Journal of Colloid and Interface Science 433 (2014) 204–210
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Facile fabrication of Pd nanoparticle/Pichia pastoris catalysts through adsorption–reduction method: A study into effect of chemical pretreatment Huimei Chen a, Dengpo Huang b, Liqin Lin a, Tareque Odoom-Wubah b, Jiale Huang b,⇑, Daohua Sun b, Qingbiao Li a,b,c,⇑ a
Environmental Science Research Center, College of the Environment & Ecology, Xiamen University, Xiamen 361005, PR China Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China c College of Chemistry & Life Science, Quanzhou Normal University, Quanzhou 362000, PR China b
a r t i c l e
i n f o
Article history: Received 19 May 2014 Accepted 25 July 2014 Available online 6 August 2014 Keywords: Adsorption Chemical modification Palladium nanoparticles 4-Nitrophenol
a b s t r a c t Based on rapid adsorption and incomplete reduction in Pd (II) ions by yeast, Pichia pastoris (P. pastoris) GS115, the effects of pretreatment on adsorption and reduction of Pd (II) ions and the catalytic properties of Pd NP/P. pastoris catalysts were studied. Interestingly, the results showed that the adsorption ability of the cells for Pd (II) ions was greatly enhanced after they were pretreated with aqueous HCl, aqueous NaOH and methylation of amino group. For the reduction in the adsorbed Pd (II) ions, more slow reduction rates by pretreated P. pastoris cells were displayed compared with the cells without pretreatment. Using the reduction of 4-nitrophenol as a model reaction, the Pd NP/P. pastoris catalysts based on the cells after pretreatment with aqueous HCl, aqueous NaOH and methylation of amino group exhibited higher stability than the unpretreated cells. The enhanced stability of the Pd catalysts can be attributed to smaller Pd NPs, better dispersion of the Pd NPs, and stronger binding forces of the pretreated P. pastoris for preparing the Pd NPs. This work exemplifies enhancing the stability of Pd catalysts through pretreatments. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction With the proliferation of nanotechnology in modern society, metal nanoparticles (NPs) as building blocks are of great importance owing to their unique properties, which are different from bulk materials [1–4]. Hence, a variety of physical, chemical and biological methods have been developed to synthesize NPs with tunable shapes and sizes [5]. Driven by the growing impetus of green chemistry, considerable effort has been geared toward the synthesis of metal nanoparticles using microorganisms under mild conditions [6–8]. For example, Macaskie et al. showed the adsorption and rapid reduction in Pd (II) by using Desulfovibrio desulfuricans at the expense of formate or H2 as electron donor [9,10]. Besides the synthesis of noble metal NPs using microorganisms, their potential catalytic applications have also intrigued great ⇑ Corresponding authors at: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China (Q. Li). Fax: +86 592 2184822. E-mail addresses: [email protected] (J. Huang), [email protected] (Q. Li). http://dx.doi.org/10.1016/j.jcis.2014.07.038 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
interest. Nevertheless, the isolation of the NPs formed employing intracellular synthesis for catalytic applications is difficult and the microorganisms used for the extracellular synthesis of NPs must be extensively screened [11,12]. Hence, it is not easy to load microbially synthesized NPs onto inorganic supports. As adsorption and reduction of metal ions in tandem by microorganisms can lead to metal/microorganism composites, dual functions of microorganisms as reductive scaffolds for NPs and supports for catalytic applications could circumvent the isolation of NPs from the microorganisms. For instance, several applications of Pd/microorganism in dehalogenation [13–20], reduction [21–24], hydrogenation [13,23,25,26] and CAC bond forming [23,27,28] reactions have been demonstrated. Windt et al. also showed that such Pd catalysts for the dechlorination of polychlorinated biphenyls were superior to those commercialized [29]. The above synthetic protocol by tandem adsorption and reduction with microorganisms exemplifies the promising applications of metal/microorganism composites as catalysts. However, effect of chemical pretreatment on fabrication and catalytic performance of metal/microorganism catalysts has not been reported hitherto. In this study, using the easily cultivated Pichia pastoris (P. pastoris) GS115, rapid adsorption and
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incomplete reduction in Pd (II) ions were introduced to synthesize Pd NP/P. pastoris catalysts. In particular, the effect of chemical pretreatment of P. pastoris cells on the adsorption rates of Pd (II) ions and the properties of the Pd NP/P. pastoris were studied. The catalytic performances of the as-prepared Pd NP/P. pastoris catalysts were finally investigated by using the reduction in 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) as a model reaction. 2. Experimental section 2.1. Materials and reagents P. pastoris GS115 was purchased from Invitrogen Corporation, USA. Soya peptone (BR) was purchased from Guangdong Huan Kai microbial technology Co., Ltd, China. Yeast extract powder was purchased from Oxoid Ltd, England. Other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd, China. In addition, deionized water was used for preparing all aqueous solutions. 2.2. Preparation of the dried powder of P. pastoris GS115 cells P. pastoris GS115 cells were grown at 30 °C for 48 h in YPD medium, in which the percentage (weight/volume) of glucose, soya peptone and yeast were 2%, 2% and 1%, respectively. Then the cells were harvested by centrifugation (3500 rpm, 10 min) at room temperature, washed thrice with deionized water and dried in the oven at 60 °C for 24 h. Finally, the dried cells were ground into fine powder and screened with a 100 mesh sieve.
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equilibrium liquid-phase concentrations (mg/L), V is the volume of solution (L) and m is the weight of dry cell (g). 2.4. Characterizations of Pd NP/P. pastoris catalysts Transmission electron microscopy (TEM) images were obtained on an electron microscope (Tecnai F30, the Netherlands) at 300 keV. X-ray diffraction (XRD) was conducted on an X’pert PRO diffractionmeter (PANalytical B.V., the Netherlands) with Cu Ka radiation at 40 kV and a current of 30 mA with Cu Ka radiation. X-ray photoelectron spectroscopy (XPS) was performed on a Quantum 2000 spectrometer, and the Al Ka line was used as the excitation source. The binding energy was calibrated by C1 s as reference energy (C1 s = 284.5 eV). Fourier transform infrared spectroscopy (FTIR, Nicolet 6700; USA) was used to analyze component changes before and after reduction. 2.5. Catalytic reduction of 4-nitrophenol The catalytic reaction was conducted on the UV–Vis spectrophotometer (Evolution 220, Thermo scientific) with a thermocontroller and rectangular cell of 1 cm optical path length under stirring. The 2.2 mL reaction solution containing aqueous NaBH4 solution (9 mM), aqueous 4-NP solution (0.09 mM) and Pd NP/P. pastoris catalysts (1 mg, 1.78 wt.%) were monitored every 10 s at 400 nm and 30 °C. 3. Results and discussion
2.3. Adsorption of Pd (II)
3.1. Optimization of the adsorption condition
Solid palladium chloride of 1.0 g was dissolved in 50 mL aqueous HCl of 1 M and then preserved in the refrigerator (4 °C) for later use. In a typical adsorption experiment, carefully weighted dried cell powder was firstly dispersed into 100 mL deionized water (the cell concentration, 4 g/L) in a 250 mL Erlenmeyer flask. Then the aqueous Pd (II) solution (1 mM) was added. After the aqueous solution was adjusted to a pH value of 2, the flask was finally shaken on a rotary shaker at 150 rpm at 30 °C. Samples were taken at intervals to monitor the Pd (II) concentration in the solution, which were analyzed by the atomic absorption spectrophotometer (AAS) (Pgeneral, China). And the analyses were performed in triplicate. The pretreatment procedures used for P. pastoris cell were as follows. Firstly, the carefully weighted dried cell powders of 2.0 g were treated with different concentrations (0.05 M, 0.2 M or 0.5 M) of aqueous HCl or NaOH solutions (25 mL) for 24 h. The carboxyl groups of the cells were esterified with 21 mL mixed solvents of methanol and HCl (20:1, volume ratio) for 24 h. The amino groups of the cells were methylated with 60 mL mixed solvents of formic acid and 88% formaldehyde (2:1, volume ratio) for 24 h. After each pretreatment, the samples were centrifuged, washed thrice and completely dried in the oven at 60 °C. The adsorption rate of the adsorption of Pd (II) was calculated according to Eq. (1).
To optimize the adsorption condition, effects of P. pastoris cell concentration, adsorption temperature and initial pH value on the adsorption rate of Pd (II) ions were first investigated. The variation curves of the adsorption rates of the Pd (II) ions over time at different P. pastoris cell concentrations (Fig. S1, Supporting information) show that, the adsorption rates all increased over time at the beginning and tended to be stable after 2 h. Moreover, the adsorption rates increased largely with the increase in the cell concentration. Usually at the higher cell concentration, more binding sites of the cells are available for the Pd (II) ions. At the cell concentrations of 4 and 8 g/L, the adsorption rates significantly increased at the initial stage, indicating that the adsorption of Pd (II) ions by P. pastoris cells was fast. For initial Pd (II) concentration of 1 mM, only 68 ± 7% of Pd (II) ions was adsorbed at the cell concentration of 4 g/L. It should be noted that Pd (II) ions were not completely bound by the cells even when the cell concentration was doubled (i.e. 8 g/L). Adsorption rates of Pd (II) ions at different adsorption temperatures (30–80 °C, Fig. S2) indicated that the adsorption temperature had little effect on the adsorption of Pd (II) ions. While the solution pH plays an important role in metal adsorption. Variations of the adsorption rates of Pd (II) ions over time at different initial pH values (pH values of 1–4, Fig. S3) show that higher adsorption rates were achieved at pH 2. Macaskie et al. also found that the optimum pH for biosorptive Pd (II) uptake was 2–4 [30]. Previous studies had shown that metal ions were first adsorbed and immobilized on the cell surface via electrostatic effect or coordination complexation in an acidic environment [31,32]. However, at pH 1, much more H+ could compete with Pd (II) ions for binding sites. As a result, lower adsorption rates of Pd (II) ions at pH 1 were obtained. In the cases of pH 3 and pH 4, the decreased adsorption rates may be due to the electrostatic repulsion between surface sites of adsorbent and metal ions [33]. To the higher pH solution (pH > 6), palladium hydroxide could be formed and the solution color turned to green–brown from yellow [30].
Adsorption rate=% ¼
Co Ct 100% Co
ð1Þ
The equilibrium adsorption capacity of the cell, qe (mg/g) was calculated according to Eq. (2).
qe ¼
ðCo CeÞ V m
ð2Þ
where Co and Ct are the concentrations of Pd (II) at the initial time and the other specific interval time, respectively. Ce is the
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Given the cell concentration of 4 g/L, adsorption temperature of 30 °C and pH 2, effect of the initial Pd (II) concentration on the adsorption of Pd (II) by the cells was further examined to measure the adsorption isotherms. Fig. 1(a) shows that variations of the adsorption rates increased over time at different initial Pd (II) concentrations. Considering the same adsorption time, the adsorption rates gradually decreased with the increase in the initial Pd (II) concentration. Correlation of the adsorption capacity with the initial Pd (II) concentration is presented in Fig. 1(b), showing that the adsorption capacity increased from 21.2 to 265 mg/L and then decreased to 371 mg/L with the increase in the initial Pd (II) concentration. Furthermore, the maximum adsorption capacity of the cells was 34.88 mg Pd per gram dry cells. In addition, Langmuir model was used to describe the above adsorption data. The results showed that qmax was 38.58 mg Pd per gram dry cells, which was close to the experimental value of 34.88 mg g1.
3.2. Effect of the cell pretreatment on adsorption rate of Pd (II) ions Fig. 2 shows the adsorption rates of Pd (II) ions by the cells at 30 °C before and after pretreatments, i.e. modification by aqueous HCl, NaOH, esterification of carboxyl group, and methylation of amino group. As shown, the pretreatments had a significant influence on the adsorption rates. The adsorption rates of the cells for Pd (II) ions were greatly enhanced after they were pretreated by aqueous HCl, NaOH (except the case of 0.05 M) and methylation of amino group. In contrast, it was minimally affected by the pretreatments with esterification of carboxyl group. SEM was used to observe the cells before and after treatments. As shown in Fig. S4, to some extent, the elliptic morphology of P. pastoris cells changed and cell structural integrity was altered after treatment, which may lead to higher surface area and adsorption of more metal ions by the cells. It was also thought that the charged groups in the wall after modification appeared to be involved in direct interactions with cations or in regulating metal ion-ligand complex formation [34]. Change in H+ concentrations markedly altered the strength of the association between the cell wall and the metals. Based on the better adsorption abilities of the pretreated cell (0.5 M HCl, 0.2 M NaOH and methylation of amino group), subsequent reduction in Pd (II) was discussed at higher temperature (80 °C) (As known, high temperature is favorable to the reduction in Pd (II) compared with lower temperature 30 °C.) and the resulting Pd NP/P. pastoris catalysts were characterized next. Fig. 3 shows the adsorption rates of Pd (II) by the cells before
Fig. 2. The adsorption rates of Pd (II) by the pretreated cells at 30 °C. (Adsorption conditions: cell concentration, 4 g/L; Pd (II) concentration, 1 mM; pH 2; 180 min.).
Fig. 3. The adsorption rates of Pd (II) by the cells before and after pretreatment. (Adsorption conditions: cell concentration, 4 g/L; Pd (II) concentration, 1 mM; pH 2; 180 min; Pretreatment: 0.5 M HCl, 0.2 M, NaOH and methylation of amino group).
and after pretreatment at 30 and 80 °C, respectively. As shown, the adsorption rates of Pd (II) by the unpretreated cells little changed by switching the adsorption temperature from 30 to 80 °C while those by the pretreated cells slightly decreased.
Fig. 1. The adsorption curves at different initial Pd (II) concentration: (a) Adsorption rate as a function of time, (b) Adsorption capacity as a function of initial Pd (II) concentration. (Adsorption conditions: cell concentration 4 g/L, pH 2, 30 °C).
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3.3. Characterization of Pd NP/P. pastoris catalysts The adsorption of metal ions onto the microbial biomass might be accompanied by the reduction in metal ions [35]. Herein, the color change of the solution from light yellow to black could be observed [36]. To compare the properties of the Pd NPs synthesized by the unpretreated and pretreated cells, the initial Pd (II) concentration was adjusted to ensure the same Pd loading over the cells according to the adsorption results in Fig. 3. TEM images of the Pd NPs synthesized by the cells with no pretreatment are presented in Fig. 4. As shown, the NPs were spherical in shape and most of the Pd NPs were well distributed on the cell surface. According to the insert histogram of size distribution, the particle size ranged from 2 to 17 nm and the statistic size was 8.7 ± 2.5 nm. By contrast, Fig. 5 shows TEM images of the Pd NPs synthesized by the pretreated cells, indicating that the shape of prepared NPs was also spherical. However, the statistic sizes of the as-obtained Pd NPs reduced to 6.3 ± 1.0, 4.0 ± 0.7 and 3.3 ± 0.8 nm, respectively, which was smaller than those in the case of the unpretreated cells (Fig. 4). Therefore, by comparing Figs. 4 and 5, it can be found that the size of the Pd NPs was affected by the cell pretreatment. As discussed above (Figs. 2 and 3), the adsorption ability of the cells for Pd (II) ions was greatly enhanced after the pretreatments by aqueous HCl, NaOH and methylation of amino group. The enhanced adsorption led to more Pd NPs over the cells. Therefore, given the same Pd loading, the size of the Pd NPs by the unpretreated cells was larger than those pretreated by aqueous HCl, aqueous NaOH and methylation. The mechanism on the effect of the adsorbed Pd (II) amount on the Pd NP formation was further clarified. More nucleation sites were resulted in the case of higher adsorbed Pd (II) amount. As a result, more Pd nuclei were formed over the cell surface while less unadsorbed Pd (II) ions were available for the growth of the nuclei, given the same initial Pd (II) concentration. Hence, the Pd NPs with smaller size and narrower size distribution were produced after the cell pretreatment. In order to confirm the crystalline phase of the as-synthesized Pd NPs on the cells, Pd NP/P. pastoris catalysts were characterized by XRD. When the Pd loading was as low as 1.78 wt.%, the Bragg reflections were not observed for small size and high dispersion of Pd NPs on the cell surface (Fig. 6(a)). However, if the Pd loading was increased to 10 wt.%, according to the standard XRD patterns of face-centered cubic (fcc) Pd (PDF-2 card No. 01-087-0643), prominent Bragg reflections at 2h of 40.0°, 46.1°, 67.9°, and 82.0° were observed, which corresponds to the Bragg reflections of (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2). Fig. 6(b) demonstrates XPS of Pd 3d peaks of the synthesized Pd NP/P. pastoris catalysts.
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The Pd (0) 3d peak consists of a doublet made of the 3d5/2 and 3d3/2 peaks. Fig. 6(b) shows 335.3 eV was the Pd (0) 3d5/2 peak, which was similar to Refs. [37,38]. The 3d3/2 peak shifted 5.25 eV respect to the 3d5/2 peak. At the same time, Pd2+ peak was also represented at the binding energy of 337.5 eV, indicating that still some Pd (II) ions were bound and not reduced to Pd (0) by the cell surface. As reduction occurred on the dried cell surface, it was suggested that such reduction was non-enzymatic [39]. FTIR spectroscopic studies were carried out to investigate the plausible mechanism behind the formation of the Pd NPs and the change of functional groups on the cell surface. Fig. 7 gives FTIR spectra of the unpretreated and pretreated cells before and after reacting with Pd (II) ions. It can be seen from Fig. 7(a) that the intensity of the absorption band of the alcohol hydroxyl structure CAOH at 1239 cm1 weakened after pretreatment [40] while the appearance of carbonyl structure C@O at 1720 cm1 (red line) may be due to the reactions of ANH2 and AOH with acetic anhydride to form some amides and esters, respectively [41]. In addition, other two absorption bands centered at 1650 and 1545 cm1 were assigned to the amide I and amide II of proteins due to AC@O and ±NAH stretching vibrations in their amide linkages, respectively [41,42]. However, the intensities of the two absorption bands weakened after the formation of the Pd NPs, as shown in Fig. 7(b), implying that these functional groups were adsorbed onto the surface of Pd NPs and they played crucial roles in the reduction of the Pd (II) ions. 3.4. Catalytic activity of Pd catalysts To study the effects of the pretreatments on catalytic properties of the Pd NP/P. pastoris catalysts, the reduction of 4-NP to 4-AP with NaBH4 as the reductant was used as a model reaction [43,44]. The kinetics of 4-NP reduction in the presence of Pd NP/ P. pastoris catalysts were studied by UV–Vis spectroscopy. In Fig. 8(a) the peak at 318 nm of 4-NP shifted to an intensified peak at 400 nm shortly after the addition of NaBH4 [45]. After the Pd NP/ P. pastoris catalysts were added, the 4-NP would be gradually converted into 4-AP and the intensity of peak at 400 nm successively decrease, following gradual increase in peak intensity at 308 nm indicated the formation of 4-AP. Meanwhile, the yellow solution became colorless. Generally, the reduction in 4-NP can be considered as a pseudo-first order reaction when the concentration of BH-4 greatly exceeded that of 4-NP [41,43]. The kinetic equation could be calculated as follows:
dC t ¼ K app C t dt
ð3Þ
Fig. 4. TEM image (a) and magnified TEM image (b) of the Pd NPs synthesized by unpretreated P. pastoris cells (Reducing conditions: cell concentration, 4 g/L; Pd (II) concentration, 1 mM; pH 2; 80 °C, 24 h) The insert histogram indicates their corresponding size distributions.
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Fig. 5. TEM images of the Pd NPs synthesized by pretreated P. pastoris cells: (a) 0.5 M HCl, (b) methylation of amino group and (c) 0.2 M NaOH, respectively. The insert histograms indicate their corresponding size distributions. The initial Pd (II) concentration was (a) 0.77, (b) 0.71 and (c) 0.77 mM, respectively. (Reducing conditions: cell concentration, 4 g/L; pH 2; 80 °C, 24 h).
Fig. 6. XRD patterns (a) and XPS of the Pd 3d peaks (b) of the Pd NP/P. pastoris catalysts.
Fig. 7. FTIR spectra of the cell pretreatment before (a) and after (b) reacting with Pd (II).
ln
Ct C0
¼ K app t þ m
ð4Þ
where Ct is the concentration of 4-NP at time t and Kapp is the apparent rate constant. Fig. 8(b) shows a linear relation between ln (Ct/C0) and reactive time t, and the value of Kapp could by calculated by the slope of the fitting line. Among all the catalysts, the unpretreated showed the maximum rate constant (7.5 103 s1). The rate constants achieved by the pretreated with aqueous HCl, methylation of amino group and aqueous NaOH were 4.3 103, 4.1 103 and 2.6 103 s1, respectively. However, if these catalysts were reused for five cycles, the rate constant in the case of the unpretreated dramatically diminished after the second cycle and gradually decreased after the 3rd, 4th and 5th cycles (Fig. S5(a)). However,
the reaction rates of the pretreated tended to be stable after three cycles. In addition, control experiments with sole P. pastoris cells in the absence of the Pd NPs and without Pd NP/P. pastoris catalysts were also conducted. The results showed that the color of the solution did not change during our experimental time, indicating that 4NP cannot be reduced to 4-AP in the absence of the catalysts [46]. As discussed in Fig. 6(b), there were Pd (II) ions on the surface of the unpretreated cell. The ratio of Pd (0)/Pd (II) was 1.00, which can be estimated by the XPS peak area. The valence states of Pd on the pretreated cell were also measured. According to the results of Fig. S6, the ratios of Pd (0)/Pd (II) on the cell after pretreatment by HCl solution, methylation of amino group and NaOH solution were 0.79, 0.68 and 0.29, respectively. It could be concluded that Pd (II) is adsorbed better, but not reduced better on pretreated cell
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Fig. 8. UV–vis absorption spectra of the catalytic reduction of 4-NP to 4-AP developed with different reaction times (a) and plot of ln(Ct/C0) versus time for the reduction (b).
surfaces. That led to the lower reaction rates for the reduction in 4-NP at the first time. However after the catalysts were reused for the first time, the ratios of Pd (0)/Pd (II) increased to almost the same (Fig. S7) and the sizes of Pd NPs were also increased (Fig. S8). That is, Pd (II) was reduced to some extent by NaBH4 to make the Pd (0)/Pd (II) ratios be 3.2, 3.4, 3.0 and 3.4 and the sizes of Pd NPs were 9.6 ± 2.2, 5.9 ± 1.4, 4.9 ± 1.1 and 4.8 ± 1.1 nm, respectively. As far as the size effect of the Pd NPs was concerned, the rate constant increased with decreasing the size of the Pd NPs, which was consistent with the previous literature [43]. Smaller particles possessed higher surface area, leading to enhanced catalytic performance. Thus, the higher stability of the pretreated Pd NP/P. pastoris catalysts after three cycles may be due to the pretreatments make the Pd NPs well dispersed over the cells and strengthened the binding force of the cell surface for Pd NPs. When it comes to the effect of the adsorbed Pd (II) amount on the catalytic properties of the Pd NPs, the Pd NPs with smaller size and narrower size distribution in the case of lower adsorbed Pd (II) amount, leading to better catalytic activity because of small size effect. It seemed that much of the Pd (II) ions at the later stage were reduced by NaBH4. However, slow reduction in some Pd (II) ions by the P. pastoris cells was also important. In order to clarify the respective roles of NaBH4 and the cells in the reduction in Pd (II) ions and immobilization of Pd NPs, one catalyst was prepared by adsorption of Pd (II) and subsequent reduction with NaBH4 at 30 °C to minimize the reduction with the cells, given the same other conditions. As shown in Fig. S5(b), the durability of the catalysts was poor though the rate constant for the first run was close to that of Pd NP/P. pastoris catalysts without pretreatment. In contrast, the stability of the Pd NP/P. pastoris catalysts was enhanced after the pretreatments (Fig. S5(a)), which were prepared by slow reduction with the cells and further reduction with NaBH4. The slow reduction in Pd (II) ions with the P. pastoris cells allowed preferential nucleation over the cell surface, strengthening the interaction between Pd nanocrystals and the cell surface. Thus, stronger interaction between Pd NPs and the cells could be resulted, which might be responsible for the stability of the pretreated Pd NP/P. pastoris catalysts. The subsequent reduction with NaBH4 promoted the growth of the Pd NPs over the cell surface. Nevertheless, only rapid reduction in adsorbed Pd (II) ions with NaBH4 without slow reduction with the cells gave rise to weaker interaction, thus leading to poor durability of the catalysts. 4. Conclusions In summary, on the basis of rapid adsorption and incomplete reduction in Pd (II) ions by P. pastoris GS115, the effects of cell
pretreatments on the adsorption and reduction of Pd (II) were investigated in this work. The adsorption abilities of the cells for Pd (II) ions were greatly enhanced after they were pretreated with aqueous HCl, aqueous NaOH (except the case of 0.05 M) and methylation of amino group but little affected by the pretreatments with esterification of carboxyl group. However the reduction in the adsorbed Pd (II) ions by the pretreated cells showed lower reduction rates, compared with that by the unpretreated cells. Using the reduction of 4-NP to 4-AP as a model reaction, the asprepared Pd NP/P. pastoris catalysts based on the pretreated cells exhibited higher stability than the unpretreated cells. This can be attributed to the better dispersion of the smaller Pd NPs and stronger binding forces of the pretreated P. pastoris cells employed for fabricating the Pd NPs. This work exemplifies enhancing the stability of Pd catalysts through pretreatments. Acknowledgments This work was supported by the Fundamental Research Funds for Central Universities (2010121051) and the National Natural Science Foundation of China (21036004 and 21106117). The authors are grateful to Dr. Xianxue Li for his kind help. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.07.038. References [1] J. Huang, C. Liu, D. Sun, Y. Hong, M. Du, T. Odoom-Wubah, W. Fang, Q. Li, Chem. Eng. J. 235 (2014) 215. [2] J. Huang, G. Zhan, B. Zheng, D. Sun, F. Lu, Y. Lin, H. Chen, Z. Zheng, Y. Zheng, Q. Li, Ind. Eng. Chem. Res. 50 (2011) 9095. [3] X. Wang, D. Yang, P. Huang, M. Li, C. Li, D. Chen, D. Cui, Nanoscale 4 (2012) 7766. [4] M. Wang, T. Odoom-Wubah, H. Chen, X. Jing, T. Kong, D. Sun, J. Huang, Q. Li, Nanoscale 5 (2013) 6599. [5] B. Wu, N. Zheng, Nano Today 8 (2013) 168. [6] D. Mandal, M.E. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol. Biot. 69 (2006) 485. [7] P. Mohanpuria, N.K. Rana, S.K. Yadav, J. Nanopart. Res. 10 (2008) 507. [8] D. Hebbalalu, J. Lalley, M.N. Nadagouda, R.S. Varma, ACS Sustain. Chem. Eng. 1 (2013) 703. [9] P. Yong, N.A. Rowson, J.P.G. Farr, I.R. Harris, L.E. Macaskie, Biotechnol. Bioeng. 80 (2002) 369. [10] J.R. Lloyd, P. Yong, L.E. Macaskie, Appl. Microbiol. Biotechnol. 64 (1998) 4607. [11] T. Klaus, R. Joerger, E. Olsson, C.G. Granqvist, Proc. Natl. Acad. Sci. U S A 96 (1999) 13611. [12] N. Duran, P.D. Marcato, M. Duran, A. Yadav, A. Gade, M. Rai, Appl. Microbiol. Biot. 90 (2011) 1609. [13] N.J. Creamer, K. Deplanche, T.J. Snape, I.P. Mikheenko, P. Yong, D. Samyahumbi, J. Wood, K. Pollmann, S. Selenska-Pobell, L.E. Macaskie, Hydrometallurgy 94 (2008) 138.
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Facile fabrication of Pd nanoparticle/Pichia pastoris catalysts through adsorption–reduction method: A study into effect of chemical pretreatment Huimei Chen a, Dengpo Huang b, Liqin Lin a, Tareque Odoom-Wubah b, Jiale Huang b,⇑, Daohua Sun b, Qingbiao Li a,b,c,⇑ a
Environmental Science Research Center, College of the Environment & Ecology, Xiamen University, Xiamen 361005, PR China Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China c College of Chemistry & Life Science, Quanzhou Normal University, Quanzhou 362000, PR China b
a r t i c l e
i n f o
Article history: Received 19 May 2014 Accepted 25 July 2014 Available online 6 August 2014 Keywords: Adsorption Chemical modification Palladium nanoparticles 4-Nitrophenol
a b s t r a c t Based on rapid adsorption and incomplete reduction in Pd (II) ions by yeast, Pichia pastoris (P. pastoris) GS115, the effects of pretreatment on adsorption and reduction of Pd (II) ions and the catalytic properties of Pd NP/P. pastoris catalysts were studied. Interestingly, the results showed that the adsorption ability of the cells for Pd (II) ions was greatly enhanced after they were pretreated with aqueous HCl, aqueous NaOH and methylation of amino group. For the reduction in the adsorbed Pd (II) ions, more slow reduction rates by pretreated P. pastoris cells were displayed compared with the cells without pretreatment. Using the reduction of 4-nitrophenol as a model reaction, the Pd NP/P. pastoris catalysts based on the cells after pretreatment with aqueous HCl, aqueous NaOH and methylation of amino group exhibited higher stability than the unpretreated cells. The enhanced stability of the Pd catalysts can be attributed to smaller Pd NPs, better dispersion of the Pd NPs, and stronger binding forces of the pretreated P. pastoris for preparing the Pd NPs. This work exemplifies enhancing the stability of Pd catalysts through pretreatments. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction With the proliferation of nanotechnology in modern society, metal nanoparticles (NPs) as building blocks are of great importance owing to their unique properties, which are different from bulk materials [1–4]. Hence, a variety of physical, chemical and biological methods have been developed to synthesize NPs with tunable shapes and sizes [5]. Driven by the growing impetus of green chemistry, considerable effort has been geared toward the synthesis of metal nanoparticles using microorganisms under mild conditions [6–8]. For example, Macaskie et al. showed the adsorption and rapid reduction in Pd (II) by using Desulfovibrio desulfuricans at the expense of formate or H2 as electron donor [9,10]. Besides the synthesis of noble metal NPs using microorganisms, their potential catalytic applications have also intrigued great ⇑ Corresponding authors at: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China (Q. Li). Fax: +86 592 2184822. E-mail addresses: [email protected] (J. Huang), [email protected] (Q. Li). http://dx.doi.org/10.1016/j.jcis.2014.07.038 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
interest. Nevertheless, the isolation of the NPs formed employing intracellular synthesis for catalytic applications is difficult and the microorganisms used for the extracellular synthesis of NPs must be extensively screened [11,12]. Hence, it is not easy to load microbially synthesized NPs onto inorganic supports. As adsorption and reduction of metal ions in tandem by microorganisms can lead to metal/microorganism composites, dual functions of microorganisms as reductive scaffolds for NPs and supports for catalytic applications could circumvent the isolation of NPs from the microorganisms. For instance, several applications of Pd/microorganism in dehalogenation [13–20], reduction [21–24], hydrogenation [13,23,25,26] and CAC bond forming [23,27,28] reactions have been demonstrated. Windt et al. also showed that such Pd catalysts for the dechlorination of polychlorinated biphenyls were superior to those commercialized [29]. The above synthetic protocol by tandem adsorption and reduction with microorganisms exemplifies the promising applications of metal/microorganism composites as catalysts. However, effect of chemical pretreatment on fabrication and catalytic performance of metal/microorganism catalysts has not been reported hitherto. In this study, using the easily cultivated Pichia pastoris (P. pastoris) GS115, rapid adsorption and
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incomplete reduction in Pd (II) ions were introduced to synthesize Pd NP/P. pastoris catalysts. In particular, the effect of chemical pretreatment of P. pastoris cells on the adsorption rates of Pd (II) ions and the properties of the Pd NP/P. pastoris were studied. The catalytic performances of the as-prepared Pd NP/P. pastoris catalysts were finally investigated by using the reduction in 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) as a model reaction. 2. Experimental section 2.1. Materials and reagents P. pastoris GS115 was purchased from Invitrogen Corporation, USA. Soya peptone (BR) was purchased from Guangdong Huan Kai microbial technology Co., Ltd, China. Yeast extract powder was purchased from Oxoid Ltd, England. Other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd, China. In addition, deionized water was used for preparing all aqueous solutions. 2.2. Preparation of the dried powder of P. pastoris GS115 cells P. pastoris GS115 cells were grown at 30 °C for 48 h in YPD medium, in which the percentage (weight/volume) of glucose, soya peptone and yeast were 2%, 2% and 1%, respectively. Then the cells were harvested by centrifugation (3500 rpm, 10 min) at room temperature, washed thrice with deionized water and dried in the oven at 60 °C for 24 h. Finally, the dried cells were ground into fine powder and screened with a 100 mesh sieve.
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equilibrium liquid-phase concentrations (mg/L), V is the volume of solution (L) and m is the weight of dry cell (g). 2.4. Characterizations of Pd NP/P. pastoris catalysts Transmission electron microscopy (TEM) images were obtained on an electron microscope (Tecnai F30, the Netherlands) at 300 keV. X-ray diffraction (XRD) was conducted on an X’pert PRO diffractionmeter (PANalytical B.V., the Netherlands) with Cu Ka radiation at 40 kV and a current of 30 mA with Cu Ka radiation. X-ray photoelectron spectroscopy (XPS) was performed on a Quantum 2000 spectrometer, and the Al Ka line was used as the excitation source. The binding energy was calibrated by C1 s as reference energy (C1 s = 284.5 eV). Fourier transform infrared spectroscopy (FTIR, Nicolet 6700; USA) was used to analyze component changes before and after reduction. 2.5. Catalytic reduction of 4-nitrophenol The catalytic reaction was conducted on the UV–Vis spectrophotometer (Evolution 220, Thermo scientific) with a thermocontroller and rectangular cell of 1 cm optical path length under stirring. The 2.2 mL reaction solution containing aqueous NaBH4 solution (9 mM), aqueous 4-NP solution (0.09 mM) and Pd NP/P. pastoris catalysts (1 mg, 1.78 wt.%) were monitored every 10 s at 400 nm and 30 °C. 3. Results and discussion
2.3. Adsorption of Pd (II)
3.1. Optimization of the adsorption condition
Solid palladium chloride of 1.0 g was dissolved in 50 mL aqueous HCl of 1 M and then preserved in the refrigerator (4 °C) for later use. In a typical adsorption experiment, carefully weighted dried cell powder was firstly dispersed into 100 mL deionized water (the cell concentration, 4 g/L) in a 250 mL Erlenmeyer flask. Then the aqueous Pd (II) solution (1 mM) was added. After the aqueous solution was adjusted to a pH value of 2, the flask was finally shaken on a rotary shaker at 150 rpm at 30 °C. Samples were taken at intervals to monitor the Pd (II) concentration in the solution, which were analyzed by the atomic absorption spectrophotometer (AAS) (Pgeneral, China). And the analyses were performed in triplicate. The pretreatment procedures used for P. pastoris cell were as follows. Firstly, the carefully weighted dried cell powders of 2.0 g were treated with different concentrations (0.05 M, 0.2 M or 0.5 M) of aqueous HCl or NaOH solutions (25 mL) for 24 h. The carboxyl groups of the cells were esterified with 21 mL mixed solvents of methanol and HCl (20:1, volume ratio) for 24 h. The amino groups of the cells were methylated with 60 mL mixed solvents of formic acid and 88% formaldehyde (2:1, volume ratio) for 24 h. After each pretreatment, the samples were centrifuged, washed thrice and completely dried in the oven at 60 °C. The adsorption rate of the adsorption of Pd (II) was calculated according to Eq. (1).
To optimize the adsorption condition, effects of P. pastoris cell concentration, adsorption temperature and initial pH value on the adsorption rate of Pd (II) ions were first investigated. The variation curves of the adsorption rates of the Pd (II) ions over time at different P. pastoris cell concentrations (Fig. S1, Supporting information) show that, the adsorption rates all increased over time at the beginning and tended to be stable after 2 h. Moreover, the adsorption rates increased largely with the increase in the cell concentration. Usually at the higher cell concentration, more binding sites of the cells are available for the Pd (II) ions. At the cell concentrations of 4 and 8 g/L, the adsorption rates significantly increased at the initial stage, indicating that the adsorption of Pd (II) ions by P. pastoris cells was fast. For initial Pd (II) concentration of 1 mM, only 68 ± 7% of Pd (II) ions was adsorbed at the cell concentration of 4 g/L. It should be noted that Pd (II) ions were not completely bound by the cells even when the cell concentration was doubled (i.e. 8 g/L). Adsorption rates of Pd (II) ions at different adsorption temperatures (30–80 °C, Fig. S2) indicated that the adsorption temperature had little effect on the adsorption of Pd (II) ions. While the solution pH plays an important role in metal adsorption. Variations of the adsorption rates of Pd (II) ions over time at different initial pH values (pH values of 1–4, Fig. S3) show that higher adsorption rates were achieved at pH 2. Macaskie et al. also found that the optimum pH for biosorptive Pd (II) uptake was 2–4 [30]. Previous studies had shown that metal ions were first adsorbed and immobilized on the cell surface via electrostatic effect or coordination complexation in an acidic environment [31,32]. However, at pH 1, much more H+ could compete with Pd (II) ions for binding sites. As a result, lower adsorption rates of Pd (II) ions at pH 1 were obtained. In the cases of pH 3 and pH 4, the decreased adsorption rates may be due to the electrostatic repulsion between surface sites of adsorbent and metal ions [33]. To the higher pH solution (pH > 6), palladium hydroxide could be formed and the solution color turned to green–brown from yellow [30].
Adsorption rate=% ¼
Co Ct 100% Co
ð1Þ
The equilibrium adsorption capacity of the cell, qe (mg/g) was calculated according to Eq. (2).
qe ¼
ðCo CeÞ V m
ð2Þ
where Co and Ct are the concentrations of Pd (II) at the initial time and the other specific interval time, respectively. Ce is the
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Given the cell concentration of 4 g/L, adsorption temperature of 30 °C and pH 2, effect of the initial Pd (II) concentration on the adsorption of Pd (II) by the cells was further examined to measure the adsorption isotherms. Fig. 1(a) shows that variations of the adsorption rates increased over time at different initial Pd (II) concentrations. Considering the same adsorption time, the adsorption rates gradually decreased with the increase in the initial Pd (II) concentration. Correlation of the adsorption capacity with the initial Pd (II) concentration is presented in Fig. 1(b), showing that the adsorption capacity increased from 21.2 to 265 mg/L and then decreased to 371 mg/L with the increase in the initial Pd (II) concentration. Furthermore, the maximum adsorption capacity of the cells was 34.88 mg Pd per gram dry cells. In addition, Langmuir model was used to describe the above adsorption data. The results showed that qmax was 38.58 mg Pd per gram dry cells, which was close to the experimental value of 34.88 mg g1.
3.2. Effect of the cell pretreatment on adsorption rate of Pd (II) ions Fig. 2 shows the adsorption rates of Pd (II) ions by the cells at 30 °C before and after pretreatments, i.e. modification by aqueous HCl, NaOH, esterification of carboxyl group, and methylation of amino group. As shown, the pretreatments had a significant influence on the adsorption rates. The adsorption rates of the cells for Pd (II) ions were greatly enhanced after they were pretreated by aqueous HCl, NaOH (except the case of 0.05 M) and methylation of amino group. In contrast, it was minimally affected by the pretreatments with esterification of carboxyl group. SEM was used to observe the cells before and after treatments. As shown in Fig. S4, to some extent, the elliptic morphology of P. pastoris cells changed and cell structural integrity was altered after treatment, which may lead to higher surface area and adsorption of more metal ions by the cells. It was also thought that the charged groups in the wall after modification appeared to be involved in direct interactions with cations or in regulating metal ion-ligand complex formation [34]. Change in H+ concentrations markedly altered the strength of the association between the cell wall and the metals. Based on the better adsorption abilities of the pretreated cell (0.5 M HCl, 0.2 M NaOH and methylation of amino group), subsequent reduction in Pd (II) was discussed at higher temperature (80 °C) (As known, high temperature is favorable to the reduction in Pd (II) compared with lower temperature 30 °C.) and the resulting Pd NP/P. pastoris catalysts were characterized next. Fig. 3 shows the adsorption rates of Pd (II) by the cells before
Fig. 2. The adsorption rates of Pd (II) by the pretreated cells at 30 °C. (Adsorption conditions: cell concentration, 4 g/L; Pd (II) concentration, 1 mM; pH 2; 180 min.).
Fig. 3. The adsorption rates of Pd (II) by the cells before and after pretreatment. (Adsorption conditions: cell concentration, 4 g/L; Pd (II) concentration, 1 mM; pH 2; 180 min; Pretreatment: 0.5 M HCl, 0.2 M, NaOH and methylation of amino group).
and after pretreatment at 30 and 80 °C, respectively. As shown, the adsorption rates of Pd (II) by the unpretreated cells little changed by switching the adsorption temperature from 30 to 80 °C while those by the pretreated cells slightly decreased.
Fig. 1. The adsorption curves at different initial Pd (II) concentration: (a) Adsorption rate as a function of time, (b) Adsorption capacity as a function of initial Pd (II) concentration. (Adsorption conditions: cell concentration 4 g/L, pH 2, 30 °C).
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3.3. Characterization of Pd NP/P. pastoris catalysts The adsorption of metal ions onto the microbial biomass might be accompanied by the reduction in metal ions [35]. Herein, the color change of the solution from light yellow to black could be observed [36]. To compare the properties of the Pd NPs synthesized by the unpretreated and pretreated cells, the initial Pd (II) concentration was adjusted to ensure the same Pd loading over the cells according to the adsorption results in Fig. 3. TEM images of the Pd NPs synthesized by the cells with no pretreatment are presented in Fig. 4. As shown, the NPs were spherical in shape and most of the Pd NPs were well distributed on the cell surface. According to the insert histogram of size distribution, the particle size ranged from 2 to 17 nm and the statistic size was 8.7 ± 2.5 nm. By contrast, Fig. 5 shows TEM images of the Pd NPs synthesized by the pretreated cells, indicating that the shape of prepared NPs was also spherical. However, the statistic sizes of the as-obtained Pd NPs reduced to 6.3 ± 1.0, 4.0 ± 0.7 and 3.3 ± 0.8 nm, respectively, which was smaller than those in the case of the unpretreated cells (Fig. 4). Therefore, by comparing Figs. 4 and 5, it can be found that the size of the Pd NPs was affected by the cell pretreatment. As discussed above (Figs. 2 and 3), the adsorption ability of the cells for Pd (II) ions was greatly enhanced after the pretreatments by aqueous HCl, NaOH and methylation of amino group. The enhanced adsorption led to more Pd NPs over the cells. Therefore, given the same Pd loading, the size of the Pd NPs by the unpretreated cells was larger than those pretreated by aqueous HCl, aqueous NaOH and methylation. The mechanism on the effect of the adsorbed Pd (II) amount on the Pd NP formation was further clarified. More nucleation sites were resulted in the case of higher adsorbed Pd (II) amount. As a result, more Pd nuclei were formed over the cell surface while less unadsorbed Pd (II) ions were available for the growth of the nuclei, given the same initial Pd (II) concentration. Hence, the Pd NPs with smaller size and narrower size distribution were produced after the cell pretreatment. In order to confirm the crystalline phase of the as-synthesized Pd NPs on the cells, Pd NP/P. pastoris catalysts were characterized by XRD. When the Pd loading was as low as 1.78 wt.%, the Bragg reflections were not observed for small size and high dispersion of Pd NPs on the cell surface (Fig. 6(a)). However, if the Pd loading was increased to 10 wt.%, according to the standard XRD patterns of face-centered cubic (fcc) Pd (PDF-2 card No. 01-087-0643), prominent Bragg reflections at 2h of 40.0°, 46.1°, 67.9°, and 82.0° were observed, which corresponds to the Bragg reflections of (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2). Fig. 6(b) demonstrates XPS of Pd 3d peaks of the synthesized Pd NP/P. pastoris catalysts.
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The Pd (0) 3d peak consists of a doublet made of the 3d5/2 and 3d3/2 peaks. Fig. 6(b) shows 335.3 eV was the Pd (0) 3d5/2 peak, which was similar to Refs. [37,38]. The 3d3/2 peak shifted 5.25 eV respect to the 3d5/2 peak. At the same time, Pd2+ peak was also represented at the binding energy of 337.5 eV, indicating that still some Pd (II) ions were bound and not reduced to Pd (0) by the cell surface. As reduction occurred on the dried cell surface, it was suggested that such reduction was non-enzymatic [39]. FTIR spectroscopic studies were carried out to investigate the plausible mechanism behind the formation of the Pd NPs and the change of functional groups on the cell surface. Fig. 7 gives FTIR spectra of the unpretreated and pretreated cells before and after reacting with Pd (II) ions. It can be seen from Fig. 7(a) that the intensity of the absorption band of the alcohol hydroxyl structure CAOH at 1239 cm1 weakened after pretreatment [40] while the appearance of carbonyl structure C@O at 1720 cm1 (red line) may be due to the reactions of ANH2 and AOH with acetic anhydride to form some amides and esters, respectively [41]. In addition, other two absorption bands centered at 1650 and 1545 cm1 were assigned to the amide I and amide II of proteins due to AC@O and ±NAH stretching vibrations in their amide linkages, respectively [41,42]. However, the intensities of the two absorption bands weakened after the formation of the Pd NPs, as shown in Fig. 7(b), implying that these functional groups were adsorbed onto the surface of Pd NPs and they played crucial roles in the reduction of the Pd (II) ions. 3.4. Catalytic activity of Pd catalysts To study the effects of the pretreatments on catalytic properties of the Pd NP/P. pastoris catalysts, the reduction of 4-NP to 4-AP with NaBH4 as the reductant was used as a model reaction [43,44]. The kinetics of 4-NP reduction in the presence of Pd NP/ P. pastoris catalysts were studied by UV–Vis spectroscopy. In Fig. 8(a) the peak at 318 nm of 4-NP shifted to an intensified peak at 400 nm shortly after the addition of NaBH4 [45]. After the Pd NP/ P. pastoris catalysts were added, the 4-NP would be gradually converted into 4-AP and the intensity of peak at 400 nm successively decrease, following gradual increase in peak intensity at 308 nm indicated the formation of 4-AP. Meanwhile, the yellow solution became colorless. Generally, the reduction in 4-NP can be considered as a pseudo-first order reaction when the concentration of BH-4 greatly exceeded that of 4-NP [41,43]. The kinetic equation could be calculated as follows:
dC t ¼ K app C t dt
ð3Þ
Fig. 4. TEM image (a) and magnified TEM image (b) of the Pd NPs synthesized by unpretreated P. pastoris cells (Reducing conditions: cell concentration, 4 g/L; Pd (II) concentration, 1 mM; pH 2; 80 °C, 24 h) The insert histogram indicates their corresponding size distributions.
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Fig. 5. TEM images of the Pd NPs synthesized by pretreated P. pastoris cells: (a) 0.5 M HCl, (b) methylation of amino group and (c) 0.2 M NaOH, respectively. The insert histograms indicate their corresponding size distributions. The initial Pd (II) concentration was (a) 0.77, (b) 0.71 and (c) 0.77 mM, respectively. (Reducing conditions: cell concentration, 4 g/L; pH 2; 80 °C, 24 h).
Fig. 6. XRD patterns (a) and XPS of the Pd 3d peaks (b) of the Pd NP/P. pastoris catalysts.
Fig. 7. FTIR spectra of the cell pretreatment before (a) and after (b) reacting with Pd (II).
ln
Ct C0
¼ K app t þ m
ð4Þ
where Ct is the concentration of 4-NP at time t and Kapp is the apparent rate constant. Fig. 8(b) shows a linear relation between ln (Ct/C0) and reactive time t, and the value of Kapp could by calculated by the slope of the fitting line. Among all the catalysts, the unpretreated showed the maximum rate constant (7.5 103 s1). The rate constants achieved by the pretreated with aqueous HCl, methylation of amino group and aqueous NaOH were 4.3 103, 4.1 103 and 2.6 103 s1, respectively. However, if these catalysts were reused for five cycles, the rate constant in the case of the unpretreated dramatically diminished after the second cycle and gradually decreased after the 3rd, 4th and 5th cycles (Fig. S5(a)). However,
the reaction rates of the pretreated tended to be stable after three cycles. In addition, control experiments with sole P. pastoris cells in the absence of the Pd NPs and without Pd NP/P. pastoris catalysts were also conducted. The results showed that the color of the solution did not change during our experimental time, indicating that 4NP cannot be reduced to 4-AP in the absence of the catalysts [46]. As discussed in Fig. 6(b), there were Pd (II) ions on the surface of the unpretreated cell. The ratio of Pd (0)/Pd (II) was 1.00, which can be estimated by the XPS peak area. The valence states of Pd on the pretreated cell were also measured. According to the results of Fig. S6, the ratios of Pd (0)/Pd (II) on the cell after pretreatment by HCl solution, methylation of amino group and NaOH solution were 0.79, 0.68 and 0.29, respectively. It could be concluded that Pd (II) is adsorbed better, but not reduced better on pretreated cell
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Fig. 8. UV–vis absorption spectra of the catalytic reduction of 4-NP to 4-AP developed with different reaction times (a) and plot of ln(Ct/C0) versus time for the reduction (b).
surfaces. That led to the lower reaction rates for the reduction in 4-NP at the first time. However after the catalysts were reused for the first time, the ratios of Pd (0)/Pd (II) increased to almost the same (Fig. S7) and the sizes of Pd NPs were also increased (Fig. S8). That is, Pd (II) was reduced to some extent by NaBH4 to make the Pd (0)/Pd (II) ratios be 3.2, 3.4, 3.0 and 3.4 and the sizes of Pd NPs were 9.6 ± 2.2, 5.9 ± 1.4, 4.9 ± 1.1 and 4.8 ± 1.1 nm, respectively. As far as the size effect of the Pd NPs was concerned, the rate constant increased with decreasing the size of the Pd NPs, which was consistent with the previous literature [43]. Smaller particles possessed higher surface area, leading to enhanced catalytic performance. Thus, the higher stability of the pretreated Pd NP/P. pastoris catalysts after three cycles may be due to the pretreatments make the Pd NPs well dispersed over the cells and strengthened the binding force of the cell surface for Pd NPs. When it comes to the effect of the adsorbed Pd (II) amount on the catalytic properties of the Pd NPs, the Pd NPs with smaller size and narrower size distribution in the case of lower adsorbed Pd (II) amount, leading to better catalytic activity because of small size effect. It seemed that much of the Pd (II) ions at the later stage were reduced by NaBH4. However, slow reduction in some Pd (II) ions by the P. pastoris cells was also important. In order to clarify the respective roles of NaBH4 and the cells in the reduction in Pd (II) ions and immobilization of Pd NPs, one catalyst was prepared by adsorption of Pd (II) and subsequent reduction with NaBH4 at 30 °C to minimize the reduction with the cells, given the same other conditions. As shown in Fig. S5(b), the durability of the catalysts was poor though the rate constant for the first run was close to that of Pd NP/P. pastoris catalysts without pretreatment. In contrast, the stability of the Pd NP/P. pastoris catalysts was enhanced after the pretreatments (Fig. S5(a)), which were prepared by slow reduction with the cells and further reduction with NaBH4. The slow reduction in Pd (II) ions with the P. pastoris cells allowed preferential nucleation over the cell surface, strengthening the interaction between Pd nanocrystals and the cell surface. Thus, stronger interaction between Pd NPs and the cells could be resulted, which might be responsible for the stability of the pretreated Pd NP/P. pastoris catalysts. The subsequent reduction with NaBH4 promoted the growth of the Pd NPs over the cell surface. Nevertheless, only rapid reduction in adsorbed Pd (II) ions with NaBH4 without slow reduction with the cells gave rise to weaker interaction, thus leading to poor durability of the catalysts. 4. Conclusions In summary, on the basis of rapid adsorption and incomplete reduction in Pd (II) ions by P. pastoris GS115, the effects of cell
pretreatments on the adsorption and reduction of Pd (II) were investigated in this work. The adsorption abilities of the cells for Pd (II) ions were greatly enhanced after they were pretreated with aqueous HCl, aqueous NaOH (except the case of 0.05 M) and methylation of amino group but little affected by the pretreatments with esterification of carboxyl group. However the reduction in the adsorbed Pd (II) ions by the pretreated cells showed lower reduction rates, compared with that by the unpretreated cells. Using the reduction of 4-NP to 4-AP as a model reaction, the asprepared Pd NP/P. pastoris catalysts based on the pretreated cells exhibited higher stability than the unpretreated cells. This can be attributed to the better dispersion of the smaller Pd NPs and stronger binding forces of the pretreated P. pastoris cells employed for fabricating the Pd NPs. This work exemplifies enhancing the stability of Pd catalysts through pretreatments. Acknowledgments This work was supported by the Fundamental Research Funds for Central Universities (2010121051) and the National Natural Science Foundation of China (21036004 and 21106117). The authors are grateful to Dr. Xianxue Li for his kind help. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.07.038. References [1] J. Huang, C. Liu, D. Sun, Y. Hong, M. Du, T. Odoom-Wubah, W. Fang, Q. Li, Chem. Eng. J. 235 (2014) 215. [2] J. Huang, G. Zhan, B. Zheng, D. Sun, F. Lu, Y. Lin, H. Chen, Z. Zheng, Y. Zheng, Q. Li, Ind. Eng. Chem. Res. 50 (2011) 9095. [3] X. Wang, D. Yang, P. Huang, M. Li, C. Li, D. Chen, D. Cui, Nanoscale 4 (2012) 7766. [4] M. Wang, T. Odoom-Wubah, H. Chen, X. Jing, T. Kong, D. Sun, J. Huang, Q. Li, Nanoscale 5 (2013) 6599. [5] B. Wu, N. Zheng, Nano Today 8 (2013) 168. [6] D. Mandal, M.E. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol. Biot. 69 (2006) 485. [7] P. Mohanpuria, N.K. Rana, S.K. Yadav, J. Nanopart. Res. 10 (2008) 507. [8] D. Hebbalalu, J. Lalley, M.N. Nadagouda, R.S. Varma, ACS Sustain. Chem. Eng. 1 (2013) 703. [9] P. Yong, N.A. Rowson, J.P.G. Farr, I.R. Harris, L.E. Macaskie, Biotechnol. Bioeng. 80 (2002) 369. [10] J.R. Lloyd, P. Yong, L.E. Macaskie, Appl. Microbiol. Biotechnol. 64 (1998) 4607. [11] T. Klaus, R. Joerger, E. Olsson, C.G. Granqvist, Proc. Natl. Acad. Sci. U S A 96 (1999) 13611. [12] N. Duran, P.D. Marcato, M. Duran, A. Yadav, A. Gade, M. Rai, Appl. Microbiol. Biot. 90 (2011) 1609. [13] N.J. Creamer, K. Deplanche, T.J. Snape, I.P. Mikheenko, P. Yong, D. Samyahumbi, J. Wood, K. Pollmann, S. Selenska-Pobell, L.E. Macaskie, Hydrometallurgy 94 (2008) 138.
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