Dyeing of Cotton with Indigo Using Alkaline Protease and Additives Barvanje bombaža z indigom z uporabo alkalne proteaze in aditivov

Indigo is invariably applied on cotton to produce an attractive blue shade, together with the desired wash-down eff ects. Because it is water insoluble, indigo is reduced and solubilised with sodium dithionite and NaOH to develop affi nity for cotton. Sodium dithionite dissociates into hazardous by-products viz. sulphate, sulphite and thiosulphate with a harmful eff ect on the environment due to their toxicity, as well as a corrosive eff ect on waste lines. To overcome these problems, the eco-friendliness of alkaline protease, together with iron (II) sulphate (FeSO4) as a reducing agent, was studied as a replacement for sodium dithionite. Dyed samples were characterised by attenuated total refl ection, using Fourier transformation infrared spectroscopy, scanning electron microscope and X-ray diff raction (XRD). It was observed that alkaline protease, together with iron (II) sulphate, is also capable of producing a comparable reduction potential in dye baths, reduction bath stability, and the surface colour strength and colour fastness properties of dyed cotton compared to those obtained using sodium dithionite.

, [187][188][189][190][191][192][193][194][195][196][197][198][199] by-products formed due to the decomposition of sodium dithionite are sulphur compounds (e.g. Na 2 S, NaHS, etc.), which pollute the atmosphere through the formation of hydrogen sulphide. At the same time, sulphur salts in the form of sulphates and sulphites (Na 2 SO 3 , NaHSO 4 , Na 2 SO 4 and Na 2 S 2 O 3 ) contaminate sewage, lower its pH and corrode concrete supply and drainage pipes [3]. Other problems associated with the use of sodium dithionite relate to costs and storage. Th e problems associated with the use of Na 2 S 2 O 4 have led to the search for alternative, non-dithionitebased reduction systems. Th ese include the application of iron (II) salts, together with gluconic acid and NaOH at 60 °C [3][4][5], and iron (II) salts in combination with tartaric or citric acid, triethanolamine and NaOH at room temperature [6][7]. All these reduction systems showed results comparable to sodium dithionite with some exceptions. In recent years, the use of eco-friendly materials, such as enzymes for sustainable textile processing has emerged [9,10]. Enzymes belonging to oxidoreductases and hydrolases categories play an important role in the reduction of dye in alkaline media. One of those enzymes, e.g. alkaline protease belonging to the hydrolases category with nomenclature 3.4.x, has been found to be useful in the reduction of sulphur dye [11,12]. It is used for various industrial purposes, such as detergents, waste management, food, leather, silver recovery and textiles, to improve the shrinkage resistance of wool, for bioblasting and for the removal of sericin [13][14][15][16][17][18][19][20][21][22][23]. Alkaline protease possesses many characteristic features, such as its stability at high temperature at an alkaline pH [18,24]. Th is work studied the application of alkaline protease, together with iron (II) sulphate as a reducing agent, for the dyeing of cotton with indigo. Pre-treated cotton was dyed with reduced and solubilised indigo using sodium dithionite and alkaline proteases separately using the '6 dip 6 nip' technique. Th e Box-Behnken response surface design was used to analyse the performance of alkaline protease, together with iron (II) sulphate, to achieve the optimised parameters and performance of both reduction systems in terms of pH, the reduction potential (mV) at various stages of dyeing, the surface colour strength (K/S) of dyed cotton, the stability of the reduction bath, and the fastness properties and tensile strength of dyed cotton. Th e characteristics and surface morphology of dyed cotton were evaluated using ATR-FTIR, XRD and SEM.

Preparation of padding liquor a) Sodium dithionite and NaOH system
A stock vat and dilution liquor were used to prepare the required concentration of indigo padding liquor (Table 1). To prepare the stock vat, the required amount of sodium hydroxide (NaOH) and indigo were added to 100 ml water and heated to 50 °C. Th e required amount of sodium dithionite (Na 2 S 2 O 4 ) was then added and the solution left to sit for 15-20 minutes to complete the reduction of the indigo. To prepare the dilution liquor, the required amount of NaOH and Na 2 S 2 O 4 was added to 1 litre of water at room temperature and stirred well until a clear solution was obtained. A padding liquor with 3 g/l indigo was prepared from these two solutions. A total of 567 ml of dilution liquor was added to 100 ml of the reduced stock vat to make 667 ml of padding liquor solution. Th e concentrations of dye, NaOH and Na 2 S 2 O 4 to prepare the stock vat and dilution liquor were as detailed below (as per guidelines of BASF) to prepare control. b) Alkaline protease and iron (II) sulphate system In this case, the padding liquor was prepared in the same manner used for the dithionite system, with only diff erence being the use of protease, together with iron (II) sulphate, instead of dithionite.

Dyeing of cotton with indigo
Cotton was dyed with reduced indigo from both reduction systems using the '6 dip 6 nip' padding technique. Th is included the dipping of cotton in the dye liquor for 30 seconds, followed by padding at a pressure of 1 kg/cm 2 for 75-80% of pick up and airing for 1 minute to complete the '1 dip 1 nip' cycle. Th e cotton fabric was dyed in six such consecutive cycles with fi nal airing for 3 minutes to convert the reduced dye on the fabric to its oxidised form. Th e dyed samples were then thoroughly washed in hot water.

Statistical analysis of dyed cotton
Th e Box-Behnken response surface design was used to analyse and optimise process parameters. Th is included identifying the best suitable combinations of parameters and the levels thereof to achieve a dye strength (K/S) equivalent to that obtained in the dithionite system. Five dyeing parameters were studied: the concentration of FeSO 4 , NaOH, alkaline protease, indigo and temperature. Th ese factors, with their coded values according to the 3 5 Box-Behnken experimental design, are presented in Table 2. Using these fi ve parameters (factors), each with three levels, a 3 5 Box-Behnken design was run to obtain a set of data (run), consisting of a total of 46 runs with six replicates at the central point. Th e design run is presented in Table 3. Th e results were analysed using response surface plots and equations were formed for a response at a 95% confi dence level. Response surface fi gures were analysed to understand   the eff ect of an individual parameter (factor) on dye strength (K/S). A regression equation was formed accordingly. All design formations and statistical analysis were carried out using Design Expert 7 soft ware. A quadratic polynomial was used to analyse the relationship of dye strength (K/S) (response) with fi ve independent variables (factors) for Box-Behnken design runs. Th e accuracy of the model was verifi ed using the coeffi cient of determination (R 2 ) to the measure the goodness of fi t to the model. When R 2 approaches unity, the empirical model fi ts the actual data. P-values of less than 0.05 were considered to be statistically signifi cant. Th e lack-of-fi t test was analysed to check the adequacy of the model. Two techniques, i.e. the response surface fi gures technique and the regression equation technique, were used to predict the optimised combination of the factors that result in maximum colour strength (K/S).

Evaluation of dye bath and dyed cotton
Th e surface colour strength (K/S) of dyed cotton was evaluated using a computer colour match (Datacolor Check, Datacolor International, US), while colour fastness properties, such as light, wash and rubbing, were evaluated using AATCC test methods 16-2004 (light), 61-2007 (wash), 8-2007 (rubbing) respectively. Reduction dye baths were evaluated in terms of pH and reduction potential using a digital pH cum ORP meter (Century Instruments, Chandigarh, India) at various stages of dyeing i.e. before and aft er the reduction of the dye, as well as aft er the completion of the dye.

Estimation of dye uptake
To study the amount of indigo uptake on cotton fabric aft er each dip and nip, the known weight of dyed cotton fabric was dissolved in dimethyl sulfoxide (DMSO) to extract indigo from the dyed cotton fabric. Th e extract was analysed using a UV-Vis spectrophotometer (Perkin Elmer) to evaluate the mass of indigo (g) per 100 g of cotton fabric aft er each padding-nipping-airing cycle.

Stability of reduction dye baths
Reduction baths in sodium dithionite and alkaline protease, together with iron (II) salt, were prepared in the absence of dye and stored for a specifi c period (0-24 hours) at room temperature, aft er which pH and reduction potential (-mV) were noted. Dye was added and the cotton fabric was dyed aft er reduction and solubilisation. To study the stability of reduction baths in the presence of dye, dye baths were prepared and stored for a specifi c period, and pH and mV were noted, followed by dyeing of the cotton fabric in these baths.

X-ray diff raction
Diff ractograms were generated using a Malvern Pan analytical XRD. A small sample was clamped into a sample holder on a goniometer (radius 240 mm) in a scanning range of 10-60 with a step size of 0.008° and an X-ray radiation wavelength of λ = 0.15406 nm using Cu K α . Th e X-ray generator was operated at 40 mA and 45 kV. ATR-FTIR spectra were obtained using an ALPHA FT-IR spectrophotometer (Bruker, USA).

Tensile strength of cotton fabric
A universal testing machine (Aimil, Delhi) was used to measure the tensile strength of cotton fabric using an ASTM D5035 test method aft er dyeing in both reduction systems.

Dyeing of cotton with sodium dithionite
Cotton fabric was dyed with indigo using the '6 dip 6 nip' padding technique, followed by oxidation and washing. Th e K/S of the dyed cotton was determined to be 22.40 at λ max 590 nm. Th e range of pH and mV were 12.2 to 12.8 and (-640 mV to -780 mV) respectively at various stages of dyeing.

Dyeing of cotton with alkaline protease
Eff orts to dye cotton fabric with indigo using alkaline protease instead of sodium dithionite could not generate the required reduction potential nor any reduction of dye leading to no dye strength (K/S) on cotton. Th e reduction potential of the bath was in the range of -330 mV to -370 mV. In contrast, indigo requires a reduction potential of around -650 mV and more for reduction. It was determined that enzyme activity is enhanced by the addition of metals to the bath [25,26]. For this reason, indigo reduction baths were formulated with alkaline protease, together with iron (II) sulphate. Cotton fabric was dyed in this bath in the same manner that is used in the dithionite system. Th e reduction potential of the bath was raised to around -700 mV to -720 mV with the reduction of indigo. Th e cotton fabric was then dyed with a lower K/S. To improve the K/S, the levels of dyeing parameters were varied, and the reduction baths were prepared by dyeing cotton from each bath to attempt to achieve the same dye strength on cotton that was obtained in the dithionite system. Th e more suitable ranges were concentrations of FeSO 4 (20-25 g/l), NaOH (7.5-12/5 g/l), alkaline protease (1-2 g/l), indigo (7-8 g/l) and temperature (60-80 °C). Th e actual values of levels in the Box-Behnken design were formed accordingly (Table 4). Putting these levels in the Box-Behnken design resulted in 46 separate runs. Indigo baths were prepared based on these sets of parameters and levels, while the cotton fabric was dyed and the K/S was evaluated. Th e results are presented in Table 5. It was observed that the range of K/S was 12.26 to 23.9, while the highest K/S was observed if the bath  (3), [187][188][189][190][191][192][193][194][195][196][197][198][199] was prepared according to run 33, i.e. FeSO 4 (20 g/l), NaOH (10 g/l), alkaline protease (1.5 g/l), indigo (7.5 g/l) and temperature (60 °C). It should be noted that a 3 g/l indigo bath resulted in a K/S of 22.40 in dithionite system.

Infl uence of dyeing parameters on K/S
Th e infl uence of process parameters on concentrations of FeSO 4 , NaOH, protease and indigo, and temperature on K/S was evaluated using the Box-Behnken design and response surface methodology. All of the main eff ects, two interaction factors and the cubic eff ect with R-square 0.97 obtained using ANOVA are presented in Table 6. Th e model was signifi cant at a 95% confi dence interval, as the value of 'Prob>F' was less than 0.05. In this case, the concentrations of FeSO 4 , NaOH, protease and tempera-   Table 6. Th is equation can predict the theoretical K/S of dyed samples for given dyeing parameters.

Infl uence of FeSO 4 and NaOH concentrations
Th e combined eff ect of FeSO 4 and NaOH concentrations on K/S at a constant alkaline protease concentration, indigo concentration (moderate level) and temperature (lower level) is shown in Figure  1(a). A moderate level of NaOH (10 g/l) and FeSO 4 (20 g/l) resulted a maximum K/S of 23.9. Increasing the concentration of FeSO 4 resulted in a decrease in the dye strength (K/S) of cotton. An increased concentration of FeSO 4 may have decreased the concentration of NaOH in the dye bath due to the formation of insoluble Fe(OH) 2 , which may aff ect the solubility of reduced indigo.

Infl uence of FeSO 4 and protease concentrations
Th e combined eff ect of FeSO 4 and alkaline protease concentrations on K/S at constant indigo and NaOH concentrations (medium level) and temperature (lower level) is shown in Figure 1(b). A lower level of FeSO 4 concentration (20 g/l) and a moderate level of protease concentration (1.5 g/l) resulted in a maximum K/S of 23.9 with a reduction potential in the range of (-660 to -720) mV before and aft er the reduction of the dye, and at the end of dyeing. Increasing the concentration of FeSO 4 decreased the K/S of dyed cotton to 22.6, which may be due to the partial reduction of indigo. FeSO 4 reacts with NaOH to form Fe(OH) 2 , resulting in a decrease in the effective concentration of sodium hydroxide (moderate level) in dye baths.

Infl uence of FeSO 4 and temperature
Th e combined eff ect of FeSO 4 concentration and temperature on K/S at constant alkaline protease, indigo and NaOH concentrations (moderate level) is shown in Figure 1(c). A lower level of FeSO 4 concentration and temperature resulted in a maximum K/S of 23.9. Increasing the FeSO 4 concentration and temperature resulted in a decrease in the K/S of dyed cotton to 16.69. In this case, protease was probably not completely activated to reduce indigo.

Infl uence of protease concentration and temperature
Th e combined eff ect of protease concentration and temperature on K/S at constant indigo and NaOH concentrations (moderate level) and FeSO 4 (lower level) is shown in Figure 1(d). A moderate level of protease concentration and a lower temperature level resulted in a maximum K/S of 23.9. Increasing the temperature resulted in a decrease in K/S to

Indigo uptake and surface colour strength
Th e dye uptake (g of dye/100 g cotton) aft er each dip/nip was evaluated and is shown in Figure 2(a). Th e respective K/S aft er each dip/nip against that of indigo uptake is shown in Figure 2(b). K/S and dye uptake both increased proportionately. Although fi nal K/S was nearly identical in both reduction systems, total dye uptake was found to be higher in the protease system despite a lower K/S in the protease system aft er the fi rst dip/nip compared to that in the dithionite system. Th e same K/S in both the cases with a variation in dye uptake facilitated more diff usion of dye into cotton in the protease system.

Stability of reduction baths 3.5.1 In the absence of dye
Th e stability of reduction baths with Na 2 S 2 O 4 and alkaline protease was studied in the absence of dye for up to 24 hours. Reduction baths were prepared and covered. Aft er storing them for a specifi c period of time, the pH and reduction potential of the baths were measured. Indigo was then added to the baths. Dyeing was carried out, with the results presented in Table 7. Both of the reduction systems retained their reduction capability for up to 24 hours. Th ere was a progressive drop in mV and pH in both reduction baths with the passage of time. K/S gradually decreased with an increase in storage time in both reduction systems and is shown in Figure 3(a). Although reduction baths in both systems showed good stability over a 24-hour period, maximum surface colour strength was observed for dyeing at 0 hours.

In the presence of dye
Reduction baths with Na 2 S 2 O 4 and alkaline protease were prepared, followed by the addition of indigo. Th e baths were covered and stored for up to 24 hours. Th e results were noted in terms of pH and reduction potential, and are presented in Table 8.    is also shown in fi gure 3(b). Reduced dye baths showed a maximum dye strength for dyeing at 0 hours, although good stability was observed for up to 24 hours.

SEM analysis of dyed cotton fabric
Th e surface morphology of dyed cotton was characterised using an SEM (Zeiss EVO 50) at a voltage of 10 kV and a 5,000-x magnifi cation. Th e surface of the cotton fi bre was considered damaged due to dyeing. Th e SEM images of undyed cotton as well as cotton dyed using sodium dithionite and protease systems are shown on Figure 4. Th e images show marginal damage on the surface of cotton dyed in the sodium dithionite system, which was not prominent in the protease system.

ATR-FTIR of dyed cotton
Th e spectra of dyed cotton with sodium dithionite and alkaline protease are compared with undyed cotton (control) and are shown in Figures 5(a) and 5(b) respectively. Th e absorption bands were mainly observed in the ranges of 3,869 to 2,850 cm -1 and 1,623 to 522 cm -1 . Strong band spectra were found in a range of 3,869-2,900 cm -1 , as the result of the stretching vibration of O-H and C-H bonds, while the band peak at around 3,266-3,258 cm -1 is due to the stretching vibration of R-OH in cellulose. Th is peak also includes inter-and intra-molecular hydrogen bonds [27][28][29].
Th e band peak of around 2,913 cm -1 to 2,849 cm -1 is due to the symmetrical and asymmetrical stretching of -CH 2 groups in cellulose [30,31]. A typical band in the range of 1,623 cm -1 to 522 cm -1 was observed. Th e absorption band at 1,623 cm -1 to 1,619 cm -1 is characterised for the stretching of C=C [18]. Th e band peaks at 1,459 cm -1 , 1,393 cm -1 , 1,366 cm -1 , 1,310 cm -1 , 1,161 cm -1 and 1,057 cm -1 are characterised for the deformation or stretching vibrations of C=O, C-H, C-O-C, C-O, C-N, C=C and N-H groups in cellulose, as well as indigo [32][33][34]. In Figure 5(b), diff erences were observed in the spectrum of dyed cotton using alkaline protease. Th ere are changes in the absorption band in the range of 748 cm -1 to 700 cm -1 , which is assigned to the inplane bending of the methyl group in cellulose [31]. Th e band spectra of both the sodium dithionite and alkaline protease systems were found to be nearly the same. It can thus be concluded that no chemical changes occurred in the new proposed reduction system.

XRD of dyed cotton
Th e X-ray diff ractograms of undyed and dyed cotton are shown in Figure 6. Th e purpose of x-ray diffraction was to identify the loss in crystallinity of cotton due to a loss in tensile strength aft er dyeing. Th e degree of crystallinity is one of the most important parameters for a crystalline structure and was evaluated using Herman's method [28][29][30][31][32][33][34][35]. In this method, the crystallinity index was calculated by the ratio of crystalline area to the total area of the X-ray diff raction curve, as given equation 2.
where, A Crystaline is the crystalline area and A Total is area of X-ray diff raction curve.
Th e oxidation reaction of indigo may lead to a decrease in the degree of polymerisation of cellulose and may cause a loss in the tensile strength of cotton. Th is study revealed that crystallinity remained unchanged in the warp of dyed cotton in both reduction systems, although there was a marginal (7%) drop in crystallinity in dyed cotton with dithionite, while the protease system did not result in a drop in the crystallinity of cotton (Table 9). Interestingly, the drop in tensile strength in the weft direction was similar in both systems, but marginal. Th e drop in the crystallinity index was also found to be around 7% in the dithionite system and 4% in the protease systems. Th is indicates that no significant damage occurred in the newly proposed protease-based reduction system.

Fastness performance
Th e light, rubbing and wash fastness of indigo-dyed cotton in the Na 2 S 2 O 4 and alkaline protease systems were evaluated and compared. Th e results are presented in Table 10. Light fastness remained very good to excellent in both the Na 2 S 2 O 4 and alkaline protease reduction systems. Wash fastness was also very good to excellent (4)(5) in the alkaline protease system. Rubbing fastness was excellent, and very good to excellent in dry and wet conditions respectively. It can be concluded from the data that the new proposed alkaline protease-based reduction system proved to be a good and comparable match with the commercial sodium dithionite reduction system.

Conclusion
Th e work presented in this study illustrates the use of alkaline protease, together with iron (II) sulphate, as a promising reducing agent for the dyeing of cotton with indigo. Both sodium dithionite (Na 2 S 2 O 4 ) and alkaline protease demonstrated comparable K/S, with few variations. Dye strength with 3 g/l indigo in the sodium dithionite system was a complete match with that of cotton dyed in the protease system for an indigo concentration of 7.5 g/l, although dye uptake with an increased concentration of indigo was found to be on the higher side. Th e stored baths in both the absence and presence of indigo showed good stability for up to 24 hours. However, the maximum dye strength was obtained at 0 hours of dyeing, i.e. just aft er the reduction and solubilisation of the dye. Damage on the surface of dyed cotton was less prominent in the alkaline protease-based reduction system. Th e change in the crystallinity index was around 4%, meaning no significant damage was observed. Th e drop in the tensile strength of dyed cotton using alkaline protease was less signifi cant than that using dithionite, as less damage to the cotton occurred. Th e colourfastness of dyed cotton was similar in both reduction systems. Th us, alkaline protease, together with iron (II) salt, could serve as a substitute for sodium dithionite.