Improving the Adsorption Properties of Keratin-Based Goat Hair Toward Reactive Dyes in Dyeing Wastewater by Steam Explosion

ABSTRACT Keratin-based goat hair could be used bioadsorbent due to its large availability, biodegradability and low cost. Unfortunately, most available keratin adsorbents are either low in adsorption capacity or high in preparation cost. In this study, goat hair was steam exploded, frozen and disintegrated to prepare the exploded goat hair powder (EGHP), and was further used for the adsorption of reactive blue 19 (RB 19) in dyeing wastewater. Steam explosion significantly increased the adsorption capacity of keratin-based goat hair toward RB 19. Adsorption capacity of RB 19 in EGHP increased from 55 to 427 mg/g after a steam explosion under 1.8 MPa for 150 s. The increased adsorption capacity was mainly due to decreased crystallinity, increased specific surface area from 0.678 to 8.583 m2/g and the increased free NH 2 groups for EGHP. These structure changes resulted in that more dye molecules could easily and fast entered into the amorphous region of EGHP, thereby improving the adsorption rate and amount of RB 19 dyes. Desorption and regeneration study indicated that the adsorption capacity still maintained 79% after 6 cycles. These results demonstrated that EGHP could be considered as promising bioadsorbent to treat the dyeing wastewater.


Introduction
Keratin-rich animal by-products such as bristles, feathers, wool and hairs, etc., constitute the third most abundant renewable polymeric material present in nature and have attracted much attention (Falco et al. 2019). It was reported that chicken feathers could be a protein source by thermal alkaline pretreatment followed by enzymatic hydrolysis (Cheong et al. 2022). Pig bristles were studied to in N-N dimethylformamide then precipitated with chloroform in order to purify the dye. The purified RB 19 had a purity of 95% followed by the purified method (Chen et al. 2016) and was used in the following experiments.
N-N dimethylformamide, chloroform, sodium hydroxide, hydrochloric acid and anhydrous sodium carbonate were purchased from China National Pharmaceutical Group Co., Ltd.

Preparation of goat hair bioadsorbents
Three kinds of goat hair bioadsorbents were prepared as shown in Figure 1 and compared. Goat hair was cut into about 1 cm with a scissor, which was simply called CGH. The cut goat hair without steam explosion were frozen with liquid nitrogen and disintegrated with the freezing disintegrator (DC3-1, Hebei Beichen Technology Co., Ltd, China) at the rotational speed of 25,000 r/min, which was simply called FDGH. The cut goat hair was steam exploded with QBS-200B test bed (Gentle Science & Technology Co., Ltd., Henan Province) then was frozen with liquid nitrogen and disintegrated into powder with the same disintegrator and the powder was simply called EGHP. The used test bed can complete an explosion within 0.0875 s with the catapult mode. The wet goat hair was set for 5 h after being wetted by spraying about 100% water (w/w, based on the weight of goat hair) then was placed in the chamber of the steam explosion test bed. According to our previous investigation (Hou et al. 2017), the steam pressure was changed from 1.3 to 1.8 MPa (the corresponding temperature was changed from 192 to 207°C) and the treatment time was changed from 30 to 240 s in this study.

Preparing dyeing wastewater
In consideration of hydrolyzation during the dyeing process, RB 19 was dissolved in sodium carbonate solution with a concentration of 5 g/L and the dye solution was then heated at 80°C for 1 hour to simulate the hydrolysis of the dye during the dyeing process (Xu et al. 2013). Then, the dye solution was adjusted to neutral using hydrochloric acid, which simulated the dyeing wastewater. The dyeing wastewater with the initial dye concentration from 40 to 5000 mg/L was used to adsorption experiments.

Determination of dye concentration in dye solution
The absorbance spectra sof RB 19 dye solution was measured with the UV-visible spectrophotometer TU-1901 (Purkinje General Instrument Co. Ltd., Beijing, China) using a quartz cell with path length of 1 cm. RB 19 dye solution has a strong absorbance peak at 594 nm. The concentration of RB 19 in the dye solution was measured at 594 nm based on the Lambert-Beer law. The calibration curve had the relation of y = 0.0152× (y was absorbance and x was dye concentration(mg/L)) with R 2 value of 0.999.

Characterization of adsorption kinetics and thermodynamics of bioadsorbents
In all adsorption experiments, the amount of adsorbents was 10 g for 1 liter of dyeing wastewater and the adsorption processes were studied by changing the concentration of the dye solution at room temperature in triplicate. The pH value of the adsorption bath was adjusted ranging from 2.0 to 9.5 (Song et al. 2017) using disodium hydrogen phosphate and citric acid buffer solution, borax and sodium hydroxide buffer solution, respectively.
The adsorption kinetics and thermodynamics of RB 19 onto goat hair were investigated. The pseudo-first-order and the pseudo-second-order models follow the Equations (1) and (2) (Song et al. 2017), respectively.
In Equations (1) and (2), q t (mg/g)and q e (mg/g) are the amount of dye adsorbed by the adsorbent at time t(h) and equilibrium, respectively. k 1 and k 2 are pseudo-first-order and pseudo-second-order rate constants, respectively. q t and q e were calculated by the Equations (3) and (4) respectively.
Where C 0 , C t and C e (mg/L)are the concentration of RB 19 dye in solution at the beginning, the time t and the equilibrium of adsorption, respectively. V(L) is the volume of dye solution. m is the weight of the used bioadsorbent. The Langmuir isotherm model is expressed as the Equation (5) (Song et al. 2017).
where C e is the concentration of RB 19 dye in solution at the equilibrium of adsorption. q e is the amount of adsorbed RB 19 per gram of goat hair at equilibrium. q max (mg/g) is the maximum adsorption amount of the adsorbent. b is the Langmuir constant. The Freundlich isotherm model is expressed as the Equation (6) (Song et al. 2017).
Where K F (((mg·g −1 )(L·mg −1 ) 1/n )) is the Freundlich constant and n is the heterogeneity factor, which are related to the adsorption capacity and adsorption intensity, respectively.
The dye removal efficiency at the equilibrium of adsorption was calculated according to the equation (7) (Song et al. 2017).
Where C 0 and C e are the concentration of RB 19 dye in solution at the beginning and the equilibrium of adsorption, respectively.
The adsorption properties of three kinds of goat hair as bioadsorbents, i.e., CGH, FDGH and EGHP were measured and compared.

Morphology
The morphologies of samples were measured using a SU1510 scanning electron microscope (SEM, Hitachi, Japan) and SEM was operated at an accelerating voltage of 5 kV. All samples were sputtercoated with a thin layer of gold prior to scanning.

Crystalline structures
A D2 PHASER X-ray Diffractometer (Bruker AXS Co., Germany; wavelength 1.54 Å, Cu Kα radiation) was used to analyze the crystallinity of goat hair. The intensity and current of the generator were 40 kV and 40 mA, respectively. The powdered samples were scanned from 5° to 40° at a rate of 4°/min and a step size of 0.02°. The crystal index (CI) of the samples was calculated based on the Segal's Equation (8) (Zhang et al. 2019).
Where I 9 � is the intensity of absorption peak at 2θ = 9° andI 14 � is the intensity of absorption valley at 2θ = 14°.

Specific surface area and porosity
The Brunauer-Emmett-Teller (BET) specific surface area was measured by TriStar II3020 Analyzer (American Micromeritics instrument Ltd., America). A 100 mg of each sample were degasificated for 13 h at 200°C. The nitrogen adsorption-desorption isotherm was analyzed in the range of 0.05-0.95 relative pressures.

Conductometric and potentiometric titration of free amine groups
Concentrations of free amine groups of samples were quantified using a Mettler Toledo FE28 pH meter equipped with an LE38 electrode and Metetler Toledo Sven 2 Go S3 conductivity meter equipped with an InLab® 738-ISM, respectively. About 1 g of samples was precisely weighed, and dispersed in 40 g of de-ionized water. Standardized 0.1 mol/L HCl solution was added into the dispersion to adjust pH to 2, under which the amine groups in samples were protonated. Standardized 0.1 mol/L NaOH solution was used in titration.

Statistical analysis
The experimental data were subjected to variance analysis and Tukey's test using SAS program (SAS Institute, Raleigh, NC, USA) at 95% significance level (p < .05). Figure 2 shows the effect of the adsorption bath pH on adsorption quantity of RB 19 onto EGHP. As the pH value of the adsorption bath increased from pH 2.0 to pH 9.5, the adsorption quantity q e of RB19 on EGHP was significantly decreased. As shown in Figure 3, RB 19 molecules carried negative charge -SO 3 -groups in solution. Goat hair had an isoelectric point of around pH 4.1. Goat hair carried positive charge when pH was below 4.1 and carried negative charge when pH was above 4.1. Dye adsorption is a combination of electrostatic attraction between -SO 3 -and -NH 3 + and electrostatic repulsion between -SO 3 -and -COO-. As the pH value raised from 2.0 to pH 9.5, more -NH 3 + and -COOH groups on EGHP became -NH 2 and -COO-. Therefore, the surface charge of EGHP turned  from being positive (pH < 4.1) to being negative (pH > 4.1). Therefore, less dye molecules were attracted onto EGHP because electrical attraction between dye and EGHP molecules decreased and electrical repulsion increased with the increased pH value, which resulted in the decreased adsorption of RB19 onto EGHP. The high removal efficiency of the keratin adsorbent occurs in acid conditions, which is the disadvantage of keratin adsorbents when adsorbing RB 19 because the usual pH value of the textile wastewater is 8-9. However, the pH value of 2 was used in the subsequent adsorption experiments in order to compare the effect of steam explosion on adsorption. Figure 4 shows adsorption kinetic curves and fitted adsorption kinetic curves of RB 19 onto CGH, FDGH and EGHP with pseudo-first-order and pseudo-second-order model. As shown in Figure 4a, the amount of RB19 dye onto EGHP was highest and the required time to reach equilibrium sorption for FDGH and EGHP was significantly lower than that for CGH. The amount of RB19 dye onto goat hair q t increased quickly during the initial process. Then, the curve flattened after 6 h for EGHP and 8 h for FDGH, indicating that the adsorption reached equilibrium. However, the adsorption reached equilibrium after 26 h for CGH, indicating that CGH had the lowest adsorption rate. The experimental amounts of dye adsorbed onto EGHP at equilibrium (286.3 mg/g) were about 2.5 and 11.4 times of that onto FDGH and CGH, respectively. Steam explosion and freezing disintegration increased the  Table 1, the higher correlation coefficients (≥0.980) and q e,cal which matched better with q e,exp indicated that the adsorption of RB 19 onto goat hair was in accord with pseudo-second-order model.

Effect of steam explosion on adsorption isotherms of goat hair
As can be seen from Figure 5 and Table 2, the adsorption capacity of EGHP toward RB 19 is significantly higher than that of CGH and FDGH. The Langmuir equation agreed well with the equilibrium isotherm for CGH, FDGH and EGHP toward RB19 owing to the high correlation coefficient (R 2 = 0.99), indicating a monolayer adsorption at the binding sites on the surface of goat hair due to electrostatic interaction between adsorbent group -NH 3 + and adsorbates group -SO 3 -. The intraparticle diffusion model will be studied in the future. As shown in Table 2, the adsorption capacity q max of EGHP was highest (286.30 mg/g), which was 2.3 and 5.2 times that of FDGH and CGH, respectively. EGHP showed highest removal efficiency for RB 19 in dye wastewater.

Effect of steam explosion conditions on adsorption capacity of EGHP
As can be seen from Figure 6a, the adsorption capacity of goat hair toward RB19 obviously increased with the increased steam pressure from 1.3 to 1.8 MPa. Under the same steam treatment time of 150s, the adsorption capacity of EGHP toward RB19 under the steam pressure of 1.8 MPa increased to 427 mg/g, which was 7.7 times that of CGH (55 mg/g). As shown in Figure 6b, the adsorption capacity slightly increased with the extended steam treatment time from 30-240s under the same steam pressure of 1.5MPa. The results indicated that the steam pressure had more important effect to the adsorption capacity of goat hair toward RB 19 than the steam treatment time.

Effect of steam explosion on the structures of goat hair
Morphology SEM images of goat hair after being steam exploded under different conditions are presented in Figure 7. Compared with CGH, the exploded goat hair presented the smaller scale spaces (due to the contraction of goat hair) and their scales were seriously damaged. When the steam pressure increased from 1.3 to 1.8 MPa at the same steam treatment time of 150s, the scales of goat hair were damaged more seriously and some scales almost fell off. Without the hindrance of scales, more adsorption sites of goat hair were exposed and the dye molecules could easily and fast entered into the amorphous region of EGHP thereby improving the adsorption rate and amount of EGHP. As shown in Figure 7, the damage of scales slightly increased with the extended steam treatment time from 30 to 240s. Electric photo of CGH and SEM images of FDGH and EGHP are presented in Figure 8. It shows that FDGH has a length from 50 to 150 um and a fineness from 4 to 40 μm and EGHP was particle form which had a diameter from 2 to 20 um, indicating that the goat hair is not only broken in the axial direction but also is cleaved in the radial direction. The bioadsorbent with a smaller size resulted in the higher adsorption.   Figure 9 and Table 3 shows the Effect of steam pressure and steam treatment time on the crystal structures of CGH and EGHP after the different steam explosion conditions, respectively. EGHP after the different steam explosion conditions show significantly lower crystal index (CI) than CGH. Moreover, the CI of EGHP decreased from 8.1 to 2.6 with the increasing steam pressure from 1.3 to 1.8 MPa. It indicated that the amorphous region of goat hair expanded after the steam explosion. Therefore, more RB19 molecules could be adsorbed into the expanded amorphous region of EGHP and the adsorption capacity improved. Table 3 also shows that CI of EGHP at 1.5 MPa slightly decreased from 8 to 5.2 with the increased steam treatment time from 30 to 240s. In general, steam pressure has a more significant effect on the crystalline structure and the adsorption capacity of goat hair.  Figure 10 shows nitrogen adsorption-desorption isotherm curves for different bioadsorbents. It shows that all EGHP have a higher quantity of adsorbed nitrogen than CGH and FDGH. The quantity of adsorbed nitrogen of EGHP significantly increased with increasing the steam pressure from 1.3 to 1.8 MPa. As shown in Table 4, BET specific surface area and the pore size of EGHP increased with increasing the steam pressure. BET specific surface area, BJH adsorption cumulative surface area and volume of pores for EGHP (1.8MPa,150s) are, respectively, 12.7, 20.6 and 31.9 times that of CGH, indicating that there are significantly more pores between 17 Å and 3000 Å width for EGHP. However, FDGH and CGH have a near BET specific surface area and the pore size. The   adsorption capacity has a positive and linear correlation with the BET specific surface area. However, the BET specific surface area does not necessarily stand for the real surface area under the adsorption conditions since goat hair swell in aqueous environment. Here, the BET specific surface areas are used to compare adsorption capability of three different goat hair with similar chemical structures, assuming that their swelling behaviors are also similar to each other (Li, Mu, and Yang 2019). Compared with CGH and FDGH, EGHP after the steam explosion (1.8 MPa, 150 s) has highest adsorption capacity (427 mg/g) toward RB19, which was 7.8 times that of CGH (55 mg/ g), as shown in Figure 3a. It was reported that several RB19 molecules aggregated into an ellipsoid with three axes 33.4 Å*11.4 Å *8.8 Å (Shimode Mitsuo et al. 1996). Most dyes have an average size around 10-20 Å (Li, Mu, and Yang 2019). It was reported that pores with diameters less than 20 Å in a swollen cellulosic material take up approximately 20% of its total surface area (Li, Mu, and Yang 2019). Therefore, a certain proportion of the total surface area are not able to be entered by dye molecules (Li, Mu, and Yang 2019). By increasing the steam pressure from 1.3 to 1.8 MPa, some pores were enlarged as shown in Table 4 and more dye molecules could enter the pores of the EGHP more easily. In general, high BET specific surface area and available porosity of the bioabsorbent enable the rapid removal of RB 19 dyes from wastewater (Ibrahim et al. 2017).   Table 5 shows the concentration of free amine groups for CGH, FDGH and EGHP. The concentration of free NH 2 groups for EGHP was increased, which was 1.60 times higher than that of CGH and 1.01 times higher than that of FDGH. The increased free NH 2 groups resulted from the degradation of the keratin biomolecules of goat hair during the steam explosion. The temperatures of saturated steam corresponding to the pressure of 1.3, 1.5 and 1.8 MPa are 192, 198 and 207°C respectively, which could result in the degradation of goat hair. Therefore, more NH 2 from EGHP can be used to adsorb -SO 3 -of RB 19 molecules.

Recyclable adsorption ability of EGHP toward RB19
As shown in Figure 11, the first adsorption capacity of EGHP was 286.4 mg/g, and the adsorption capacity after 6 adsorption-desorption cycles decreased to 226.2 mg/g due to that a small quantity of the bonded dye molecule could not be removed. Although the adsorption capacity decreased with increasing the cycle number, the adsorption capacity still maintained 79% of the first adsorption capacity after 6 cycles, indicating that EGHP which had adsorbed RB 19 could be easily regenerated in NaOH solution (0.1 g/L) and had good reusability. The adsorption and desorption performance of keratin-based goat hair could be simply controlled by adjusting the charges on the sorption sites for good reusability.  11. The adsorption capacity of EGHP after different adsorption-desorption cycles toward RB 19 (Conditions of steaming explosion: 1.3 MPa, 150 s; Adsorption conditions: initial dye concentration 3500 mg/L, pH 2.0, amount of EGHP 10 g/L).

Comparison of adsorption capacities with other adsorbents
As shown in Table 6, compared to the some recently reported adsorbents for adsorbing anionic dyes in dyeing wastewater, such as cationic Zr-based metal-organic framework, nanoporous silica hydrogel by cross-linking method, nanolayer-constructed TiO(OH)2 microstructures, modified resin DK 110 with loading ionic liquid. Compared with these adsorbents, EGHP has a higher adsorption capacity and it has a great potential for anionic dye adsorption.

Conclusions
Steam explosion significantly increased the adsorption capacity toward RB 19 of goat hair. Compared with the steam treatment time, the steam pressure had a more important effect on the adsorption capacity. Steam explosion of 1.8 MPa and 150 s increased adsorption capacity from 55 to 427 mg/g at room temperature. The increased adsorption of EGHP was mainly due to the broken scales, decreased CI from 30.1% to 2.1%, the increased BET specific surface area from 0.678 to 8.583 m 2 /g and the increased free NH 2 groups. In addition, EGHP became small particles which had a diameter from 2 to 20 um and the pores enlarged in EGHP. These structure changes resulted in more anionic dye molecules could easily and fast entered into the amorphous region of EGHP and were adsorbed, thereby improving the adsorption rate and amount of RB 19 dyes. The time required to reach the equilibrium of adsorption decreased from 26 to 6 h. The adsorption of RB 19 was in accord with the second-order kinetic model and the Langmuir thermodynamic model for CGH, FDGH and EGHP. The adsorption capacity still maintained 79% of the first adsorption capacity after 6 adsorption-desorption cycles. Waste-keratin via steam explosion could be considered as promising bioadsorbent to treat wastewater from dyeing industry.