Production of Silk Fibroin Membrane for Heavy Metal Removal in Water


 Thermodynamically driven salts and annealing processes developed the pure silk fibroin (SF) membranes from Bombyx mori cocoons. For more generation, the silk fibroin solution 5 wt% mixed the sodium chloride (NaCl) and polyethylene glycol (PEG). The top surface and cross-section of silk fibroin mixed with NaCl (SF/NaCl) showed pore in the surface membrane and hydrophilic property. The β-sheet content in the SF/NaCl membrane is higher than pure silk fibroin (SF) and the silk fibroin mixed with PEG (SF/PEG) membranes. Then cadmium (Cd), lead (Pb), and mercury (Hg) metals in water filtered pass through only SF/NaCl experimented. The pressure, pH, and temperature were effect by the flow rate efficiency of SF/NaCl membranes. The results showed that the highest % removal of Cd, Pb, and Hg were 45.36%, 61.43%, and 86.87%, respectively. The best condition of absorption capacity was 8.50, 6.42, and 41.14 mg/g for Cd, Pb, and Hg. As a natural biopolymer, SF/NaCl possibly apply for Hg removal for water treatment, its nontoxicity, excellent biocompatibility, and mechanical toughness.


Introduction
Silk cocoons are biopolymers, the long lament to protect larvae from humidity, bacteria, molds, and ultraviolet from sunlight during completed metamorphosis (Floren et al. 2016). Silk bers are consist of two types of protein micro laments that the broin coated by the sericin ( In previous studies, there are many methods for silk broin membranes and lms and applied to remove the heavy metals-silk broin modi ed to natural biomaterial membrane for heavy metal removals (Gao et al. 2017). Gao et al., 2017, used silk broin membrane to remove lead (Pb 2+ ) and cadmium (Cd 2+ ) ions, effective between 82% and 56%, respectively. Furthermore, Silk broin/chitosan (SF/CS) blend membranes which result shown that the permeability of SF/CS blend membranes was in the sequence K + > Ca 2+ > Cd2 + > Pb 2+ > Cu 2+ > Ni 2+ (Du et al. 2006). In addition, Rastogi et al. 2020 produced silk broin blended with different biomaterials for metals adsorption, which they found e ciencies equivalent to 81.1% for Cd and 93.75% for Pb ions. There were sever studies that produced lm and membrane to remove and adsorb metals ions from water by using silk broin combined with biomaterial hybrids For past decades, heavy metals, such as cadmium (Cd), lead (Pb), and mercury (Hg), have been highly toxic and hazardous pollutants from industry, agriculture, and community activities. Therefore, Cd, Pb, Hg are toxicity, persistency, and bioaccumulation in water bodies has brought great harm to aquatic living. It is complicated to eliminate low concentration heavy metals from the wastewater. The wastewater must take the new methods to remove heavy metals from e uent water before discharge to the environment.
The membrane technology is processed to treat the ability of silk broin to adsorb heavy metals from wastewater. Water treatment technology requires safe polymers and materials to focus on natural materials. Natural silk broin has become used for water treatment because of its nontoxicity, good biocompatibility, biodegradability, and mechanical toughness (Yazawa et al. 2016;Shome et al. 2021;Yao et al. 2021, Chen et al. 2021. Membrane technology has been used as an effective technology for removing water contaminants. A variety of membrane materials have been developed for speci c purposes and to improve puri cation e ciency. A nontoxic natural biopolymer, biocompatibility, biodegradability, and toughness, all is the advantage of silk broin can apply for water treatment. This experiment aimed to produce an alternative membrane from natural material to remove heavy metals from water.

Chemical and materials
All chemicals used in this study are in analytical grade. Bombyx mori cocoon was purchased from Buriram province, Thailand, and it was used without any puri cation. Polyethylene glycol (PEG), sodium chloride (NaCl), and lithium bromide (LiBr) were purchased from Union Science, Ltd. co (Chiang Mai, Thailand). The metal salts of Cd, Pb, and Hg ions were obtained from Loba Chemie Pvt. Ltd (Mumbai, India).

Silk broin preparation
The Silk broins (SF) were prepared in the laboratory following standard procedures reported in the literature (Rockwood et al. 2011, Sudsandee et al. 2019). Brie y, B. mori cocoon was boiled for 30 min in 0.02 M Na 2 CO 3 aqueous solution to extract the sericin protein. The bers were rinsed several times with distilled water and air-dried at room temperature overnight. LiBr aqueous solution at 9.3 M poured on the degummed silk bers and oven at 60 o C for 4 h. The silk broin solution was dialyzed against distilled water for 72 h using a dialysis tube (MWCO 3,500). The puri ed solution was centrifuged at 6000 rpm for 30 min to eliminate the insoluble brous. The nal SF concentration was measured by weight balance, and it was diluted with distilled water to obtain an SF concentration of 5 to 8 wt.% for further use in this study.

Synthesis of Silk Membrane
In this study, the silk broin membranes were prepared by a drop-casting method. Ten grams of sodium chloride (NaCl) and 4 mL of polyethylene glycol (PEG) were separately mixed with 50 mL of 5.0 wt% silk broin solution to generate a blended silk broin/sodium chloride (SF/NaCl) and silk broin/polyethylene glycol (SF/PEG) membrane. The mixed solutions were poured into glass plates with a diameter of 80 mm, and the solution was dried in an oven at 60 o C for 24 h. The dried membrane was soaked in the distilled water until uses. New distilled water is changed daily.

Characterization
The morphology of surface and cross-sectional of pure and blended SF membranes were studied by using a scanning electron microscope (LEO Electron Company, USA) with a magni cation of 100k and a voltage of 3.0 kV. All infrared membranes were collected using a Fourier transform infrared spectroscopy (ThermoFisher SCIENCETIFIC, Waltham, Massachusetts, United States). For each measurement, the scan was recorded with a resolution of 4 cm -1, and the measurement range was between 400 and 4000 cm -1 .
The secondary structure of SF, including beta sheets and alpha helices, was evaluated using a software peak t. The con rmation of silk II structure (β-sheet) analyzed a distinct shift in the β-sheet peak at 1612-1624 cm −1 (Litvinov et al. 2012). The surface hydrophilicity properties of the membranes were inspected by water contact angle (FACE, Automatic Interfacial Tensiometer, PD-VP) measurement.
Pure water ux ( ow rate) analyses for the prepared membranes were carried out in a dead-end mode.
The newly prepared pristine or blended SF membranes were placed on the membrane ltration cell, which has a diameter of 5 cm and a volume capacity of 1,000 mL. The membrane ltration cell was connected to the vacuum pump, as shown in Fig. 1. Pure water ux of every membrane sample was monitored at the operating pressure between 100 -500 mbars.

Membrane performance of metal removal
The separation performance of the blended SF membrane was analyzed using metal solutions of Cadmium (Cd), Lead (Pb), and Mercury (Hg) as a feed solution at the concentration of 10 ppm. The membrane ltration of the metal solution was carried out at a pressure between 100 -500 mbars. The permeate was collected over declined intervals in the sample container, and the solution were tested for metal concentration using atomic absorption spectrophotometer, AAS (Hitachi High Technologies, Tokyo, Japan) for Cd and Pb. At the same time, the Hg was measured using an MA-3000 mercury analyzer (Nippon Instruments, Tokyo, Japan). Each experiment was carried duplicated while the removal rate of studied metals can be calculated using equation 1 (Gao et al., 2017).
Where C 0 is initially concentration of metal (mg/L) C t is nal concentration of metal after pass membrane (mg/L) The effect of pH on membrane performance for metal removal was tested between 7 and 13. The effect of temperature (5,10,20,30, and 40 o C) on membrane ow rate was also measured.
The adsorption capacity (q) was measured by Equetion 2 (Ouyang et al.2919; Rastogi et al. 2020): Where C 0 is metal concentration in stock solution (mg/L) C t is metal concentration after ltration test (mg/L) V s is metal solution volume (L) m is membrane mass (g)

Morphology of silk broin membranes
Pure SF membranes and blended SF membranes of NaCl and PEG were simply prepared by the dropcasting method. The physical characteristic (i.e., color) of pure SF membrane slightly differed from the blended membranes as presented in Fig. 2  The FTIR spectra of SF, SF/NaCl, and SF/PEG membranes were collected to investigate the change of surface chemistry of the prepared silk membranes. As presented in Fig. 3, the characteristic structure of silk II at the adsorption peak of 1620 cm −1 , were found in all membranes. This peak refers to the formation of β-sheet structure in the silk membrane.
The amount of β-sheet crystals found on SF/NaCl membrane was higher than that on SF and SF/PEG membranes. Silk broin is consists of β-sheet crystallites and amorphous domains, and these can The hydrophilicity of the studied SF membranes was also investigated using the water contact angle method. As shown in Fig. 4, the contact angle of the SF/NaCl and SF/PEG blended membranes was decreased due to the addition of NaCl and PEG to SF matrix. Among the studied membranes, SF/NaCl hydrophilicity can make the membrane fouling resistant due to the water's easy diffusion through the membrane thickness. Hence, the SF/NaCl blended membrane has better chances of antifouling ability and higher water ux.

Pure water ux ( ow rate)
The deionized water was treated by keeping the temperature (20ºC) and ow rate varying the pressure as 100 mbars, 200 mbars, 300bars, 400 mbars, and 500 mbars. As shown in Fig. 5, the ow rate of membrane permeation was increased from 120 mL/min to 1033.3 mL/min when pressure was increased from 100 mbars to 500 mbars. The increasing pressure during the membrane contact with water can reduce the number and strength of hydrogen bonds between β-chains, thus dramatically weakening the strength of silk broin (Cheng et Fig. 6. The water temperature between 5 -20 o C showed ow rate stability to increase, but after 20 oC was the ow rate instability. The water molecules in uence the glass transition of silk (Yazawa et al. 2016), the effect of thermal water can be changing the ow rate properties. In addition, heating solution cast broin can be converted to the silk II β -sheet structure commonly found in B. mori cocoon bers (Drummy et al. 2005).
The water pH range was 7-13. The pH effect of the owing rate of SF/NaCl membrane, the metal wastewater sample was treated by keeping the pressure (200 mbars) constant. The average ow rate of membrane permeation was increased from 180.0 mL/min to 597.3 mL/min when pH was increased from 7 to 13. Then it was decreased to 540.0 mL/min at pH 13, as shown in Fig. 7. 3.3 Heavy metal removal and e ciency.
The SF/NaCl membranes were applied to remove the three metals Cd, Pb, and Hg in deionized waters. It was the concentration 10 ppm. Various pH of metal waters of Cd, Pb, and Hg has shown the % removal in Fig. 8. pH Cd removal was found in the range of 10.76 -45.36 %, as shown in Fig. 8. The highest % removal for Cd was 45.36% at pH 12. At the same time, Pb % removal was found to range from 10.80 -61.43%. The highest % removal for Pb was 61.43% at pH 12. The highest % removal for Hg was 78.18% at pH 9. The mean concentration of Hg removal was range 70.20 -78.18 % for pH 7-12. Comparing the % removals of Cd, Pb, and Hg found that Hg was high e ciency for metal removal by use SF/NaCl higher than Cd and Pb at pH 7-12.

Temperature
The various metal water temperature was range from 5 -40 o C, as shown in Fig. 9. Cd removal was found in the range of 6.90 -15.04 %. The highest % removal for Cd was 15.04% at 5 o C. At the same time, Pb % removal was found the range of 13.93 -16.27%. The highest % removal for Pb was 16.27 % at 40 o C. The highest % removal for Hg was 72.72 % at 30 o C. The mean concentration of Hg removal was range 61.26 -72.72 % for temperatures 5 -40 o C. Comparing the % removals of Cd, Pb, and Hg found that Hg was high e ciency for metal removal by use SF/NaCl higher than Cd and Pb at temperature 5 -40 o C.

Pressure
The various metal water pressure in the ltrated column was range 100 -500 mbars as shown in Fig. 10. Cd removal was found to range from 2.90 -16.34 %. The highest % removal for Cd was 16.34 % at pressure 500 mbars. At the same time, Pb % removal was found to the range to 2.50 -16.13%. The highest % removal for Pb was 16.13% at pressure 200 mbars. The highest % removal for Hg was 86.87 % at pressure100 mbars. The mean concentration of Hg removal was range to 62.71 -86.87 % for pressure 100-500 mbars. Comparing the % removals of Cd, Pb, and Hg found that Hg was high e ciency for metal removal by use SF/NaCl higher than Cd, and Pb at pressure 100-500 mbars.
Comparison of the % of metal removal is shown in Table 1. This study showed that the highest % removal of Cd, Pb, and Hg were 45.36%, 61.43%, and 86.87%, respectively. This study, apply SF/NaCl shown that % Cd removal is lower than research studies by using the MWSF (modi ed water-insoluble silk broin) and chitosan/silk broin lms (Ramya and Sudha 2013; Gao et al., 2017). In addition, the % Pb removal is lower than the studies of Gao et al., 2017 andZhao et al., 2020. SF/NaCl membrane is excellent for removal Hg greater than Metallic molybdenum disul de (MoS2)/Silk nano bril (Zhao et al., 2020).
The average membrane mass (m: unit, gram (g)) was 1.76 g, and the metal solution volume (Vs: unit, liter(L)) was range 0.12 -1.04 liters. The adsorption capacity (mg/g) of Cd, Pb, and Hg for SF/NaCl membrane is shown in Table 2. In this study, the highest adsorption capacity of Cd was 8.50 mg/g of metal water at 5 o C, at pH 7 and, pressure 200 mbars. For Pb found that the highest adsorption capacity was 6.42 mg/g of metal water at pH 12, at 20 o C, and pressure 200 mbars. Finally. The highest adsorption capacity of Hg was 41.14 mg/g of metal water at 100 mbars, at pH 7, and at 20 o C. The pH of the metal solution found that Cd and Pb were high e ciencies of adsorption capacity at pH 12, and Hg was pH 9. In addition, the appropriate temperature of a metal solution of Cd, Pb, and Hg was at 5 o C, 40 o C, 30 o C, respectively. The best condition of absorption capacity was 8.50, 6.42, and 41.14 mg/g for Cd, Pb, and Hg, but the result was lower than the study of Cd and Pb absorption capacity of Rastogi et al. 2020.

Conclusion
The possibility of silk broin membranes for heavy metal removal was evaluated in this work. SF membrane, SF/PEG membrane, and SF/NaCl were developed by casting 5 wt% of Bombyx mori cocoon. The SF/NaCl membrane produced pore size on membranes, water permeate and showed higher crystalline formation (β-sheet) that enhanced the structured membrane. The contact angle ( o ) of SF/NaCl was lower than SF and SF/PEG membranes. The SF/NaCl removed the Cd, Pb, and Hg. Deference of pressure, pH, and the temperature was effect by the ow rate e ciency of SF/NaCl membranes. The results showed that the highest % removal of Cd, Pb, and Hg were 45.36%, 61.43%, and 86.87%, respectively. The best condition of absorption capacity was 8.50, 6.42, and 41.14 mg/g for Cd, Pb, and Hg. As a natural biopolymer, SF/NaCl possibly apply for Hg removal for water treatment, its nontoxicity, excellent biocompatibility, and mechanical toughness.

Declarations Acknowledgment
The authors would like to express their sincerest gratitude to Mae Fah Luang University, Chiang Rai, Thailand, and the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand laboratory analysis support.
Availability of data and materials The data are available upon a reasonable request to only the corresponding author.

Authors Contributions
SS involved in conceptualization, laboratory analysis, writing -original draft, editing, and project administration. PD, PB, and HC-C are involved in conceptualization. RM, NK involved laboratory analysis. SW and SL involved nal editing.

Funding
The study was funded by the Mae Fah Luang University, Thailand.
Ethical Approval Not applicable.

Consent to Participate
Not applicable.

Consent to participate
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Consent to publish
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Figure 1
Membrane ltration experimental set-up