MnO2 Submicroparticles from Chinese Brush and Their Application in Treatment of Methylene Blue Contaminated Wastewater

Eggshell membrane (ESM) is selected as biotemplate to prepare MnO2 submicroparticles (SMPs) using Chinese Brush with sodium hydroxide solution. The size with average 710 nm of the obtained materials is in good consistency with the microsructured biotemplate. An efficient and convenient absorbent for methylene blue (MB) is developed. The removal efficiency could reach up to 93% in 35 min under room temperature without pH adjusting owing to the excellent adsorption from ESM itself and hydroxyl group formed on the surface of MnO2 crystal in the aqueous solution. Materials on the membrane can be separated from the wastewater simply to avoid the secondary pollution caused by the leak of material. This interesting approach to MnO2 SMPs and facile operation for MB adsorption could open a new path to the submicro-materials based wastewater treatment.

Eggshell membrane (ESM) is a kind of biomaterial with great imperative though it is generally considered as a domestic waste. 35 This microscopic biopolymeric fibrous net is composed mainly of proteins (80-85%), 10% of which are collagens and 70-75% are other proteins and glycoproteins. 36 Due to the unique structure and property, ESM has been utilized as a biotemplate for synthesis of inorganic materials. Novel metal materials such as gold nanoparticles, silver nanoparticles, macroporous silver network, Pt-Ag/polymers, have been constructed through ESM templating. [37][38][39][40] On the other hand, sulfide, 41 selenide, 42 oxidide 43,44 have been synthesized using ESM as a template. Besides, other kinds of material based on ESM have been studied. [45][46][47] As a kind of inorganic nanomaterials, MnO 2 have drawn much attention because of their flexible structures and unique properties and have been applied to catalysis, ion exchange, supercapacitors, molecule adsorption, biosensors and so on. 48 One of the recent applications has fo-Wang et al.: MnO 2 Submicroparticles from Chinese Brush ... cused on the MnO 2 based micromotors. [49][50][51][52] And micromotors containing manganese oxide and noble metal or graphene have also been studied. [53][54][55] In this work, by consideration of its special microstructure, abundant component of protein, we choose ESM as the biotemplate to help synthesize MnO 2 . Most important of all, ESM could be obtained expediently and free of charge. Furthermore, on the basis of the interaction between protein and metal ions, a novel and interesting procedure with Chinese Brush to grow MnO 2 submicroparticles (MnO 2 SMPs) on ESM is developed. As reported by Furuichi et al, hydroxyl groups could be formed on the surface of MnO 2 in aqueous solutions. 56 Cao et al confirmed that the hydroxyl groups were involved in the adsorption. 48 Therefore, combining the adsorption capacities of both ESM itself 57 and hydroxyl groups formed on the surface of MnO 2 in the aqueous media, these accessible Mn-O 2 SMPs are applied successfully to the treatment of methylene blue (MB) wastewater.

1. Reagents and Apparatus
Deionized water with conductivity of 18.2 mΩ cm -1 was used in this experiment from a water purification system (ULUPURE, Chengdu, China). Manganese acetate (MnAc 2 , M w = 245.09, AR) and methylene blue (MB) were purchased from Kemiou Chemical Co. Ltd. (Tianjin, China). Sodium hydroxide (NaOH, AR) and all the other reagents were at least of analytical grade. Eggshell was obtained from Hongye student mess hall of Taiyuan Institute of Technology, and eggshell membrane was peeled off from the shell carefully. Diluents with different pH values were prepared by titrating with 0.1 mol L -1 sodium hydroxide or hydrochloric acid solution to the required p-H values.
Scanning electron microscopy (SEM) of ESM and MnO 2 SMPs were carried out on a Quanta 200 FEG scanning electron microscope. The size distribution of as-prepared nanomaterial was performed at a laser particle sizer (Malvern Nano-ZS90). The X-ray photoelectron spectroscopy (XPS) was measured with an AXIS ULTRA DLD electron spectrometer (Kratos) using monochromatic Al Kα radiation for analysis of the surface composition and chemical states of the product. Thermogravimetry (TG) measurement was carried out in air at a heating rate of 10°C min -1 on a Rigaku TG thermal analyzer (Rigaku Co., Japan). The UV-vis absorption spectra were recorded on a TU-1901 UV-vis spectrophotometer (Puxi, China).

Synthesis of MnO 2 SMPs
MnO 2 SMPs in this experiment were synthesized through a simple and interesting method. In a typical process, eggshell membrane (ESM) was firstly peeled off ca-refully from a fresh eggshell and cleaned 10 times with deionized water to remove residual egg white and then dried at room temperature. The clean ESM was cut into small pieces and soaked into 0.1 mol L -1 manganese acetate solution with a certain proportion (0.5 g to 100 mL). After 12 hours, the adsorbed ESM pieces were taken out and washed 5 times with deionized water and placed onto a watch glass to dry. At last, a Chinese Brush was dipped in 0.1 mol L -1 NaOH solution for 20 seconds. NaOH solution as ink was brushed evenly on the adsorbed ESM. Five minutes later, the color change of the membrane from white to light brown indicated that the MnO 2 SMPs were synthesized successfully. The MnO 2 /ESM piece was washed and dried to preserve for characterization and practical use.

3. Treatment of Methylene Blue Wastewater
15 mg MnO 2 /ESM materials and equal amounts of ESM, as a control experiment, were placed in the 4 mL MB solution with the concentration of 8 mg L -1 under stirring. After 35 min, materials and ESM were taken out to stop the adsorption. The UV-vis spectra of MB solutions after adsorption were recorded immediately at room temperature. All of the absorption intensity of MB measurement was set at wavelength 664 nm. The removal efficiency (R, %) and adsorption capacity (q e , mg g -1 ) were calculated using the equations below: (1) (2) where C 0 and C e (mg L -1 ) stand for the initial and final concentrations of MB in the treatment solutions, respectively, V is the volume of the mixture solution (L), and W is the mass of adsorbent used (g).

1. Synthesis Mechanism
Scheme 1 displays the schematic diagram of the synthesis process of submicro-structured MnO 2 on ESM using Chinese Brush. As reported, eggshell membrane is composed of fibrous proteins with different kinds of acidic/basic amino acid residues like -OH, -COOH, -NH 2 , -SH, etc on the surface. When ESM pieces were soaked into the manganese acetate solution, Mn 2+ showed a trend (from lone electron pair of heteroatom and unoccupied orbital in Mn atom) to adsorb onto the "active site" on the ESM, which resulted in a uniformly dispersive distribution of Mn 2+ on the fibrous proteins. After washing and drying

2. 2. UV-Vis Spectroscopy and X-ray Photoelectron Spectroscopy
The UV-Vis spectrum of as-prepared MnO 2 SMPs is shown in Figure S2. A single absorption peak at 360 nm is found. To investigate the surface composition and elemental analysis for the resultant MnO 2 SMPs, the X-ray photoelectron spectroscopy (XPS) was carried out. In the full scan spectrum ( Figure S3), it shows that the synthesized material is composed of elements Mn 2p, O 1s, C 1s and N 1s. The elements C 1s, N 1s and partial O 1s come from proteins in ESM. To examine the details, XPS spectra of Mn 2p and O 1s were measured. As shown in Mn 2p spectrum (Figure 2a), two peaks are observed at 654.2 and 642.4 eV, which can be assigned to Mn 2p 1/2 and Mn 2p 3/2 , respectively. Meanwhile, the O 1s spectrum (Figure 2b) can be resolved into three peaks. The strongest peak at  at the room temperature, Chinese Brush with NaOH solution was brushed on the adsorbed ESM. This step caused a reaction in situ between Mn 2+ and OHaround these "active site" and as a result MnO 2 were obtained after 5 min. 57 Owing to the uniformly dispersion of Mn 2+ on the membrane, MnO 2 particles were generated and grew along with the fibrous proteins to form a biomimetic material.

2. 1. Scanning Electron Microscopy
Morphologies of ESM before and after MnO 2 preparation were investigated for comparison. Figure 1a displays the scanning electron microscopy (SEM) images of ESM, in which multilayer and overlapping fibrous proteins are observed. After the reaction with MnO 2 , by contrast, plenty of spherical particles array densely on the adsorbed membrane (Figure 1b and Figure S1a). Interestingly, particles arraying along with the original fiber-like protein is observed, and it is more obvious and straightforward in SEM image with smaller amplification factor ( Figure S1a). To measure the particle size of synthesized material, a Nano Particle Analyzer testing was carried out. The results are shown in Figure S1b. And an average diameter of ∼710 nm is obtained, which is a good consistency with the microstructured biotemplate.

2. Thermogravimetry Analysis
Furthermore, a thermogravimetry (TG) analysis was carried out to illustrate the content of the composite (Figure 3). Blue and red curves indicate the mass changes of ESM only and synthesized MnO 2 /ESM material, respectively. It can be seen that ESM, as a kind of protein, is burnt out at about 600 °C and the quality is almost zero (blue curve in Figure 3). To study the relative amount of MnO 2 SMPs coated on ESM, dotted portion in Figure 3 is zoomed in. It is vividly shown that the curves remain unchanged with the temperature rising afterwards. However, the horizontal part of MnO 2 /ESM is obviously higher than that of ESM only, which is attributed to the inorganic material existence. The difference of two horizontal curves stands for the relative amount of MnO 2 SMPs in ESM, which is calculated to be 2.77%.

Methylene Blue Wastewater Treatment
The detailed characterization and measurement demonstrate that the synthesized material is ESM coated   MnO 2 SMPs (MnO 2 /ESM). Owing to the handy operation of "put in" and "take out", these materials were further applied to removal of MB. Figure 4a displays the photographs of ESM and MnO 2 /ESM before and after adsorption of MB. Two sets of contrastive pictures show that ESM itself is capable of adsorbing for MB. Light pink ESM (1) turns into blue (2) after adsorption of a certain amount of MB. However the color change degree of MnO 2 /ESM before and after adsorption is bigger: brown MnO 2 /ESM (3) becomes dark green (4). The UV-Vis absorption spectra of MB before and after adsorption by ESM only and MnO 2 /ESM are recorded in Figure 4b. It is evidently indicated that the absorption intensity of MB at 664 nm after MnO 2 /ESM adsorption is significantly smaller than the one treated with ESM only. Figure S4a exhibits the equation of linear regression of MB solutions, by which the removal efficiencies of ESM and MnO 2 /ESM adsorption are calculated in Figure S4b. Inset photographs shows the color change of MB solution before and after adsorption: (5) is original MB solution; (6) and (7) are MB solution after ESM and MnO 2 /ESM adsorption, respectively. The color gap between (5) and (7) keeps pace with the removal efficiency of 93% by MnO 2 /ESM adsorption.

4. Investigation of Time and pH for Adsorption
Adsorption time for MB by MnO 2 /ESM adsorption was investigated by UV-Vis spectroscopy as shown in Figures 5a and S5a. Under different adsorption time the absorption intensity decreases gradually as a function of time and remains the same after 35 min, which represents the whole adsorption process. Figure S5a shows the time dependent removal efficiency curve for MB, it can be seen that the removal efficiency increases rapidly at first 10 min and flats out gradually afterwards. A maximal removal efficiency of 93% is obtained at 35 min. Moreover, Figure S5b demonstrates the effect of pH condition on the adsorption by MnO 2 /ESM. It turns out that the removal efficiency is kept in the range of 50%-62% under different pH values. The pH is not a factor to influence within the experimental error. It is worth noting that the removal efficiency under a certain pH condition is not as high as that in the distilled water solution. The additional adsorption for ions, which was used to adjust the acidity of the solution, took charge of this phenomenon. The desorption of MB was performed by placing the adsorbed Mn-O 2 /ESM into deionized water. Figure 5b shows the absorption spectra of MB by adsorption for 35 min and desorption for 24 h. It is seen obviously that the shape and position of absorption peak are the same before and after adsorption, which indicates that the molecule structure of MB keeps unchanged during the removal procedure. Therefore, the removal procedure is an adsorption-desorption equilibrium process.

5. Study of Kinetics and Adsorption Isotherm
In order to better understand the adsorption behavior of MB on MnO 2 /ESM, the adsorption capacities at different time (q t ) were recorded. As shown in Figure 6a, the adsorption of MB increases gradually with the time prolonged and becomes balanced after 35 min. Based on this, experimental data are calculated and organized in Figure  6b to investigate the adsorption kinetics. Two kinetic models are generally used to evaluate the adsorption, 59 and it can be concluded that the adsorption process of MB on MnO 2 /ESM is in accordance with the pseudo-second-order model (correlation coefficient of 0.99508 for pseudofirst-order model and 0.99915 for pseudo-second-order model).
Moreover, the effect of initial MB concentration on equilibrium adsorption capacity (q e ) is shown in Figure 7a, where the adsorption capacity steadily enhances with increasing the initial concentration of MB ad- was further studied through Langmuir and Freundlich isotherms models (Figure 7b), which were common adsorption isotherms models for evaluating the adsorption process. 59 According to the data calculation and linear fitting, it is concluded that Langmuir model is able to interpret the MB adsorption process better (correlation coefficients are 0.9967 and 0.9787 for Langmuir isotherms and Freundlich isotherms models, respectively).

6. Effect of Hydrogen Peroxide on Removal of MB
The effect of H 2 O 2 on the dye MB as a function of time monitored by UV-visible spectra was investigated in Figure S6a. We observed that the presence of H 2 O 2 affected the absorbance of dye itself about 5% in 35 min and the shape of the peaks underwent no change. Then the effect of H 2 O 2 with various concentrations on the removal efficiency of dye MB decontamination by MnO 2 was examined. The results are showed in Figure S6b. It is straightforward that H 2 O 2 decreases the removal efficiency of dye MB by MnO 2 .

Conclusions
The MnO 2 submicroparticles were prepared through an eggshell membrane based biotemplating method. The size of MnO 2 SMPs kept correspondence with the diameter of the fibrous proteins, which indicated the bio-inspired growth of MnO 2 SMPs. Taking advantages of macrooperability and adsorption performance stemmed from both protein membrane and hydroxyl on the surface of MnO 2 in the aqueous solution, the ESM coated MnO 2 SMPs was applied to the MB wastewater treatment. The adsorption process followed the pseudo-second-order kinetic model and Langmuir isotherms model, and the removal efficiency could reach up to 93% under room temperature without pH adjustment. This simple, green and interesting approach gives a facile concept of metal oxide materials synthesis, which is considered of great potential applications in wastewater treatment area.  and the Program for the (Reserved) Discipline Leaders of Taiyuan Institute of Technology. We thank Institute of Coal Chemistry, Chinese Academy of Science for the material characterization.