 Elsevier B.V. All rights reserved.
catalytic properties. They have broad applications in the fields of electronics, environmental sensing, biomedicine and fine chemical synthesis [1,2]. Various physical and chemical methods have been successfully used to produce AuNPs, but most of them are either of high-cost or involved with the usage of hazardous chemicals [3,4]. The biological methods are considered to be the green and ecofriendly alternatives for the synthesis of nanoparticles owing to their nature of rich diversity, cost-effective, and mild condition [5].
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Biological systems, such as bacteria, fungi and plants, have been shown to possess the ability of producing AuNPs via intra- and/or extra-cellular ways [4]. Among them, fungi are gaining more interests for the green synthesis of nanoparticles due to their practical advantages of easy handling, large amounts of secreted proteins and high yields of biomass, which make them more promising for industrial AuNPs production [3,6]. Up to now, a few fungal species have been reported for biogenic synthesis of AuNPs, and these nanoparticles are found to be multi-shaped, e.g. sphere, triangle, hexagon, cube and rod [3,4]. Some proteins or biomolecules secreted by fungi may act as reducing, capping and stabilizing agents responsible for the synthesis of AuNPs, while NAD(P)Hdependent reductases are considered to be the most common type [3,4,7]. Yeasts such as Candida albicans [8], Hansenula anomala [9], Pichia jadinii [10] and Yarrowia lipolytica [11] have also been reported to produce AuNPs, which have a good potential for the bulk production of nanoparticles [4]. However, other yeast species for AuNPs synthesis are less investigated. The reduction of nitrophenols to aminophenols in the presence of NaBH4 has been generally reported to examine the catalytic behaviors of AuNPs [12,13] Nitrophenols, like 4-nitrophenol (4-NP), are widely used in the manufacture of dyes, pharmaceuticals and pesticides, and they pose a great threat to human and the environment due to the carcinogenic and mutagenic properties [14]. Previous studies have already indicated that AuNPs can efficiently catalyze the hydrogenation of nitroarene to anilinem at ambient temperature [2,12]. The reduction product of nitrophenols, like 4-aminophenol (4-AP), have a broad application in various industries, including photographic developer, corrosion inhibitor, drying agent and the manufacture of analgesic and antipyretic drugs [15]. The catalytic activity of AuNPs is generally related to the particle size, and the functional groups on the surface of AuNPs may also affect the catalytic behavior [15]. Therefore, developing a facile and green approach to synthesize AuNPs with high catalytic activities for nitrophenols reduction is of environmental and industrial importance. Magnusiomyces ingens (synonym of Dipodascus ingens) is a nonconventional yeast, but the green synthesis of metal nanoparticles by Magnusiomyces ingens has been rarely described. In previous study, a yeast strain Magnusiomyces ingens LH-F1 was isolated from sea mud, which exhibited a good capability in decolorizing various azo dyes under aerobic condition [16]. Herein, the biogenic synthesis of AuNPs from HAuCl4 was investigated using the yeast cells of Magnusiomyces ingens LH-F1. The effects of reaction conditions on AuNPs synthesis were investigated, and the resulting nanoparticles were characterized by UV–vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the catalytic activities of the as-synthesized AuNPs were also determined for the reduction of nitrophenols to aminophenols. To the best of our knowledge, this is the first study regarding the biogenic synthesis of AuNPs by yeast Magnusiomyces ingens.
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Fig. 1. Time evolution of UV–vis spectra during the formation of AuNPs by the cells of Magnusiomyces ingens LH-F1. The insert shows the intensity change of SPR peak during the formation of AuNPs. Strain LH-F1 cells (OD600 2.0) were incubated with 1.0 mM HAuCl4 at 30 ◦ C for 24 h under continuous shaking.
3-aminophenol (3-AP), 2-aminophenol (2-AP). All other chemicals were of analytical grade. The yeast strain, Magnusiomyces ingens LH-F1 (CGMCC No. 10367), was isolated previously from the sea mud of a harbor industrial zone in Dalian, China, which was routinely cultivated in the culture medium containing KH2 PO4 1.0 g/L, (NH4 )2 SO4 1.0 g/L, MgSO4 ·7H2 O 0.5 g/L and glucose 4.0 g/L [16]. 2.2. Biosynthesis of AuNPs by yeast Magnusiomyces ingens LH-F1 Strain LH-F1 was grown aerobically at 30 ◦ C in the culture medium until it reached the late log-phase. Cells were harvested by centrifugation (10,000g for 10 min at 4 ◦ C), washed twice with double distilled water (ddH2 O), and stored at −80 ◦ C prior to use. Then, cells were re-suspended in the same ddH2 O to the optical density at 600 nm (OD600 ) of 2.0 (corresponding to the dry cell weight of 2.2 mg/mL). For AuNPs synthesis, the HAuCl4 stock solution (50 mM) was added to the cell suspension with a final concentration of 1.0 mM, and the reaction mixture was incubated at 30 ◦ C for 24 h under continuous shaking. Subsequently, the mixture was centrifuged at 3000g for 5 min, and the supernatant was collected, which was then filtered through 0.45 m syringe Millipore filters to remove the cell debris before further characterization. Effects of different parameters on AuNPs synthesis by strain LHF1 were investigated, including the concentrations of biomass and initial gold ion. To check the influences of biomass concentration, strain LH-F1 cells were re-suspended to OD600 1.0, 1.5, 2.0, 2.5 and 3.0, which were then mixed with 1.0 mM HAuCl4 . As for the effects of initial gold ion, strain LH-F1 cells (OD600 2.0) were mixed with 0.1, 0.5, 1.0, 2.0 and 5.0 mM HAuCl4 . The mixtures were incubated at 30 ◦ C for 24 h under continuous shaking, and UV–vis spectra were monitored to evaluate the formation of AuNPs. 2.3. Characterization of AuNPs
2. Experimental 2.1. Materials Hydrogen tetrachloroaurate (III) hydrate (HAuCl4 ·3H2 O) was purchased from J&K Scientific Ltd. (China). Nitrophenols and aminophenols were obtained from Sinopharm Chemical Regent Beijing Co., Ltd. (China), including 4-nitrophenol (4-NP), 3nitrophenol (3-NP), 2-nitrophenol (2-NP), 4-aminophenol (4-AP),
Spectral analysis of AuNPs was performed using a UV–vis spectrophotometer (Metash UV-9000, China). Gold concentrations were determined using inductively coupled plasma optical emission spectrometer (ICP-OES, Perkin-Elmer Optima 2000 DV, USA). SEM (Hitachi SU8020, Japan) and TEM (FEI Tecnai G220 S-Twin, USA) were used to evaluate morphology of AuNPs. DLS measurement was carried out by Zetasizer Nano ZS (Marvin Instruments, UK) to analyze the average particle size of AuNPs. FTIR spectra of AuNPs and strain LH-F1 cells were obtained using a Shimadzu
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IRPrestige-21 FTIR spectrophotometer (Japan) with the wavelength ranging from 750 to 4000 cm−1 . 2.4. SDS-PAGE analysis of AuNPs To investigate the possible proteins involved in AuNPs synthesis by strain LH-F1, the SDS-PAGE analysis were performed according to the methods by Das et al. [17] with some modifications. Briefly, after AuNPs synthesis by strain LH-F1, the unbound proteins on AuNPs surface were separated by centrifugation at 12,000g for 30 min. The supernatant containing the unbound proteins was collected and electrophoresed by SDS-PAGE. The pellet containing the nanoparticle-bound proteins was washed twice with ddH2 O, re-suspended in ddH2 O, and then subjected to SDS-PAGE. The SDSPAGE was performed with 12% acrylamide gels as described by Laemmli [18]. To obtain the cell-free protein extract of strain LH-F1, the frozen cells were re-suspended in phosphate sodium buffer (PBS, 50 mM, pH 7.0) and lysed by freezing and thawing followed by sonication at 4 ◦ C for 30 min (Ultrasonic Processor CPX 750, USA). After centrifugation at 15,000g for 30 min (4 ◦ C), the supernatant was filtered through 0.45 m syringe Millipore filters and used as the cellfree protein extract. Bradford assays were performed to determine the concentrations of protein in the cell-free extract. The protein extract (0.2 mg/mL) was then incubated with 1.0 mM HAuCl4 and 100 M NADH at 30 ◦ C for 24 h under continuous shaking. The formation of AuNPs was examined by UV–vis spectrophotometer. The experiment was also carried out under identical conditions excepting the addition of NADH.
amounts of strain LH-F1 cells (OD600 1.0–3.0) with 1.0 mM HAuCl4 . As shown in Fig. S2, the SPR peak of AuNPs was observed in the range of 539–565 nm with different concentrations of biomass, suggesting the sizes and shapes of the as-synthesized nanoparticles could be different [20]. Meanwhile, the intensity of SPR peaks increased with biomass concentration, reaching maximum at OD600 2.0, but then decreased when further increasing the biomass concentration (OD600 > 2.0). The effects of initial gold ion concentration (0.1–5.0 mM) on AuNPs synthesis were also investigated. Results indicated that almost no SPR peaks were observed when incubating strain LH-F1 cells with 0.1 or 5.0 mM HAuCl4 (Fig. S3). Furthermore, the AuNPs synthesized by strain LH-F1 had a higher SPR intensity when using 1.0 mM HAuCl4 as the substrate. Thus, the biomass concentration of OD600 2.0 (corresponding to the dry cell weight of 2.2 mg/mL) and HAuCl4 concentration of 1.0 mM were chosen for AuNPs synthesis in the further studies. In previous studies, only a few kinds of yeasts were shown to have a good potential in AuNPs synthesis, such as Candida albicans [8], Hansenula anomala [9], Pichia jadinii [10] and Yarrowia lipolytica [11]. Little information was available concerning the AuNPs synthesis by Magnusiomyces ingens strains. Magnusiomyces ingens LH-F1 was reported to be capable of aerobic decolorization and degradation of various azo dyes [16]. Herein, strain LH-F1 showed an excellent activity for AuNPs synthesis, possessing a promising application in the green synthesis of noble metal nanoparticles.
2.5. Catalytic activity of AuNPs The catalytic activity of AuNPs was measured using nitrophenols as the substrates. In a typical assay, 150 L of 2 mM stock solution of nitrophenols were mixed with 1 mL of 30 mM freshly prepared NaBH4 solution and 1.75 mL ddH2 O. To initiate the reaction, 100 L of biogenic AuNPs (2.6 × 10−5 mmol, which was determined by ICP-OES) was added, and the evolution of the UV–vis spectra was recorded. The apparent rate constants (kapp ) of catalytic reaction were determined through measuring the decrease in absorbance at 400, 396 and 415 nm for 4-NP, 3-NP and 2-NP, respectively, over an appropriate time period. Control experiments were performed under identical conditions without AuNPs or using strain LH-F1 cells in place of AuNPs. All the reactions were carried out in triplicate at ambient temperature. 3. Results and discussion 3.1. Biosynthesis of AuNPs by Magnusiomyces ingens LH-F1 The biosynthesis of AuNPs was carried out using the yeast cells of Magnusiomyces ingens LH-F1. As shown in Fig. S1, the color of the reaction supernatant changed from yellow to light purple in 4 h, and then turned to deep purple after 6 h, indicating the rapid synthesis of AuNPs by Magnusiomyces ingens LH-F1. According to the UV–vis spectra (Fig. 1), no significant peaks were observed within 2 h when incubating strain LH-F1 cells with HAuCl4 . Then, a distinct surface plasmon resonance (SPR) peak at ∼540 nm appeared after 4 h of incubation, and the SPR peak intensity increased rapidly within 8 h, followed by steadily increasing to saturation in 24 h. It could be noted that the SPR peak (540 ± 2 nm) had no significant shift during the process of AuNPs synthesis, suggesting the presence of some capping agents secreted by strain LH-F1 that might prevent the agglomeration of AuNPs [19,20]. To investigate the effects of biomass concentration on the biosynthesis of AuNPs, assays were performed using different
3.2. Characterization of AuNPs biosynthesized by Magnusiomyces ingens LH-F1 The morphology of AuNPs synthesized by strain LH-F1 was examined by SEM and TEM (Fig. 2). The results showed that a mixture of sphere, plates (triangle, hexagon, pentagon), and irregular shaped nanoparticles were obtained. The plate-shaped nanoparticles, with the edge length ranging from 16 to 420 nm based on TEM image analysis, seemed to be larger than the spherical-like particles, whose diameter was in the range of 10–80 nm. The average particles size of AuNPs was also calculated and found to be 80.1 ± 9.8 nm. However, DLS data indicated that the average size of as-synthesized AuNPs was 137.8 ± 4.6 nm (Fig. S4), which was larger than that observed in TEM. Previous studies indicated the sizes of nanoparticles recorded by DLS were generally larger in comparison to TEM measurements, probably due to the interference during DLS measurements resulting from the overlapping of nanoparticles or the enveloping of bioorganic compounds on the core of AuNPs [7,12] FTIR analysis was used to characterize the strain LH-F1 and its biogenic synthesized AuNPs. As shown in Fig. 3, the IR spectrum of AuNPs closely resembled the one of strain LH-F1 cells, suggesting the AuNPs might combine with some biomolecules from the yeast strain during the synthetic process. The intense broad band at ∼3400 cm−1 should be attributed to the stretching vibrations of hydroxide (-OH) or amide (-NH) groups [10]. The bands at ∼1650 and ∼1540 cm−1 might correspond to the amide I and amide II, respectively [19]. The two bands observed at ∼1730 and ∼1400 cm−1 could be assigned to the carboxyl groups [21,22]. The bands at ∼2900 and ∼1040 cm−1 were probably corresponding to C H stretching and C N stretching, respectively [19,23]. These results suggested that some proteins containing amide and carboxyl groups might be adsorbed on the surface of AuNPs, which could be involved in the reduction of Au3+ and the stabilization of AuNPs [19,21].
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Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of strain LH-F1 proteins responsible for gold biomineraliazaition. Lane M, standard protein molecular weight marker; lane 1, unbound proteins after AuNPs biosynthesis; lane 2, bound proteins absorbed on AuNPs. Arrows indicate ∼42 and ∼50 kDa proteins.
Fig. 2. Scanning electron microscopy (SEM) (a) and transmission electron microscopy (TEM) (b) images of AuNPs synthesized by strain LH-F1 cells.
Fig. 3. Fourier transform infrared spectra (FTIR) of strain LH-F1 and the biogenic AuNPs.
3.3. SDS-PAGE analysis of AuNPs biosynthesized by Magnusiomyces ingens LH-F1 To investigate the possible proteins associated with the synthesis of AuNPs by strain LH-F1, SDS-PAGE analysis was performed.
After AuNPs biosynthesis using the yeast cells of strain LH-F1, the unbound and bound proteins were separated by centrifugation (12000g for 30 min). The supernatant containing the unbound proteins was then subjected to SDS-PAGE, while the pellet containing the bound proteins was washed twice with ddH2 O before SDS-PAGE analysis to get rid of free proteins that were not capping the AuNPs. The profile of unbound proteins exhibited two prominent bands with molecular weight of ∼42 and ∼50 kDa (Fig. 4, lane 1), while the profile of the bound proteins showed a number of bands (Fig. 4, lane 2), which further confirmed the adsorption of proteins on the surface of AuNPs. Previous study by Das et al. has shown that two unbound proteins with molecular weight of ∼42 and ∼45 kDa were observed by SDS-PAGE, which could act as reducing agents for the synthesis of AuNPs by Rhizopous oryzae, while the ∼80 kDa bound protein could act as capping agent for the stabilization of AuNPs [17]. Furthermore, Malhotra et al. found that some low molecular weight proteins (∼36.9, ∼17.6 and ∼14.9 kDa) could be involved in the biosynthesis of AuNPs and AgNPs by Stenotrophomonas spp. [7], while Reddy et al. suggested that proteins with molecular weight between 25 and 66 kDa released by Bacillus Subtilis could be responsible for Au3+ ions reduction [24]. Thus, the two unbound proteins (∼42 and ∼50 kDa) were also probably involved in formation of AuNPs by strain LH-F1, which might play a key role in the reduction process of Au3+ ions. In the meanwhile, since a variety of proteins were detected on the surface of AuNPs, there should be plenty of biomolecules secreted by strain LH-F1 that could act as the capping agents, especially the proteins with molecular weights below 30 kDa. Although various proteins could be involved in the biosynthesis of AuNPs by fungal or bacterial strains, the NAD(P)H-dependent reductases were suggested to be the common type of functional enzymes responsible for the reduction process of Au3+ ions [3,4,25]. The NADH-dependent reductases in the protein extract of Fusarium oxysporum were found to be involved in the reduction of Au3+
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Fig. 5. Time-dependent UV–vis spectra during the catalytic reduction of nitrophenols to aminophenols by NaBH4 using AuNPs as the catalysts. (a) 4-nitrophenol (4-NP); (b) 3-nitrophenol (3-NP); (c) 2-nitrophenol (2-NP).
ions to Au0 [26], and the NADPH-dependent enzyme from the cellfree extract of Sclerotium rolfsii had the ability to synthesize AuNPs at ambient temperature [23]. Since strain LH-F1 could efficiently decolorize various azo dyes through biodegradation process, the azoreductases, typical kinds of NAD(P)H-dependent enzymes, were suggested to be responsible for the reduction of azo dyes [16], which might be involved in AuNPs biosynthesis. Further investigation was carried out using the cell-free protein extract of strain LH-F1 for the synthesis of AuNPs. The purple product with an intense SPR peak at ∼540 nm was obtained when incubating the cell-free extract (0.2 mg/mL) with 1.0 mM HAuCl4 in the presence of NADH (100 M) (Fig. S5), suggesting the formation of AuNPs. However, similar result was also observed even without NADH (Fig. S5), indicating that some NAD(P)H-independent enzymes or other biomolecules could be also secreted by strain LH-F1 and played important roles in AuNPs biosynthesis. Chauhan et al. found that the glutathione-like compound, phytochelatin, synthesized by Candida albicans could synthesize AuNPs from Au3+ [27], while Apte et al. found that the melanin isolated from Yarrowia lipolytica NCIM 3590 could be used as a reducing and capping agent for the synthesis of AuNPs [28]. However, the specific biomolecules involved in AuNPs synthesis by strain LH-F1 were unable to be identified. According to the results shown above, the mechanism of AuNPs synthesis by Magnusiomyces ingens LH-F1 could be proposed. Au3+ ions were firstly reduced to Au0 by some specific biomolecules in the cell surface or cytoplasm of strain LH-F1. The small Au0 nanocrystals could be capped and stabilized by the biomolecules, leading to the formation of dispersed AuNPs. In the meanwhile, the as-synthesized AuNPs were released to the outside of cells, thus the nanoparticles could be easily separated from the cells through centrifugation.
3.4. Catalytic activity of AuNPs for the reduction of nitrophenols The catalytic activity of AuNPs synthesized by strain LH-F1 was quantitatively determined for the reduction of nitrophenols, i.e. 4NP, 3-NP and 2-NP, with excess amount of NaBH4 . For 4-NP (Fig. 5a), when mixing with NaBH4 , the absorption peak underwent a red shift from 317 nm to 400 nm, and the color of the solution changed from light yellow to deep yellow correspondingly, which could be resulted from the formation of 4-nitrophenolate ion in the alkaline condition. Then, after the addition of 100 L nanoparticles with an Au content of 2.6 × 10−5 mmol measured by ICP-OES, the absorption intensity of 4-NP at 400 nm decreased immediately with a new absorption peak appeared at ∼300 nm, indicating the formation of the reduction product, 4-AP [19]. 4-NP could be completely reduced within 3 min, and the concomitant yellow color of the solution faded and turned light purple due to the presence of AuNPs (Fig. 5a). According to the first-order rate model [29,30], the apparent kinetic rate constant of 4-NP (kapp-4NP ) was calculated to be 32.5 × 10−3 s−1 . Similarly, the absorption peaks of 3-NP and 2-NP shifted from 329 and 349 nm to 396 and 415 nm, respectively, when NaBH4 was added, both of which decreased rapidly after the addition of AuNPs in company with the appearance of new peaks at ∼290 and ∼291 nm due to the formation of 3-AP and 2-AP, respectively (Fig. 5b and c). The apparent kinetic rate constants of 3-NP (kapp-3NP ) and 2-NP (kapp-2NP ) were also determined as 13.2 × 10−3 and 13.8 × 10−3 s−1 , respectively. It is well-recognized that the size of nanoparticles may play an important role in catalytic reduction, and smaller particles generally exhibit higher activities [15,31]. Compared with previous studies concerning the AuNPs catalysts of 4-NP reduction (Table S1), the AuNPs synthesized by strain LH-F1 with relatively large particle size seemed to exhibit a much better catalytic activity.
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For instance, AuNPs synthesized by the stem extract of Breynia rhamnoides (5% vol fraction) with an average sizes of ∼25 nm could catalyze the reduction of 4-NP at a rate constant of 9.2 × 10−3 s−1 [19], while the AuNPs synthesized by Escherichia coli K12 cells with the particle size around 50 nm could be used as a heterogeneous catalyst in 4-NP reduction with a rate constant of 0.2 × 10−3 s−1 [32]. Due to the “naked” AuNPs might not act as catalysts in some reactions, an appropriate stabilizer could not only help to stabilize the AuNPs but also improve the catalytic properties [15]. Various ligands have been introduced into AuNPs synthesis as function of stabilizer, including polymers [33], dendrimer [13], carbon nanotubes [34], silica nanotubes [35] and natural extracts [19,32], and the as-synthesized AuNPs exhibited a good catalytic activity for 4-NP reduction [15]. In the present study, biomolecules absorbed on the surface of AuNPs might act as organic ligands, which could improve the binding or reducing of substrates on nanoparticles, leading to excellent catalytic activities for nitrophenols reduction under mild conditions. Nevertheless, further investigation on biosynthesis of AuNPs should be performed to control the size, shape and stability of nanoparticles in order to improve their catalytic activities. 4. Conclusions A simple and eco-friendly method for the biogenic synthesis of AuNPs by the yeast cells of Magnusiomyces ingens LH-F1 was developed for the first time. The conditions for the synthesis of AuNPs were optimized and the nanoparticles were characterized. Some biomolecules were probably absorbed on the surface of AuNPs, which could play an important role in AuNPs synthesis. These biomolecules also enhanced the catalytic activity of as-synthesized AuNPs, which could serve as efficient catalysts for the reduction of nitrophenols. The present study should provide a novel candidate for green synthesis of noble metal nanoparticles. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21176040 and 51508068), the Program for New Century Excellent Talents in University (No. NCET-13-0077), and the Fundamental Research Funds for the Central Universities (No. DUT14YQ107). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2016.02. 033. References [1] E.C. Dreaden, A.M. Alkilany, X. Huang, C.J. Murphy, M.A. El-Sayed, The golden age: gold nanoparticles for biomedicine, Chem. Soc. Rev. 41 (2012) 2740–2779. [2] Y. Zhang, X. Cui, F. Shi, Y. Deng, Nano-gold catalysis in fine chemical synthesis, Chem. Rev. 112 (2012) 2467–2505. [3] M. Kitching, M. Ramani, E. Marsili, Fungal biosynthesis of gold nanoparticles: mechanism and scale up, Microb. Biotechnol. 8 (2015) 904–917. [4] U. Shedbalkar, R. Singh, S. Wadhwani, S. Gaidhani, B.A. Chopade, Microbial synthesis of gold nanoparticles: current status and future prospects, Adv. Colloid Interface Sci. 209 (2014) 40–48. [5] O.V. Kharissova, H.V.R. Dias, B.I. Kharisov, B.O. Pérez, V.M. Pérez, The greener synthesis of nanoparticles, Trends Biotechnol. 31 (2013) 240–248. [6] G.S. Dhillon, S.K. Brar, S. Kaur, M. Verma, Green approach for nanoparticle biosynthesis by fungi: current trends and applications, Crit. Rev. Biotechnol. 32 (2012) 49–73. [7] A. Malhotra, K. Dolma, N. Kaur, Y.S. Rathore, Ashish, S. Mayilraj, A.R. Choudhury, Biosynthesis of gold and silver nanoparticles using a novel marine strain of Stenotrophomonas, Bioresour. Technol. 142 (2013) 727–731.
285
[8] T. Ahmad, I.A. Wani, N. Manzoor, J. Ahmed, A.M. Asiri, Biosynthesis, structural characterization and antimicrobial activity of gold and silver nanoparticles, Colloids Surf. B Biointerfaces 107 (2013) 227–234. [9] K. Sathish Kumar, R. Amutha, P. Arumugam, S. Berchmans, Synthesis of gold nanoparticles: an ecofriendly approach using Hansenula anomala, ACS Appl. Mater. Interfaces 3 (2011) 1418–1425. [10] M. Gericke, A. Pinches, Microbial production of gold nanoparticles, Gold Bull. 39 (2006) 22–28. [11] P.S. Pimprikar, S.S. Joshi, A.R. Kumar, S.S. Zinjarde, S.K. Kulkarni, Influence of biomass and gold salt concentration on nanoparticle synthesis by the tropical marine yeast Yarrowia lipolytica NCIM 3589, Colloids Surf. B Biointerfaces 74 (2009) 309–316. [12] A. Mishra, M. Kumari, S. Pandey, V. Chaudhry, K.C. Gupta, C.S. Nautiyal, Biocatalytic and antimicrobial activities of gold nanoparticles synthesized by Trichoderma sp, Bioresour. Technol. 166 (2014) 235–242. [13] A. Kannan, P. Rajakumar, Synthesis and catalytic application of glycodendrimers decorated with gold nanoparticles—reduction of 4-nitrophenol, RSC Adv. 5 (2015) 46908–46915. [14] P.K. Arora, A. Srivastava, V.P. Singh, Bacterial degradation of nitrophenols and their derivatives, J. Hazard. Mater. 266 (2014) 42–59. [15] P.X. Zhao, X.W. Feng, D.S. Huang, G.Y. Yang, D. Astruc, Basic concepts and recent advances in nitrophenol reduction by gold- and other transition metal nanoparticles, Coord. Chem. Rev. 287 (2015) 114–136. [16] L. Tan, H. Li, S.X. Ning, B.W. Xu, Aerobic decolorization and degradation of azo dyes by suspended growing cells and immobilized cells of a newly isolated yeast Magnusiomyces ingens LH-F1, Bioresour. Technol. 158 (2014) 321–328. [17] S.K. Das, J. Liang, M. Schmidt, F. Laffir, E. Marsili, Biomineralization mechanism of gold by zygomycete fungi Rhizopous oryzae, ACS Nano 6 (2012) 6165–6173. [18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [19] A. Gangula, R. Podila, M. Ramakrishna, L. Karanam, C. Janardhana, A.M. Rao, Catalytic reduction of 4-nitrophenol using biogenic gold and silver nanoparticles derived from Breynia rhamnoides, Langmuir 27 (2011) 15268–15274. [20] S.K. Das, A.R. Das, A.K. Guha, Microbial synthesis of multishaped gold nanostructures, Small 6 (2010) 1012–1021. [21] J.Y. Song, H.K. Jang, B.S. Kim, Biological synthesis of gold nanoparticles using Magnolia kobus and Diopyros kaki leaf extracts, Process Biochem. 44 (2009) 1133–1138. [22] S.K. Das, M.M.R. Khan, A.K. Guha, N. Naskar, Bio-inspired fabrication of silver nanoparticles on nanostructured silica: characterization and application as a highly efficient hydrogenation catalyst, Green Chem. 15 (2013) 2548–2557. [23] K.B. Narayanan, N. Sakthivel, Facile green synthesis of gold nanostructures by NADPH-dependent enzyme from the extract of Sclerotium rolfsii, Colloids Surf. A Physicochem. Eng. Asp. 380 (2011) 156–161. [24] A.S. Reddy, C.Y. Chen, C.C. Chen, J.S. Jean, H.R. Chen, M.J. Tseng, C.W. Fan, J.C. Wang, Biological synthesis of gold and silver nanoparticles mediated by the bacteria Bacillus subtilis, J. Nanosci. Nanotechnol. 10 (2010) 6567–6574. [25] N. Durán, P.D. Marcato, M. Durán, A. Yadav, A. Gade, M. Rai, Mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides bacteria, fungi, and plants, Appl. Microbiol. Biotechnol. 90 (2011) 1609–1624. [26] P. Mukherjee, S. Senapati, D. Mandal, A. Ahmad, M.I. Khan, R. Kumar, M. Sastry, Extracellular synthesis of gold nanoparticles by the fungus Fusarium oxysporum, ChemBioChem 3 (2002) 461–463. [27] A. Chauhan, S. Zubair, S. Tufail, A. Sherwani, M. Sajid, S.C. Raman, A. Azam, M. Owais, Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer, Int. J. Nanomed. 6 (2011) 2305–2319. [28] M. Apte, G. Girme, A. Bankar, A. Ravikumar, S. Zinjarde, 3,4-Dihydroxy-l-phenylalanine-derived melanin from Yarrowia lipolytica mediates the synthesis of silver and gold nanostructures, J. Nanobiotechnol. 11 (2013) 2. [29] S. Wunder, F. Polzer, Y. Lu, Y. Mei, M. Ballauff, Kinetic analysis of catalytic reduction of 4-nitrophenol by metallic nanoparticles immobilized in spherical polyelectrolyte brushes, J. Phys. Chem. C 114 (2010) 8814–8820. [30] S. Gu, S. Wunder, Y. Lu, M. Ballauff, Kinetic analysis of the catalytic reduction of 4-nitrophenol by metallic nanoparticles, J. Phys. Chem. C 118 (2014) 18618–18625. [31] S. Panigrahi, S. Basu, S. Praharaj, S. Pande, S. Jana, A. Pal, S.K. Gosh, T. Pal, Synthesis and size-selective catalysis by supported gold nanoparticles: study on heterogeneous and homogeneous catalytic process, J. Phys. Chem. C 111 (2007) 4596–4605. [32] S.K. Srivastava, R. Yamada, C. Ogino, A. Kondo, Biogenic synthesis and characterization of gold nanoparticles by Escherichia coli K12 and its heterogeneous catalysis in degradation of 4-nitrophenol, Nanoscale Res. Lett. 8 (2013) 70. [33] K. Kuroda, T. Ishida, M. Haruta, Reduction of 4-nitrophenol to 4-aminophenol over Au nanoparticles deposited on PMMA, J. Mol. Catal. A Chem. 298 (2009) 7–11. [34] H. Li, J.K. Jo, L. Zhang, C.-S. Ha, H. Suh, I. Kim, A general and efficient route to fabricate carbon nanotube-metal nanoparticles and carbon nanotube-inorganic oxides hybrids, Adv. Funct. Mater. 20 (2010) 3864–3873. [35] Z. Zhang, C. Shao, P. Zou, P. Zhang, M. Zhang, J. Mu, Z. Guo, X. Li, C. Wang, Y. Liu, In situ assembly of well-dispersed gold nanoparticles on electrospun silica nanotubes for catalytic reduction of 4-nitrophenol, Chem. Commun. 47 (2011) 3906–3908.