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Recovery of gold from industrial wastewater by immobilized gold-binding proteins on porous silica carriers grafted with amino group
Biochemical Engineering Journal 152 (2019) 107388
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Recovery of gold from industrial wastewater by immobilized gold-binding proteins on porous silica carriers grafted with amino group Yin-Lung Hana, Pei-Jyuan Gaoa, Chieh-Lun Chenga, , Pong-Yee Wub, Jo-Shu Changb,c,d, ⁎⁎
T
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a
Green Energy and Environment Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan c Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung 407, Taiwan d Center for Nanotechnology, Tunghai University, Taichung 407, Taiwan b
HIGHLIGHTS
Au-binding proteins were used for Au recovery from industrial wastewater. • Immobilized protein immobilization capacity on the amino-grafted porous silica was optimized. • The silica carrier was pelleted at 700 °C to improve its mechanical strength to 19.47 kg. • The Au biosorption was conducted with a max. capacity of 1259 mg Au/mg protein. • Fixed-bed • Max. Au adsorption rate of the fixed beds was 33.4 mg/g/h with 40 mg Au/L wastewater. ARTICLE INFO
ABSTRACT
Keywords: Biosorption Protein immobilization Porous silica carrier Au-binding protein
Our recent work demonstrated excellent gold biosorption potential of extracellular proteins obtained from a thermophilic bacterium Tepidimonas fonticaldi AT-A2. In this study, the gold-binding proteins were immobilized on amino-grafted porous silica carriers to further enhance the feasibility of using them for Au recovery from real industrial wastewater. The maximum protein immobilization capacity on the amino-grafted porous silica reached the highest level of 13.83 mg protein/g carrier when the protein loading concentration was 400 mg/L and the amino groups content on the silica carrier was 16%. The mechanical strength of the amino-grafted porous silica could be improved to 19.47 kg by converting the silica powder into pellet form using a calcination temperature of 700 °C. Continuous biosorption of Au (III) from prepared Au solution and from real industrial wastewater was carried out using fixed beds packed with the protein-loaded amino-grafted porous silica pellets. The maximum Au adsorption capacity of the fixed beds with a feeding rate of 0.12 ml/min was 1259 and 448 mg Au/mg protein from the prepared Au solution and an electroplating wastewater (Au concentration was approximately 40 mg/L), respectively. The maximum Au adsorption rate of the fixed beds when using the real wastewater was 33.4 mg/g/h, which is similar to the results of using the prepared Au solution.
1. Introduction Precious metals can be found in nature in atmosphere, solid and liquid phases. In particular, precious metals are found in natural ores. The usual way to find and extract them is through exploration and mining from nature. Because of the limited amount of resources of precious metals in nature, researchers are now looking for efficient ways to recover precious metals from other resources, such as electronic and catalytic wastes, solid wastes, liquid wastes and geothermal sites.
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Reclaiming the precious metals of very low concentration from various waste stream solutions often relies on unconventional technologies for metal recovery, such as biosorption and bioaccumulation techniques [1]. Nevertheless, the literature shows that bio-reclamation of such metals is still limited to lab-scale research. Hence, more efforts are needed to get the real benefit of microbial strategy for treating the urban mined secondary materials [2]. Gold, a most important precious metal whose market in intertwined with global economy, has a demand and supply of 35,000 tons per year
Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan. Corresponding author. E-mail addresses: [email protected] (C.-L. Cheng), [email protected] (J.-S. Chang).
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https://doi.org/10.1016/j.bej.2019.107388 Received 14 May 2019; Received in revised form 20 September 2019; Accepted 23 September 2019 Available online 24 September 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
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and the largest source of supply (25,000 tons per year) is still from mining [3]. Because of the limited amount of resources of precious metals in nature, researchers are now looking for alternative ways to procure gold. Gold recovery from secondary sources, such as industrial wastewater, electronic waste and metal scraps is being explored currently to meet the growing demand. Industrial wastes are recognized as a good source for the recovery of precious metals. In Taiwan, the gold is mainly used in the electroplating process of printed circuit boards (PCB) manufacturing factory and the resulting wastewater contains around 5–40 ppm of Au. In order to increase the overall process benefit, recovery of the gold from the process wastewater seems to be necessary [4]. Methods currently in use for gold recovery from waste solutions include solvent extraction, ion exchange, chemical precipitation, cementation, electro-wining, coagulation and pyro-metallurgical processes [5]. Nevertheless, most of the above methods are time consuming and energy intensive, while they also generate a chemical laden sludge to be disposed of. Biosorption is considered a suitable way to recover gold from industrial wastewaters with numerous advantages including high biodegradability, high selectivity, environmental compatibility, high biodegradability, low cost, and simple operating procedures. Biosorbents used for metal biosorption cover a wide range of bio-materials, such as biomass, protein and other renewable materials, while bioaccumulation typically applies live microbial cells for metal uptake and accumulation [6]. Biosorption is a quick process independent of the presence of specific nutrients, while bioaccumulation is slow and nutrient dependent [7]. Our recent work showed that extracellular proteins secreted from a thermophilic bacterium Tepidimonas fonticaldi AT-A2 had an excellent gold binding performance with a high gold binding capacity of 9.7 mg Au/mg protein and a good selectivity against co-existing metals [3]. Thus, the metal-binding proteins seem to be very effective on gold recovery from aqueous waste streams in terms of adsorption capacity per biosorbent weight and the selectivity. However, to assist the use of the protein-based biosorbent in practical cases, the proteins usually should be immobilized on appropriate matrix for easier operation and recovery of proteins after use. Moreover, the immobilization matrix used should be mechanically and chemically strong to withstand actual process conditions [8]. Protein immobilization technique has been widely used in biosensor, biofuel, agro-industry, pharmaceutical, waste treatment and precious metal recovery [9]. Until now, protein immobilization has been carried out by adsorption on inert supports, entrapment in a polymeric matrix, covalently bound to vector compounds, and by cells cross-linking [10]. Chemical binding and physical adsorption are frequently employed to immobilize protein on/in the support material. Chemical methods are associated with the formation of covalent bonding or cross linking to capture the protein on the surface or inside the supporter, while physical methods are related to the ionic adsorption between protein and support. The binding strength of covalent bonding is much higher than that of physical adsorption. This requires the presence of two mutually reactive chemical groups in the protein and support material. Some studies have indicated that the primary amine (eNH3), carboxylic acid (eCOOH), thiol (-SH), hydroxyl (eOH), phenol (eArOH), thioether (R¹-S-R²), imidazole and guanidine groups present in the protein can be used for covalent immobilization [11]. In order to increase the immobilization efficiency, surface modification of the support material is necessary, and the SH- (thiol), NH- (amino) and COOH- (carboxylic) are general functional groups applied for modification of surface properties of the support material. Recently, microarrays used the thiol-ene reaction to immobilize biotin, streptavidin and pTyr-peptide on thiol-modified silicon wafers [12]. Al-Dhrub et al. [13] showed that amino functionalized magnetic nanoparticles (modified with 3-(aminopropyl)triethoxysilane (APTES)) is an effective adsorbent for human carbonic anhydrase I (hCA I), and 61% of the immobilized hCA I activity was retained after repeated use for 13 cycles. Deka et al. [14] also used Cage-type cubic mesoporous silica (FDU-12)
as the adsorbent modified with carboxylic functionalization for lysozyme immobilization, and a maximum lysozyme adsorption capacity of 895 mg/g was obtained. In this study, the gold-binding proteins from T. fonticaldi AT-A2 were immobilized for practical applications in the removal of gold from an electroplating wastewater. Porous silica was chosen as the support material to immobilize the Au-binding protein due to its great mechanical strength and extremely high binding surface area. To further enhance protein immobilization efficiency, amine group was grafted onto the surface of silica. The developed biosorbent was used for the recovery of Au from the industrial wastewater using continuous fixedbed operations to evaluate the feasibility of the protein-based biosorbent. After metal adsorption, the metal recovery from loaded adsorbent via desorption process was also discussed. According to the study by Dwivedi et al. [15], three kinds of desorption agents were chosen in the process, which were sodium hydroxide, hydrogen chloride and thiourea. The best desorption performance was obtained when using 1 M thiourea solution as the desorption agent, resulting in an 87% desorption efficiency of Au metal from the loaded protein biosorbent. Thus, biosorption is currently considered top ranked technology that can be used for the recovery of metals or sequestration of toxic and pollutant metals even under trace metal concentrations. 2. Materials and methods 2.1. Microorganisms, protein and mediums The Au-binding proteins were produced from a thermophilic bacterium, Tepidimonas fonticaldi AT-A2, which isolated from the Antun Hot Spring, Hualien County, Taiwan [3,16,17]. The composition of culture medium consisted of: acetic acid, 3.30 g/L; yeast extract, 1.00 g/L; KH2PO4, 0.53 g/L; (NH4)2SO4, 1.00 g/L; MgCl2, 0.10 g/L; trace element solution, 1 ml/L. The composition of trace element solution is as follows: ZnSO4·7H2O, 0.10 g/L; MnCl2·4H2O, 0.03 g/L; H3BO3, 0.30 g/L; CoCl2·6H2O, 0.20 g/L; CuCl2·2H2O, 0.01 g/L; NiCl2·6H2O, 0.02 g/L; Na2MoO4·2H2O, 0.03 g/L. Tepidimonas fonticaldi AT-A2 was cultured at 50 °C, 200 rpm for 48 h and the extracellular proteins of AT-A2 were immobilized on the porous silica grafted with amino group as described in the following section. The extracellular proteins contained multiple proteins. Analysis of the SDS PAGE gel with a small molecular weight (MW) marker indicates the existence of proteins smaller than 10 kDa, and there are two strong bands corresponding to a MW of 4 and 8 kDa. The Au-binding capacity of 3–10 kDa proteins (1.50 mg Au/mg protein) appeared to be nearly 12.5-fold higher than the proteins > 10 kDa (0.12 mg Au/mg protein) [3]. The amino acid composition of the acid hydrolyzed samples is different for the > 10 kDa protein fraction and the 3–10 kDa protein fraction. The presence of Cys and His could be a possible reason for the better Au adsorption efficiency of the proteins with the size of 3–10 kDa [3]. 2.2. Characterization of materials- amino-grafted porous silica The octenyl succinic anhydride (OSA-pretreated starch (0.36 g) was added into the CTAB solution (cetyltrimethylammonium bromide, 7.3 g of CTAB in 100 ml of 35% ethanol). Then, 22 mL of TEOS (Tetraethyl orthosilicate) and 1.5 mL of sulfuric acid were then added into the mixture, mixed well and incubated for 6 h for hydrolysis. After hydrolysis, the mixture was titrated with 12.5% ammonium hydroxide solution at 25 °C for 2 h. The precipitated particles were filtered and washed with water several times to completely remove any un-adsorbed components and dried at 50 °C for 24 h. The particles were calcined at 550 °C for 2 h to obtain porous silica with a particle size of 20–50 μm. Porous silica with an amount of 1.0 g was added into 20 mL deionized water and the pH was adjusted to 10 by 12.5% ammonium hydroxide solution. A 3.6 g of the APTES ((3-Aminopropyl) 2
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triethoxysilane) was added to the mixture with vigorous mixing and the reaction was carried out at 100 °C and 600 rpm for 3.0 h. The aminografted porous silica thus obtained was filtered and washed with water several times to completely remove un-adsorbed components and dried at 50 °C for 24 h.
Table 1 Major element composition of the wastewater obtained from an electroplating factory.
2.3. Preparation of protein-based biosorbent (protein immobilization) Amino-grafted porous silica (0.2 g) was added into 20 ml prepared protein with different protein loadings of 40–400 mg/L from AT-A2 in 50 ml serum bottle. The immobilization was conducted at a stable temperature of 25 °C, stirring rate of 100 rpm, pH of 7, and adsorption time of 6 h. Residual Au binding activity and adsorbed protein concentration on amino-grafted porous silica was regularly monitored at designated time intervals until immobilization reached equilibrium state. Then the immobilized Au-binding proteins were used for further experiments.
Component elements
Concentration (mg/L)
Au Al Ca Fe K Mg Na Ni P S Si Zn
39.1 2.4 7.4 0.1 133.9 0.7 11.2 0.7 2.6 2.4 2.3 0.2
2.6. Measurement of Au concentration
2.4. Fixed-bed column biosorption by immobilized protein
The 1000 mg/L Au(III) standard solution of was obtained from High Purity Standard (HPS) Inc., USA. Working standards were prepared by progressive dilution of standard Au(III) solutions using deionized water. Au content in solutions was measured by Inductively Coupled Plasma (ICP), which detected Au intensity at an emission wavelength of 267.595 nm (Jobin-Yvon ICP Optical Emission Spectrometer JY 2000-2, Horiba Scientific. Inc.). Prior to ICP measurement, the precious metal solutions were appropriately treated with HNO3 to ensure that the metal concentration in the sample was linearly dependent on the detected absorbance. In other words, the pH value of sample solution was below 2.0. The Au adsorption capacity was calculated as follows.
The immobilized protein-based biosorbent (totally 4.0 g of silica pellets, which is 2.5 mm in diameter) was packed into three columns (Height = 5.5 cm, Inner diameter = 1.2 cm) for continuous biosorption of Au (III) (Fig. 1). The influent tank contains pure Au (III) solution at a concentration of 37.5 mg/L prepared with HAuCl 4·3H 2O or industrial wastewater from an electroplating factory containing 39.1 mg/L Au(III). Composition of the industrial wastewater used is indicated in Table 1. The influent tank was mixed by a magnetic stirrer at a stirring rate of 100 rpm. The influent was passed through the fixed-bed columns by a peristaltic pump with a volumetric flow rate of 0.12 ml/min.
Au adsorption capacity: [(Au conc. (adsorption before) - Au conc. * V(operation volume)]/ W(weight of Protein)
before))
2.5. Measurement of protein concentration The protein concentration in the solution was determined by Bradford assay using commercialized Bio-Red Protein Assay Dye Reagent. Briefly, 100 μL reagent was mixed with 150 μL sample and incubated in 96 well plates at 25 °C for about 20 min. At the end of incubation, absorbance of 595 nm (OD595) of the mixture was measured by ELISA (BioTek). Bovine Serum Albumin (BSA, Sigma) was used as protein standard. The calculation of protein loss percentage and protein immobilization efficiency are performed as follows.
(adsorption
2.7. Estimation of amino group content of the immobilized carrier The amino acids present on the support was determined by thermogravimetric analysis, assuming that the weight loss between 400 °C and 600 °C was associated with the desorption of the amino acids adsorbed on the support surface. The thermogravimetric curves were recovered in N2 flow on a TGA 4000 (PerkinElmer) instrument with a heating rate of 20 °C/min from 50 °C to 880 °C.
Protein loss percentage: [Protein conc. (wash solution) * V(wash volume)] /[(Protein conc. (immobilization before) - Protein conc. (immobilization before)) * V(operation volume)] *100
2.8. Surface area analysis Brunauer Emmett Teller (BET) instrument was used to measure the surface area, pore size, pore volume, and pore diameter of the support material and catalysts. The BET machine used in this study is ASAP 2020 Micromeritics. The samples were degassed at 200 °C for 2 h.
Protein immobilization efficiency: [(Protein conc. (immobilization before) Protein conc. (immobilization before)) * V(operation volume)]/ W(weight of carrier)
Fig. 1. An illustration for the setup of continuous biosorption fixed-bed columns. a total amount of 4 g of Silica immobilized proteins was evenly packed in the three columns. The influent tank contains 40 mg/L of Au (III) solution prepared with HAuCl4·3H2O or industrial wastewater containing 39.1 mg/L of Au(III). 3
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Barrett-Joyner-Halenda (BJH) method was used to determine the pore volume and the pore size.
match the Langmuir Isotherm type model (Eq. 1) with a qmax and K d value of 15.6 mg protein/g silica and 46.1 mg/L, respectively. Note that the protein immobilization capacity (mg/g) is calculated with qmax C , where qmax : maximum protein immobilization efficiency (mg Kd + C protein/g silica); K d : half saturation constant (mg/L)
2.9. Hardness analysis The hardness of silica pellets was measured by the tablets hardness tester (TH-C). The sample was prepared as a 1.0 cm × 0.8 cm × 1.2 cm cube on the testing platform (1.0 cm × 1.0 cm) to analyze the hardness.
3.2. Effect of amino group content on silica matrix on protein immobilization efficiency
3. Results and discussion
Our previous work showed the detailed Au-binding properties of the extracellular proteins obtained from the AT-A2 strain. Under the optimal adsorption conditions of 50 °C and pH 5, the maximal Au adsorption capacity of the AT-A2 proteins was 9.7 mg Au/mg protein [3]. When further using the AT-A2 proteins for Au removal from industrial wastewater containing 15 mg/L Au, a maximum adsorption capacity of 1.45 mg Au/mg protein was obtained with a removal efficiency of 71% [3]. For practical applications of the protein biosorbents, an enhanced protein biosorbent concentration is vital. Therefore, in this study, the AT-A2 proteins were immobilized with amino-grafted porous silica (prepared as mentioned in Section 2.2) support to make them more applicable in real cases. The first step in the protein immobilization process is to maximize the protein attached onto the support material. The adsorbates (proteins) loading concentration for the reaction with the aminografted porous silica was chosen as the key parameter to be optimized. A protein loading concentration of 40–400 mg/L was used and the immobilization reaction was performed at 25 °C and pH 7. The protein immobilization efficiency increased from 7.22 to 12.7 mg/g, as the protein loading concentration was increased from 46 mg/L to 170 mg/L, while it increases only slightly when the protein loading concentration was further increased from 170 to 400 mg/L (Fig. 2). The maximum immobilization efficiency attained was 12.7 mg protein/g silica when a protein loading concentration of 400 mg/L was used. The results show that a higher protein loading concentration could enhance the efficiency of protein immobilization on the silica matrix. However, when protein loading was higher than 170 mg/L, the immobilization efficiency reached a maximum. This could be due to the insufficiency of protein binding sites on the support material when higher protein loading concentration was used. The trend shown in Fig. 2 seems to
Surface characteristics of the support material that define the binding of the protein biosorbent (e.g., functional groups, structure, etc.) is a major factor affecting immobilization efficiency. Also, in turn this could affect the adsorption preference of the biosorbent for adsorbates based on the adsorbate’s properties. Previous studies suggested that proteins could be immobilized efficiently with surface modification of the porous silica [18]. Hence, to increase the immobilization efficiency, the porous silica matrices with different amino group content was examined to examine their performance as the protein immobilization support [19]. Three silica matrices grafted with amino group were prepared; namely, F14, L11, L41, which contains an amino group content of 20.3, 16.0, and 11.4%, respectively. The different amino group content was achieved by using different mol ratio of SiO2 and NH 2. For instance, F14 was prepared with a SiO2 and NH2 mol ratio of 0.8:1, while L11 and L41 was prepared with a ratio of 1:1 and 4:1, respectively. The protein immobilization efficiency obtained from using SiO 2 only (no amino group) and porous silica with three different amino content (i.e., F14, L11, L41) was assessed. As indicated in Fig. 3, the protein immobilization efficiency can be enhanced by 50% when the amino content was 16–20%, compared to original porous silica (amino content: 0%). In addition, the maximum protein immobilization efficiency was around 13.83 mg/g with 16.0% amine containing porous silica. The protein loss decreased with an increase in the amino content of porous silica, since the efficient binding sites in the form of amino groups increased. As shown in Fig. 3, the protein loss decreased from 20.89 to 4.24%, as amino content increased from 0 to 16%. However, the protein immobilization capacity of the porous silica slightly decreased when the amino content increased from 16.0% to 20.3%, and the protein immobilization capacity of amino-grafted porous silica at 20.3% amino content was also slightly lower (12.69 mg/g) when compared with that obtained from using 16.0% amino content. These results suggest that an excess of amino group on the porous silica could cause more crosslinking leading to a decrease in the amount of protein binding sites.
Fig. 2. The effect of protein loading concentration on protein immobilization efficiency and fallen rate. The amount of silica matrix was 0.2 g. The immobilization temperature and pH was 25 °C and 7, respectively.
Fig. 3. The effect of amino group content of porous silica on protein immobilization efficiency and protein loss percentage. The amino group content of SiO2 only, F14, L11, and L41 was 0, 20.3, 16.0, and 11.4%.
3.1. Effect of protein loading concentration on protein immobilization efficiency
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on continuous Au (III) biosorption was investigated. Continuous biosorption experiments were conducted at a bed depth of 5.5 cm, an influent Au (III) concentration of approximately 40 mg/L, and a fixed flow rate of 0.12 ml/min. When feeding with prepared Au solution at a concentration of 37.5 mg/L, the maximum Au adsorption capacity was nearly 1259 mg Au/g protein, the maximum adsorption rate was 33.2 mg Au/g protein/h and the maximum cumulative Au recovery was 14.9 mg, which was achieved within a feeding time of 64 h (Table 4). In contrast, when the influent was changed to real wastewater (containing an Au concentration of 39.1 mg/L) obtained from an electroplating factory, the maximum adsorption rate (33.4 mg/g/h) was similar to that of using prepared Au solution (33.2 mg/g/h), but the maximum adsorption capacity and the total Au recovery were 448 mg/g and 5.0 mg, respectively, which is 2.8 and 3.0 fold lower, respectively, when compared with those obtained from using the prepared Au solution (Table 4). The much lower Au recovery performance from the real wastewater is somewhat expectable, even though the real wastewater and the prepared Au solution contain a similar Au concentration. This is because the real wastewater contains several other metals (Table 1) that could compete for the adsorption sites on the biosorbent leading to lower adsorption efficiency. In addition, the Au (III) adsorption rate at the time of equilibrium adsorption for the real wastewater and pure Au solution reached around 7.3 mg/g/h (at 17.5 h) and 20.5 mg/g/h (at 38.8 h), respectively. The maximum adsorption rate obtained from the immobilized protein is markedly lower than that obtained from the suspended protein biosorbent (3300 mg/g/h at concentration of 46.38 mg Au/L) (Data not show). The major reason for this could be due to the decrease in the surface area and pore size of silica pellets after sintering treatment at 700 °C (Table 2), leading to poorer diffusion of Au (III) from the solution to silica pellets. After the initial rapid adsorption period, the diffusion of Au (III) becomes the rate limiting step for adsorption. Therefore, the Au (III) adsorption rate of industrial wastewater and pure Au solution were maintained a low level when compared with that of suspended protein biosorbent. Nevertheless, the experimental results obtained from this work still demonstrated the feasibility of using the AT-A2 protein immobilized on the porous silica pellets to recover Au from real industrial wastewater from electroplating factory with satisfactory performance (Table 4), while the mass transfer efficiency of the immobilized protein biosorbents should be much improved to enhance the Au (III) adsorption rate and maximum adsorption capacity of the fixed-bed columns.
Table 2 The mechanical strength and density of silica pellets synthesized at different calcination temperatures. Calcination temperature (oC)
Average hardness (kg)
Density (g/cm3)
600 700 800
9.35 ± 0.14 19.47 ± 0.39 5.05 ± 0.05
0.46 ± 0.01 0.81 ± 0.02 0.57 ± 0.01
Therefore, the optimal amino group content on the silica matrix for the immobilization of the Au-binding protein is 16.0%. 3.3. Effect of particle size and mechanical strength of pelleted silica matrix on protein immobilization efficiency Using the power form silica-immobilized protein described above for Au biosorption has some technical problems, such as difficulty to separate proteins after adsorption, significant proteins loss after regeneration, low mechanical strength, and particle size too small to be used in the fixed bed columns. These problems significantly limit the use of the biosorbents in industrial application for extended operation time [20–22]. To overcome these issues, the silica matrix was pelleted to improve its mechanical strength and increase its particle size. To achieve this, the pretreated starch was added to the amino-grafted silica matrix (with 16% amino content) to synthesize the silica pellets with a height of 2.0 mm and a diameter of 1.5 mm. The effect of calcination temperature on the mechanical strength of silica pellets is shown in Table 2. The average hardness of the silica pellets increased from 9.35 kg at 600 °C to 19.47 kg at 700 °C, and then declined to 5.05 kg at 800 °C. The highest hardness obtained from calcination at 700 °C is 2 fold higher than that obtained from using 600 °C calcination. In the density analysis, a similar performance was seen at 600 to 800 °C, and the highest density (0.81 g/cm3) was obtained at 700 °C. These could be attributed to the sintering phenomenon which could decrease the pore size and increase the density, creating a heavier structural arrangement. However, the porous structure was easily disintegrated at higher temperatures due to sudden structural change or partial melting. As shown in Table 3, the surface area of silica pellet decreased to 235.1 m2/g after sintering at 700 °C, which is 4.5 times lower than that of silica powder. Meanwhile, the pore size of silica pellet also decreased to 2.8 nm. Due to decreased structural surface area and pore size, immobilization efficiency of the silica pellets was comparatively lower than that of silica powder. As shown in Fig. 3, the protein immobilization capacity of silica pellet was 3.5 mg/g, which is 3.9 times lower than silica powder. Apparently, the silica pellet synthesis methods should be much improved in order to avoid structural disintegration but also maintain immobilization efficiency. This could be done by optimizing the components ratio in the synthesis and pelletization of amino-grafted porous silica.
4. Conclusion Amino grafting of porous silica was performed using APTES, which enhanced the immobilization efficiency of Au binding proteins of AT-A2 by 50%. The maximum immobilization efficiency of protein was 13.83 mg/g using the amino-grafted porous silica with 16% amino group and 400 mg/L protein loading. The mechanical strength of the biosorbents increased by 2 fold at a high calcination temperature of 600–700 °C. Continuous biosorption of Au (III) from electroplating wastewater containing 39.1 mg/L of Au was successfully operated with fixed bed columns containing pelletized amino-grafted silica with immobilized Au binding proteins. The maximum Au adsorption capacity and adsorption rate of 448.0 mg Au/g protein and 33.4 mg Au/g protein/h, respectively, were
3.4. Recovery of Au metal from industrial wastewater by continuous biosorption Continuous biosorption of Au (III) was conducted using fixedbed columns packed with Au-binding proteins immobilized on amino-grafted porous silica pellets. The effect of different sources of Au (wastewater from electroplating factory and pure Au solution)
Table 3 Comparison of proteins immobilization efficiency and protein loss ratio with different form of silica carriers. Matrix type
Surface area (m2/g)
Pore size (nm)
Immobilization efficiency (mg/g)
Protein loss percentage (%)
SiO2 powder SiO2 pellet
1065.3 ± 19.2 235.1 ± 1.9
10.2 ± 0.2 2.8 ± 0.1
13.83 ± 0.43 3.48 ± 0.41
4.24 ± 0.25 1.52 ± 0.34
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Table 4 The performance of continuous Au recovery from pure Au solution and industrial wastewater (obtained from an electroplating factor) with ATA2 proteins immobilized on amino-grafted porous silica pellet packed in fixed-bed columns. Type of influent
Au concentration in the influent (mg/L)
Maximum Au adsorption (mgAu/g-protein)
Maximum Au adsorption rate (mg-Au/g-protein/h)
Equilibrium adsorption rate (mg-Au/g-protein/h)
Total cumulative Au recovery (mg)
Prepared Au solution Electroplating Wastewater
37.5 ± 0.1 39.1 ± 0.1
1259.0 ± 13.8 448.0 ± 16.1
33.2 ± 0.7 33.4 ± 1.1
20.5 ± 0.4 7.3 ± 0.2
14.9 ± 0.4 5.0 ± 0.2
obtained at a flow rate of 0.12 ml/min, demonstrating a good performance for the continuous Au recovery from industrial wastewater.
[7] J. Kaduková, E. Virčíková, Comparison of differences between copper bioaccumulation and biosorption, Environ. Int. 31 (2005) 227–232. [8] K.M. Khoo, Y.P. Ting, Biosorption of gold by immobilized fungal biomass, Biochem. Eng. J. 8 (2001) 51–59. [9] N. Hilal, V. Kochkodan, R. Nigmatullin, V. Goncharuk, L. Al-Khatib, Lipase-immobilized biocatalytic membranes for enzymatic esterification: comparison of various approaches to membrane preparation, J. Membr. Sci. 268 (2006) 198–207. [10] F. Veglio, F. Beolchini, Removal of metals by biosorption: a review, Hydrometallurgy 44 (1997) 301–316. [11] E. Steen Redeker, D.T. Ta, D. Cortens, B. Billen, W. Guedens, P. Adriaensens, Protein engineering for directed immobilization, Bioconjug. Chem. 24 (2013) 1761–1777. [12] M. Köhn, Immobilization strategies for small molecule, peptide and protein microarrays, J. Pept. Sci. 15 (2009) 393–397. [13] A.H.A. Al-Dhrub, S. Sahin, I. Ozmen, E. Tunca, M. Bulbul, Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles, Process Biochem. 57 (2017) 95–104. [14] J.R. Deka, D. Saikia, Y.S. Lai, C.H. Tsai, W.C. Chang, H.M. Kao, Roles of nanostructures and carboxylic acid functionalization of ordered cubic mesoporous silica in lysozyme immobilization, Microporous Mesoporous Mater. 213 (2015) 150–160. [15] A.D. Dwivedi, S.P. Dubey, S. Hokkanen, R.N. Fallah, M. Sillanpaa, Recovery of gold from aqueous solutions by taurine modified cellulose: an adsorptive–reduction pathway, Chem. Eng. J. 255 (2014) 97–106. [16] W.M. Chen, H.W. Huang, J.S. Chang, Y.L. Han, T.R. Guo, S.Y. Sheu, Tepidimonas fonticaldi sp. nov., a slightly thermophilic betaproteobacterium isolated from a hot spring, Int. J. Syst. Evol. Microbiol. 63 (2013) 1810–1816. [17] Y.L. Han, Y.C. Lo, C.L. Cheng, W.J. Yu, D. Nagarajan, C.H. Liu, Y.H. Li, J.S. Chang, Calcium ion adsorption with extracellular proteins of thermophilic bacteria isolated from geothermal sites-a feasibility study, Biochem. Eng. J. 117 (2017) 48–56. [18] M. Piao, D. Zou, X. Ren, S. Gao, C. Qin, Y. Piao, High efficiency biotransformation of bisphenol A in a fluidized bed reactor using stabilized laccase in porous silica, Enzyme Microb. Technol. 126 (2019) 1–8. [19] S. Sahin, Stability evaluation of 6-phosphogluconate dehydrogenase immobilized on amino-functionalized magnetic nanoparticles, Prep. Biochem. Biotechnol. 49 (2019) 590–596. [20] R.S. Bai, T.E. Abraham, Studies on chromium(VI) adsorption-desorption using immobilized fungal biomass, Bioresour. Technol. 87 (2003) 17–26. [21] K.M. Khoo, Y.P. Ting, Biosorption of gold by immobilized fungal biomass, Biochem. Eng. J. 8 (2001) 51–59. [22] E. Rosales, J. Meijide, M. Pazos, M.A. Sanroman, Challenges and recent advances in biochar as low-cost biosorbent: from batch assays to continuous-flow systems, Bioresour. Technol. 246 (2017) 176–192.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Acknowledgements This work was supported by a grant from the Industrial Technology Research Institute of Taiwan. References [1] S. Ilyas, R. Chi, H.N. BhattiI, A. Bhatti, M.A. Ghauri, Column bioleaching of lowgrade mining ore containing high level of smithsonite, talc, sphaerocobaltite and azurite, Bioprocess. Syst. Eng. 35 (2012) 433–440. [2] S. llyas, M.S. Kim, J.C. Lee, A. Jabeen, H.N. Bhatti, Bio-reclamation of strategic and energy critical metals from secondary resources, Metals 7 (2017) 207. [3] Y.L. Han, J.H. Wu, C.L. Cheng, D. Nagarajan, C.R. Lee, Y.H. Li, Y.C. Lo, J.S. Chang, Recovery of gold from industrial wastewater by extracellular proteins obtained from a thermophilic bacterium Tepidimonas fonticaldi AT-A2, Bioresour. Technol. 239 (2017) 160–170. [4] R. Sattar, S. Ilyas, H.N. Bhatti, A. Ghaffar, Resource recovery of critically-rare metals by hydrometallurgical recycling of spent lithium ion batteries, Sep. Purif. Technol. 209 (2019) 725–733. [5] S. Syed, Recovery of gold from secondary sources-a review, Hydrometallurgy 115 (2012) 30–51. [6] Y.C. Lo, C.L. Cheng, Y.L. Han, B.Y. Chen, J.S. Chang, Recovery of high-value metals from geothermal sites by biosorption and bioaccumulation, Bioresour. Technol. 160 (2014) 182–190.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Recovery of gold from industrial wastewater by immobilized gold-binding proteins on porous silica carriers grafted with amino group Yin-Lung Hana, Pei-Jyuan Gaoa, Chieh-Lun Chenga, , Pong-Yee Wub, Jo-Shu Changb,c,d, ⁎⁎
T
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a
Green Energy and Environment Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan c Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung 407, Taiwan d Center for Nanotechnology, Tunghai University, Taichung 407, Taiwan b
HIGHLIGHTS
Au-binding proteins were used for Au recovery from industrial wastewater. • Immobilized protein immobilization capacity on the amino-grafted porous silica was optimized. • The silica carrier was pelleted at 700 °C to improve its mechanical strength to 19.47 kg. • The Au biosorption was conducted with a max. capacity of 1259 mg Au/mg protein. • Fixed-bed • Max. Au adsorption rate of the fixed beds was 33.4 mg/g/h with 40 mg Au/L wastewater. ARTICLE INFO
ABSTRACT
Keywords: Biosorption Protein immobilization Porous silica carrier Au-binding protein
Our recent work demonstrated excellent gold biosorption potential of extracellular proteins obtained from a thermophilic bacterium Tepidimonas fonticaldi AT-A2. In this study, the gold-binding proteins were immobilized on amino-grafted porous silica carriers to further enhance the feasibility of using them for Au recovery from real industrial wastewater. The maximum protein immobilization capacity on the amino-grafted porous silica reached the highest level of 13.83 mg protein/g carrier when the protein loading concentration was 400 mg/L and the amino groups content on the silica carrier was 16%. The mechanical strength of the amino-grafted porous silica could be improved to 19.47 kg by converting the silica powder into pellet form using a calcination temperature of 700 °C. Continuous biosorption of Au (III) from prepared Au solution and from real industrial wastewater was carried out using fixed beds packed with the protein-loaded amino-grafted porous silica pellets. The maximum Au adsorption capacity of the fixed beds with a feeding rate of 0.12 ml/min was 1259 and 448 mg Au/mg protein from the prepared Au solution and an electroplating wastewater (Au concentration was approximately 40 mg/L), respectively. The maximum Au adsorption rate of the fixed beds when using the real wastewater was 33.4 mg/g/h, which is similar to the results of using the prepared Au solution.
1. Introduction Precious metals can be found in nature in atmosphere, solid and liquid phases. In particular, precious metals are found in natural ores. The usual way to find and extract them is through exploration and mining from nature. Because of the limited amount of resources of precious metals in nature, researchers are now looking for efficient ways to recover precious metals from other resources, such as electronic and catalytic wastes, solid wastes, liquid wastes and geothermal sites.
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Reclaiming the precious metals of very low concentration from various waste stream solutions often relies on unconventional technologies for metal recovery, such as biosorption and bioaccumulation techniques [1]. Nevertheless, the literature shows that bio-reclamation of such metals is still limited to lab-scale research. Hence, more efforts are needed to get the real benefit of microbial strategy for treating the urban mined secondary materials [2]. Gold, a most important precious metal whose market in intertwined with global economy, has a demand and supply of 35,000 tons per year
Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan. Corresponding author. E-mail addresses: [email protected] (C.-L. Cheng), [email protected] (J.-S. Chang).
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https://doi.org/10.1016/j.bej.2019.107388 Received 14 May 2019; Received in revised form 20 September 2019; Accepted 23 September 2019 Available online 24 September 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
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and the largest source of supply (25,000 tons per year) is still from mining [3]. Because of the limited amount of resources of precious metals in nature, researchers are now looking for alternative ways to procure gold. Gold recovery from secondary sources, such as industrial wastewater, electronic waste and metal scraps is being explored currently to meet the growing demand. Industrial wastes are recognized as a good source for the recovery of precious metals. In Taiwan, the gold is mainly used in the electroplating process of printed circuit boards (PCB) manufacturing factory and the resulting wastewater contains around 5–40 ppm of Au. In order to increase the overall process benefit, recovery of the gold from the process wastewater seems to be necessary [4]. Methods currently in use for gold recovery from waste solutions include solvent extraction, ion exchange, chemical precipitation, cementation, electro-wining, coagulation and pyro-metallurgical processes [5]. Nevertheless, most of the above methods are time consuming and energy intensive, while they also generate a chemical laden sludge to be disposed of. Biosorption is considered a suitable way to recover gold from industrial wastewaters with numerous advantages including high biodegradability, high selectivity, environmental compatibility, high biodegradability, low cost, and simple operating procedures. Biosorbents used for metal biosorption cover a wide range of bio-materials, such as biomass, protein and other renewable materials, while bioaccumulation typically applies live microbial cells for metal uptake and accumulation [6]. Biosorption is a quick process independent of the presence of specific nutrients, while bioaccumulation is slow and nutrient dependent [7]. Our recent work showed that extracellular proteins secreted from a thermophilic bacterium Tepidimonas fonticaldi AT-A2 had an excellent gold binding performance with a high gold binding capacity of 9.7 mg Au/mg protein and a good selectivity against co-existing metals [3]. Thus, the metal-binding proteins seem to be very effective on gold recovery from aqueous waste streams in terms of adsorption capacity per biosorbent weight and the selectivity. However, to assist the use of the protein-based biosorbent in practical cases, the proteins usually should be immobilized on appropriate matrix for easier operation and recovery of proteins after use. Moreover, the immobilization matrix used should be mechanically and chemically strong to withstand actual process conditions [8]. Protein immobilization technique has been widely used in biosensor, biofuel, agro-industry, pharmaceutical, waste treatment and precious metal recovery [9]. Until now, protein immobilization has been carried out by adsorption on inert supports, entrapment in a polymeric matrix, covalently bound to vector compounds, and by cells cross-linking [10]. Chemical binding and physical adsorption are frequently employed to immobilize protein on/in the support material. Chemical methods are associated with the formation of covalent bonding or cross linking to capture the protein on the surface or inside the supporter, while physical methods are related to the ionic adsorption between protein and support. The binding strength of covalent bonding is much higher than that of physical adsorption. This requires the presence of two mutually reactive chemical groups in the protein and support material. Some studies have indicated that the primary amine (eNH3), carboxylic acid (eCOOH), thiol (-SH), hydroxyl (eOH), phenol (eArOH), thioether (R¹-S-R²), imidazole and guanidine groups present in the protein can be used for covalent immobilization [11]. In order to increase the immobilization efficiency, surface modification of the support material is necessary, and the SH- (thiol), NH- (amino) and COOH- (carboxylic) are general functional groups applied for modification of surface properties of the support material. Recently, microarrays used the thiol-ene reaction to immobilize biotin, streptavidin and pTyr-peptide on thiol-modified silicon wafers [12]. Al-Dhrub et al. [13] showed that amino functionalized magnetic nanoparticles (modified with 3-(aminopropyl)triethoxysilane (APTES)) is an effective adsorbent for human carbonic anhydrase I (hCA I), and 61% of the immobilized hCA I activity was retained after repeated use for 13 cycles. Deka et al. [14] also used Cage-type cubic mesoporous silica (FDU-12)
as the adsorbent modified with carboxylic functionalization for lysozyme immobilization, and a maximum lysozyme adsorption capacity of 895 mg/g was obtained. In this study, the gold-binding proteins from T. fonticaldi AT-A2 were immobilized for practical applications in the removal of gold from an electroplating wastewater. Porous silica was chosen as the support material to immobilize the Au-binding protein due to its great mechanical strength and extremely high binding surface area. To further enhance protein immobilization efficiency, amine group was grafted onto the surface of silica. The developed biosorbent was used for the recovery of Au from the industrial wastewater using continuous fixedbed operations to evaluate the feasibility of the protein-based biosorbent. After metal adsorption, the metal recovery from loaded adsorbent via desorption process was also discussed. According to the study by Dwivedi et al. [15], three kinds of desorption agents were chosen in the process, which were sodium hydroxide, hydrogen chloride and thiourea. The best desorption performance was obtained when using 1 M thiourea solution as the desorption agent, resulting in an 87% desorption efficiency of Au metal from the loaded protein biosorbent. Thus, biosorption is currently considered top ranked technology that can be used for the recovery of metals or sequestration of toxic and pollutant metals even under trace metal concentrations. 2. Materials and methods 2.1. Microorganisms, protein and mediums The Au-binding proteins were produced from a thermophilic bacterium, Tepidimonas fonticaldi AT-A2, which isolated from the Antun Hot Spring, Hualien County, Taiwan [3,16,17]. The composition of culture medium consisted of: acetic acid, 3.30 g/L; yeast extract, 1.00 g/L; KH2PO4, 0.53 g/L; (NH4)2SO4, 1.00 g/L; MgCl2, 0.10 g/L; trace element solution, 1 ml/L. The composition of trace element solution is as follows: ZnSO4·7H2O, 0.10 g/L; MnCl2·4H2O, 0.03 g/L; H3BO3, 0.30 g/L; CoCl2·6H2O, 0.20 g/L; CuCl2·2H2O, 0.01 g/L; NiCl2·6H2O, 0.02 g/L; Na2MoO4·2H2O, 0.03 g/L. Tepidimonas fonticaldi AT-A2 was cultured at 50 °C, 200 rpm for 48 h and the extracellular proteins of AT-A2 were immobilized on the porous silica grafted with amino group as described in the following section. The extracellular proteins contained multiple proteins. Analysis of the SDS PAGE gel with a small molecular weight (MW) marker indicates the existence of proteins smaller than 10 kDa, and there are two strong bands corresponding to a MW of 4 and 8 kDa. The Au-binding capacity of 3–10 kDa proteins (1.50 mg Au/mg protein) appeared to be nearly 12.5-fold higher than the proteins > 10 kDa (0.12 mg Au/mg protein) [3]. The amino acid composition of the acid hydrolyzed samples is different for the > 10 kDa protein fraction and the 3–10 kDa protein fraction. The presence of Cys and His could be a possible reason for the better Au adsorption efficiency of the proteins with the size of 3–10 kDa [3]. 2.2. Characterization of materials- amino-grafted porous silica The octenyl succinic anhydride (OSA-pretreated starch (0.36 g) was added into the CTAB solution (cetyltrimethylammonium bromide, 7.3 g of CTAB in 100 ml of 35% ethanol). Then, 22 mL of TEOS (Tetraethyl orthosilicate) and 1.5 mL of sulfuric acid were then added into the mixture, mixed well and incubated for 6 h for hydrolysis. After hydrolysis, the mixture was titrated with 12.5% ammonium hydroxide solution at 25 °C for 2 h. The precipitated particles were filtered and washed with water several times to completely remove any un-adsorbed components and dried at 50 °C for 24 h. The particles were calcined at 550 °C for 2 h to obtain porous silica with a particle size of 20–50 μm. Porous silica with an amount of 1.0 g was added into 20 mL deionized water and the pH was adjusted to 10 by 12.5% ammonium hydroxide solution. A 3.6 g of the APTES ((3-Aminopropyl) 2
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triethoxysilane) was added to the mixture with vigorous mixing and the reaction was carried out at 100 °C and 600 rpm for 3.0 h. The aminografted porous silica thus obtained was filtered and washed with water several times to completely remove un-adsorbed components and dried at 50 °C for 24 h.
Table 1 Major element composition of the wastewater obtained from an electroplating factory.
2.3. Preparation of protein-based biosorbent (protein immobilization) Amino-grafted porous silica (0.2 g) was added into 20 ml prepared protein with different protein loadings of 40–400 mg/L from AT-A2 in 50 ml serum bottle. The immobilization was conducted at a stable temperature of 25 °C, stirring rate of 100 rpm, pH of 7, and adsorption time of 6 h. Residual Au binding activity and adsorbed protein concentration on amino-grafted porous silica was regularly monitored at designated time intervals until immobilization reached equilibrium state. Then the immobilized Au-binding proteins were used for further experiments.
Component elements
Concentration (mg/L)
Au Al Ca Fe K Mg Na Ni P S Si Zn
39.1 2.4 7.4 0.1 133.9 0.7 11.2 0.7 2.6 2.4 2.3 0.2
2.6. Measurement of Au concentration
2.4. Fixed-bed column biosorption by immobilized protein
The 1000 mg/L Au(III) standard solution of was obtained from High Purity Standard (HPS) Inc., USA. Working standards were prepared by progressive dilution of standard Au(III) solutions using deionized water. Au content in solutions was measured by Inductively Coupled Plasma (ICP), which detected Au intensity at an emission wavelength of 267.595 nm (Jobin-Yvon ICP Optical Emission Spectrometer JY 2000-2, Horiba Scientific. Inc.). Prior to ICP measurement, the precious metal solutions were appropriately treated with HNO3 to ensure that the metal concentration in the sample was linearly dependent on the detected absorbance. In other words, the pH value of sample solution was below 2.0. The Au adsorption capacity was calculated as follows.
The immobilized protein-based biosorbent (totally 4.0 g of silica pellets, which is 2.5 mm in diameter) was packed into three columns (Height = 5.5 cm, Inner diameter = 1.2 cm) for continuous biosorption of Au (III) (Fig. 1). The influent tank contains pure Au (III) solution at a concentration of 37.5 mg/L prepared with HAuCl 4·3H 2O or industrial wastewater from an electroplating factory containing 39.1 mg/L Au(III). Composition of the industrial wastewater used is indicated in Table 1. The influent tank was mixed by a magnetic stirrer at a stirring rate of 100 rpm. The influent was passed through the fixed-bed columns by a peristaltic pump with a volumetric flow rate of 0.12 ml/min.
Au adsorption capacity: [(Au conc. (adsorption before) - Au conc. * V(operation volume)]/ W(weight of Protein)
before))
2.5. Measurement of protein concentration The protein concentration in the solution was determined by Bradford assay using commercialized Bio-Red Protein Assay Dye Reagent. Briefly, 100 μL reagent was mixed with 150 μL sample and incubated in 96 well plates at 25 °C for about 20 min. At the end of incubation, absorbance of 595 nm (OD595) of the mixture was measured by ELISA (BioTek). Bovine Serum Albumin (BSA, Sigma) was used as protein standard. The calculation of protein loss percentage and protein immobilization efficiency are performed as follows.
(adsorption
2.7. Estimation of amino group content of the immobilized carrier The amino acids present on the support was determined by thermogravimetric analysis, assuming that the weight loss between 400 °C and 600 °C was associated with the desorption of the amino acids adsorbed on the support surface. The thermogravimetric curves were recovered in N2 flow on a TGA 4000 (PerkinElmer) instrument with a heating rate of 20 °C/min from 50 °C to 880 °C.
Protein loss percentage: [Protein conc. (wash solution) * V(wash volume)] /[(Protein conc. (immobilization before) - Protein conc. (immobilization before)) * V(operation volume)] *100
2.8. Surface area analysis Brunauer Emmett Teller (BET) instrument was used to measure the surface area, pore size, pore volume, and pore diameter of the support material and catalysts. The BET machine used in this study is ASAP 2020 Micromeritics. The samples were degassed at 200 °C for 2 h.
Protein immobilization efficiency: [(Protein conc. (immobilization before) Protein conc. (immobilization before)) * V(operation volume)]/ W(weight of carrier)
Fig. 1. An illustration for the setup of continuous biosorption fixed-bed columns. a total amount of 4 g of Silica immobilized proteins was evenly packed in the three columns. The influent tank contains 40 mg/L of Au (III) solution prepared with HAuCl4·3H2O or industrial wastewater containing 39.1 mg/L of Au(III). 3
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Barrett-Joyner-Halenda (BJH) method was used to determine the pore volume and the pore size.
match the Langmuir Isotherm type model (Eq. 1) with a qmax and K d value of 15.6 mg protein/g silica and 46.1 mg/L, respectively. Note that the protein immobilization capacity (mg/g) is calculated with qmax C , where qmax : maximum protein immobilization efficiency (mg Kd + C protein/g silica); K d : half saturation constant (mg/L)
2.9. Hardness analysis The hardness of silica pellets was measured by the tablets hardness tester (TH-C). The sample was prepared as a 1.0 cm × 0.8 cm × 1.2 cm cube on the testing platform (1.0 cm × 1.0 cm) to analyze the hardness.
3.2. Effect of amino group content on silica matrix on protein immobilization efficiency
3. Results and discussion
Our previous work showed the detailed Au-binding properties of the extracellular proteins obtained from the AT-A2 strain. Under the optimal adsorption conditions of 50 °C and pH 5, the maximal Au adsorption capacity of the AT-A2 proteins was 9.7 mg Au/mg protein [3]. When further using the AT-A2 proteins for Au removal from industrial wastewater containing 15 mg/L Au, a maximum adsorption capacity of 1.45 mg Au/mg protein was obtained with a removal efficiency of 71% [3]. For practical applications of the protein biosorbents, an enhanced protein biosorbent concentration is vital. Therefore, in this study, the AT-A2 proteins were immobilized with amino-grafted porous silica (prepared as mentioned in Section 2.2) support to make them more applicable in real cases. The first step in the protein immobilization process is to maximize the protein attached onto the support material. The adsorbates (proteins) loading concentration for the reaction with the aminografted porous silica was chosen as the key parameter to be optimized. A protein loading concentration of 40–400 mg/L was used and the immobilization reaction was performed at 25 °C and pH 7. The protein immobilization efficiency increased from 7.22 to 12.7 mg/g, as the protein loading concentration was increased from 46 mg/L to 170 mg/L, while it increases only slightly when the protein loading concentration was further increased from 170 to 400 mg/L (Fig. 2). The maximum immobilization efficiency attained was 12.7 mg protein/g silica when a protein loading concentration of 400 mg/L was used. The results show that a higher protein loading concentration could enhance the efficiency of protein immobilization on the silica matrix. However, when protein loading was higher than 170 mg/L, the immobilization efficiency reached a maximum. This could be due to the insufficiency of protein binding sites on the support material when higher protein loading concentration was used. The trend shown in Fig. 2 seems to
Surface characteristics of the support material that define the binding of the protein biosorbent (e.g., functional groups, structure, etc.) is a major factor affecting immobilization efficiency. Also, in turn this could affect the adsorption preference of the biosorbent for adsorbates based on the adsorbate’s properties. Previous studies suggested that proteins could be immobilized efficiently with surface modification of the porous silica [18]. Hence, to increase the immobilization efficiency, the porous silica matrices with different amino group content was examined to examine their performance as the protein immobilization support [19]. Three silica matrices grafted with amino group were prepared; namely, F14, L11, L41, which contains an amino group content of 20.3, 16.0, and 11.4%, respectively. The different amino group content was achieved by using different mol ratio of SiO2 and NH 2. For instance, F14 was prepared with a SiO2 and NH2 mol ratio of 0.8:1, while L11 and L41 was prepared with a ratio of 1:1 and 4:1, respectively. The protein immobilization efficiency obtained from using SiO 2 only (no amino group) and porous silica with three different amino content (i.e., F14, L11, L41) was assessed. As indicated in Fig. 3, the protein immobilization efficiency can be enhanced by 50% when the amino content was 16–20%, compared to original porous silica (amino content: 0%). In addition, the maximum protein immobilization efficiency was around 13.83 mg/g with 16.0% amine containing porous silica. The protein loss decreased with an increase in the amino content of porous silica, since the efficient binding sites in the form of amino groups increased. As shown in Fig. 3, the protein loss decreased from 20.89 to 4.24%, as amino content increased from 0 to 16%. However, the protein immobilization capacity of the porous silica slightly decreased when the amino content increased from 16.0% to 20.3%, and the protein immobilization capacity of amino-grafted porous silica at 20.3% amino content was also slightly lower (12.69 mg/g) when compared with that obtained from using 16.0% amino content. These results suggest that an excess of amino group on the porous silica could cause more crosslinking leading to a decrease in the amount of protein binding sites.
Fig. 2. The effect of protein loading concentration on protein immobilization efficiency and fallen rate. The amount of silica matrix was 0.2 g. The immobilization temperature and pH was 25 °C and 7, respectively.
Fig. 3. The effect of amino group content of porous silica on protein immobilization efficiency and protein loss percentage. The amino group content of SiO2 only, F14, L11, and L41 was 0, 20.3, 16.0, and 11.4%.
3.1. Effect of protein loading concentration on protein immobilization efficiency
4
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on continuous Au (III) biosorption was investigated. Continuous biosorption experiments were conducted at a bed depth of 5.5 cm, an influent Au (III) concentration of approximately 40 mg/L, and a fixed flow rate of 0.12 ml/min. When feeding with prepared Au solution at a concentration of 37.5 mg/L, the maximum Au adsorption capacity was nearly 1259 mg Au/g protein, the maximum adsorption rate was 33.2 mg Au/g protein/h and the maximum cumulative Au recovery was 14.9 mg, which was achieved within a feeding time of 64 h (Table 4). In contrast, when the influent was changed to real wastewater (containing an Au concentration of 39.1 mg/L) obtained from an electroplating factory, the maximum adsorption rate (33.4 mg/g/h) was similar to that of using prepared Au solution (33.2 mg/g/h), but the maximum adsorption capacity and the total Au recovery were 448 mg/g and 5.0 mg, respectively, which is 2.8 and 3.0 fold lower, respectively, when compared with those obtained from using the prepared Au solution (Table 4). The much lower Au recovery performance from the real wastewater is somewhat expectable, even though the real wastewater and the prepared Au solution contain a similar Au concentration. This is because the real wastewater contains several other metals (Table 1) that could compete for the adsorption sites on the biosorbent leading to lower adsorption efficiency. In addition, the Au (III) adsorption rate at the time of equilibrium adsorption for the real wastewater and pure Au solution reached around 7.3 mg/g/h (at 17.5 h) and 20.5 mg/g/h (at 38.8 h), respectively. The maximum adsorption rate obtained from the immobilized protein is markedly lower than that obtained from the suspended protein biosorbent (3300 mg/g/h at concentration of 46.38 mg Au/L) (Data not show). The major reason for this could be due to the decrease in the surface area and pore size of silica pellets after sintering treatment at 700 °C (Table 2), leading to poorer diffusion of Au (III) from the solution to silica pellets. After the initial rapid adsorption period, the diffusion of Au (III) becomes the rate limiting step for adsorption. Therefore, the Au (III) adsorption rate of industrial wastewater and pure Au solution were maintained a low level when compared with that of suspended protein biosorbent. Nevertheless, the experimental results obtained from this work still demonstrated the feasibility of using the AT-A2 protein immobilized on the porous silica pellets to recover Au from real industrial wastewater from electroplating factory with satisfactory performance (Table 4), while the mass transfer efficiency of the immobilized protein biosorbents should be much improved to enhance the Au (III) adsorption rate and maximum adsorption capacity of the fixed-bed columns.
Table 2 The mechanical strength and density of silica pellets synthesized at different calcination temperatures. Calcination temperature (oC)
Average hardness (kg)
Density (g/cm3)
600 700 800
9.35 ± 0.14 19.47 ± 0.39 5.05 ± 0.05
0.46 ± 0.01 0.81 ± 0.02 0.57 ± 0.01
Therefore, the optimal amino group content on the silica matrix for the immobilization of the Au-binding protein is 16.0%. 3.3. Effect of particle size and mechanical strength of pelleted silica matrix on protein immobilization efficiency Using the power form silica-immobilized protein described above for Au biosorption has some technical problems, such as difficulty to separate proteins after adsorption, significant proteins loss after regeneration, low mechanical strength, and particle size too small to be used in the fixed bed columns. These problems significantly limit the use of the biosorbents in industrial application for extended operation time [20–22]. To overcome these issues, the silica matrix was pelleted to improve its mechanical strength and increase its particle size. To achieve this, the pretreated starch was added to the amino-grafted silica matrix (with 16% amino content) to synthesize the silica pellets with a height of 2.0 mm and a diameter of 1.5 mm. The effect of calcination temperature on the mechanical strength of silica pellets is shown in Table 2. The average hardness of the silica pellets increased from 9.35 kg at 600 °C to 19.47 kg at 700 °C, and then declined to 5.05 kg at 800 °C. The highest hardness obtained from calcination at 700 °C is 2 fold higher than that obtained from using 600 °C calcination. In the density analysis, a similar performance was seen at 600 to 800 °C, and the highest density (0.81 g/cm3) was obtained at 700 °C. These could be attributed to the sintering phenomenon which could decrease the pore size and increase the density, creating a heavier structural arrangement. However, the porous structure was easily disintegrated at higher temperatures due to sudden structural change or partial melting. As shown in Table 3, the surface area of silica pellet decreased to 235.1 m2/g after sintering at 700 °C, which is 4.5 times lower than that of silica powder. Meanwhile, the pore size of silica pellet also decreased to 2.8 nm. Due to decreased structural surface area and pore size, immobilization efficiency of the silica pellets was comparatively lower than that of silica powder. As shown in Fig. 3, the protein immobilization capacity of silica pellet was 3.5 mg/g, which is 3.9 times lower than silica powder. Apparently, the silica pellet synthesis methods should be much improved in order to avoid structural disintegration but also maintain immobilization efficiency. This could be done by optimizing the components ratio in the synthesis and pelletization of amino-grafted porous silica.
4. Conclusion Amino grafting of porous silica was performed using APTES, which enhanced the immobilization efficiency of Au binding proteins of AT-A2 by 50%. The maximum immobilization efficiency of protein was 13.83 mg/g using the amino-grafted porous silica with 16% amino group and 400 mg/L protein loading. The mechanical strength of the biosorbents increased by 2 fold at a high calcination temperature of 600–700 °C. Continuous biosorption of Au (III) from electroplating wastewater containing 39.1 mg/L of Au was successfully operated with fixed bed columns containing pelletized amino-grafted silica with immobilized Au binding proteins. The maximum Au adsorption capacity and adsorption rate of 448.0 mg Au/g protein and 33.4 mg Au/g protein/h, respectively, were
3.4. Recovery of Au metal from industrial wastewater by continuous biosorption Continuous biosorption of Au (III) was conducted using fixedbed columns packed with Au-binding proteins immobilized on amino-grafted porous silica pellets. The effect of different sources of Au (wastewater from electroplating factory and pure Au solution)
Table 3 Comparison of proteins immobilization efficiency and protein loss ratio with different form of silica carriers. Matrix type
Surface area (m2/g)
Pore size (nm)
Immobilization efficiency (mg/g)
Protein loss percentage (%)
SiO2 powder SiO2 pellet
1065.3 ± 19.2 235.1 ± 1.9
10.2 ± 0.2 2.8 ± 0.1
13.83 ± 0.43 3.48 ± 0.41
4.24 ± 0.25 1.52 ± 0.34
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Biochemical Engineering Journal 152 (2019) 107388
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Table 4 The performance of continuous Au recovery from pure Au solution and industrial wastewater (obtained from an electroplating factor) with ATA2 proteins immobilized on amino-grafted porous silica pellet packed in fixed-bed columns. Type of influent
Au concentration in the influent (mg/L)
Maximum Au adsorption (mgAu/g-protein)
Maximum Au adsorption rate (mg-Au/g-protein/h)
Equilibrium adsorption rate (mg-Au/g-protein/h)
Total cumulative Au recovery (mg)
Prepared Au solution Electroplating Wastewater
37.5 ± 0.1 39.1 ± 0.1
1259.0 ± 13.8 448.0 ± 16.1
33.2 ± 0.7 33.4 ± 1.1
20.5 ± 0.4 7.3 ± 0.2
14.9 ± 0.4 5.0 ± 0.2
obtained at a flow rate of 0.12 ml/min, demonstrating a good performance for the continuous Au recovery from industrial wastewater.
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