Fibrous keratin protein bio micro structure for efficient removal of hazardous dye waste from water: Surface charge mediated interfaces for multiple adsorption desorption cycles

Journal Pre-proof Fibrous keratin protein bio micro structure for efficient removal of hazardous dye waste from water: Surface charge mediated interfaces for multiple adsorption desorption cycles Nadeeka D. Tissera, Ruchira N. Wijesena, Harini Yasasri, K.M. Nalin de Silva, Rohini M. de Silva PII:

S0254-0584(20)30169-3

DOI:

https://doi.org/10.1016/j.matchemphys.2020.122790

Reference:

MAC 122790

To appear in:

Materials Chemistry and Physics

Received Date: 9 December 2019 Revised Date:

1 February 2020

Accepted Date: 10 February 2020

Please cite this article as: N.D. Tissera, R.N. Wijesena, H. Yasasri, K.M.N. de Silva, R.M. de Silva, Fibrous keratin protein bio micro structure for efficient removal of hazardous dye waste from water: Surface charge mediated interfaces for multiple adsorption desorption cycles, Materials Chemistry and Physics (2020), doi: https://doi.org/10.1016/j.matchemphys.2020.122790. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier B.V. All rights reserved.

Fibrous keratin protein bio micro structure for efficient removal of hazardous dye waste from water: Surface charge mediated interfaces for multiple adsorption desorption cycles

Nadeeka D. Tissera,1,2 Ruchira N. Wijesena,1,2 Harini Yasasri2, K.M. Nalin de Silva,1 Rohini M. de Silva1* 1

Centre for Advanced Materials and Devices (CAMD), Department of Chemistry, University of

Colombo, Colombo 00300, Sri Lanka. 2

Sri Lanka Institute of Nanotechnology (SLINTEC), Nanotechnology and Science Park,

Mahenwatte, Pitipana, Homagama 10200, Sri Lanka.

*Corresponding Author: Rohini M. de Silva Email: [email protected]

Fibrous keratin protein bio micro structure for efficient removal of hazardous dye waste from water: Surface charge mediated interfaces for multiple adsorption desorption cycles

Nadeeka D. Tissera,1,2 Ruchira N. Wijesena,1,2 Harini Yasasri2, K.M. Nalin de Silva,1 Rohini M. de Silva1* 1

Centre for Advanced Materials and Devices (CAMD), Department of Chemistry, University of Colombo, Colombo 03, Sri Lanka

2

Sri Lanka Institute of Nanotechnology (SLINTEC), Nanotechnology & Science park, Mahenwatta, Pitipana, Homagama, Sri Lanka

ABSTRACT For the first time we report fabrication of a keratin protein based bio material for efficient removal of hazardous dye waste from water using merino wool fibers. Surface hydrolyzed keratin protein fibers were prepared by subjecting merino wool fibers to controlled hydrolysis. During the hydrolysis the cuticle cells which sheath the hierarchical microstructure of the wool fibers were denatured. This has resulted in exposing ortho cortex of the fibers with comparatively higher degree of free amine and other surface functional groups which were confirmed by the SEM, EDX, AFM, FT-IR, NMR and CHNS analysis. The material showed exceptional adsorption capacities for carcinogenic Rhodamine B dye in the presence of acetic acid. Under optimized conditions, the surface hydrolyzed keratin protein fibers showed ~95% of dye adsorption with maximum adsorption capacity of 294 mg/g at 298 K, in the presence of 3.5 % acetic acid. This is the highest reported for the Rodhamine dye adsorption at similar conditions using other dye adsorption methods. Adsorption isotherm studies and Scatchard analysis of the isotherms were carried out to understand the binding mechanism of the dye molecules to the keratin protein fiber material. The main chemical attractions were found to be due to the free amine and carboxylic functionalities presence in the hydrolyzed wool fiber material. Also it was observed to have charge induced adsorption and desorption which were driven mainly due to the protonation and deprotonation of the amine groups present in the keratin protein fibers. It is also confirmed that keratin protein fiber can be used for multiple times for adsorption of the dye molecules from an aqueous medium. Combining the high adsorption capacity and recyclability, surface hydrolyzed keratin protein fibers prepared from merino wool can be a promising bio material to remove Rhodamine B dye from an aqueous medium modulated by acetic acid.

Key words: wool, Rhodamine dye, adsorption, desorption, hydrolysis, protonation

INTRODUCTION Contamination of water with dye waste has become a major issue globally. Over 10 000 varieties of dye types are used by many industries for coloration purpose. Specifically In the textile industry approximately 12 % of dye is lost during the manufacturing and dyeing processes. From these 20 % of the lost dyes enters in to water stream as industrial waste, threatening the aquatic eco system and human health [1-3]. Rhodamine B (C.I 4570) dye is a highly water soluble xanthene dye and used heavily in the textile industry. It is believed to be a carcinogenetic compound to both humans and animals with various other health implications such as respiratory track issues, irritation to skin and eyes. Therefore, Rodhamine B, has been a center of focus, for many scientific studies which aim to remove this dye from water before they enter natural water systems. . Among these, chemical precipitation, ion exchange, membrane filtration, physical adsorption, chemical oxidation/ reduction or bio-removal are the most commonly practiced methodologies for the removal of dyes and other similar hazardous dye compounds form the waste water [4-10]. From such technologies bio based adsorption or filtration membranes have attracted many interest due to its attributes as an efficient, cheap and eco-friendly approach [11-14] . On this regard, wool consist of fibrous cross-linked keratin protein consisting of highly conserved 19 amino acid sequence, rich with carbon, hydrogen, nitrogen, oxygen and sulfur , linked together by peptide bonds [15-17] has been the focal point for many research due to its biocompatibility. Material forming the bulk of keratin fibers consists of polypeptide chains which is a biological polymer. These polypeptides are formed from different individual amino acids. Wool keratin protein is high in cysteine, glycine, proline and serine, but it is low in lysine, histidine and methionine, and tryptophan is barely present [18]. Cysteine present in wool has an important role in determining its physicochemical properties. The keratin protein fibers contains diamine acid residue and cysteine in its structure which distinguishes them from other protein fibers. Also hydrogen, hydrophobic and ionic bonds play a major role for the stability and properties of the wool keratin. The ionic bonds present in wool observed to have pH dependent bond behavior where at the isoelectric point of wool which is pH 4.9 the ionic bonding is highest. At the isoelectric point protein is in the state of zwitterion (+H3N–CHR– COO–). In the presence of high acidic or basic conditions, the ionic bonds in wool are at their lowest level. The ionic bond in wool occurs between carboxylic anions and ammonium cations of the polypeptide backbone. Therefore these bonds can be reduced by protonation of the carboxylic acid groups at low pH values or deprotonation of the amine group at high pH or alkaline condition [19].

From different types of wool fibers available in the industry merino wool is a fine fiber having comparative higher fiber length [20, 21]. Also the material has high fiber strength and has a natural crimp which is due to the inter molecular crosslinking of polypeptide chains through –SS linkages [22]. This allow this specific wool material to be used in many research studies including preparation of activated carbon for waste water treatment, heavy metal adsorption from waste water and use as a low cost adsorbent for removal of methylene blue from aqueous solutions [23-25] . Therefore in this work, we report the possibility of surface modification of merino wool fibers using alkaline hydrolysis for efficient adsorption of Rodhamine B dye. Hierarchical architecture of the wool fiber was exploited through controlled alkaline hydrolysis of cuticle cells to expose inner α- keratin rich matrix. This has introduced – NH2 and –COOH groups on to the wool fiber surface. Out of these chemical functional groups amines can be protonated in the presence of acetic acid. The protonated amines in the presence of acetic acid and carboxylic acid found to be the main interaction moieties in the proposed system. Interestingly the proposed system showed an outstanding ability for the efficient removal of Rodhamine B dye. The main advantage of this system is the reusability of the material through multiple number of adsorption desorption cycles while maintaining the same efficiency of adsorption in each cycle.

EXPERIMENTAL SECTION Material Merino wool fibers (average diameter: 15 µm), Glacial acetic acid (99% purity) and sodium hydroxide with 99.8 % purity level was obtained from sigma Aldrich. Rhodamine B (C.I 4570) dye was a kind gift from Stretchline Pvt Ltd (Sri Lanka). Deionized water was used in all experiments. Preparation of absorbent Merino wool fibers was first washed using distilled water to remove any dust particles or impurities. Wool fibers (0.125 g) was hydrolyzed in 40 ml of 0.1 M, 0.3 M, 0.5 M, 0.8 M and 1 M NaOH solutions separately for 10 min at room temperature. The resulting dispersions were centrifuged at 9000 rpm, supernatant was discarded. The precipitate was washed with distilled water using centrifugation for multiple times until the pH of the wool dispersion became neutral. Washed wool samples were dried at 60 ° C to remove excess water. Hydrolyzed washed and dried wool samples were labelled as 0.1 M wool, 0.3 M wool, 0.5 M wool, 0.8 M wool and 1 M wool according to the molarity of the alkaline medium used for hydrolysis.

Physio chemical characterization The surface morphology of the wool was characterized using a Hitachi SU6600 schottky field emission scanning electron microscope (FE-SEM) operating at 15 kV and a Park Systems Atomic Force Microscope (AFM). AFM images were obtained using an XE-100 microscope. The measurements were performed in air at room temperature using non- contact mode, with Si tips of the 1650–00 type scanning at a rate of 0.5Hz. Elemental analysis of the wool samples was performed using Energy Dispersive X-Ray spectroscopy (EDX) and C, H, N & S composition of the wool samples was analyzed using Perkin Elmer 2400 Series II CHNS Analyzer (USA). Fourier transformed infrared spectra (FT-IR) of wool fibers and hydrolyzed wool fibers were obtained using Bruker, Vertex 80. Attenuated Total Reflectance (ATR) measurement mode was used with the sample mounted on a ZnSe crystal. Absorption spectra was recorded in 32 scans in the 800 cm-1 to 4000 cm-1 wave number range with 4 cm-1 resolution. UV-Vis spectrophotometer (Shimadzu UV-Vis 3600) was used for the dye adsorption studies. UV-Vis spectra of the dye samples were obtained using the transmittance mode of the spectrophotometer in the wave length range of 400 nm - 800 nm. 13C NMR spectra of the wool sample and hydrolyzed wool samples were recorded using CP MAS (crosspolarization magic angle spinning and dipolar coupling ) using a Bruker AVANCE-III spectrometer operating at 100.62 MHz. The spectra were recorded at room temperature using a rotor spinning at 5 kHz with 2 ms contact time for cross-polarization

Rhodamine dye adsorption study Wool fibers with different degree of hydrolysis and a control was first evaluated for rhodamine dye adsorption. For this study 0.04 g of hydrolysed wool fibers was added in to a 1500 ppm Rhodamine dye solution which contain 3 % acetic acid. Dye solution which contain the hydrolysed wool fibers was stirred at room temperature for 40 minutes. A wool fibers sample of 0.04 g without hydrolysis was also used for the experiment as the control. After 40 minutes dye solution was centrifuged and supernatant was analysed using UV-Vis spectrophotometer. The residual amount of dye in the dye solution was calculated by the absorbance at the λmax of 540 nm and the concentration was back calculated using a calibration curve. Wool sample which has the highest adsorption was selected for further adsorption studies.

Effect of adsorbent dosage on dye removal The effect of adsorbent dosage on the dye removal efficiency was investigated in a range from 0.1 to 1 g with a dye initial concentration of 100 mg/L (100 ppm) at 25 °C. The effect of the amount of acetic acid presence in the dye solution on the dye removal efficiency was investigated in a acetic acid concentration range of 0- 3.5 % with a dye initial concentration of 100 mg/L at 25 °C with an absorbent loading of 0.8 g/L. Effect of initial dye concentration on the adsorption capacity was investigated by using a Rhodamine dye solution in the range of 80 –

500 mg/L with adsorbent dosage of 0.8 g/L, with 3.5 % acetic acid and at 25 °C. For all experiment 100 ml of the dye solution was used and the experiments were carried out in triplicate. Percentage of dye removal and adsorption capacity was calculated using equation 1 and 2 below.

=

=

100 %

(1) (2)

Where, E is the percentage dye removal, Co and Ce (ppm) are the initial and equilibrium concentration of the dye respectively. Ct (ppm) is the dye concentration in aqueous solution at time t (min). qe (mg/g) is the amount of dye adsorbed at equilibrium per unit mass of the adsorbent, m is the mass of adsorbent (g) used and V is the volume of solution (L). RESULTS AND DISCUSSION: Surface morphological features of the wool fibers and alkaline hydrolysed wool fibers are shown in Figure 1. According to the SEM micrographs the fibers surfaces showed distinguishable surface features emerging due to the alkaline hydrolysis.

Figure 1. SEM image of (a) wool fibers (b) 0.1 M wool fibers (c) 0.3 M wool fibers (d) 0.5 M wool fibers (e) 0.8 M wool fibers (f) 1 M wool fibers As shown in Figure 1a pristine wool fibers surface has a tubular structure which consist of a scale structure on the outer most layer, which are the cuticle cells. In the hierarchical microstructure of wool fibers, cuticle cells covers the cortex and medulla [18]. These cells are

overlapped and are arranged to one direction along the length of the fibers (Figure 1a). Surface of the individual cells observed to have a smooth structure. After subjecting to alkaline hydrolysis, fibers surface showed a denaturing of its outermost layer. Gradual increment of denaturing is clearly with increase in concentration of NaOH (Figure 1a-f). For 0.1 M wool, surface of the cuticle cells are observed to be shrunken to have parallel ridges running along the length of the fibers (Fig 1 (b)). This observation is more apparent for 0.3 M wool fibers (Fig 1 (c)). According to the SEM micrograph the traces of the scales are observed on the 0.3 M wool fibers surface (Fig 1 (c)). With the increasing alkaline conditions (0.5 M wool) fiber surface showed a fibriler morphology in the sub-micrometre range and the cuticle cells were less prominent as shown in Fig 1 (d). At higher alkaline conditions, (0.8 M wool & 1 M wool) cuticle cells were all diminished (Fig 1 (e) & (f)) and fibriler like morphology on fiber surfaces was more prominent. A noticeable shrinkage of these fibrils in their diameter was observed for 0.8 M wool (Figure 2e) compared to 0.5 M wool (Fig 1 (d)). According to the morphological analysis using SEM it is clear that, with the controlled alkaline hydrolysis cuticle cells of the wool fibers could be removed in order to expose the cortex which is the 2nd hierarchical structure of the wool fibers.

Figure 2. AFM image of (a) wool fibers (b) 0.5 M wool fibers surface Surface topography of the wool fibers before alkaline hydrolysis and after subjecting for alkaline hydrolysis (0.5 M wool fibers) were further analysed using AFM. Surface topography the pristine wool fibers shows the presence of scales on the surface of wool fibers (Fig 2 (a)). For 0.5M wool the cuticle cells was not observed (Fig 2 (b)) which was also observed in SEM analysis (Fig 1). AFM image clearly showed the hydrolysed wool fibers consist of bundle of fibrils parallel arranged along the length of the fibers (Fig 2 (b)). This study further confirmed, the ability of controlled alkaline hydrolysis to remove of cuticle cells which consist the outer most surface of the wool fibers. With the removal of - sheet (cuticle cells) the internal fibrils of

the wool fibers was opened. Figure 3 depicts the FT-IR spectra and images of the pristine wool and wool fibers hydrolyzed under alkaline conditions.

Figure 3. (A) FT-IR spectra (4000 – 800 cm -1) of (a) pristine wool (b) hydrolysed wool fibers (B) Second derivative infrared spectra of (a) wool (b) 0.1M wool (c) 0.3M wool (d) 0.5M wool (C) images of the pristine wool and hydrolysed wool fibers samples In Figure 3 (A) FT-IR spectra of pristine wool and hydrolysed wool sample showed absorption band in the wavelength range 4000 –800 cm-1. These absorption bands correspond to N-H stretching of amide-A (3272 cm-1) and amide-B (3065 cm-1). These bands are influenced by the fermi resonance between the first overtones of Amide-II[19]. The band at 2926 cm-1 is related to the stretching vibration mode of C-H presence in pristine wool and hydrolysed wool fibers. The mid-infrared spectrum with keratin absorption bands at 1629 cm-1, 1515 cm-1 and 1238 cm-

1

can be designated respectively as amide-I, amide-II and amide-III[20]. These are often used to study the conformational arrangement of the protein in the wool fibers. Amide-III band consist of more complex vibration modes derived from in-plane bending from N-H, C-N stretching, C-C stretching and C-O bending[15]. Therefore it is not easy to identify a correlation between the amide-III band shape and the protein secondary structure. Instead amide-I and amide-II band can be more effectively used to describe secondary structure of the protein presence in wool[21]. Amide-I absorption band occur due to the stretching vibration of C=O while amide-II derives mainly from in-plane bending of N-H with minor contribution from C-N and C-C stretching vibrations. In Figure 3B, the broad amide-I and amide-II bands were resolved using the second derivative of the FT-IR spectrum of pristine wool and alkaline hydrolysed wool fibers. In this second-order derivative spectrums it is to be noted that the peak positions are inverted.

The band at 1734 cm-1 (Figure 3B) corresponds to the C=O stretching vibration from free carboxyl groups belonging to glutamic and aspartic acids of the wool protein [22]. For the pristine wool sample (Figure 3B-a), absorption at 1652 cm-1, and at 1541 cm-1 is due to the presence of amide-I and amide-II presence in α – helix protein which characterizes the intermediate filament structure of the wool fibers [23]. The second weaker absorption band at 1637 cm-1 and 1697 cm-1 for pristine wool sample (Figure 3B-a) corresponds to the amide-I groups presence in the - sheet and disordered keratin conformations [24]. These - sheet are typical constituent of the cuticle cells (outer layer of the wool fibers) and the matrix that cover the intermediate filament structure of the wool fibers [25]. Compared with the pristine wool fibers (Figure 3B-a) there is a significance decrease in the absorptions bands at 1697 cm-1 and 1637 cm-1 (Figure 3B-b, c, d) for all alkaline hydrolysed wool fibers. This indicate the gradual removal of - sheet structure of the (cuticle cells) wool fibers with increasing hydrolysis conditions. The broad band at 1627 cm-1 with a shoulder peak at 1615 cm-1 also correspond to amide I of the - sheet structure (Figure 1B-a). In this broad band, the shoulder peak at 1615 cm-1 was not observed for 0.5 M wool or 0.3 M wool (Figure 3B-c & d) and showed a decrease in the intensity for 0.1 M wool (Figure 3B-b), which further confirm the diminishing of sheath structure of the wool fibers. The shoulder peak at 1578 cm-1 (Figure 3B) attributes to the asymmetric COO- stretch which originate due to the aspartic and glutamic acid side chains. The strong peak at 1514 cm-1 and 1541 cm-1 (Figure 3B) are the bands that originate from parallel and perpendicular amide II groups of the α – helix protein present in both pristine wool and alkaline hydrolysed wool samples [26]. For amide II, the decoupled C-N stretch was observed at 1437 cm-1 and 1418 cm-1 for pristine wool and alkaline hydrolysed wool fibers (Figure 3B). From the information obtained using FT-IR, it can be confirmed the alkaline hydrolysis has caused a gradual removal of the - sheets. For all the alkaline hydrolysed wool fiber systems α – helix protein structures were found to be intact and present in the sample.

Figure 3C clearly shows the physical changes apparent in the wool sample with the increasing hydrolysed conditions. First, it could be observed that distended nature of the wool fibers shows a gradual decrease with respect to alkaline hydrolysis conditions. Secondly, hydrolyzed wool fibers appeared slightly yellowish and matte compared to control fibers indicating a surface chemical change. NMR, EDX & CHNS analysis of the wool samples Figure 4A shows the energy dispersive X- ray element mapping of the pristine wool and surface hydrolysed wool fibers. According to the element map the samples mainly consisted of C, N, O and S (H is not within the detectable limit of the EDX). The outer surface of the wool fibers is abundant with S [24]. The CHNS analysis of these wool samples shows a decrease in S content with the increase in the hydrolysis, where pristine wool sample showed 4.03% of S while 0.5 M wool only showed 2.43% of S (Figure 4A). The presence of S in [27] wool is mainly due to amino acid cysteine and thioester lipids in cuticle cells . The decrease in sulphur content of the wool samples can be attributed to the removal of outer most surface which are the cuticle cells. Therefore from the information obtained using EDX and CHNS analysis it is clear that the alkaline hydrolysis has led to the decrease of cuticle layer of the wool samples.

Figure 4. (A) EDX & CHNS analysis of the wool samples (B) Dye removal percentage of wool and hydrolysed wool samples Pristine wool and 0.5 M wool sample were further analysed using NMR spectrophotometer. The spectra obtained using the study (Supplementary data Figure S4) was expanded, deconvoluted and fitted with Gaussian function to identify the individual peaks in the NMR study (Figure 5). For wool & 0.5 M wool samples 13C peaks of carbonyl, aromatic , C α methane and side chain aliphatic carbons and 13C spinning side bands were observed in 170-185, 115-165, 50-70, 10-45 & 224-228 ppm respectively [28]. As shown in Figure 4 and Table 1, methyl group side chain of alanine and beta carbon of leucine can be observed at 18 ppm & 23 ppm

respectively. The intense peak at 27 ppm is due to the presence of beta carbons in glutamic acid and glutamine residues in wool and 0.5 M wool sample. Also additional peaks were observed at 32, 35 and 38 ppm due to the glutamic acid, glutamine and Proline residues in wool samples. Peak at 43.1 ppm for wool sample and 43.7 ppm for 0.5 M wool sample is attributed to the β - carbon in cross-linked cysteine residues (S-S disulphide link between neighbouring keratin molecules). It is apparent that, peak area and FWHM of this peak have reduced in 0.5 M wool sample compared with wool sample (Table 1). This may be due to the chemical reduction of these cysteine groups during the alkaline hydrolysis which was further confirmed by the CHNS analysis results. The intense peak at 56.1 ppm for wool sample is associated with the βsheets confirmations. Compared with peak area and FWHM of the peak at 56.1 ppm for wool sample, 0.5 M wool sample showed a reduction in peak broadness and peak area. This further confirms the decrease of β-sheets concentration during the hydrolysis process. Peak at 58.5 ppm, 60 ppm and 63 ppm is due to the α-helical confirmation in the wool samples. For the 0.5 M wool and wool samples the peak area and FWHM values of these peaks showed similar values indicating α-helical confirmations are preserved during the alkaline hydrolysis. Peaks observed at 173 ppm, 175 ppm and 177 ppm attributes to the carbonyl carbon of β-sheets molecular confirmations of Serine, carbonyl carbon of Cysteine and Glutamine in α-helical confirmations respectively [29]. The peak area ratio of 173 ppm and 175 ppm in wool sample and 0.5 M wool is calculated to be 3.48 & 1.31. This shows in 0.5 M wool sample, the amount of β-sheet confirmation in Serine has reduced due to the alkaline hydrolysis. Also with relative to the α-helical carbonyl components of Glutamine, the β-sheet confirmation of Serine showed a reduction in hydrolysed wool sample. Therefore the NMR study further confirm the alkaline hydrolysis has led to a decrease of the β-sheet structures in the wool sample.

Figure 5. Expanded regions of the NMR spectrum of wool and 0.5 M wool sample, deconvoluted and fitted with Gaussian function

Table 1. 13C chemical shift, peak area and full width at half maximum (FWHM) peak height of de-convoluted Gaussian peaks of NMR spectra for wool and 0.5 M wool samples wool 13

0.5M wool

Peak area

FWHM

(x 106) (a.u)

(ppm)

14.73

0.259

1.79

18.33

1.23

2.38

22.91

5.07

27.53

5.96

C Chemical shift in ppm

13

Peak area

FWHM

(x 106) (a.u)

(ppm)

18.87

0.531

1.85

3.87

23.30

2.87

3.37

3.00

27.93

4.67

3.10

C Chemical shift in ppm

31.99

7.13

4.89

32.33

5.76

4.65

35.61

0.559

1.59

35.82

0.306

1.53

38.03

1.89

3.22

38.40

4.01

6.23

43.07

5.91

6.45

43.86

2.09

3.98

51.27

0.050

0.71

56.10

3.18

3.37

56.19

1.55

2.59

58.57

1.69

1.69

58.58

1.37

1.82

60.23

2.25

2.27

60.37

2.00

2.52

62.95

2.53

3.09

63.28

2.70

3.55

118.61

0.449

2.96

118.65

0.276

3.19

125.03

1.73

4.70

125.80

1.25

4.51

128.66

0.209

2.20

128.42

0.196

2.00

130.57

2.98

6.62

130.38

0.988

3.56

139.24

0.219

2.54

133.25

0.361

2.96

158.24

0.196

1.73

157.36

0.0117

0.76

160

0.661

2.50

159.98

0.618

3.04

173.78

2.223

2.69

173.57

1.15

2.63

175.38

0.638

1.71

175.18

0.877

2.15

177.78

11.06

6.26

177.8

7.88

6.10

224.46

1.11

3.48

224.46

0.807

3.57

228.04

2.64

5.93

227.94

2.41

6.92

Removal of dye by the absorbent Efficiency of the hydrolysed wool fibers for the removal of rhodamine B (chemical information is given in supplementary data Figure S1) from aqueous solutions were studied. According to Figure 4B hydrolysed wool fibers have very high dye removal capability which is 74% compared

to pristine wool fibers hydrolysed wool fibers of 4%. Further, 0.5 M wool fibers showed ~96 % dye removal from a 100 ml of 100 ppm dye solution (in 3 % acetic acid medium) within 40 minutes time. Therefore from different alkaline hydrolysed wool fibers and pristine wool fibers, 0.5 M wool sample showed the highest adsorption for rhodamine dye and hence selected for further adsorption studies. The surface charge build on pristine wool and 0.5 M wool sample in acetic acid medium was analysed by measuring the zeta potential of the material dispersed in distilled water (0.8 g/L) having different acetic acid concentrations. Zeta potential measurement of the material at different acetic acid concentrations is given in Figure 6a. In water the surface charge of pristine wool and 0.5 M wool sample remained at -3 mV & -25 mV respectively. For 0.5 M wool, a maximum positive surface charge of +36 mV was obtained at 3.5 % acetic acid concentration having a dispersion pH of 2. Compared with the pristine wool fibers 0.5 M wool fibers showed a gradual increment of the surface charge with respect to the increase in acetic acid concentration.

Figure 6. (a) ζ potential of pristine wool and 0.5 M wool dispersion at different acetic concentrations (b) effect of acetic acid in dye solution for dye removal efficiency (0.5 M wool sample dosage = 0.8 g/L , 100 ml of 100 ppm solution, T = 27 ° C, pH = 2) Pristine wool fibers did not show a positive charge build on the surface with increasing acetic acid concentration and material remain in its negative surface charge of -11.47 mV at 3.5% acetic acid concentration. However, 0.5 M wool fibers show net positive zeta potential which indicate predominant presence of positively charged groups on the surface of the 0.5 M wool at

acetic acid concentration higher than 1%. Therefore, it is found to be most suitable for the adsorption of rhodamine B. Charge induced adsorption behavior of dye, to 0.5 M wool fibers was analysed. For this dye solutions having different concentrations of acetic acid was used. Figure 6b shows the adsorption of rhodamine dye on 0.5 M wool sample was dependent on the acetic acid concentration in the dye solution. In water, the surface charge of the 0.5 M wool sample was at negative value of -25.57 mV, and it was observed to have an almost negligible dye removal percentage (~ 3%). surface charge of the 0.5 M wool sample reached +20.67 mV at 1% acetic acid concentration and sample’s dye removal percentage was 57% after 90 minutes of contact time. Increase in acetic acid concentration to 1.5 % has allowed the sample to build a higher positive surface charge of +33.47 mV which allowed the sample to remove 68% dye within 90 minutes of contact time. Samples having 2.5% , 3% and 3.5 % acetic acid concentration showed a further increase in the surface charge (approximately +36 mV), have removed 63%, 72%, and 86 % rhodamine dye respectively within a short contact time period (after 15 minutes). For a sample concentration of 0.8 g/L the maximum surface charge build on the 0.5 M wool was +36 mV at 3.5% acetic acid concentration and reached its maximum adsorption of 91 % within 30 minutes of contact time. This can be due to the abundant cationic group presence in the 0.5 M wool fibers in the presence of acetic acid and for further adsorption studies therefore, 3.5% acetic acid concentration was used. Adsorption equilibrium and kinetic study Adsorbent dosage was varied to determine the minimal amount required for achieving the maximum adsorption. Adsorption kinetics of rhodamine dye on varying 0.5 M wool dosage was also investigated to understand the mechanism of adsorption.

Figure 7. (a) effect of 0.5M wool dosage on rhodamine dye removal (100 ml of 100 ppm solution, T = 27 ° C) (b) Pseudo-second-order kinetic plot of rhodamine dye adsorption at different 0.5 M wool sample loading. As shown in Figure 7a the increase in 0.5 M wool loading increased the dye removal percentage. For loading of 0.8 g/L and above, a dye removal percentage of > 90% was reached after 30 minutes of contact time. Also according to Figure 7a for loading of 0.8 g/L and above, adsorption capacity of the sample increased instantly within the initial 30 minutes contact time and thereafter proceeded at lower rate until the steady state was reached within 160 min. To investigate the detailed characteristics of the adsorption behaviour of rhodamine dye on 0.5 M wool, two kinetic models, pseudo-first order and pseudo-second were used. The results in these was used to study the adsorption mechanism of rhodamine dye on 0.5 M wool and the rate controlling steps involved in the adsorption of rhodamine dye on the sample. Pseudo-first order kinetic equation can be expressed as; ln − = ln − (1) Pseudo-second order kinetic equation can be expressed as; =

(2)

+

In both equations, qe and qt are the amount of dye adsorbed (mg/g) at equilibrium and adsorption time t (min) respectively. k1 (1/min) and k2 (g/ (mg. min)) are defined as the adsorption rate constant. For pseudo-first order kinetic model, the slope and intercept value of the plot between ln (qe - qt) versus t (min) was used to arrive at the values of qe and k1 (Supplementary data Figure S1). For pseudo-second order kinetic model, values of qe and k2 were calculated from the slope and intercept of linear plot of t/qt versus t (Figure 7b). Kinetic parameters and correlation coefficient of the fitted plot was (R2) is given in table 2 below. Table 2. Kinetic parameters obtained, using adsorption study of rhodamine dye on 0.5M wool at different adsorbent loading levels Pseudo – first order

Pseudo-second order

Adsorbent loading (g/L)

qe (exp.) qe (cal.) k1) min- R2 1 (mg/g) (mg/g) )

qe (cal.) k2 (g/mg. R2 (mg/g) min)

0.1

294

157.99

0.08188

0.89131

294.98

0.001442 0.99944

0.2

232

176.19

0.04934

0.98378

239.23

0.00076

0.99818

0.4

203

204.52

0.07598

0.98967

212.31

0.000833 0.99706

0.8

116

33.99

0.04959

0.77102

117.51

0.006015 0.99981

1

95

15.79

0.04823

0.51412

95.51

0.013144 0.99987

For the adsorbent loading of 0.8 g/L and 1 g/L, R2 values of the pseudo- second order kinetic model were 0.99981, 0.99987 respectively and the calculated values of qe using this model is in close comparison with the experimental values of qe (Table 2). For the same adsorbent loading R2 values of the pseudo-first- order kinetic model were 0.77102 and 0.51412.Thus the calculated values of qe deviated greatly from the experimental values of qe. Compared with the pseudo-first- order kinetic model the pseudo-second-order model again showed close comparison with the calculated qe and experimental qe value (Table 1) for the adsorption loading of 0.1 g/L, 0.2 g/L 0.4 g/L, 0.8 g/L and 1 g/L. Therefore pseudo-second-order kinetic model best fit the adsorption of rhodamine dye on 0.5 M wool for all the loading amounts used for the study. Therefore it can be concluded that surface adsorption is the rate limiting step for the removal of rhodamine dye from the liquid bath using 0.5 M wool. According to the results obtained the optimized adsorbent loading was selected as 0.8 g/L and used for further adsorption experiments.

Figure 8. (a) Effect of initial dye concentration on dye removal efficiency (0.5 M wool sample loading at 0.8 g/L, acetic acid 3.5% and T = 23 °C). (b) Effect of initial dye concentration on equilibrium adsorption capacity of 0.5 M wool sample (c) Langmuir adsorption isotherm model for adsorption of Rhodamine dye on 0.5 M wool sample According to Figure 8a an increase in the contact time has increase the amount of dye adsorbed in all wool samples. At lower initial dye concentrations (80 & 100 ppm) during the initial contact time of 30 minutes the dye adsorption occurred at a faster rate and became slower as the time progress. This is because during the initial stage, due to the availability of vacant sites on the surface, the adsorption is very fast [38]. As the time progress these vacant sites will be occupied

by dye molecules and adsorption will be more difficult due to the repulsive forces between the adsorbed dye molecules and dye molecules in the dye solution[30]. Also according to Figure 8a as the initial dye concentration increased the time taken to equilibrate the dye adsorption is increased. Further Figure 8a reveals as the initial dye concentration increase from 80 ppm to 500 ppm the dye removal efficiency decreased from 95% to 37% after 60 minutes of contact time. Figure 8b reveals, an increase in the initial dye concentration increased the dye adsorption capacity per unit mass of the adsorbent. This was further explored in equilibrium adsorption study and different adsorption isotherm models were used to understand the dominant adsorption mechanism [40]. Adsorption isotherm study Langmuir and Freundlich isotherm models were used to analyse interaction between the absorbent and the adsorbate when the adsorption process reached equilibrium. Langmuir isotherm model assumes that the adsorption takes place as a homogeneous monolayer coverage on the surface of the adsorbate and do not process beyond its surface. Conversely Freundlich isotherm model assumes multilayer adsorption on heterogeneous surface[31, 32]. The equations with respect to linearized Langmuir isotherm and Freundlich isotherm models are given in equation 5 and 6 below. = ln

(5)

+ = ln ! + ln

"

(6)

In these equations Ce is the concentration of dye in the solution at equilibrium stage. Adsorbed dye amount per gram of the adsorbent is given by qe (mg/g). Maximum adsorption capacity of the adsorbent is qm. KL and KF are the Langmuir and Freundlich constants. For the Langmuir adsorption isotherm models the values of qm and KL was obtained using the slope and intercept of the linear plot of Ce/qe versus Ce. In Freundlich isotherm model n represents the adsorption intensity, where n > 1 indicates a favourable condition for adsorption. Also KF and n values were obtained from the intercept and slope of the linear plot of ln (qe) against ln (Ce).The isotherms of both models based on the experimental data are shown in Figure 8c and Figure S2 of the Supporting Information. The parameters obtained for these models using the isotherms are summarized in Table 3. Table 3. Parameters calculated from data obtained for different isotherm models Langmuir isotherm model Freundlich isotherm model

Adsorbent

T(K)

qm (mg/g)

KL )L/mg)

R2

KF

n

R2

0.5 M wool

298

304.88

8.79

0.99978

23227

3.75

0.83141

Based on the results obtained, R2 value of the Langmuir and Freundlich isotherm models were 0.99978 and 0.83141 respectively (Table 3). Therefore it appears the present adsorption process does not follow the Freundlich isotherm model but it follows the Langmuir isotherm model over the entire concentration range. Based on this model, for 0.5 M wool sample, a monolayer adsorption of the rhodamine dye molecules has taken place. As shown in Figure 8c the adsorption capacity increase with increase in initial dye concentration. The calculated maximum adsorption capacity of the 0.5 M wool for rhodamine dye is 304.88 mg/g (Table 3). This value is in good agreement with the experimental value of 298 mg/g. The adsorption capacity of the 0.5 M wool sample for rhodamine dye was compared with the adsorption capacity of other carbon- nitrogen based material for rhodamine dye (Table 4). Based on the information obtained 0.5 M wool sample showed higher adsorption capacity compared to other materials. Table 4. Maximum adsorption capacity of Rhodamine dye on other carbon-nitrogen based state-of-the –art adsorbents Absorbent

Rice husk

Maximum Adsorbent Adsorption loading Capacity (g/l) qm(mg/g)

pH at qm

Temp (o C)

Reference

2

16.0

2.1

60

[33]

H2SO4 acid activated sludge

3.4

0.4

6.5

-

[34]

Activated carbon from Sago waste

16.2

1.0

5.7

30

[35]

Modified baker’s yeast

59.2

0.5

6.5

25

[36]

71

0.3

2.1

60

[33]

91.1

0.2

5.5

25

[37]

Activated carbon carbonaceous adsorbent from industrial waste

Bagasse pith activated carbon

103.6

1.0

3.5

50

[38]

Rice husk porous carbon

215.5

0.8

6.5

25

[39]

Tannery residual biomass

250

1.0

3.5

50

[40]

Surface hydrolysed keratin protein fibers (0.5M wool)

294

0.4

4.0

27

This work

307.2

0.16

4.0

45

[41]

Activated carbon from tires

Adsorption and desorption of dye for reuse of 0.5 M wool sample The fibrous nature of the 0.5 M wool sample with a fibre diameter of ~30 µm and length > 2 mm allows the material to be separated from an aqueous medium and easily regenerated. The reusability of the adsorbent was assessed by performing multiple adsorption-desorption cycles. Adsorption of rhodamine B dye was carried out for 80 ppm, 100ml of dye at fixed 0. 8 g/L loading of 0.5 M wool for a predetermined equilibrium time (30 minutes) period. For the adsorption cycle the dye solution is charged with 3.5% acetic acid at pH 2. The desorption of the dye molecules from 0.5 M wool was achieved in 100 ml aqueous solution with 0.5% NaOH at pH 10 for 30 minute time period. The adsorption and desorption percentage of the dye to and from the 0.5 M wool sample for three adsorption-desorption cycle is given in Figure 9a. The corresponding photographs of the dye solution in each adsorption-desorption cycle is given in Figure 9a. During first three cycles the dye removal percentage was at 95% while after the fourth cycle it slightly reduced to 94%. During desorption 97% (78 ppm) of dye was eluted to the aqueous solution. The desorption of Rhodamine dye from 0.5 M wool can be due to the deprotonating of cation and amine groups present on the sample under alkaline condition. During the desorption Negatively charged surface of the sample will increase thereby repelling dye molecules to the aqueous medium, which allow the functional groups to be vacant for the next adsorption cycle. The results show that 0.5 M wool sample’s dye adsorption ability remain effective after multiple number of use, thus can be reused.

Figure 9. (a) Adsorption and desorption behaviour of 0.5 M wool sample under acidic and alkaline conditions (b) Scatchard plot of the equilibrium isotherm data for rhodamine dye on 0.5 M wool sample

Surface binding mechanism of rhodamine dye on 0.5M wool sample

Surface adsorption of Rhodamine dye molecules on 0.5 M wool sample can be assisted through ionic, chemical and physical interaction. The Scatchard plot is a widely used method to investigate the mechanism of surface adsorption of an adsorbate information on the binding of an adsorbate by the biomolecules such as proteins[42]. It is common that the surface adsorption of chemical species on to an adsorbate can be influenced by the surface functional moieties [43]. The equilibrium isotherm data obtained in this study were further analysed using the Scatchard plot to investigate the surface binding mechanism of the dye molecules on to the adsorbent. Figure 9b clearly shows non linearity in the binding mechanism of rhodamine dye molecules to the 0.5M wool sample. The non-linear Scatchard plot like this, present in Figure 9b provide strong evidence for the presence of multiple binding sites such as phosphate, carboxyl, amino and hydroxyl groups in the hydrolyzed fiber for the adsorption of rhodamine dye molecules[44, 45]. These binding sites on 0.5 M wool were chemically modified to analyse the contribution from each for the adsorption of dye molecules. Table 5 below show contribution of each binding site for the adsorption of the dye molecules. For the chemical modification of amine groups 1 g of 0.5 M wool sample was refluxed for 4h with 15 ml of formaldehyde and 30 ml formic acid. This resulted the methylation of the amine groups as shown in the reaction given in equation 2. The methylation blocks the primary and secondary amines presence in the 0.5 M wool sample. As given in Table 4, the methylation has markedly inhibited the rhodamine dye adsorption by mean values of 40% .therefore amine groups present in the sample has majorly contributed to the rhodamine dye molecule adsorption. R NH2 +2HCHO + 2HCOOH → R N (CH3)2 +2CO2 +2H2O

(2)

For the 0.5 M wool sample it was assumed to have free carboxylic acids due to the alkaline hydrolysis of peptides in proteins. For the modification of these acids, an amino acid coupling reaction was carried out using N,N’- Dicyclohexyl carbodiimid (DCC). 0.1 g of DCC was dissolved in 10 ml of methanol with 0.48 ml of Diethyl amine as the catalysis for the coupling reaction. 0.25 g of 0.5 M wool sample was added to the solution and reaction was carried out at room temperature for 24 hours. The sample was rinsed thoroughly with distilled water and dried at 45 C before adsorption studies. The amino acid coupling reaction has blocked the carboxylic acid groups present in the 0.5 M wool sample as shown in the reaction given in equation 3. Through this reaction carboxylic acid presence in the 0.5 M wool sample was blocked for the adsorption of dye molecules and has decreased the mean adsorption value by 35% (Table 4). R COOH + C13H22N2 → R COOCH3 +H2O

(3)

Table 5. Effect of chemical treatments on 0.5M wool for rhodamine dye uptake

Chemical modification method for 0.5M wool sample

Percentage of dye uptake after 90 minutes

Percentage reduction in dye uptake

No chemical modification

95%

-

Amine groups blocked 0.5M wool

54.2%

40.8%

Carboxyl groups blocked 0.5M wool

60.5%

34.5%

Based on above observations a mechanism was suggested to explain structural and chemical change of the pristine wool fibers after subjecting for alkaline hydrolysis. Also a mechanism was suggested to explain the, increased adsorption of Rodhamine dye in the presence of acetic acid in the aqueous medium.

Figure 10. Suggested structural and chemical changes of the (a) pristine wool fibers (b) after subjecting for alkaline hydrolysis (c) hydrolysed wool fibers interaction with rhodamine dye in acetic acid medium After subjecting wool fibers (Figure 10a) for alkaline hydrolysis, cuticle cells were removed and most of the - sheet keratin protein was hydrolysed (Figure 10b). Hydrolysed wool fibers consist of parallel arranged fibril bundle with free amine and carboxyl groups (Figure 10b) where amine groups can be protonated and positive charge was built in acetic acid medium (Figure 10c) which allows to form chemical interaction with rhodamine dye molecules. From

these functional groups protonated amine has played a major role in adsorption of dye molecules to the 0.5 M wool sample. Therefore surface hydrolysis and acetic acid induced protonation of the merino wool fibers was effectively used for the adsorption of dye molecules from an aqueous solution.

Conclusion: A keratin protein based bio adsorbent was prepared via controlled alkaline hydrolysis of wool fibers. Fiber treatment has allowed to remove the outer surface of the wool fiber which allow more dye adsorption compared to the unmodified wool fiber. It can be observed that surface hydrolysed wool material had ~90 times higher rhodamine dye adsorption compared to an unmodified wool fibers. The alkaline hydrolysis also has introduced chemical functional groups on the fiber surface which was found to be contributing towards the high adsorption. Among others reported materials in literature, 0.5 M wool sample ranks at high level with an adsorption capacity of 294 mg/g at room temperature. Another interesting property of the prepared keratin protein based wool material is that, same system can be regenerated in alkaline medium and used in several dye adsorption cycles with adsorption efficiencies close to 95%. In view of the simple preparation method of the adsorbent, its reusability, high adsorption capacity and decomposability, it is expected that surface hydrolysed wool material can be applied for dye wastewater remediation through different techniques

Acknowledgements We would like to thank Dr. Nuwan de Silva, Dr. Laksiri Weerasinghe, Ms. Rashmi Kumarasinghe and Mr. Maduka Vithaksha for numerous support.

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Highlights •

Surface hydrolyzed keratin protein fibers were prepared using merino wool.

•

Hydrolyzed wool fibers showed exceptional adsorption of Rhodamine.

•

Equilibrium capacity is best aligned with the Langmuir isotherm model.

•

The keratin protein fibers showed an adsorption capacity of 294 mg/g at 298 K.

•

Up to now, the highest reported for the Rodhamine adsorption.

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.