Prospects of Silk Sericin as an Adsorbent for Removal of Ibuprofen

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Prospects of Silk Sericin as an Adsorbent for Removal of Ibuprofen from Aqueous Solution Vishal Kumar Verma, and Senthilmurugan Subbaih Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b01827 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 28, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Industrial & Engineering Chemistry Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Industrial & Engineering Chemistry Research

Prospects of Silk Sericin as an Adsorbent for Removal of Ibuprofen from Aqueous Solution 1

Vishal Kumar Verma, 2*Senthilmurugan S

1, 2

Department of Chemical Engineering, Indian Institute of Technology, Guwahati – 781039,

India E-mail: [email protected]/[email protected]; 2 [email protected] * Corresponding author: [email protected] Corresponding Address: Dr. Senthilmurugan S Associate Professor Chemical Engineering Department Indian Institute of Technology, Guwahati India-781039 Email: [email protected] Contact No: +919945213864, +913612583527

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 45

Prospects of Silk Sericin as an Adsorbent for Removal of Ibuprofen from Aqueous Solution Abstract: This paper presents removal of Ibuprofen from aqueous solution using commercially available silk Sericin as an adsorbent in an integrated adsorbent-membrane process. The adsorption study was performed at different physiological condition such as adsorbent concentration (1-10g), Ibuprofen concentration (10-70 mg/L), temperatures (20, 30 and 40 °C) and pH (5, 6, 7, and 8). The occurrence of adsorption before membrane separation was confirmed by performing analysis of Sericin-ibuprofen interaction and complex formation. Sericin-ibuprofen interaction and complex formation was investigated using FTIR, FESEM, Fluorescence spectroscopy, XRD, and ITC. Sericin and Ibuprofen interaction is spontaneous and endothermic in nature while random coil transition of Sericin governed the adsorption system. ITC analysis exhibited a binding affinity (Kb) value of 2.51 x 104±1.4 and one binding site (n≈1) per molecule at 27°C, revealing moderate binding of Ibuprofen to the Sericin protein. Complete removal of Ibuprofen (at 10mg/L, pH 8) was achieved using 10 g of Sericin (pH 4) and at a temperature of 40°C in reverse osmosis membrane process. The results established in this work concludes that Sericin may be used as an adsorbent for the removal of micro-pollutants such as Ibuprofen from drinking water. Keywords: Ibuprofen, Silk Sericin, adsorption, membrane, micro-pollutants 1. Introduction Micro-pollutants are generated from many sources and they are called as contaminants, which are persistent and bioactive nature. These contaminants are finally getting dissolved in waste water streams from multiple sources such as, pharmaceuticals and personal care products


Page 3 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(PPCPs), endocrine disrupting chemicals (EDCs), steroid hormones, industrial chemical waste, pesticides and many other recalcitrant compounds. These micro-pollutants cannot be removed with conventional wastewater treatment technologies. Therefore, removal of these substances during municipal waste water treatment has been found to be incomplete

1–3

. The continuous

discharges of micro pollutants in waste water stream is expected to cause long term hazards since the contaminants are bio-accumulating nature and even forming new mixtures in our water reuse cycle. The exact impact on ecological system by micro-pollutants are yet to be fully discovered. As per the recent lab analysis 4, the levels of drug pollutants measured in streams, lakes and well water near pharmaceutical factories in India are 100,000 to 1,000,000 times higher in comparison to the levels measured in waters that receive sewage effluent in the US or China. Many studies report the adverse effects of pharmaceuticals to the aquatic organisms, chronic long term exposure to these drugs may have detrimental effects on metabolism of non-target organisms including microbes fish and other aquatic organisms

5–7

. Due to profound use of diclofenac (a

widely used Nonsteroidal anti-inflammatory drug, NSAID) an unusual high death rate among three different species of vulture was reported in India and Pakistan 8. The extent of the impact of pharmaceutical waste on human beings is not as defined and well studied. Still it has been a topic of debate recently and even in low dosages, long term exposure to pharmaceutical drug remnants may be hazardous to humans 5. Both conventional and advanced water treatment technologies such as coagulation, flocculation, chlorination, advance oxidation process (AOP), adsorption with activated carbon and membrane filtration are widely used for drinking water treatment application 9. Coagulation and flocculation are primary treatment process and cannot be used for removal of pharmaceutical micro pollutants 10

. While free chlorine was found to oxidize approximately half of the pharmaceuticals



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 45

investigated, and chloramine was found to be less efficient 11. AOP such as ozonation, UV/H2O2, UV/O3, UV/TiO2, and UV/Fenton are very effective however, these process may lead to formation of potentially harmful by-products by reaction with background compounds in water, incomplete degradation products and interference of radical scavengers 12–15. Pressure-driven membrane filtration processes such as, Nanofiltration (NF), and Reverse Osmosis (RO) are widely used for drinking water treatment application to improve taste of the water by removing dissolved salts

16

. The complete removal of salt from drinking water is also

not desirable because that leads to mineral deficit issue. Both RO and NF process highly effective alternative methods for removal of organic micro-pollutants

17,18

. However, selective

and complete removal of micro pollutants are not possible and it is limited by membrane separation efficiency 17,19–22. Adsorption process have earned preference as a sustainable solution with respect to complete removal of micro pollutants from drinking water source 23. In addition, adsorption process enables complete capturing and safe disposal without producing toxic byproducts. PAC (powdered activated carbon) and GAC (granular activated carbon) has been reported to have high removal of pharmaceutical compounds, especially hydrophobic compounds

10,24

. However, the selective removal and isolation of micro pollutant may be

difficult task with PAC and GAC. Therefore different materials have been investigated for their characteristics and adsorption capability for pharmaceutical drugs, some of the reported adsorbents includes soil minerals, metal oxide, siliceous material, zeolites, agricultural waste, industrial waste, and other materials

25–28

. Still the identification of adsorbents with following

properties are very important in the field of drinking water treatment: (i) nontoxic, (ii) low-cost, and (iii) selective removal capacity of pharmaceutical micro pollutants. Moreover, most of the existing adsorbents are not compatible for surface modification of membrane (via coating;


Page 5 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


blending or grafting) which becomes major issue for specific removal of desired micropollutants29. In this paper, we have evaluated the feasibility of Sericin uses as a material for surface modification in UF-membrane for selective removal of drug-based micro-pollutants. Sericin is a highly hygroscopic globular protein derived from silkworm cocoons with molecular weight in the range of 10-400 kDa, depending on the method of extraction 30. It comprises of 18 amino acids, majority of which have strong polar side groups, such as hydroxyl, carboxyl and amino groups

31

. Sericin is a by-product during silk degumming process. Recently, Sericin has

found its application in biomedical science, regenerative medicine, textile industry and membrane fabrication

32

. Sericin exhibits inherent properties like hydrophilicity, amphoteric,

antioxidant, anti-fouling, antimicrobial and are non-toxic in nature

33,34

. These physicochemical

properties make it very suitable as a bio-sorbent for enhancing membrane surface properties (via coating immobilization or blending) for selective removal of drug based micro-pollutants from aqueous environment. Even though NF and RO are regarded as highly effective in removal of pharmaceuticals with varying degree of rejection (from 45-90%)

19,20

, trace quantities of target pharmaceutical

compounds have been found to breach membrane barrier. But, due to higher molecular weight of Sericin molecule, RO membrane is enabling complete removal of Sericin while passing aqueous Sericin solution

35

. Therefore, the integrated Sericin based adsorption with RO process can be

enable dual activity i.e. (i) selective adsorption of micro pollutant with Sericin molecule and (ii) removal of combined molecule in RO membrane. This novel approach will enable complete removal of micro pollutant from aqueous solution. This project aims harnessing the physical and chemical properties of Sericin for synthesis of membrane filtration unit with desired qualities targeting removal of Ibuprofen from drinking water. Though, there has been few recent studies 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 45

on use of Sericin for removal of micro-pollutants like heavy metals and textile dyes, there has been no report of any study involving Sericin utilization in a membrane process for removal of drug based micro-pollutants from drinking water. This work will establish viability of Sericin as a potent candidate for membrane modification and deliver a modified membrane-adsorption system to remove Ibuprofen from drinking water. Six different methods were used to study the adsorption behaviour of Ibuprofen in Sericin. 2. Experimental 2.1.Materials Ibuprofen is a prototypical Nonsteroidal anti-inflammatory, over the counter (OTC) drugs. Its wide therapeutic application for pain administration, high consumption rate, reported persistent occurrence in water, eco-toxicity, physicochemical properties, and validated analytical methods have prompted us to select it for current study

36,37

. The target pharmaceutical compound

Ibuprofen (CAS-No 15687-27-1) was obtained from Sigma-Aldrich Chemicals Pvt. Limited. The stock solutions for Ibuprofen was prepared in Millipore water. The stock solutions were used to obtain the calibration curves and for batch adsorption studies. All dilutions were done in Millipore water. The calibration curve for High performance Liquid Chromatography (HPLC) analysis was prepared (see supporting information S1) and was found linear up to 10mg/l of concentrations with R2 value of 0.99. Acetonitrile and Buffer solution used for mobile phase preparation were of HPLC grade. All other chemicals used in the study were of analytical grade and were procured from Merck®, India. Commercially available pharmaceutical grade silk Sericin was used in this study and was procured from Swapnaroop drugs and pharmaceuticals, India.


Page 7 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.2.Methods 2.2.1. Characterisation 2.2.1.1.Sericin adsorbent Sericin used in this study had a molecular weight of ≈250 KDa. The structural morphology of the Sericin powder used was visually analysed using (FE-SEM) field emission scanning electron microscopy (Make: Zeiss, Model: Sigma) instrument. Powder sample was fixed on top of the stub using carbon tape and layered with gold using an auto fine coating instrument (JEOL JFC1300) prior to morphology analysis. Surface area and pore size for Sericin powder was analysed using BET method in Quantachrome surface area and pore size analyser (Model: Autosorb-IQ MP). 2.2.1.2.RO-membrane Flat Sheet RO-membrane from DOW FILMTEC™ of dimension 7.007 X 3.293 cm with an effective area of 22.935cm2 was used for filtration purpose (see supporting information S2). FESEM of RO-membrane sheet was analysed before and after filtration process to see the fouling and deposition on to the membrane. Membrane sheet was dried at room temperature and was fixed on top of the stub using carbon tape and double layer coated with gold preceding the morphology analysis. 2.2.2. Methods used to study Sericin and Ibuprofen interaction 2.2.2.1.Method 1: Integrated adsorption-cum-RO process Generally, the MWCO (molecular weight cut-off) value of DOW FilmtecTM RO membrane lies in the range of ~200-400 Da. Ibuprofen has a molecular weight of 206 dalton, and hence RO membrane are not supposed to retain it efficiently. On the other hand, the MWCO for Sericin is found to be 10-400 kDa, therefore Sericin is expected to be retained completely by RO 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

membrane. Fabiani et al. (1996)

38

Page 8 of 45

used UF/RO filtration method for filtration of waste water

from silk degumming processes and the overall rejection of Sericin was found to be more than 97 %. Li et al. (2015) 35 performed Sericin separation experiment using both silk degumming waste water and commercial grade purified Sericin in NF hollow fibre membrane to study effect of Sericin molecular weight. They observed complete removal with commercial grade pure Sericin and partial removal achieved for silk degumming waste water and similar results are observed by Capar et al. (2012)

39

. Further, to achieve complete removal of Sericin by RO membrane, the

purified higher molecular weight Sericin has to be used. Based on these facts we have designed an integrated method where Sericin specific affinity towards ibuprofen has been coupled with membrane filtration for its removal from aqueous solution. Schematics of size based separation of Ibuprofen using Sericin adsorbent together with RO-membrane shown in Figure 1. The drug molecules are supposed to adsorb on Sericin and thereafter based on size based steric exclusion mechanism, efficient removal of these drugs can be expected from RO-membrane with almost complete rejection of Sericin.


Page 9 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Schematic of size based separation of Ibuprofen using Sericin adsorbent and ROmembrane In this method, initially batch adsorption studies were carried out on a magnetic stirrer operated at a constant speed of 350 rpm, coupled with digital temperature controller for temperature regulation. In each test, two litre of Ibuprofen solution (of required concentration) was placed in a 5-litre beaker and required concentration of Sericin was added from stock solution (1000 ppt). Ibuprofen has a pKa value of ≈4.9 and hence, exist in anionic form when released in to the environment

26

. While, Sericin has an isoelectric point (pI) close to pH≈5-6 where it has zero

overall charge and the smallest intermolecular repulsive force39. Since, proteins have net negative charge above their pI values (pH > pI) and net positive charge below their pI values (pH < pI). Sericin used throughout adsorption experiment was kept fixed at pH 4 (to keep it in protonated state) while Ibuprofen pH was changed from 5 to 8 to facilitate charged based interaction. The solution was stirred continuously for required time and temperature was monitored using temperature controller. Sericin-ibuprofen solution was passed as feed through a 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 45

RO-flat sheet membrane at regulated flowrate using bypass valve and dampner (see Figure 2). The setup was run in cross flow filtration mode and recirculation mode i.e. permeate and reject recirculated back to the feed tank. As per the membrane manufacture’s guideline, a feed flow rate of 100 L/Hr and transmembrane pressure of 75 psi was maintained throughout the process. Permeate samples were taken at regular intervals of time to measure permeate flux and Ibuprofen concentration.

Figure 2. Integrated adsorption-cum-membrane filtration setup The feed and permeate samples were analysed using HPLC analyser (Shimadzu, model UFLC SPD-20A) equipped with a reverse-phase C-18 column (5 µm, 4.6 mm ×250 mm) and UVDetector to quantify the concentration of Ibuprofen at λmax = 222 nm. The mobile phase was a mixture of acetonitrile and 10mM Phosphate Buffer, pH 2.6 (70:30 v/v) with a flow rate of 1.8 mL/min in isocratic mode.


Page 11 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.2.2.2.Method 2: ATR- Fourier transform infrared spectroscopy The absorption of IR-radiation causes vibrational transition in polypeptide units of protein (Sericin) and this phenomenon gives nine characteristics bands named as: amide-A, Amide-B, and amide I to VII. Among these the Amide-I (1600 to 1700 cm-1) is most sensitive to composition of protein secondary structure. The variation in corresponding IR vibrational frequencies can provide information related to structural conformation of Sericin due to Sericin and Ibuprofen interaction

40

. Hence ATR-FTIR spectra (4000-400 cm-1) were acquired using

ATR spectrophotometer (Model: Spectrum TWO from Perkin Elmer) to confirm structural conformation of Sericin after complex formation with ibuprofen. Experiments were conducted at room temperature with 32 scans per second, at a resolution of 4cm-1 and obtained data were analysed using OMNIC software. 2.2.2.3.Method 3: Fluorescence Spectroscopy The protein folding and unfolding is expected while interacting with ligand. This phenomenon leads to exposure of tyrosine and tryptophan present in the protein from core to solvent phase and similar phenomenon is expected to happen while Sericin and Ibuprofen interaction. The fluorescence intensity of both tyrosine and tryptophan can be measured by fluorescence quenching mechanism

40

. Therefore, the drug mediated Sericin conformational changes were

monitored by tyrosine intrinsic fluorescence. Drug (Ibuprofen)-induced tyrosine intrinsic fluorescence quenching studies were carried out on spectrofluorometer (model: Fluoromax-4) at room temperature. 1 µM of Sericin protein was incubated with diverse range of Ibuprofen drug starting from 0.1mM to 1mM while maintaining pH 7.5 using 10 mM of phosphate buffer at room temperature. Samples were excited at 280nm (2nm slit width) and collect emissions between 300 and 450nm and averaging three scans.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 45

2.2.2.4.Method 4: Isothermal Titration Calorimeter (ITC) ITC measures the changes in the power needed to maintain the isothermal conditions between the reference and sample cell, which gives the measures of heat absorbed or generated when molecules interact 41. These heat changes are very low level (sub-millionths of a degree), but can be detected using high sensitivity thermocouple. The binding of ibuprofen drug with Sericin protein is expected to be endothermic/exothermic in nature. The heat of adsorption during complex formation was measured by isothermal titration calorimetry using MicroCal iTC-200 (MicroCal, Northampton, MA, USA). Sericin protein and drug sample were prepared with 10 mM phosphate buffer (pH 7.5) and they were degassed through thermovac vacuum pump prior to use in ITC. In this experiment, 50 µM of Sericin protein was filled in the sample cell and titrated with 4.5 mM of Ibuprofen drug at 27°C. Typically, 25 consecutive injections of 1.5 µl in two min interval were injected into the sample cell with adequate mixing. Heat of mixing due to the buffer solution is measured in separate experiment and were subtracted from total heat of adsorption. 2.2.2.5.Method 5: FE-SEM FE-SEM images for powder sample of Ibuprofen, Sericin and Ibuprofen-Sericin complex were acquired and analysed for structural changes. TheSericin-ibuprofen complex aqueous solution was prepared by mixing them in distilled water, then using open pan dry method ( 40 °C) the aqueous solution was dried to remove the water molecules from Sericin and Ibuprofen-Sericin complex. Adsorption of Ibuprofen on Sericin was visually confirmed using FE-SEM. All the samples were dried at room temperature prior to analysis.


Page 13 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.2.2.6.Method 6: X-ray diffractometer Amorphous and crystalline structure of Sericin, Ibuprofen and complex were examined using XRD and analysed for any change noticed after complex formation. X-ray diffraction pattern for native Sericin, Ibuprofen and Sericin-ibuprofen complex was obtained at ambient temperature using an X-ray diffractometer (XRD; make: Bruker; model: D8 Advance) using a step size of 0.05°/sec in the range of 2θ = 5 to 80° under the acceleration voltage of 40 kV and 40 mA. 3. Results and Discussion: 3.1.Sericin Characterisation The results obtained from BET analysis of Sericin powder (Table 1) showed surface area of 5.47 m2g-1 with average pore diameter of 4.055 nm and pore volume of 5.550x10-03 cc/g, indicating a non-porous morphology. In a study involving removal of dye, Chen et al. (2012)

42

reported a

surface area of 1.5 m2g-1 for Sericin powder obtained from a commercial source. Table 1. BET analysis of Sericin Sample

Silk Sericin

Multi-Point BET

Surface Area

5.473 m2/g

pore volume

0.005 cc/g

pore Diameter

4.05592 nm

Surface Area

2.939 m2/g

Pore Volume

0.006 cc/g

Pore Diameter

7.111 nm

Surface Area

4.542 m2/g

BJH adsorption

BJH desorption



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Pore Volume

0.007 cc/g

Pore Diameter

1.872 nm

Page 14 of 45

3.2.Experimental studies in integrated adsorption-cum-RO process 3.2.1. Flux through the RO membrane Pure water flux (see supporting information S3) curve was obtained for the RO- membrane sheet and the estimated membrane permeability is 0.281017 x 10-12 m3/m2.s.Pa. The membrane was preconditioned by operating for one hr at 50 Psig with distilled water before conducting the experiments with Sericin-ibuprofen solution. Permeate flux with respect to time for Ibuprofen (10 mg/L), Sericin (10g/L), and Sericin-ibuprofen complex (1000:1) is shown in Figure 3. The flux study shows constant decline in water flux with time for Sericin and Sericin-ibuprofen complex, which may be attributed to deposition of Sericin/complex on to the membrane surface.


Page 15 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Flux behaviour for Sericin (10g/l), Ibuprofen (10 mg/L) and Sericin-ibuprofen complex (1000:1 mg/l) at 517 kPa, pH 6 and 30°C, (Sericin was removed completely in permeate in all cases) 3.2.2. Effect of Sericin concentration Effect of Sericin concentration was evaluated by adding different concentration of Sericin (1 to 10 g/L) with fixed initial concentration of Ibuprofen (at 10 ppm) solution. The solution was stirred for 3 hrs on a magnetic stirrer. Experiment were conducted at 25 °C and without altering ambient pH. After 3 hrs the solution was filtered through RO membrane in complete recirculation mode and obtained results were evaluated. Sericin solution gives a pH of 6 with 10g/L of concentration without any alteration (see supporting information S4). Figure 4a shows variation observed in permeate concentration of ibuprofen with gradual increase of Sericin



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 45

concentration in feed. It was noticed that removal of Ibuprofen increases with increasing amount of Sericin and around 10g/L of Sericin was needed for complete removal of Ibuprofen at 10 ppm. Since, commercial ibuprofen is a racemic mixture and contains equal quantities of R(-)ibuprofen and S(+)-ibuprofen enantiomer, and hence larger amount of Sericin is expected to be used for non-specific binding and complete removal of Ibuprofen. In another aspect, protein conformation also plays a vital role and binding is supposed to be site specific, which was later found in FTIR, ITC and fluorescence studies that single binding site (n~1) is responsible for Sericin-ibuprofen complex formation. More than 99% rejection was noticed with 10g/L of Sericin for Ibuprofen. The observed rejection of Ibuprofen drug demonstrates the competence of Sericin as an adsorbent to remove Ibuprofen from aqueous solution. 3.2.3. Effect of Ibuprofen concentration Ibuprofen solution at different concentration were first filtered through the RO membrane and its inherent removal capacity was evaluated. Since it was found that around 10 gm/L of Sericin was required for complete removal of Ibuprofen from feed solution from 10 ppm. Solution initial concentration of Sericin is fixed at 10 gm/L and corresponding Ibuprofen rejection efficiency was assessed at different concentration of Ibuprofen (from 10 to 70 ppm). RO experiments were conducted at room temperature and with Ibuprofen solution at pH 8. Figure 4b shows the Ibuprofen concentration in RO permeate while varying RO feed concentration. Permeate Ibuprofen concentration is always found to be higher when feed solution is without Sericin. On the other hand, the rejection with Sericin is always found to be higher than in the case without Sericin.


Page 17 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Variation in permeate-Ibuprofen concentration (a) with increasing feed Sericin concentration; at 10 ppm of initial Ibuprofen conc. (b) with increasing feed Ibuprofen concentration; at 10mg/L of Sericin initial conc. 3.2.4. Effect of Ibuprofen solution pH Solution pH is supposed to affect the surface charge on solute particles and hence play an important role in adsorption. Effect of pH on Ibuprofen removal was assessed from a range of 5 to 8, to assess its effect on interaction of Ibuprofen with Sericin particles. Experiments were performed at 10g/L of Sericin (at pH 4) and room temperature, while required pH of Ibuprofen solution was adjusted using 1M NaOH and 1M HCl.

Figure 5a shows effect of pH on

adsorption of Ibuprofen and it was found that higher pH shows better removal capacity. This was reasonable because Ibuprofen has Pka value of 4.9 and hence exist as neutral species below this value and above this value Ibuprofen attains a negative charge and hence exist in anionic form. Sericin solubility has been found to decrease with increasing acidity of water. Moreover, it has



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 45

been reported that Sericin solubility in water is least at low pH, slowly escalate at a pH 5-8 and it has high solubility from 8 onwards

43

. Hence, higher pH must have escalated charged based

interaction and non-specific binding of negatively charged ibuprofen enantiomers with protonated Sericin particles, resulting in better removal capacity. 3.2.5. Effect of solution Temperature The separation efficiency for Ibuprofen with respect to temperature at constant presence of Sericin (at 10g/L) is shown in Figure 5b. Since, temperature is one of the important factor that affects heat of mixing and heat of adsorption. The experiments were performed at three different temperatures (20, 30, and 40°C) to assess the effect on seasonal removal capacity. It was observed that the adsorption of Ibuprofen on Sericin was a fast process and spontaneously almost 80 % of drug was adsorbed within 5 minutes of contact time. However, after 5 minutes, the adsorption rate decreases over time until saturation was achieved in around 25 minutes (see supporting information S5 a-c). The filtration process exhibited better removal capacity with increase in temperature. Since Sericin solubility also increases with temperature due to destabilization of the protein conformation, random coil structure become more dominant with temperature, which may have contributed to better binding capability. Moreover, since the binding of Ibuprofen with Sericin was found to be endothermic in nature (as found in ITC), increase in temperature must have assisted in improved binding capacity. The adsorption process at 40°C shows the best performance with almost complete removal of Ibuprofen from solution after 25 minutes of contact time for a period of 15 minutes of filtration. For 20°C and 30°C a maximum of 91 % and 96 % Ibuprofen separation efficiency was achieved respectively.


Page 19 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Variation in permeate-Ibuprofen concentration (a) with increasing pH of Ibuprofen feed solution (b) at different temperature (20, 30 and 40 °C) with increasing time of contact Based on results obtained from current study applicability of Sericin for membrane modification and specific removal of micro-pollutants has been compared with existing adsorbents in Table 2. Sericin shows very spontaneous binding and fast removal compare to other adsorbents, highly specific, suitable for membrane modification owing to its functional groups, antifouling and antibacterial properties. Table 2: Comparison of different adsorbents for removal of drug based micro-pollutants Sl. No.

Adsorbents

Efficiency

Remarks

References

1.

Granular Activated

80-98%

not specific towards drugs,

10,44–46

Carbon

quick breakthrough, regeneration issue, not suitable for polymeric membrane modification,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 45

prone to fouling 2.

Powdered Activated

>90%-99%

Carbon


10,47,48

high doses, long contact time, not suitable for polymeric membrane modification, prone to fouling

3.

Carbon nanotubes

>90%


49

not suitable for polymeric membrane modification 4.

Chitosan

>90%

Specific towards some

50

drug, high reusability, suitable for polymeric membrane application, antifouling properties 5.

Cyclodextrin

>80%

Specific towards drug,

51

high reusability, suitable for polymeric membrane application 6.

Zeolites

99%

Nonspecific, Highly pH

52

dependent, not suitable for polymeric membrane modification 7.

Clay

88-90%

Long contact time,

53,54

strongly affected by temp. and pH 8.

Silica

>95%

Drug specific, Long

55

contact time, suitable for membrane grafting 9.

Sericin

Complete

Highly specific, very

*current

removal

spontaneous fast removal,

study 20

ACS Paragon Plus Environment

Page 21 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


applicable at neutral pH, suitable for membrane application, antifouling and antibacterial properties

3.2.6. RO-membrane sheet characterisation FE-SEM images were obtained to study the morphological structure of the top surface of the RO sheet (Figure 6). The FE-SEM images before filtration (see Figure 6a, b) shows densely packed, interconnected, sponge-like structure with mesoporous voids in between the structure. While, the FE-SEM images of RO-sheet after filtration (see Figure 6c, d) clearly shows deposition of Sericin-ibuprofen complex on to the membrane.

Figure 6. FE-SEM images for RO membrane before filtration at (a); 25KX, (b); 50KX and after filtration (c) at 25KX, (d) 50 KX 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 45

3.3.Sericin and Ibuprofen interaction study 3.3.1. ATR-FTIR The ATR-FTIR Spectra of Sericin, Ibuprofen and Sericin-ibuprofen complex was analysed for binding confirmation and to study molecular interaction between Ibuprofen and Sericin (see Figure 7a-c). Ibuprofen spectra shows characteristic peaks at 1700 cm-1 for C=O (carbonyl stretching), 3345 cm-1 for O-H stretching of the COOH group, 2800-3100 cm-1 for alkyl stretching (3052 and 3026 cm-1 for C-H symmetric and antisymmetric stretching; 2960 cm-1 for C-H stretching; 2950 cm-1 for CH2 antisymmetric stretching; 2926 cm-1 for CH3 in-phase symmetric stretching; 2905 and 2842 cm-1 for CH stretching, 1500-1600 cm-1 for C-C stretching, 1400-1500 for CH2 deformation, 1300-1400 cm-1 for C-H bend, 1100-1200 cm-1 for C-H and CO-H in plane bending and 1000-400 cm-1 for aromatic and alkene bends. Ibuprofen characteristic peak at 1700 for C=O (carbonyl stretch) and at 3345 cm-1 for OH-stretch of carboxyl group was found to be missing from the Sericin-ibuprofen complex spectra, which validates interaction between carboxyl group of Ibuprofen and amide groups present in the Sericin. The OH-stretch peak of Sericin at 3260 cm-1 shifted to 3280 cm-1 while, Ibuprofen peaks at 3052 cm-1, 2950 cm1

, 2926 cm-1 and 2868 cm-1 re-appeared together with peaks for aromatic and alkene bends (500-

1000 cm-1) in Sericin-ibuprofen complex and hence confirms Ibuprofen adsorption on Sericin. Further to calculate the variation in corresponding IR vibrational frequencies in Amide -1 region the following analysis is presented. Amide-I bands has been widely used to identify the changes in protein secondary structure

56–58

. Considering the complexity involved in analysis of other

types of amide bands, they are generally not used for protein structure conformational analysis. Native protein generally consists of more than one secondary structure and hence absorbance usually overlap for amide I region and provide rise to merged featureless band

59

. Hence,


Page 23 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


quantitative information about secondary structure modification was obtained by linear curvefitting analysis of the component bands. Fourier self-deconvulation and second derivative procedure was used to characterize individual overlapping absorption

59,60

for native Sericin as

well as Sericin–ibuprofen complex. The broad amide I curve was resolved in to five distinct band using second derivative spectrum peak position which are attributable to different secondary structure folding. To determine the proportion of resolved components gaussian curves were iteratively fitted to deconvolved spectra.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 45

Figure 7. ATR-FTIR spectra of Sericin-ibuprofen complex, Sericin and Ibuprofen


Page 25 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Curve-fitted spectrum of the protein amide I region. (a) Pure Sericin (b) Sericinibuprofen complex. Five gaussian curves were fitted iteratively to the deconvolved curve using the peak position obtained from the second derivative spectrum as initial parameters. Prior to curve fitting, baseline correction and seven-point savaitzky-golay smoothing was used for the amide I region curve. Excellent agreement was noticed for band positions of the fitted curves with those obtained by second derivative spectrum. Secondary structure conformation to overlapped five single bands were assigned as follows and according to the previous studies 56,58: band at 1629 cm−1 as β-sheet, 1648 cm−1 as random coil, 1659 cm−1 as α-helices, 1668 cm−1 and 1680cm−1 as β-turns, and 1697 cm-1 as antiparallel β-sheet. The percent content of individual secondary structure components in Sericin and Sericin-ibuprofen complex were calculated (see Table 3 and Table 4) and their distribution were shown in Figure 8a-b. It was found that in native state Sericin has higher content of random coil (39.17%) and β-sheet (34.35%), similar trend was reported in earlier literatures

61–63

. In the Sericin-ibuprofen complex random coil

conformation (55.24%) increased in content while β-sheet (6.20%) was noticed to decrease 25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 45

subsequently. These indicate that Ibuprofen can bind to the exposed unfolded polypeptide chain of Sericin. Hence, disordered random coil structure became dominant characteristic of complex after the adsorption of Ibuprofen on to Sericin. Table 3. Assignment of Individual Amide I components (Sericin native protein) Assignment

Frequency (cm-1)

Peak area (%)

β-sheet

1629

34.35506

Random coil

1648

39.17869

α-Helix

1659

5.43163

β-turn

1670, 1682

8.478, 11.43774

Antiparallel β-sheet

1697

1.11887

Table 4. Assignment of Individual Amide I components (Sericin-Ibuprofen complex after adsorption) Assignment

Frequency (cm-1)

Peak area (%)

β-sheet

1629

6.20341

Random coil

1648

55.24681

α-Helix

1659

7.73929

β-turn

1670, 1682

12.25749, 16.92279

Antiparallel β-sheet

1697

1.63021


Page 27 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3.2. Steady state fluorescence spectroscopy Although individual content of the tyrosine and phenylalanine vary somewhat across reported literatures,

40,62

their presence in Sericin can be identified by measuring fluorescence level.

Quenching studies employing tyrosine intrinsic fluorescence of Sericin is an indispensable approach to analyze the interior location of fluorophore and subsequent structural alteration after Ibuprofen binding. Sericin exhibited intrinsic fluorescence when excited at 280 nm to show emission spectra in the 290–400 nm range with highest intensity peak at 308±1 nm (see Figure 9a). As shown in Figure 9a, the fluorescence intensity is found to be decreasing while adding Ibuprofen. This phenomenon confirms that, fluorophores (i.e. tyrosine) buried inside protein structure is coming out in solvent phase and that leads to decrease in fluorescence intensity64. Fluorescence quenching can be dynamic, which results from collision of fluorophore and quencher, or static, when ground state complex forms between the fluorophore and quencher. Fluorescence quenching is explained by most studied Stern-Volmer equation 64; = 1 + Where F0 and F represents the fluorescence intensity in absence and presence of quencher (Ibuprofen), respectively, Ksv is the Stern-Volmer quenching constant, [Q] is the concentration of quencher. The Stern–Volmer plot (see Figure 9b) of the quenching of Sericin fluorescence with Ibuprofen show a good linear relationship between Q & (F/F0) (R2= 0.9937) and estimated slope (Ksv ) is 9.196 x 104 ± 0.02581 Lmol-1. The observed fluorescence quenching intensity data can also be used to obtain the binding constant (Ka) and the number of binding sites (n). When drug molecules bind independently to a set of equivalent sites on a protein macro-molecule, the equilibrium between free and bound molecules is given by below given relationship 65,66; 27 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 45

( − ) = +

The value of Ka and n were obtained from intercept and slope of double-logarithm curve (log [(F0 − F)/F] vs. log [ibu]), as shown in Figure 9c. The calculates number of binding sites (n) ~0.98, indicating that there was one Sericin binding site for Ibuprofen and Ka was found to be 9.09 x 104 ± 0.01654 Lmol-1. Protein can be unfolded by disturbing the weak interactions that maintains the folded structure (i.e. hydrogen bonding, electrostatic interactions, and hydrophobic interactions). Ibuprofen binding must have modified the polypeptide conformation in an unfolded structure and the same was confirmed in FTIR, using amide I spectrum, where β-sheet conformation decreased and random coil conformation was found to be increased after Sericinibuprofen complex formation. The binding thermodynamics of Ibuprofen-Sericin was further investigated through ITC.


Page 29 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. (a) The quenching effect of Ibuprofen on Sericin fluorescence intensity, λmax=308±2 (b) Stern-volmer plot (c) double logarithmic plot; for quenching of Sericin by Ibuprofen. 29 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 45

3.3.3. Isothermal Titration Calorimetry ITC is an efficient method to study the binding affinity between protein and drug molecules and provide binding constant (Kb), binding stoichiometry (n), total enthalpy change (∆H) as well as total entropy change (∆S) involved during the binding reaction

41

. The ITC thermogram of

Ibuprofen titration with Sericin has shown best fitting in one set of binding model after subtraction of buffer and highlighted the presence of single binding site in Sericin for Ibuprofen with endothermic pattern in thermogram (Figure 10). ITC analysis also revealed the binding affinity (Kb) value 2.51 x 104±1.4 at 27°C that conclude moderate binding of Ibuprofen to the Sericin protein. Diverse potential interaction forces such as hydrophobic, electrostatic, hydrogen bonds and van der Waal’s are usually involved in such interaction. Therefore, ITC analysis also can reveal the driving force of corresponding interaction based on binding thermodynamics. If ∆H0, hydrophobic interaction is dominant while ∆H0 signify the dominancy of electrostatic force

41

.

Therefore, positive values of ∆H and ∆S also has revealed that hydrophobic interactions are the predominant driven force in present binding. Thermodynamic parameters of drug binding with Sericin protein has been listed in Table 5. The values of -T∆S were determined by ∆Gapp = ∆HT∆S, while ∆H and ∆S values were obtained directly from multi-injection mode ITC experiment.


Page 31 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. ITC thermogram for the binding of Ibuprofen to Sericin at 27 °C in 10mM phosphate buffer (a) primary raw data (b) binding curve derived from the raw data Table 5. Thermodynamic parameters of the Sericin and Ibuprofen interaction obtained by ITC at 27°C temperature.

Stoichiometry(n)

∆H

∆S

∆Gapp

(Kcal/mol)

(Kcal/mol.K)

(Kcal/mol)

4.62±0.36

36.18

-31.9

Kb (mole-1)

Sericin-Ibuprofen 1.15±0.021

2.51E4±1.4

interaction



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 45

The binding of Ibuprofen to Sericin protein was found to be overall endothermic in nature, the upward peaks indicates heat intake while adding Ibuprofen into Sericin solution. ∆H provide information about total energy of process and include contribution from solute as well as solvent and it is barely plausible to form favourable interaction without affecting each other. Indeed the ∆H of Sericin-ibuprofen binding involves formation and disruption of individual interactions, which includes (i) the loss of hydrogen bonds and van der walls interaction (between Sericin and solvent and between Ibuprofen and solvent), (ii) the formation of non-covalent interaction (between Sericin and Ibuprofen) and (iii) solvent restructuring near the complex surface 67. These phenomenon lead to net enthalpy change. Similarly, binding entropy ∆S is contributed by following three phenomenon such as, (i) solvent entropy change (solvent release upon binding), (ii) conformational entropy change (conformational freedom of both Sericin and Ibuprofen upon binding), and (iii) change in rotational and translational entropy (reduction in degrees of freedom upon complex formation)

67

. These three entropic terms represent the net entropy change, with

negative or positive contribution to binding free energy. A negative binding free energy (∆Gapp = -31.9Kcal/mol) shows spontaneous binding mediated by enthalpy-entropy compensation for Sericin-ibuprofen complex formation. Ibuprofen adsorption on Sericin can be considered to be an analog of protein unfolding, since Ibuprofen adsorption and protein denaturation are coalesced with each other. As revealed in FTIR the protein unfolds from beta sheet to unordered random coil structure, which may be related to the endothermic effect. Hydrophobic interactions play main role to the binding interaction and presence of polar group may be related to the positive values of ∆H° and ∆S°. Hence it was assumed that Sericin protein unfolding to random coil conformation was dominant factor for Sericin-ibuprofen complex formation i.e. adsorption process.


Page 33 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3.4. FESEM FE-SEM images of Sericin, Ibuprofen and Sericin-ibuprofen complex revealed the adsorption of Ibuprofen on Sericin protein (see Figure 11). Surface texture and morphology of Ibuprofen crystals are visualized (in Figure 11b, e), which can be seen very clearly to be deposited on the protein surface (in Figure 11c, f). Pure Sericin without Ibuprofen, as a control, is shown in Figure 11a, d. The complex particles were found to have dotted chiselled Ibuprofen structure adsorbed on the native Sericin protein surface.

Figure 11. FE-SEM of Sericin, Ibuprofen and Sericin-ibuprofen complex at 25 KX (a, b and c), at 50 KX (e, f, and g), respectively



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 45

3.3.5. XRD The XRD results for Sericin exhibited a large diffraction peak at around 2θ=21.15°, the broad curve indicates amorphous nature (see Figure 12). Similar XRD profile with peak at 2θ=19.2º has been reported

68

. This peak is characteristic of the β-sheet structure due to intermolecular

hydrogen bonding between the hydroxyl groups of the amino acid present in Sericin. Similar results have been reported in earlier studies with Sericin obtained from different sources

30,63

.

Ibuprofen XRD peaks shows its characteristic crystallinity and was found to be in good agreement with previous reported literatures

69

. The Sericin-ibuprofen complex XRD plot was

crystalline in nature and retained most of the ibuprofen characteristic peaks hence depicting adsorption of Ibuprofen on Sericin.

Figure 12. XRD profile for Ibuprofen, Sericin and Sericin-ibuprofen complex


Page 35 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. Conclusion This work deals with evaluation of silk Sericin as an adsorbent for removal of Ibuprofen from aqueous solution using an integrated adsorption-cum-membrane filtration setup. The preliminary studies on Sericin characterization was done to assess its physiochemical properties relative for adsorption. The non-porous Sericin was found to have low surface area and conformational change were noticed with exposure to increasing temperature. Its adsorption ability was analysed at different operating conditions (concentration, temperature and pH). High pH and increasing temperature was found to assist in better adsorption of Ibuprofen. Complete removal for Ibuprofen was achieved at pH 8, 40ºC and using 10g/L of Sericin concentration. Ibuprofen interaction with Sericin was investigated and endothermic peaks in ITC together with FTIR study provided information about protein unfolding playing dominant role in adsorption. Ibuprofen binding was related to Sericin transition to random coil structure. Sericin- ibuprofen binding and complex formation was established using FTIR, ITC, Fluorescence spectroscopy and XRD analysis. Use of Sericin with membrane process seems to enhance and achieve expected removal capacity. We hope this study will help in modification of existing filtration processes and provide a better solution for removal of micro-pollutants from aqueous solution. Supporting Information: Standard HPLC Calibration curve for Ibuprofen, Flat Sheet RO-membrane dimension and effective area, Pure water flux (Jw) for RO-sheet membrane, Variation in pH with Sericin Concentration, Ibuprofen removal at different temperature with time of contact. Acknowledgments: The authors are thankful to Ajeet Singh, Department of Bio Sciences & Bioengineering, IIT-Guwahati, India, for his kind help in Fluorescence Spectroscopy and ITC



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 45

analysis and for helpful discussions, and to the Central Instrument Facility, IIT-Guwahati, India. This research was financially supported by Indian Institute of Technology, Guwahati, India. References (1)

Halling-Sorensen, B.; Nielsen, S. N.; Lanzky, P. F.; Ingerslev, F.; Holten Lutzhoft, H. C.; Jørgensen, S.E. Occurence, Fate and Effects of Pharmaceuticals Substance in the Environment - A Review. Chemosphere 1998, 36, 357.

(2)

Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Pharmaceuticals, Hormones, and Other Organic Wastewater Contaminants in U.S. Streams, 1999-2000: A National Reconnaissance. Environ. Sci. Technol. 2002, 36, 1202.

(3)

Heberer, T. Occurrence, Fate, and Removal of Pharmaceutical Residues in the Aquatic Environment: A Review of Recent Research Data. Toxicol. Lett. 2002, 131, 5.

(4)

Fick, J.; Soderstrom, H.; Lindberg, R. H.; Phan, C.; Tysklind, M.; Larsson, J. D. G. Contamination of Surface, Ground, and Drinking Water From Pharmaceutical Production. Environ. Toxicol. Chem. 2009, 28, 2522.

(5)

Fent, K.; Weston, A. A.; Caminada, D. Ecotoxicology of Human Pharmaceuticals. Aquat. Toxicol. 2006, 76, 122.

(6)

Thibaut, R.; Schnell, S.; Porte, C. The Interference of Pharmaceuticals with Endogenous and Xenobiotic Metabolizing Enzymes in Carp Liver: An in-Vitro Study. Environ. Sci. Technol. 2006, 40, 5154.

(7)

De Lange, H. J.; Noordoven, W.; Murk, A. J.; Lürling, M.; Peeters, E. Behavioural


Page 37 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Responses Of< I> Gammarus pulex(Crustacea, Amphipoda) to Low Concentrations of Pharmaceuticals. Aquat. Toxicol. 2006, 78, 209. (8)

Oaks, J. L.; Gilbert, M.; Virani, M. Z.; Watson, R. T.; Meteyer, C. U.; Rideout, B. a; Shivaprasad, H. L.; Ahmed, S.; Chaudhry, M. J. I.; Arshad, M.; Mahmood, S.; Ali, A.; Khan, A. A. Diclofenac Residues as the Cause of Vulture Population Decline in Pakistan. Nature 2004, 427, 630.

(9)

Gadipelly, C.; Pérez-González, A.; Yadav, G. D.; Ortiz, I.; Ibáñez, R.; Rathod, V. K.; Marathe, K. V. Pharmaceutical Industry Wastewater: Review of the Technologies for Water Treatment and Reuse. Ind. Eng. Chem. Res. 2014, 53, 11571.

(10)

Westerhoff, P.; Yoon, Y.; Snyder, S.; Wert, E. Fate of Endocrine-Disruptor, Pharmaceutical, and Personal Care Product Chemicals during Simulated Drinking Water Treatment Processes. Environ. Sci. Technol. 2005, 39, 6649.

(11)

Pinkston, K. E.; Sedlak, D. L. Transformation of Aromatic Ether- and Amine-Containing Pharmaceuticals during Chlorine Disinfection. Environ. Sci. Technol. 2004, 38, 4019.

(12)

Gupta, V. K.; Jain, R.; Mittal, A.; Saleh, T. A.; Nayak, A.; Agarwal, S.; Sikarwar, S. Photo-Catalytic Degradation of Toxic Dye Amaranth on TiO2/UV in Aqueous Suspensions. Mater. Sci. Eng. C 2012, 32, 12.

(13)

Boorman, G. A.; Dellarco, V.; Dunnick, J. K.; Chapin, R. E.; Hunter, S.; Hauchman, F.; Gardner, H.; Cox, M.; Sills ’, R. C. Drinking Water Disinfection Byproducs: Review and Approach to Toxicity Evaluation. -Environ Heal. Perspect 1999, 107, 207.

(14)

Rajendran, S.; Khan, M. M.; Gracia, F.; Qin, J.; Gupta, V. K.; Arumainathan, S. Ce3+-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 45

Ion-Induced Visible-Light Photocatalytic Degradation and Electrochemical Activity of ZnO/CeO2 Nanocomposite. 2016, 6, 31641. (15)

von Gunten, U.; Hoigne, J. Bromate Formation during Ozonation of Bromide-Containing Waters: Interaction of Ozone and Hydroxyl Radical Reactions. Environ. Sci. Technol. 1994, 28, 1234.

(16)

Gupta, V. K.; Ali, I.; Saleh, T. A.; Nayak, A.; Agarwal, S. Chemical Treatment Technologies for Waste-Water Recycling-an Overview. RSC Adv. 2012, 2, 6380.

(17)

Yoon, Y.; Westerhoff, P.; Snyder, S. A.; Wert, E. C. Nanofiltration and Ultrafiltration of Endocrine Disrupting Compounds, Pharmaceuticals and Personal Care Products. J. Memb. Sci. 2006, 270, 88.

(18)

Muldar, M. Basic Principles of Membrane Technology; Springer Science & Business Media, 1997.

(19)

Yangali-quintanilla, V.; Maeng, S. K.; Fujioka, T.; Kennedy, M.; Li, Z.; Amy, G. Nanofiltration vs. Reverse Osmosis for the Removal of Emerging Organic Contaminants in Water Reuse. Desalin. Water Treat. 2011, 34, 50.

(20)

Kimura, K.; Toshima, S.; Amy, G.; Watanabe, Y. Rejection of Neutral Endocrine Disrupting Compounds (EDCs) and Pharmaceutical Active Compounds (PhACs) by RO Membranes. J. Memb. Sci. 2004, 245, 71.

(21)

Vergili, I. Application of Nanofiltration for the Removal of Carbamazepine, Diclofenac and Ibuprofen from Drinking Water Sources. J. Environ. Manage. 2013, 127, 177.

(22)

Radjenović, J.; Petrović, M.; Ventura, F.; Barceló, D. Rejection of Pharmaceuticals in


Page 39 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nanofiltration and Reverse Osmosis Membrane Drinking Water Treatment. Water Res. 2008, 42, 3601. (23)

Gupta, V. K.; Saleh, T. A. Sorption of Pollutants by Porous Carbon, Carbon Nanotubes and Fullerene- An Overview. Environ. Sci. Pollut. Res. 2013, 20, 2828.

(24)

Tiek Wong, K.; Yoon, Y.; Jang, M. Enhanced Recyclable Magnetized Palm Shell WasteBased Powdered Activated Carbon for the Removal of Ibuprofen: Insights for Kinetics and Mechanisms. PLoS One 2015, 10, e0141013.

(25)

Attia, T. M. S.; Hu, X. L.; Qiang, Y. Da. Synthesized Magnetic Nanoparticles Coated Zeolite for the Adsorption of Pharmaceutical Compounds from Aqueous Solution Using Batch and Column Studies. Chemosphere 2013, 93, 2076.

(26)

Behera, S. K.; Oh, S. Y.; Park, H. S. Sorptive Removal of Ibuprofen from Water Using Selected Soil Minerals and Activated Carbon. Int. J. Environ. Sci. Technol. 2012, 9, 85.

(27)

Bui, T. X.; Choi, H. Adsorptive Removal of Selected Pharmaceuticals by Mesoporous Silica SBA-15. J. Hazard. Mater. 2009, 168, 602.

(28)

Liu, Y.; Guo, Y.; Zhu, Y.; An, D.; Gao, W.; Wang, Z.; Ma, Y.; Wang, Z. A Sustainable Route for the Preparation of Activated Carbon and Silica from Rice Husk Ash. J. Hazard. Mater. 2010, 186, 1314.

(29)

Saleh, T. A.; Gupta, V. K. Synthesis and Characterization of Alumina Nano-Particles Polyamide Membrane with Enhanced Flux Rejection Performance. Sep. Purif. Technol. 2012, 89, 245.

(30)

Gupta, D.; Agrawal, A.; Rangi, A. Extraction and Characterization of Silk Sericin. Indian



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 45

J. Fibre Text. Res. 2014, 39, 364. (31)

Wu, J. H.; Wang, Z.; Xu, S. Y. Preparation and Characterization of Sericin Powder Extracted from Silk Industry Wastewater. Food Chem. 2007, 103, 1255.

(32)

Zhang, Y. Q. Applications of Natural Silk Protein Sericin in Biomaterials. Biotechnol. Adv. 2002, 20, 91.

(33)

Chun, M. K.; Choi, H. K.; Kang, D. W.; Kim, O. J.; Cho, C. S. A Mucoadhesive Polymer Prepared by Template Polymerization of Acrylic Acid in the Presence of Poly(ethylene Glycol) Macromer. J. Appl. Polym. Sci. 2002, 83, 1904.

(34)

Nagura, M.; Ohnishi, R.; Gitoh, Y.; Faculty, O.; Science, T.; October, R.; February, A. Structures and Physical Properties of Cross-Linked Sericin Membranes. J Insect Biotechnol Sericol 2001, 70, 149.

(35)

Li, H.; Shi, W.; Wang, W.; Zhu, H. The Extraction of Sericin Protein from Silk Reeling Wastewater by Hollow Fiber Nanofiltration Membrane Integrated Process. Sep. Purif. Technol. 2015, 146, 342.

(36)

Kasprzyk-Hordern, B. Pharmacologically Active Compounds in the Environment and Their Chirality. Chem. Soc. Rev. 2010, 39, 4466.

(37)

Wang, Y.; Shen, C.; Li, L.; Li, H.; Zhang, M. Electrocatalytic Degradation of Ibuprofen in Aqueous Solution by a Cobalt-Doped Modified Lead Dioxide Electrode: Influencing Factors and Energy Demand. RSC Adv. 2016, 6, 30598.

(38)

Fabiani, C.; Pizzichini, M.; Spadoni, M.; Zeddita, G. Treatment of Waste Water from Silk Degumming Processes for Protein Recovery and Water Reuse. Desalination 1996, 105, 1.


Page 41 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(39)

Capar, G. Separation of Silkworm Proteins in Cocoon Cooking Wastewaters via Nanofiltration: Effect of Solution pH on Enrichment of Sericin. J. Memb. Sci. 2012, 389, 509.

(40)

Wang, Z.; Zhang, Y.; Zhang, J.; Huang, L.; Liu, J.; Li, Y.; Zhang, G.; Kundu, S. C.; Wang, L. Exploring Natural Silk Protein Sericin for Regenerative Medicine: An Injectable, Photoluminescent, Cell-Adhesive 3D Hydrogel. Sci. Rep. 2014, 4, 7064.

(41)

Du, X.; Li, Y.; Xia, Y.-L.; Ai, S.-M.; Liang, J.; Sang, P.; Ji, X.-L.; Liu, S.-Q. Insights into Protein-Ligand Interactions: Mechanisms, Models, and Methods. Int. J. Mol. Sci. 2016, 17, 144.

(42)

Chen, X.; Lam, K. F.; Mak, S. F.; Ching, W. K.; Ng, T. N.; Yeung, K. L. Assessment of Sericin Biosorbent for Selective Dye Removal. Chinese J. Chem. Eng. 2012, 20, 426.

(43)

Drake, J. A.; Kenny, D. A.; Voskuil, T. Environmental Biotechnology. In Bioscience; Kumar, A., Ed.; Daya Books: New Delhi, 1988, 38, 420.

(44)

Grover, D. P.; Zhou, J. L.; Frickers, P. E.; Readman, J. W. Improved Removal of Estrogenic and Pharmaceutical Compounds in Sewage Effluent by Full Scale Granular Activated Carbon: Impact on Receiving River Water. J. Hazard. Mater. 2011, 185, 1005.

(45)

Rossner, A.; Snyder, S. A.; Knappe, D. R. U. Removal of Emerging Contaminants of Concern by Alternative Adsorbents. Water Res. 2009, 43, 3787.

(46)

Snyder, S. A.; Adham, S.; Redding, A. M.; Cannon, F. S.; DeCarolis, J.; Oppenheimer, J.; Wert, E. C.; Yoon, Y. Role of Membranes and Activated Carbon in the Removal of Endocrine Disruptors and Pharmaceuticals. Desalination 2007, 202, 156.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(47)

Page 42 of 45

Koley, P.; Sakurai, M.; Aono, M. Controlled Fabrication of Silk Protein Sericin Mediated Hierarchical Hybrid Flowers and Their Excellent Adsorption Capability of Heavy Metal Ions of Pb(II), Cd(II) and Hg(II). ACS Appl. Mater. Interfaces 2016, 8, 2380.

(48)

Li, X.; Li, L.; Zheng, M.; Fu, G.; Lar, J. S. Anaerobic Co-Digestion of Cattle Manure with Corn Stover Pretreated by Sodium Hydroxide for Efficient Biogas Production. Energy & Fuels 2009, 23, 4635.

(49)

Heo, J.; Joseph, L.; Yoon, Y.; Park, Y.-G.; Her, N.; Sohn, J.; Yoon, S.-H. Removal of Micropollutants and NOM in Carbon Nanotube-UF Membrane System from Seawater. Water Sci. Technol. 2011, 63, 2737.

(50)

Zhang, Y.-L.; Zhang, J.; Dai, C.-M.; Zhou, X.-F.; Liu, S.-G. Sorption of Carbamazepine from Water by Magnetic Molecularly Imprinted Polymers Based on Chitosan-Fe₃O₄. Carbohydr. Polym. 2013, 97, 809.

(51)

Alsbaiee, A.; Smith, B. J.; Xiao, L.; Ling, Y.; Helbling, D. E.; Dichtel, W. R. Rapid Removal of Organic Micropollutants from Water by a Porous β-Cyclodextrin Polymer. Nature 2016, 529, 190.

(52)

Ötker, H. M.; Akmehmet-Balcıoğlu, I. Adsorption and Degradation of Enrofloxacin, a Veterinary Antibiotic on Natural Zeolite. J. Hazard. Mater. 2005, 122, 251.

(53)

Bekçi, Z.; Seki, Y.; Yurdakoç, M. K. Equilibrium Studies for Trimethoprim Adsorption on Montmorillonite KSF. J. Hazard. Mater. 2006, 133, 233.

(54)

Putra, E. K.; Pranowo, R.; Sunarso, J.; Indraswati, N.; Ismadji, S. Performance of Activated Carbon and Bentonite for Adsorption of Amoxicillin from Wastewater:


Page 43 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Mechanisms, Isotherms and Kinetics. Water Res. 2009, 43, 2419. (55)

Kim, Y.-H.; Lee, B.; Choo, K.-H.; Choi, S.-J. Selective Adsorption of Bisphenol A by Organic–inorganic Hybrid Mesoporous Silicas. Microporous Mesoporous Mater. 2011, 138, 184.

(56)

Teramoto, H.; Miyazawa, M. Molecular Orientation Behavior of Silk Sericin Film as Revealed by ATR Infrared Spectroscopy. Biomacromolecules 2005, 6, 2049.

(57)

Jackson, M.; Mantsch, H. H. Protein Secondary Structure from FT-IR Spectroscopy: Correlation with Dihedral Angles from Three-Dimensional Ramachandran Plots. Can. J. Chem. 1991, 69, 1639.

(58)

Zhang, H.; Deng, L.; Yang, M.; Min, S.; Yang, L.; Zhu, L. Enhancing Effect of Glycerol on the Tensile Properties of Bombyx Mori Cocoon Sericin Films. Int. J. Mol. Sci. 2011, 12, 3170.

(59)

Byler, D. M.; Susi, H. Examination of the Secondary Structure of Proteins by Deconvolved FTIR Spectra. Biopolymers 1986, 25, 469.

(60)

Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Determination of Protein Secondary Structure by Fourier Transform Infrared Spectroscopy: A Critical Assessment. Biochemistry 1993, 32, 389.

(61)

Teramoto, H.; Kakazu, A.; Asakura, T. Native Structure and Degradation Pattern of Silk Sericin Studied by 13C NMR Spectroscopy. Macromolecules 2006, 39, 6.

(62)

Huang, J.; Valluzzi, R.; Bini, E.; Vernaglia, B.; Kaplan, D. L. Cloning, Expression, and Assembly of Sericin-like Protein. J. Biol. Chem. 2003, 278, 46117.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(63)

Page 44 of 45

Dash, R.; Ghosh, S. K.; Kaplan, D. L.; Kundu, S. C. Purification and Biochemical Characterization of a 70??kDa Sericin from Tropical Tasar Silkworm, Antheraea Mylitta. Comp. Biochem. Physiol. - B Biochem. Mol. Biol. 2007, 147, 129.

(64)

Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Springer Science & Business Media, 2006.

(65)

Yu, X.; Yang, Y.; Shiyu, L.; Yao, Q.; Heting, L.; Xiaofang, L.; Pinggui, Y. The Fluorescence Spectroscopic Study on the Interaction between imidazo[2,1-B]thiazole Analogues and Bovine Serum Albumin. Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 2011, 83, 322.

(66)

Mandal, G.; Bardhan, M.; Ganguly, T. Interaction of Bovine Serum Albumin and Albumin-Gold Nanoconjugates with L-Aspartic Acid. A Spectroscopic Approach. Colloids Surfaces B Biointerfaces 2010, 81, 178.

(67)

Perozzo, R.; Folkers, G.; Scapozza, L. Thermodynamics of Protein–Ligand Interactions: History, Presence, and Future Aspects. J. Recept. Signal Transduct. 2004, 24, 1.

(68)

Silva, V. R.; Hamerski, F.; Weschenfelder, T. A.; Ribani, M.; Gimenes, M. L.; Scheer, A. P. Equilibrium, Kinetic, and Thermodynamic Studies on the Biosorption of Bordeaux S Dye by Sericin Powder Derived from Cocoons of the Silkworm Bombyx Mori. Desalin. Water Treat. 2016, 57, 5119.

(69)

Dudognon, E.; Danède, F.; Descamps, M.; Correia, N. T. Evidence for a New Crystalline Phase of Racemic Ibuprofen. Pharm. Res. 2008, 25, 2853.


Page 45 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract:


Prospects of Silk Sericin as an Adsorbent for Removal of Ibuprofen

Recommend Documents