Microorganism-Templated Nanoarchitectonics of Hollow TiO2-SiO2 Microspheres with Enhanced Photocatalytic Activity for Degradation of Methyl Orange

In this study, hollow SiO2 microspheres were synthesized by the hydrolysis of tetraethyl orthosilicate (TEOS) according to the Stober process, in which Pichia pastoris GS 115 cells were served as biological templates. The influence of the preprocessing method, the TEOS concentration, the ratio of water to ethanol, and the aging time on the morphology of microspheres was investigated and the optimal conditions were identified. Based on this, TiO2-SiO2 microspheres were prepared by the hydrothermal process. The structures and physicochemical properties of TiO2-SiO2 photocatalysts were systematically characterized and discussed. The photocatalytic activity for the degradation of methyl orange (MO) at room temperature under Xe arc lamp acting as simulated sunlight was explored. The result showed that the as-prepared TiO2-SiO2 microspheres exhibited a good photocatalytic performance.


Introduction
The rapid development of the textile industry not only brings considerable economic benefits, but also aggravates environmental pollution. Due to the complex composition of textile wastewater and its high content of organic substances, harmful substances, and deep chroma, which cause serious harm to water bodies, the treatment of textile wastewater is imperative [1,2]. Water-soluble azo dyes including MO are the main targets of pollution control. Many treatment methods of dye removal have been investigated, including adsorption [3,4], photo-Fenton oxidation [5][6][7], H 2 O 2 /UV (ultraviolet) treatment [8,9], photocatalysis [10,11], and biological treatment [12][13][14]. Among these methods, photocatalysis is considered an effective method to degrade dyes in wastewater [15]. TiO 2 is considered to be one of the most promising photocatalysts for the removal of organic pollutants in textile wastewater due to its low cost, strong oxidizing properties, non-toxicity, and biochemical inertness [16][17][18]. However, there are some imperfections, such as its large energy gap, high photoelectric hole recombination rate, etc. [19][20][21][22]. The small nanoparticles have a high surface energy and readily form agglomerates, and their wide band gap (3.2 eV) makes them inactive under visible light irradiation. Moreover, the application in high temperature and high-pressure reactions is limited due to the poor mechanical strength and thermal stability of TiO 2 . Therefore, researchers are looking for more effective methods with which to improve the surface-active sites of TiO 2 , the photoelectron-hole separation rate, the solar energy usage efficiency, and the spectral response to enhance its photocatalytic performance [23]. One of the most promising methods to enhance the activity of TiO 2 under visible light and sunlight irradiation is to dope non-metals such as N, C, and Si. Due to the difference in properties between Si and Ti, mesoporous SiO 2 is more stable than mesoporous TiO 2 . Mesoporous TiO 2 can easily cause the collapse of the mesoporous structure when the template is removed by calcination at high temperatures, while silicon-based materials have a good thermal stability. Taking advantage of the good thermal stability of silicon-based materials, titanium-silicon composites could be prepared by loading titanium onto mesoporous silicon-based materials, which can effectively solve the pore structure instability of mesoporous TiO 2 [20].
The methods used for combining TiO 2 and SiO 2 can be roughly divided into two categories. One involves the mechanical mixture of TiO 2 and SiO 2 . The other involves the use of chemical methods such as co-precipitation and sol-gel, where the composite oxide TiO 2 -SiO 2 with Ti-O-Si bonds is obtained [24][25][26]. It is generally believed that TiO 2 -SiO 2 oxides with Ti-O-Si bonds perform better as catalyst supports than mechanically mixed TiO 2 -SiO 2 [27]. Among various composite oxides, TiO 2 -SiO 2 composite oxides, especially in the mesoporous structure, exhibit a good chemical stability, availability, reusability, and controllability of pore structure [28]. Compared with other chemical methods, hydrothermal methods are often applied for the preparation of metal oxide materials because of their characteristics of a fast reaction speed, an adjustable structure, and crystallinity. In this study, microorganism cells-templated TiO 2 -SiO 2 hollow microspheres were synthesized by the combination of the Stober process and the hydrothermal process and the photocatalytic activity for the degradation of methyl orange was investigated (see Figure A1).

Preparation of Hollow SiO 2 Microspheres
Hollow SiO 2 microspheres were synthesized using microorganism cells as templates according to the Stober process. In a typical experiment, 0.2 g of P. pastoris GS115 cells were suspended in 4.2 mL of ultrapure water and 10.5 mL of absolute ethanol (the ratio of water-ethanol was 1/2). The mixture was placed on a magnetic stirrer to stir at 25 • C for 1 h. Then, 6.4 mL of TEOS (1.2 mol/L, if not specified) was added and allowed to react for 2 h, followed by 2.85 mL of ammonia for another 1 h. The suspension was agitated at 25 • C for 12 h (if not specified). The product was separated by centrifugation (3500 r/min, 10 mins) and washed with ethanol and water several times. The precipitate was dried and calcinated at 550 • C for 2 h with a heating rate of 2 • C/min.

Preparation of TiO 2 -SiO 2
A total of 2 g of the as-synthesized hollow SiO 2 microspheres was dispersed in 25 mL of anhydrous ethanol and 0.25 mL of TBOT. The solution was labeled as solution A. Then, 0.2 mL of nitric acid was added into the mixture of anhydrous ethanol (25 mL) and ultrapure water (10 mL). The solution was labeled as solution B. Solution A was continuously stirred at 25 • C for 5 min, and then solution B was added drop by drop and stirred for another 2 h. The mixture was transferred to a reaction kettle and heated at 180 • C for 24 h. After the hydrothermal reaction was completed, the samples were separated by centrifugation (3500 r/min, 10 min) and washed alternately with water and ethanol several times. The obtained samples were dried in an oven at 80 • C for 10 h and calcinated at 550 • C for 1 h with a heating rate of 2 • C/min.

Determination of Photocatalytic Performance
The photocatalytic experiments were carried out in a glass vessel by a 300 W Xe arc lamp acting as simulated sunlight. The initial concentration of the methyl orange was 10 mg/L. A total of 80 mg of the photocatalyst was taken in 80 mL of MO solution. Illumination was implemented after dark treatment for 1 h to reach adsorption-desorption equilibrium. At specific time intervals (every 20 min), 4 mL of the sample was taken from the suspensions and centrifuged to remove the catalyst prior to spectral measurement.

Characterization Methods
The crystal structure of the samples was determined by an X-ray diffractometer (XRD, X'Pert Pro MPD, Panalytical, The Netherlands) operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation. The observations of morphology and microstructure were performed on a scanning electron microscope (SEM, ZEISS Sigma, Oberkochen, Germany) and a transmission electron microscope (TEM, Philips Tecnai F30, Eindhoven, The Netherlands) operated at an accelerating voltage of 300 kV. The specific surface area, pore volume, and pore size distribution of the samples were determined by Tristar-type low-temperature N 2 physical adsorption and desorption (BET, NOVA2200e, Quantachrome, Boynton Beach, FL, USA). The thermogravimetric (TG) studies were carried out on Netzsch TG209F1 thermobalance (NETZSCH Scientific Instruments Co., Selb, Germany) under a flowing-air atmosphere at a heating rate of 10 • C/min. X-ray Photoelectron Spectroscopy (XPS) measurements were performed on a PHI 5000 versa probe-II microprobe (Ulvac-Phi, Kanagawa, Japan). The UV-DRS analysis was performed on a UV-VIS Cary 5000 instrument (Varian, Palo Alto, CA, USA). BaSO 4 was served as a reference. The Fourier Transform Infrared Spectroscopy (FTIR) analysis was carried out on a Nicolet 6700 instrument (Thermo Fisher Scientific, Waltham, MA, USA).

Preparation of Hollow SiO 2 Microspheres
In recent years, inorganic hollow micro/nanostructures have attracted extensive attention due to their unique morphologies, unique physicochemical properties, and potential applications in dyes, drug delivery, and efficient catalysis [29,30]. The template method is one of the most commonly used methods for the synthesis of hollow nanomaterials [31]. The application of microorganisms as a template is considered to be an economical and green method [32][33][34][35]. Herein, the hollow SiO 2 was prepared by using the P. pastoris GS115 cells as a template and the influence of different reaction conditions on the structure were investigated.

Effect of Preprocessing Methods
Templates are vital for the preparation of hollow material. As shown in Figure 1a, solid SiO 2 microspheres with a particle size of 200-300 nm were obtained when no template was introduced. When P. pastoris GS 115 cells with the size of 1-2 µm were introduced, the obtained microspheres had successfully replicated the template structure (Figure 1b-d). The solvent has an obvious influence on the morphology. When P. pastoris GS 115 cells were suspended in ethanol or a hybrid system of ethanol and ammonia, there were many nano SiO 2 particles on the surface of hollow SiO 2 , making the surface more rough and still agglomerate, which was shown in Figure 1b,c. While hollow, SiO 2 with a smooth surface and a good dispersion could be prepared if P. pastoris GS115 cells were firstly suspended in a water-ethanol mixture (the ratio of water-ethanol is 1/2, Figure 1d). (c) P. pastoris GS 115 were suspended in a hybrid system of ethanol and ammonia and followed by TEOS and ammonia; (d) P. pastoris GS 115 were suspended in an ethanol-water mixture and followed by TEOS and ammonia.

Effect of TEOS Concentration
The concentration of TEOS has an effect on the morphology of microspheres. When the TEOS concentration was low, small SiO 2 particles agglomerated and the concentration was not enough to form the complete hollow structure, as shown in Figure 2a. As the TEOS concentration gradually increased, more complete microspheres with hollow structure could successfully be prepared (Figure 2b-d). As the concentration of TEOS continued to increase, the excess SiO 2 particles continued to grow on the surface of the hollow SiO 2 microspheres due to the limited amount of templates. The surface of the prepared microspheres was relatively rough and agglomerated together to form larger clusters (Figure 2e). Therefore, to prepare hollow SiO 2 microspheres with a smooth surface and good dispersibility, the optimal TEOS concentration is between 1.0 and 1.2 mol/L.

Effect of the Ratio of Water to Ethanol
As shown in Figure 3, when the ratio of water to ethanol was low, the generated SiO 2 particles would continue to grow on the surface of hollow SiO 2 microspheres, which made the surface of the prepared microspheres relatively rough and caused the hollow microspheres to have a certain degree of agglomeration (Figure 3a). With the increase in water/ethanol, the surface of the microspheres became smoother (Figure 3b-d).

Effect of Aging Time
The aging time also has an obvious effect on the surface of microspheres. As shown in Figure 4a, when the aging time was 6 h, the hollow SiO 2 microsphere structure was irregular and the dispersion was poor. With the aging time extended to 12 h, a complete hollow SiO 2 microsphere structure was formed with an even dispersion (Figure 4c). Complete hollow microsphere structures could be formed by extending the aging time (Figure 4d,e). The TG and FTIR characterizations were performed and the results were shown in Figures A2 and A3. The results confirm the formation of SiO 2 and show that there may be some residual biomass on the microsphere.

Preparation of Hollow TiO 2 -SiO 2
Based on the above, TiO 2 was coated on the surface of hollow SiO 2 microspheres to prepare TiO 2 -SiO 2 by the hydrothermal method.
XRD patterns of SiO 2 and TiO 2 -SiO 2 were displayed in Figure 5. The hollow SiO 2 microsphere was amorphous (curve a in Figure 5). The main peaks at 25.  [36]. Generally, the anatase TiO 2 will change to rutile TiO 2 after high-temperature roasting. However, no rutile formation was found in this sample because of the spatial grid effect after silicon addition, which improved the structural thermal stability of mesoporous TiO 2 and inhibited the transformation of anatase to rutile [37]. To observe the microscopic morphology and internal structure of the prepared TiO 2 -SiO 2 , TEM characterization was carried out and the results were shown in Figure 6. In Figure 6a, a layer of material was successfully coated on the surface of SiO 2 . The spacing of the lattice plane in Figure 6b was 0.35 nm, which was consistent with the d value of the (101) plane of the anatase TiO 2 , confirming that the surfaces of the SiO 2 were successfully coated by TiO 2 . In order to confirm the elemental composition and distribution of the TiO 2 -SiO 2 catalyst, Si, O, and Ti elements were selected for an EDX surface scan. As shown in Figure 7, the distribution ranges of the O, Si, and Ti elements are consistent with the positions occupied by SiO 2 and the distribution is very uniform. This reflects not only the O and Si element properties of SiO 2 , but also the fact that the titanium layer is successfully coated on the surface of the hollow SiO 2 microspheres. The full XPS spectra of SiO 2 and TiO 2 -SiO 2 were shown in Figure 8a and several peaks corresponding to Si, C, Ti, and O elements could be observed. Figure 8b showed the XPS spectrum of O1s of the sample SiO 2 . The characteristic peak appeared at 532.3 eV and could be assigned to the binding energy of O 1s in Si-O-Si. The peak centered at around 103.3 eV in Figure 8c confirmed the presence of the Si element in SiO 2 . A Ti 2p XPS spectrum of TiO 2 -SiO 2 in Figure 8d was fitted into three peaks. The peaks located at 458.1 eV and 463.4 eV were assigned to Ti 2p3/2 and Ti 2p1/2 of TiO 2 , respectively. A minor peak at 455.46 eV might be attributed to the low valence states of Ti [38]. The coating of TiO 2 exerted a great influence on the O 1s XPS spectrum (Figure 8e), which could be split into three peaks. The peak located at 529.6 eV and 532.7 eV were related to Ti-O-Ti and Si-O-Si. The peak at 532.0 eV could be assigned to the binding energy of the Si-O-Ti species, indicating the bonding of TiO 2 to SiO 2 [39]. Figure 8f showed the XPS spectrum of Si 2p of the sample TiO 2 -SiO 2 . There were two characteristic peaks of Si 2p located at 100.6 eV and 103.3 eV, showing that the coating of TiO 2 had an effect on the binding energy of the Si element. The specific surface area (S BET ), pore volume (V P ), and pore diameter (D P ) of different samples are summarized in Table 1. Compared with SiO 2 without a template, the specific surface area, pore volume, and pore diameter of SiO 2 produced by a yeast template increased by different degrees, and the increase in the specific surface area from 10.95 m 2 g −1 to 15.97 m 2 g −1 was mainly due to the successful formation of a hollow microsphere structure. When the hollow SiO 2 microspheres were coated with titanium, the specific surface area, pore volume, and pore diameter increased, which may be beneficial by providing more active sites and increasing the catalytic activity of the catalyst.  Figure 9a shows the N 2 adsorption-desorption isothermal curve and Barret Joyner Halenda (BJH) pore diameter distribution of SiO 2 prepared by the P. pastoris GS115 template. The N 2 adsorption-desorption isothermal curve belongs to the Langmuir-type IV mesoporous channel adsorption curve. At P/Po = 0.8-1.0, small hysteresis rings appears, which maybe have been caused by slight changes in the pore diameter of SiO 2 and the phenomenon of different pore sizes, and it could also have been caused by a small number of interstices between particles. The BJH pore diameter distribution diagram of SiO 2 inserted in Figure 9a shows that the mesopore diameter is in the range of 5 to 18 nm.  Figure 9b shows the N 2 adsorption-desorption isothermal curve and the BJH pore diameter distribution of TiO 2 -SiO 2 . At the low-pressure stage (P/Po < 0.8), there is a certain linear relationship between the adsorption amount and partial pressure, which may occur in a single layer of physical adsorption. When the partial pressure P/Po is approximately 0.8, the adsorption amount increases sharply and the adsorption enters the abrupt phase. The reason is that N 2 condenses the capillary in the mesoporous channel. When the partial pressure P/Po continues to increase, another abrupt jump occurs and a hysteresis ring appears under high partial pressure. At this time, N 2 condenses between material particles. It can be seen from the BJH pore diameter distribution curve inserted in Figure 9b that the sample pore diameter is mainly distributed between 5 and 20 nm. There are also concentrated holes, possibly caused by gaps between spherical particles of varying sizes.

Photocatalytic Activity
It could be seen from the Figure 10a that the absorbance of the solution was basically unchanged when the catalyst was not added. After the catalyst was introduced, the absorbance of MO gradually decreased with the extension of time. Moreover, the hollow TiO 2 microspheres were also prepared using P. pastoris GS115 as a template (see Figure A4). Comparing Figure 10b,c, it could be seen that the photocatalytic degradation ability of TiO 2 -SiO 2 was higher than that of pure TiO 2 prepared with P. pastoris GS115 as a template. The probable cause was that the introduction of SiO 2 to TiO 2 could reduce its surface energy to a certain extent and reduce its agglomeration, forming active hydroxyl radicals, and thereby enhancing the photocatalytic ability of TiO 2 [40]. Jiang et al. [39] reported the preparation of hierarchical hollow TiO 2 @SiO 2 composite microspherse and studied their photocatalytic performance on MO. The degradation rate was 99.7% after 3 h. Zhang et al. [41] prepared TiO 2 /SiO 2 by the sol-gel method. The degradation rate of MO was 98.03% within 180 min using the 250 W mercury lamp as the light source. As shown in Figure 10c, the absorbance of MO was reduced to zero, indicating that MO was completely degraded in 100 min. The result suggests that the as-prepared TiO 2 -SiO 2 microspheres exhibit an excellent photocatalytic activity. The estimated band gaps of pure TiO 2 and TiO 2 -SiO 2 prepared with P. pastoris GS115 as a template are 3.23 eV and 3.63 eV, respectively ( Figure 11). The band gap of TiO 2 -SiO 2 is more than that of pure TiO 2 , indicating that the introduction of silicon leads to an increase in the band gap of the semiconductor and enhances the redox capacity of holes and electrons. Hence, the photocatalytic activity can be improved. Figure 11. Estimated band gaps of (a) TiO 2 prepared from P. pastoris GS115 as a template and (b) TiO 2 -SiO 2 prepared from P. pastoris GS115 as a template based on the Tauc/Davis-Mott model.

Conclusions
In summary, P. pastoris GS115 was employed as a typical microbe to demonstrate its potential in synthesizing high-efficient photocatalysts for the degradation of organic contaminants. Hollow SiO 2 microspheres with a spherical morphology were successfully synthesized using the microbe template. The morphology and surface roughness of the hollow particles could be controlled by the reaction conditions. TiO 2 -SiO 2 microspheres were successfully prepared by the hydrothermal process. Results indicated that TiO 2 -SiO 2 kept in the favorable anatase phase of TiO 2 . The as-prepared TiO 2 -SiO 2 exhibited good photocatalytic activity for the degradation of MO and the degradation rate could reach 99.9% in 100 min because of an increase in the band gap. This work is of great significance for employing microbes in the preparation of promising photocatalysts for large-scale practical application. When TEOS is added to a mixture of water and ethanol containing microorganism, TEOS is attached to the cell wall. After the addition of ammonia, TEOS begins to hydrolyze and the resulting SiO 2 particles grow in the cell wall. Due to the slow hydrolysis rate of weak alkali in ammonia water, the adsorbed SiO 2 nanoparticles on the cell wall surface have enough time to grow. After calcination, the template is removed and the hollow SiO 2 microspheres are synthesized. TiO 2 -SiO 2 microspheres are prepared by the hydrothermal process and the photocatalytic activity for the degradation of methyl orange (MO) at room temperature under Xe arc lamp acting as simulated sunlight was explored.
In order to investigate the influence of microbe templates on the preparation of SiO 2 , the samples were characterized by TG. Figure A2 shows the TG characterization diagrams of Pichia pastoris GS115 and uncalcined SiO 2 . From the TG spectrum of P. pastoris GS115, it can be seen that there is a small weight loss peak at 85 • C, which is mainly caused by the desorption of water on the surface of the sample; in the range of 200-700 • C, in the spectrum a distinct weight loss step appeared, which was caused by the gradual breakdown of the biomass molecules of the P. pastoris GS115. The TG spectrum of uncalcined SiO 2 microspheres is similar to the weight loss peak of P. pastoris GS115, which indicates that some biomass may remain on the surface of the uncalcined SiO 2 microspheres. From the spectrum of uncalcined SiO 2 in Figure A3, the structural water -OH anti scaling vibration peak appears at 3450 cm −1 , and the peak near 1638cm −1 is the H-OH bend vibration peak of water. The peak at 955 cm −1 belongs to the bending vibration absorption peak of Si-OH, which is consistent with the literature reports. After calcination, the strong and wide absorption band at 1095 cm −1 is attributed to Si-O-Si anti scaling vibration peak, and the peak at 798cm −1 is attributed to Si-O symmetric stretching vibration peak.