Abstract
As some synthetic dyes are regarded to be toxic, mutagenic and carcinogenic, the search for eco-friendly alternatives for the synthesis of dyes and coloration has gained importance. For this reason, this study focused on finding new eco-friendly alternatives for coloring cotton. 100% cotton knitted fabrics were subjected to enzymatic coloration using a commercial laccase enzyme and various precursors. After determining the colors, the effect of pH on the enzymatic dyeing process was investigated. Then the optimization of reaction conditions was also realized statistically for the precursors giving the best results in terms of color. With the aim of obtaining further improvements in color-yield values obtained in enzymatic dyeings, the effect of the pretreatment process and the use of ultrasound were also investigated. Furthermore, the reaction pathways in enzymatic coloration were explained and results were confirmed by means of Fourier Transformed Infrared analysis. As a result of experimental studies, red and lilac colors could be successfully obtained on cotton for the first time in the literature. In this way, the theoretical basis of enzymatic dye synthesis and dyeing of cotton was clarified comprehensively. Furthermore, technical (color reproducibility; washing, rubbing, light and perspiration-fastness values; and UV protection factor), economical (chemical, energy and water consumption required for dyeing (including aftertreatments) of 1 kg fabric) and ecological aspects of enzymatic dyeings were compared with reactive dyeing. According to the experimental results it was found that biological treatment alone was enough for wastewater of enzymatic coloring, while chemical treatment will also be needed in reactive dyeing wastewater. Furthermore, color reproducibility, evenness and UV protection properties of dyed samples were comparable with that of reactive dyeings. However, in terms of the fastness levels achieved, the enzymatic coloring was far behind the reactive dyeing.
The use of synthetic dyes in the textile industry has resulted in the generation of highly polluted effluents. The complex structure of these dyes makes them recalcitrant, causing serious environmental concern. 1 This has driven the search for environmentally acceptable alternatives to chemical synthesis of dyes and coloration. 2 Green catalytic alternatives are particularly needed in the production of dyes. For this reason, in the last decades, bio-based alternatives with the least environmental impact have been widely researched for dye synthesis. In this context, enzymatic processes with laccases represent one of the most promising tools. 3
Laccases (EC 1.10.3.2) are multi-copper oxidoreductases that can catalyze the oxidation of various aromatic compounds.
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They are generally extracellular and hence easy to handle, and can be derived from bacteria or fungi on a large scale.
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The ability of laccases to generate color in situ from originally non-colored, low-molecular substances makes their use an alternative to the conventional dyeing processes.
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Enzymatic polymerization can be defined as the in vitro synthesis of polymers by enzymes via nonbiosynthetic pathway.7 Since 1985, enzymatic oxidative polymerization of aromatic compounds has been widely investigated.
2
Laccases catalyze one-electron oxidation of a wide range of inorganic and organic substances, coupled with one four-electron reduction of oxygen to water (O2 + 4H+ + 4e− → 2H2O).
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The range of substrates oxidized by laccases can be expanded through a mediator-involved reaction mechanism.
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The reactions catalyzed by laccase can be divided into three groups, in all which colored products are formed:
direct oxidation of simple phenolic derivatives, mediated oxidation of phenolic and non-phenolic substrates in the presence of mediators, coupling of reactive radicals formed by the action of laccase.
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The first type of reaction is oxidation of simple organic compounds such as mono- and diphenols and their derivatives, having amino-, carboxy-, methoxy- and sulfonate functional groups. 9 The principal mechanism of laccases is one-electron oxidation of phenolic compound to form phenoxy radicals using oxygen. The phenoxy radical may react with a second radical to form o-quinones and further spontaneously polymerize in a non-enzymatic pathway, which is the third type of reaction 2 including covalent coupling to form dimers, oligomers and polymers through C–C, C–O and C–N bonds. 10 On the other hand, the use of mediators for the oxidation occurs in case of some non-phenolic compounds, aromatic amines, and large molecules, such as the lignin complex, which do not have access to the active center of laccase. 9
Colored compounds, obtained as a result of catalysis of the precursors by laccase, can be used in the dyeing of textile fibers. When the studies on the coloring of textile materials with laccase enzyme are examined, it is understood that most of the research has been on protein fibers and studies on cotton are very few. Table 1 summarizes the articles on the enzymatic coloring of cotton to date.
Previous literature on enzymatic coloration of cotton
As can be seen from Table 1, there are very limited studies on enzymatic coloration of cotton, and a very limited range of colors could be obtained to date. At this point, the aim of this study was to obtain various colors on cotton fabrics by performing both dye synthesis and dyeing in the same bath, using a wide variety of precursors individually or in binary combinations. For this aim, 100% cotton knitted fabrics were subjected to enzymatic coloration using a commercial laccase enzyme and various precursors. As is known, various phenol or amine compounds can be used as precursor in enzymatic dyeing. When the literature is examined, it is seen that mainly phenolic compounds have been used, and and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt were investigated on cotton. However, in this study seven different amine and seven different phenol compounds, which have not been previously investigated with Cerrena unicolor as laccase enzyme, were used. This fact makes the current study novel.
After determining the colors that can be obtained with the laccase enzyme by using various precursors alone or in combination, the effect of pH in enzymatic dyeing process was investigated. The optimization of reaction conditions (enzyme concentration, precursor concentration, duration and temperature) was also realized statistically for the precursors giving the best results in terms of color. In this study, possible mechanisms of enzymatic dye synthesis with various precursors were also explained and results confirmed by means of Fourier transform infrared (FT-IR) analysis. As publications on enzymatic coloration of cotton which also explain the mechanism in detail are very limited, it is believed that this study is original.
Although in the literature it was stated that enzymatic coloration with laccases is an environmentally friendly alternative to synthetic dyes, to the best of the authors’ knowledge none of these studies investigated the ecological aspects of enzymatic dyeing. For this reason, in this study, we also aimed to compare enzymatic coloration of cotton fabric with reactive dyeing, which is currently the most common process, in terms of ecology. For this aim, the best colors obtained via enzymatic coloring were matched under laboratory conditions with reactive dyes. Then, chemical oxygen demand (COD), biological oxygen demand (BOD), total dissolved solids (TDS), ammonia nitrogen, and chloride analyses were performed on the dyeing wastewaters to compare the environmental loads caused by enzymatic coloring and reactive dyeing. Furthermore, technical (color reproducibility; washing, rubbing, light and perspiration-fastness values; and UV protection factor) and economical (chemical, energy and water consumption required for dyeing (including aftertreatments) of 1 kg fabric) aspects of these dyeings were also compared.
Material and methods
Fabric, enzyme and precursors
100% cotton knitted (single jersey) bleached fabric (yarn count: Ne16/1) with a density of 158 g/m2 was used in the experiments. All trials were carried out on the A11610N model Thermal HT dyeing machine (rotational speed 33 rpm) using 5 g of fabric samples with pure water at a liquor ratio of 1:20. Prima Green Ecofade LT100 (Dupont) from fungal origin (Cerrena unicolor) with 5700 GLacU/g of activity was used as laccase enzyme. Various amine and phenol compounds and their combinations were used as precursor in the enzymatic colorations. The names, molecular weights and chemical formulas of the precursors used in experiments are given in Table 2.
Precursors used in enzymatic dyeings19–21
Enzymatic dyeing with various precursors
With the aim of determining the precursors which give the best results in terms of color, the fabrics were treated under standard conditions (0.4 g/L laccase enzyme, 2 mmol/L precursor, 60°C, 3 h). All trials were carried out at pH 5 (adjusted with acetic acid/sodium acetate buffer), where the activity of the laccase enzyme was the highest. Cotton fabrics were also treated with aromatic compounds in the absence of laccase and color could not be obtained or fabrics stained to the aromatic compound’s own color. Despite using only amine or phenol compounds, trials have also been made with their binary combinations as follows:
4-Nitro-1-naphthylamine
2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
Ferulic acid
3-Methyl-2-benzothiazolinone hydrazonhydrochloride monohydrate
After the enzymatic dyeing process, cold, hot and cold rinsings were applied to the fabric samples, respectively. Then fabric samples were dried and their CIE L*a*b* values were measured with a spectrophotometer.
Effect of pH on enzymatic dyeing
The effect of pH on enzymatic dyeing was also examined for A3, A7 and P4 precursors, which gave the best results in terms of color. Furthermore, the effect of pH has also been studied on the binary combinations (A2+P7 = AP). For this purpose, experiments were carried out at four different pH values: 3, 5, 7 and 9. In these experiments, enzyme concentration, precursor concentration, temperature and time was 0.4 g/L, 2 mmol/L, 60°C and 3 h, respectively. The dyeing process and aftertreatments were carried out as described above. However, the dissolving processes were carried out in the ultrasonic bath in order to prevent solubility problems. After dyeing, fabric samples were dried and color-yield (K/S) and CIE L*a*b* values were measured with spectrophotometer.
Optimization of enzymatic dyeing conditions
After determining the colors that can be obtained with the laccase enzyme by using various precursors alone or in combination, the optimization of reaction conditions (enzyme concentration, precursor concentration, duration and temperature) was realized for the precursors which gave the best result in terms of color. In all trials, the dissolving processes were carried out in the ultrasonic bath. Enzymatic dyeings were performed at various;
laccase enzyme concentrations (0.1, 0.2, 0.4, 0.8, 1.0 g/L), precursor concentrations (0.5, 1, 2, 4, 8 mmol/L), temperatures (25 (room temperature), 40, 60, 80, 100°C) and times (1, 2, 3, 4, 5 h).
All experiments were carried out at pH 5. After the enzymatic dyeing process, cold, hot and cold rinsings were applied to the fabric samples, respectively. Afterward, samples were dried and color-yield (K/S) and CIE L*a*b* values were measured with spectrophotometer. Variance analysis was performed for each of the factors affecting enzymatic dye synthesis over the color-yield values obtained in three replicates, and boxplot graphics were drawn by using Minitab 15.
Effect of fabric pretreatment process on enzymatic dyeing
In the literature
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it is stated that natural coloring pigments in cotton can be used as precursor in dye formation via enzymatic oxidation. Regarding this, 100% cotton knitted (single jersey) scoured fabric (yarn count: Ne16/1), which is not bleached (i.e. contains natural pigments), with a density of 145 g/m2 was also subjected to enzymatic coloration. In these trials, bleached fabric and scoured fabric were dyed at optimum conditions with selected precursors giving the best results. Dyeing conditions were as follows:
A3: 1 g/L enzyme, 8 mmol/L precursor, 80°C, and 4 h. AP: 1 g/L enzyme, 4 mmol/L total precursors (1:1 ratio), 20°C, and 1 h. A7: 0.1 g/L enzyme, 8 mmol/L precursor, 60°C, and 1 h. P4: 1 g/L enzyme, 8 mmol/L precursor, 60°C, and 3 h.
All experiments were carried out at pH 5. After the trials, color-yield (K/S) and CIE L*a*b* values of fabric samples were measured.
Effect of ultrasound on enzymatic dyeing
To determine the effect of ultrasonic energy on color-yield values obtained in enzymatic dyeing processes, trials under optimum conditions (mentioned in the previous section) were carried out in two different ways, with and without ultrasound. Conventional dyeings to be taken as reference were also performed in ultrasonic bath; however, ultrasound was disabled during the process. Kudos brand SK2210 HP model ultrasonic bath was used in the experiments. Dyeing baths were put in a beaker, then this beaker was attached to the clamp of a burette stand and placed in the ultrasonic bath. As the experiments were carried out in a beaker, 1 g of fabric samples was processed at a liquor ratio of 1:50 in order to avoid unevenness problem. After the trials, color-yield (K/S) and CIE L*a*b* values of fabric samples were measured.
Comparison of enzymatic coloration and reactive dyeing
In this part of the study, the colors of the yellow, red and lilac obtained via enzymatic coloring were matched under laboratory conditions with reactive dyes (cold group), which have the most important use in cotton dyeing today. Then, obtaining the same color by enzymatic coloring and reactive dyeing was compared in terms of technical, economical and ecological aspects.
Enzymatic dyeing recipes are given in the section “Effect of ultrasound on enzymatic dyeing.” Dyeing recipes for yellow, red and lilac colors with reactive dyes are given in Table 3. All reactive dyes used in experiments were bifunctional (monochlorotriazine/vinylsulphoone) as can be seen from their chemical structures given in Figure 1. Dyeing was started at 40°C with a liquor containing salt, leveling agent and dye, and after 30 min the temperature was raised to 60°C within 20 min. At this point, soda ash was added and the experiment was continued for 70 min at this temperature. After the process, cold, hot and cold rinsings were applied to the fabric samples. To observe the effect of after-treatment on fastness values in both enzymatic coloring and reactive dyeing, an after-treatment with 2% cationic fixing agent was applied to the dyed samples at 50°C for 20 min. pH was 5.5 (with acetic acid) in after-treatment process.
Reactive dyeing recipes for yellow, red and lilac colors
To determine the color reproducibility, each of the dyeings (both enzymatic and reactive dyeings) was repeated five times and coefficient of variation (CV) was calculated for K/S and CIE L*a*b* values of dyed samples.
Color measurements with spectrophotometer
Reflectance (R%) values of dyed samples were measured with X-Rite Gretag Macbeth E700 (D 65/10°) and color yields (K/S) were calculated by the Kubelka–Munk equation:
R=Reflectance value in maximum absorption wave length (nm)
K=Absorption coefficient, S=Scattering coefficient
CIE L*a*b* values of the samples were also measured by X-Rite Gretag Macbeth E700 (D 65/10°) spectral photometer.
L*: lightness value defined on scale 0 (black) to 100 (white)
±a*: red/green coordinate
±b*: yellow/blue coordinate
Color fastness assessment
Washing, rubbing (dry and wet), perspiration (acidic and alkaline) and light-fastness values of dyed samples were assessed according to ISO 105 C06, 26 ISO 105-X12, 27 ISO 105-E04 28 and ISO 105 B02 29 standards, respectively.
UV transmission analyses
UV transmission analyses of fabric samples were performed according to the AS/NZS 4399: 2017 standard using a Thermo – EV0600PC & Opt lab software program. 5*5 cm samples were cut and transmission measurements were made between 290 nm and 400 nm. The average of UVA (315–400 nm) and UVB (290–315 nm) transmission values and also UPF values were calculated over four measurements for each. According to this standard, samples with UV protection values of 15 are considered “minimum,” samples between 30 and 49 are considered “good,” and samples with 50 and above are considered “excellent.” 30
Wastewater analyses
COD, BOD, TDS, Kjeldahl nitrogen and chloride content of dyeing wastewaters were evaluated according to standard test methods SM 5220:B, 31 TS 4957-1 EN 1899-1, 32 SM 254:C, SM 4500-Norg:B and SM 4500-Cl:C, respectively. 31 Furthermore, pH values of wastewater samples were measured with Mettler Toledo Fivego pH-meter at room temperature.
FT-IR analysis
To gain insight into colored products obtained by laccase catalysis, coloring reactions were carried out using laccase enzyme and aromatic compounds in the absence of fabric. For this aim, firstly dyes were synthesized under optimum conditions given below;
A3: 1 g/L enzyme, 8 mmol/L precursor, 80°C, and 4 h. AP: 1 g/L enzyme, 4 mmol/L total precursors (1:1 ratio), 25°C, and 1 h. A7: 0.1 g/L enzyme, 8 mmol/L precursor, 60°C, and 1 h. P4: 1 g/L enzyme, 8 mmol/L precursor, 60°C, and 3 h.
Then solvent of the synthesized dye solutions was removed and solid dyes were obtained. Afterwards, these dyes were subjected to infrared analysis. For this aim, ATR/FT-IR (attenuated total reflectance Fourier transform infrared spectroscopy) spectrophotometer Shimadzu IR Prestige-21 was used over the range of 600–4000 cm−1 (measurement mode: transmittance, apodization: Happ–Genzel, number of scans: 13, resolution: 4.0). ATR device was PIKE MIRacle™. The obtained data were used to draw FT-IR spectra for each precursor and dye by using origin2019b program.
Results and discussion
Enzymatic dyeing with various precursors
CIE L*a*b* values related to the experiments on determining the colors that can be obtained in enzymatic coloring with laccase enzyme by using various phenol and amine compounds, alone or in combination, are given in Table 4. Photos of dyed fabric samples are given in Figure 2.
CIE L*a*b* values of enzymatic dyed cotton samples
Colors obtained in the enzymatic dyeing (at pH 5) of cotton fabrics.
When Table 4 and Figure 2 are examined, the first thing that draws attention is that mainly soil colors (A1, A4, A6, P1, P3, P4, P7 and PP) are obtained as a result of enzymatic coloring. In terms of color depth, it can be said from L* values and fabric photos that the best result for brownish color was obtained with P4 (catechol). The process will be more complicated when it is necessary to use two precursors for obtaining a particular color. Therefore, P4 is advantageous as it necessitates only one precursor for obtaining brown color. On the other hand, with some precursors (A2, A5, P2 and P5), color could not be obtained.
Yellow shades were obtained with A3 (4-nitro-1-naphthylamine), P6 (ferulic acid), and AA1 (4-nitro-1-naphthylamine + 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid monosodium salt). However, the yellow color obtained with P6 is very pale. On the other hand, the yellow color obtained with AA1 is dull. Therefore, in terms of both color efficiency and luster, A3 seems to be the best choice. It is also advantageous as only one precursor is used for obtaining that color.
With AP (3-methyl-2-benzothiazolinone hydrazonhydrochloride monohydrate + 4-methoxyphenol) and AA2 (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt + 4-amino-1-naphthalenesulfonic acid), red colors with a very good color yield were obtained. However, as the unevenness problem was detected in the color obtained with AA2, AP was accepted as the best alternative for obtaining red color.
The blue color could not be obtained, but a bluish color, namely lilac, was obtained in the trial where A7 (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) was used as precursor.
In terms of dyeing, not only the obtained color yield but also the fastness values are of great importance. For this reason, washing and rubbing-fastness tests were applied to the dyed fabric samples and the results are given in Table 5.
Washing and rubbing-fastness values of enzymatic dyed cotton samples using various precursors
When Table 5 is evaluated, it can be said that rubbing-fastness values are moderate to good. However, especially in some precursors washing-fastness values are problematic. For P4 and A7, which were chosen for obtaining brownish and bluish colors, respectively, both the washing and rubbing-fastness values are good. However, for A3 and AP, which were chosen for obtaining yellow and red colors, respectively, washing-fastness values are quite low. The reason for the low washing-fastness values is related to the dye–fiber interactions. As can be understood from the chemical structures of enzymatically synthesized dyes given later in Figures 11, 13, 15 and 17, only secondary interactions and hydrogen bonds can be formed between fibers and these dyes, as in the case of direct dyeing of cotton. This is thought to be the reason for the low fastness values.
Effect of pH on enzymatic dyeing
The effect of pH was also examined for selected precursors. For this purpose, experiments were carried out at four different pH values as; 3, 5, 7 and 9. Photos of fabric samples are shown in Table 6 and color-yield (K/S) and CIE L*a*b* values of dyed samples are given in Table 7.
Photos of fabric samples enzymatically dyed at four different pH values using various precursors
Color-yield (K/S) and CIE L*a*b* values for enzymatic dyeings carried out at four different pH values using various precursors
From Tables 6 and 7 it can be seen that pH affects the lightness–darkness (L*) rather than the nuance (a* and b* values) of the color. It is obvious from Table 6 that pH 5 gives the optimum results for all precursors, but the results obtained at pH 7 are also quite good. For AP, it was determined that there was a change in the nuance of the color depending on the pH. However, for pH 3 and pH 7, a shift from red to orange was observed, as the increase in yellowness (b*) value of a red color indicates that color shifted from red to orange. Since our main purpose was to obtain precise red, which is the primary color, it will be more appropriate to work at pH 5 also for AP.
Optimization of enzymatic dyeing conditions
After determining the precursors giving the best results, optimization of their reaction conditions (enzyme concentration, precursor concentration, time and temperature) was realized. Color-yield results obtained from optimization studies for A3 (yellow color) are shown in Figure 3 and Figure 4, and variance analysis results are given in Table 8.
Color-yield values for optimization of enzymatic dyeing conditions for A3 (yellow color).
Optimization of enzymatic dyeing conditions for A3 (yellow color).
Variance analysis results related to the optimization of enzymatic dyeing conditions for A3
When Table 8 is examined, it is seen that the effect of enzyme concentration, precursor concentration, time and temperature on the enzymatic dyeing with A3 precursor are statistically significant (p < 0.05). Tukey test was also performed to determine the source of the difference for each parameter, and the results are given in Table 9.
Tukey test results related to the optimization of enzymatic dyeing conditions for A3
In Table 9 the results obtained depending on the effect of the enzyme concentration are collected in four groups, and it can be seen that the best color yield is obtained at the highest enzyme concentration (1 g/L). The results obtained depending on the effect of the precursor concentration are collected in five groups, and it can be seen that the best color yield is obtained at the highest precursor concentration (8 mmol/L). Furthermore, the results obtained depending on the effect of the time are collected in four groups and the best result is obtained in 4 h. The results obtained depending on the effect of temperature are collected in four groups and the best color yield is obtained at 80°C. It is important to note that there was an unevenness problem in dyeings carried out at 25°C and 40°C, as can be seen from Table 10. For this reason, K/S values of these samples should not be taken into consideration. All the mentioned findings can be clearly seen from Table 10, in which the photographs of fabric samples related to the optimization of enzymatic dyeing conditions for A3 are given.
Photographs of fabric samples related to optimization of enzymatic dyeing conditions for A3
As a result of all these evaluations, optimum conditions of enzymatic dyeing with A3 were determined as 1 g/L enzyme concentration, 8 mmol/L precursor concentration, 80°C, and 4 h.
Color-yield results obtained from optimization studies for AP (red color) are shown in Figures 5 and 6, and variance analysis results are given in Table 11.
Color-yield values for optimization of enzymatic dyeing conditions for AP (red color).
Optimization of enzymatic dyeing conditions for AP (red color).
Variance analysis results related to the optimization of enzymatic dyeing conditions for AP
When Table 11 is examined, it is seen that the effect of enzyme concentration, total precursor concentration (1:1 mixing ratio), time and temperature parameters on enzymatic dyeing with AP are statistically significant (p < 0.05). Tukey test was also carried out to determine the source of the difference for each parameter, and the results are given in Table 12.
Tukey test results related to the optimization of enzymatic dyeing conditions for AP
In Table 12, the results obtained depending on the effect of the enzyme concentration are collected in four groups, and it is seen that the best color yield is obtained at the highest enzyme concentration (1 g/L). The results obtained depending on the effect of the total precursor concentration (1:1 mixing ratio) are collected in three groups, and it is seen that the best color yield is obtained at the highest precursor concentration (8 mmol/L). However, the optimum total precursor concentration was taken as 4 mmol/L, as the color of the dyed sample at the total precursor concentration of 8 mmol/L changed and its color turned to a yellowish-red rather than a precise red color. Furthermore, the results obtained depending on the effect of the time are collected in two groups and the best result is obtained in 1-2-3-4 h, which are in the same group. Therefore, it can be said that there is no statistically significant difference between 1-2-3-4 h, and 1 h of duration is the most suitable. In Table 12, the results obtained depending on the effect of temperature are collected in five groups and it is seen that the best color yield is obtained at 25°C (room temperature), which is the lowest temperature value.
All the mentioned findings can be clearly seen from Table 13 in which the photographs of fabric samples related to the optimization of enzymatic dyeing conditions for AP are given.
Photographs of fabric samples related to optimization of enzymatic dyeing conditions for AP
As a result of all these evaluations, the optimum conditions of enzymatic dyeing with AP were determined as 1 g/L enzyme concentration, 4 mmol/L total precursor concentration, 25°C, and 1 h.
Color-yield results obtained from optimization studies for A7 (lilac color) are shown in Figures 7 and 8, and variance analysis results are given in Table 14.
Color-yield values for optimization of enzymatic dyeing conditions for A7 (lilac color).
Optimization of enzymatic dyeing conditions for A7 (lilac color).
Variance analysis results related to the optimization of enzymatic dyeing conditions for A7
When Table 14 is examined, it is seen that the effect of enzyme concentration, precursor concentration, time and temperature parameters on enzymatic dyeing with A7 are statistically significant (p < 0.05). Tukey test was also carried out to determine the source of the difference for each parameter, and the results are given in Table 15.
Tukey test results related to the optimization of enzymatic dyeing conditions for A7
In Table 15, the results obtained depending the effect of the enzyme concentration are collected in four groups and it is seen that the best color yield is obtained at the lowest enzyme concentration (0.1 g/L). The results obtained depending on the effect of the precursor concentration are collected in four groups, and it is seen that the best color yield is obtained at the highest precursor concentration (8 mmol/L). Furthermore, the results obtained depending on the effect of the time are collected in two groups and the best result is obtained in 1-2-4-5 h, which are in the same group. There is no statistically significant difference between 1-2-4-5 h, therefore the 1 h period is the most suitable. The results obtained depending on the effect of temperature are collected in four groups, and it is seen that the best color yield is obtained at 40°C and 60°C. As the unevenness problem occurred at 40°C, 60°C was chosen as the most suitable temperature. All the mentioned findings can be clearly seen from Table 16 in which the photographs of fabric samples related to the optimization of enzymatic dyeing conditions for A7 are given.
Photographs of fabric samples related to optimization of enzymatic dyeing conditions for A7
As a result of all these evaluations, optimum conditions of enzymatic dyeing with A7 were determined as 0.1 g/L enzyme concentration, 8 mmol/L precursor concentration, 60°C, and 1 h.
Color-yield results obtained from optimization studies for P4 (brown color) are shown in Figure 9 and Figure 10, and variance analysis results are given in Table 17.
Color-yield values for optimization of enzymatic dyeing conditions for P4 (brown color).
Optimization of enzymatic dyeing conditions for P4 (brown color).
Variance analysis results related to the optimization of enzymatic dyeing conditions for P4
When Table 17 is examined, it is seen that the effect of enzyme concentration, precursor concentration, time and temperature parameters on enzymatic dyeing with P4 are statistically significant (p < 0.05). Tukey test was also carried out to determine the source of the difference for each parameter, and the results are given in Table 18.
Tukey test results related to the optimization of enzymatic dyeing conditions for P4
In Table 18, the results obtained depending on the effect of enzyme concentration are collected in three groups, and it is seen that the best color yield is obtained with 0.4-0.8-1 g/L, which are in the same group. There was no statistically significant difference between 0.4-0.8-1 g/L. As the unevenness problem was observed at lower enzyme concentrations, 1 g/L was taken as optimum. The results obtained depending on the effect of the precursor concentration are collected in five groups, and it is seen that the best color yield is obtained at the highest precursor concentration (8 mmol/L). The results obtained depending on the effect of time are collected in three groups, and the best result is obtained in 3 h. The results obtained depending on the effect of temperature are collected in three groups, and the best color yield is obtained at 25-40-60°C, which are in the same group. As the unevenness problem occurred on dyed fabrics at 25°C and 40°C, 60°C was taken as the optimum temperature. All the mentioned findings can be clearly seen from Table 19 in which the photographs of fabric samples related to the optimization of enzymatic dyeing conditions for P4 are given.
Photographs of fabric samples related to optimization of enzymatic dyeing conditions for P4
As a result of all these evaluations, optimum conditions of enzymatic dyeing with P4 were determined as 1 g/L enzyme concentration, 8 mmol/L precursor concentration, 60°C, and 3 h.
Effect of fabric pretreatment process on enzymatic dyeing
In these experiments, we aimed to determine whether natural flavonoids in the structure of unbleached cotton are included in the coupling reaction of precursors used in enzymatic dyeing. For this aim, bleached fabric and scoured fabric were dyed at optimum conditions with selected precursors. The color measurement results obtained are given in Table 20 and the photographs of fabric samples are given in Table 21.
The color measurement results of bleached fabric and scoured fabric subjected to enzymatic coloration
Photos of bleached fabric and scoured fabric subjected to enzymatic coloration
When Table 20 and Table 21 are examined, it can be seen that the colors of scoured fabrics are more yellow (higher b* value) and also slightly darker (lower L* value). As scoured fabrics are more yellowish compared with bleached fabrics, it is quite normal for scoured fabrics to have a more yellow color nuance. On the other hand, it can be said that the darker shade of scoured fabrics supports the natural flavonoids present in the structure of unbleached cotton that are also included in enzymatic coupling reaction with precursors. However, the darker ground color of scoured fabric should also be taken into consideration to some extent.
Effect of ultrasound on enzymatic dyeing
To determine the effect of ultrasonic energy on color-yield values obtained in enzymatic dyeing processes, trials under optimum conditions were carried out in two different ways, with and without ultrasound. Color-yield (K/S) and CIE L*a*b* values of fabric samples are given in Table 22 and the photographs of fabric samples are given in Table 23.
Color measurement results of fabric samples subjected to enzymatic dyeings with and without using ultrasound
Photos of fabric samples subjected to enzymatic dyeings with and without using ultrasound
When Table 22 and Table 23 are examined, it can be seen that the color yield (K/S) of the dyeings made in the presence of ultrasound is much higher than the dyeings made in the absence of ultrasound. In general, it can be said that performing the dyeing process in an ultrasonic environment increases the dyeing efficiency, and hence color-yield values obtained in dyeings. As is known, a cavitation effect occurs in the ultrasonic environment, and the dye uptake of the fibers increases as the kinetic energy in the dyeing bath increases and the aggregation of the dye molecules decreases. When the nuance of the colors of the dyeing done in the presence and absence of ultrasound is evaluated, it is noteworthy that the color nuance of the fabrics dyed in the presence of ultrasound is generally redder (higher a* values) and yellower (higher b* values).
FT-IR analyses
As phenol oxidases, laccases can efficiently catalyze polymerization of phenolic moieties. 33 Simple organic compounds such as mono- and diphenols are subjected to direct oxidation by laccase enzyme. The reaction mechanism of direct oxidation involves deprotonation of the hydroxyl group of the phenolic substrate to give phenoxy radical, which is very unstable and may undergo enzymatic oxidation to quinonoid derivatives or spontaneous non-enzymatic coupling reactions to produce mainly dimers. These dimers arise as a result of formation of new bonds between the carbon and oxygen atom (C-O) or between two carbon atoms (C-C). 9 After longer reaction times, oligomers and homomolecular polymers can also be generated from these dimers.9,33 In the light of these explanations, the proposed mechanism for laccase-catalyzed dye synthesis from catechol is given in Figure 11.
To characterize the chemical structure of catechol (P4 precursor) and the brown dye (P4 dye) obtained from it by laccase, the corresponding FT-IR spectra were determined and are given in Figure 12.
FT-IR spectra of P4 precursor and P4 dye (brown color).
As can be seen from Figure 12, there are some differences between the spectrum of P4 precursor and P4 dye. In spectra of P4 precursor, the main peaks were observed at 737, 849, 934, 1034, 1086, 1179, 1238, 1462, 1515, 1614, 1693, 2927, 3045, 3316 and 3441 cm−1. Peaks at 1462, 1515 and 1614 cm−1 are ascribed to the C=C stretchings.16,36 Also the peak detected at 1693 cm−1 can be attributed to C=C double bond. The peaks at 2927 and 3045 cm−1 are related to the Ar=C-H stretchings. The two bands observed at 3316 and 3441 cm−1 correspond to the stretching of alcohol’s free –OH group and H-bonded –OH group, respectively. 36
On the other hand, in the FT-IR spectrum of P4 dye, the main peaks were observed at 744, 796, 928, 1013, 1047, 1106, 1258, 1403, 1548, 1634, 1717, 2927, 3164, 3276 and 3401 cm−1. The C–O–C stretching of aromatic ether is seen at 1047 cm−1 and 1258 cm−1. However, it is important to note that these peaks can also assign to C–H vibrations corresponding to in-plane bending modes for aromatic ring. 16 The presence of C–O–C bond in the structure supports the homopolymerization by laccase given in Figure 11. On the other hand, the peak at 1106 cm−1 assigns to C-C stretching, 37 which supports the presence of dimers produced as a result of formation of new bonds between the two carbon atoms (C-C). Furthermore, peaks at 1403, 1548 and 1634 cm−1 are ascribed to the C=C stretchings.16,36 The peak at 1700 cm−1 corresponds to the vibration of R2C=O quinones band. 4 This finding demonstrates that also o-benzoquinone compounds are formed in the reaction media. All these findings support that with the effect of laccase, all possible products are formed in some extent and are present as a mixture in the system. In other words, P4 dye contains both o-benzoquinone compound and dimers and homopolymer of catechol. The peaks at 2927 and 3164 cm−1 are related to the Ar=C-H stretchings. The two bands observed at 3276 and 3401 cm−1 correspond to the stretching of alcohol’s free –OH group and H-bonded –OH group, respectively. 36
Laccases not only catalyze the removal of a hydrogen atom from the hydroxyl group of methoxy-substituted monophenols, ortho- and paradiphenols, but also can oxidize other substrates such as aromatic amines, syringaldazine, and non-phenolic compounds to form free radicals. 6 There has been a report on the laccase-mediated oxidation of aromatic amines to form yellow and blue dyes via C-N bond formation. 38 Similar to the mechanism proposed by Wellington, 38 the reaction pathway for the laccase-catalyzed oxidative coupling of the 4-nitro-1-naphthylamine is proposed as given in Figure 13.
Laccase-catalyzed formation of a yellow dye (A3 dye) from 4-nitro-1-naphthylamine (A3 precursor).
To characterize the chemical structure of 4-nitro-1-naphthylamine (A3 precursor) and the yellow dye (A3 dye) obtained by its catalytic polymerization with laccase, the corresponding FT-IR spectra were determined and are given in Figure 14.
FT-IR spectra of A3 precursor and A3 dye (yellow color).
As can be seen from Figure 14, there are some differences between the spectrum of A3 precursor and A3 dye. In spectra of A3 precursor, the main peaks were observed at 829, 1027, 1258, 1310, 1515, 1568, 1647, 2927, 3223, 3329 and 3401 cm−1. The presence of C–N bonds could be identified by an intensive signal at 1027 cm−1, which possibly derived from the stretching of C–N bonds.36,39 The peak observed at 1258 cm−1 indicates the presence of C–N stretching of aromatic amines. Furthermore, peaks at 1515 cm−1 and 1310 cm−1 are ascribed to the N–O asymmetric and symmetric stretching vibrations of nitro, respectively. 36 The peak at 1568 cm−1 is also a broad band, which could be due to overlapping of the absorbance of several bonds, including conjugated carbonyl and N–H bending. 11 Weaker peaks detected at 1647 and 2927 cm−1 are related to the alkene C=C stretching 16 and Ar-C-H stretching, respectively. As known, two bands from 3400–3300 and 3330–3250 cm−1 show the presence of primer amines. 36 Peaks present at 3329 and 3401 cm−1 are therefore related to the –NH2 groups in A3 precursor.
In the FT-IR spectrum of A3 dye, the main peaks were observed at 803, 1013, 1403, 1555, 1627, 1700, 2927, 3164, 3276 and 3401 cm−1. The presence of C–N bonds could be identified by an intensive signal at 1013 cm−1, which possibly derived from the stretching of C–N bonds.36,39 Peaks at 1555 cm−1 and 1403 cm−1 are ascribed to the N–O asymmetric and symmetric stretching vibrations of nitro, respectively. A peak observed near 1700 cm−1 refers to the presence of C=N stretching. 36 C=N stretching, which does not exist in A3 precursor, confirms the pathway given in Figure 13. Furthermore, the absence of primer amine bands and C–N stretching of aromatic amines in the A3 dye structure also supports the proposed mechanism. Weaker peaks detected at 1627 and 2927 cm−1 are related to the alkene C=C stretching 16 and Ar-C-H stretching, respectively.
Dye precursors can also be used in combination with a suitable modifier (coupler), which will enlarge the color palette achieved in the enzymatic dyeing. The modifier would react with the dye precursor in the presence of laccase, converting it to a colored compound. 6 Due to the ability of generating multiple forms of phenolic radicals from the same substrates, there is a high probability of formation of variety of products resulting from spontaneous and accidental coupling of these highly reactive intermediates. The dimer formation reactions are referred to as homomolecular coupling reactions. Laccase is also able to couple a typical laccase substrate with substances that are not substrates for laccase, thereby creating new heteromolecular hybrid molecules. 9 Through the coupling of phenoxy radicals with substrates such as aromatic amines by non-enzymatically 1,4-Michael-type adducts, 6 products having entirely new physical and chemical properties are obtained. Among the substrates, new chemical bonds are formed between the carbon and nitrogen atoms (C-N) as shown in Figure 14. 9 Besides the coupling reaction with amine compounds, phenoxyl radicals formed by oxidation of phenolic compounds can also react among themselves according to the similar mechanism shown in Figure 10. Moreover, amine compounds in the environment can also react among themselves. Thus, characterization of the compounds formed by laccase-mediated oxidation of the dye precursor (phenol compound) and modifier (amine compound) is very complex.6,11 The proposed reaction pathway for laccase-mediated oxidative cross-coupling of 4-methoxyphenol and 3-methyl-2-benzothiazolinone hydrazone hydrochloride is given in Figure 15.
To characterize the chemical structure of 4-methoxyphenol (P7 precursor) and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (A2 precursor) and the red dye (AP dye) obtained from them by laccase, the corresponding FT-IR spectra were determined and are given in Figure 16.
FT-IR spectra of P7 precursor, A2 precursor and AP dye (red color).
As can be seen from Figure 16, there are some differences between the spectrum of P7 and A2 precursors and AP dye. In spectra of P7 precursor, the main peaks were observed at 823, 1027, 1100, 1172, 1278, 1442, 1607, 1634, 2827, 2953, 3078 and 3335 cm−1. Peaks at 1442, 1607 and 1634 cm−1 are ascribed to the C=C stretchings.16,36 The peak at 2827 cm−1 is the characteristic stretching band of the Ar-O-CH3 bond. The peaks at 2953 and 3078 cm−1 are related to the Ar=C-H stretchings. The band observed at 3335 cm−1 corresponds to the stretching of alcohol’s free –OH group. 36
In spectra of A2 precursor, the main peaks were observed at 763, 842, 1040, 1119, 1265, 1344, 1442, 1468, 1575, 1673, 2927, 3012, 3170, 3237 and 3421 cm−1. The presence of C–N bonds could be identified by an intensive signal at 1040 cm−1 which possibly derived from the stretching of C–N bonds.39,41 The peak at 1119 cm−1 is due to N-N bond. Peaks observed at 1265 and 1344 cm−1 can be attributed to the substituted ring in-plane C–N stretching and C–S bending, respectively. 41 Furthermore, peaks at 1442 and 1468 cm−1 are ascribed to the C=C stretchings.16,36 The peak at 1575 cm−1 is also a broad band, which could be due to overlapping of the absorbance of several bonds, including conjugated carbonyl and N–H bending. 11 The peak detected at 1673 cm−1 can be attributed to =C=N- group. 37 Peaks present at 3237 and 3421 cm−1 correspond to the –NH2 groups in A2 precursor. 36
In the FT-IR spectrum of AP dye, the main peaks were observed at 796, 1047, 1126, 1244, 1403, 1555, 1700, 2861, 2927, 3170, 3269 and 3395 cm−1. The absorption band at 796 cm−1 is assigned to aromatic C–S stretching. 37 In turn, the presence of C–N bonds could be identified by an intensive signal at 1047 cm−1 which possibly derives from the stretching of C–N bonds.39,41 The peak at 1126 cm−1 can be attributed to N-N bond. Peaks observed at 1244 and 1403 cm−1 can be attributed to the substituted ring in-plane C–N stretching and C–S bending, respectively.36,41 Furthermore, the peak at 1555 cm−1 is ascribed to the C=C stretching.16,36 The peak detected at 1700 cm−1 can be attributed to =C=N- group. 37 The peak at 2861 cm−1 is the characteristic stretching band of the Ar-O-CH3 bond. The band observed at 3269 cm−1 corresponds to the secondary N-H stretching. All these findings confirm the mechanism given in Figure 15, and it can be said that mainly the compound given above is formed, rather than the one given below in that figure.
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (A7) (ABTS) is an excellent electrochemical mediator for laccases. ABTS, undergoes reversible oxidation to form a stable and intensively colored dication ABTS2+ as shown in Figure 17. 42
Formation of the cation radical and the dication by removal of one and two electrons from ABTS. 42
To characterize the chemical structure of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (A7 precursor) and the lilac dye (A7 dye) obtained by its catalytic oxidation with laccase, the corresponding FT-IR spectra were determined and are given in Figure 18.
FT-IR spectra of A7 precursor and A7 dye (lilac color).
As can be seen from Figure 18, there are some differences between the spectrum of A7 precursor and A7 dye. In spectra of A7 precursor, the main peaks were observed at 703, 810, 1013, 1119, 1231, 1331, 1575, 1620, 1739, 2927, 2979 and 3183 cm−1. The absorption band at 703 cm−1 is assigned to aromatic C–S stretching. 37 The strong peaks at 810 cm−1 and 1013 cm−1 contain the contributions from the S-O stretching (sulfoxide) and S=O bond, respectively. 43 The peak at 1119 cm−1 can be attributed to both N-N bond and C-C stretching (skeletal vibrations). 37 Peaks observed at 1231 and 1331 cm−1 can be attributed to the substituted ring in-plane C–S bending and C–N stretching, respectively. 41 Furthermore, peaks at 1575 cm−1 and 1620 cm−1 are ascribed to the aromatic C=C stretchings.16,36 One of the main differences in the structure of A7 precursor and A7 dye is the =C=N- group. However, double-bonded nitrogen groups, such as =C=N- group, exhibit absorptions close to the carbonyl (C=O) and alkene (C=C) double bond stretching region. While they are characteristic for the functional group, they are sometimes difficult to assign from first principles because of the overlap with other common functional groups in the region. 37 For this reason, the peak detected at 1739 cm−1 can be attributed to =C=N- group, but it also assigns to presence of C=C double bond.
In the FT-IR spectrum of A7 dye, the main peaks were observed at 697, 803, 1027, 1119, 1225, 1324, 1410, 1482, 1555, 1627, 1693, 2927, 2972 and 3164 cm−1. The absorption band at 697 cm−1 is assigned to aromatic C–S stretching. 37 The strong peaks at 803 cm−1 and 1027 cm−1 contain the contributions from the S-O stretching (sulfoxide) and S=O bond, respectively. 43 The peak at 1119 cm−1 can be attributed to C-C stretching (skeletal vibrations). 37 Peaks observed at 1225 and 1324 cm−1 can be attributed to the substituted ring in-plane C–S bending and C–N stretching, respectively. 41 The peaks at 1410 and 1482 cm−1 assign to the Ar-C=N and disazo (–N=N-) groups, respectively. 36 Ar-C=N and disazo (–N=N-) groups, which do not exist in A7 precursor, confirm the pathway given in Figure 17. Furthermore, peaks at 1555 cm−1 and 1627 cm−1 are ascribed to the aromatic C=C stretchings.16,36 The peak detected at 1693 cm−1 can also be attributed to C=C double bond.
Comparison of enzymatic coloration and reactive dyeing
Technical comparison
One of the critical issues for textile dyeing is the reproducibility of colors. In the studies carried out to date, the issue of reproducibility in enzymatic coloring has never been examined. Within the scope of this study, the recipes created for yellow, red and lilac colors were repeated five different times and standard deviation and variation coefficient values regarding to the color-yield (K/S) and CIE L*a*b* values obtained were calculated. The results are given in Table 24.
Standard deviation and variation coefficient values regarding to the color measurement results of enzymatic coloration and reactive dyeings repeated five times
When the photographs given in Table 24 are examined, it is seen that the colors of enzymatic dyed and reactive dyed fabrics are very close to each other for yellow, red and lilac colors. The color measurement results obtained also confirm these findings. Although there is some difference in the nuances of the colors obtained, the aim is to demonstrate how reproducibility and fastness values will change if a similar color is obtained with enzymatic coloration or reactive dyeing. When Table 20 is examined, it is seen that the standard deviation and variation coefficient of the color measurement values obtained in repetitive dyeings for both enzymatic coloring and reactive dyeing are quite low. Although standard deviation and variation coefficient values in enzymatic coloring are slightly higher than reactive dyeing, it is possible to say that both enzymatic coloring and reactive dyeing are very reproducible under laboratory conditions, as these values are very low.
Washing, rubbing and light-fastness values of similar colors obtained by enzymatic coloring and reactive dyeing are given in Table 25. Perspiration-fastness values are given in Table 26.
Washing, rubbing and light-fastness values of similar colors obtained by enzymatic coloring and reactive dyeing
Perspiration-fastness values of similar colors obtained by enzymatic coloring and reactive dyeing
Table 25 shows that the washing and rubbing-fastness values obtained in reactive dyeings are very good in all colors and the light-fastness values are of medium to good levels. Furthermore, after-treatment with fixing agent caused 1/2 point decrease in light-fastness values. Considering the fastness values obtained in enzymatic coloring, washing and rubbing-fastness values in yellow and red colors are quite low; in lilac color, it is seen that it is at medium levels. In the case of after-treatment with the fixing agent, it is not possible to achieve a significant improvement in washing-fastness values in all colors, while significant improvements have been achieved especially in wet rubbing-fastness. On the other hand, light-fastness values are generally very low in all colors and after-treatment has no effect.
Table 26 shows that the acidic and alkaline perspiration-fastness values obtained in reactive dyeing are at very good levels in all colors. Besides, the after-treatment with fixing agent led to an increase of 1/2 point, but there was no significant change as fastness levels were already 5 in many samples. When the fastness values obtained in enzymatic coloring are examined, the perspiration-fastness values in yellow and red colors are quite low; in lilac color, it is seen that it is at very good levels. In the case of after-treatment, significant improvements were achieved in all colors, especially in red color.
After comparing the color and fastness properties of enzymatic coloring and reactive dyeing, UPF tests were performed to compare the UV protection properties of dyed fabrics. The results are given in Table 27.
UV protection properties of fabric samples subjected to enzymatic coloring and reactive dyeing
Table 27 shows that the UV protection factor of fabrics dyed with reactive dyes are always higher than the fabrics subjected to enzymatic coloration. Even if the fiber, yarn and fabric properties and color and dyeing depth that affect the UV protection property of a dyed material are the same, the difference in the chemical structure of the dye formed in the enzymatic coloration and reactive dyes explains the difference. On the other hand, when considered as a UV protection class, it can be said that there is no critical difference between them; that is, the UV protection properties of all samples with both enzymatic coloring and reactive dyeing process ranged from good to excellent.
Economical comparison
Water, dye (reactive dye or dye precursor), chemical, electricity, natural gas and labor costs per 1 kg of cotton knitted fabric were calculated and the total cost of dyeing the same color (yellow, red or lilac) with enzymatic method and reactive dyes were compared. The unit price values used in these calculations are given in Table 28.
Unit prices of the consumables used in enzymatic coloring and reactive dyeing
While calculating the electricity cost, the industrial electricity price was taken as 0.08 €/kWh and the loss factor as 2.5. If the power for the dyeing machine is taken as 2.9 kW, the electricity cost can be calculated as (TL) = 2.9 kW × dyeing time (h) × 0.08 €/kWh.
Natural gas cost was calculated as follows:
While working at a ratio of 1:10, 1 kg of fabric will be treated with 10 L of water. The heat required to raise the temperature of 10 L of water from T1°C to T2°C is based on the formula Q = m (g) × c (cal/g °C) × ΔT and taking into account that the density of the water is 1 (i.e. its volume and mass are the same) Q = 10,000 (g) × 1 (cal/g °C) × ΔT (°C) = 10,000 × ΔT cal = 10 × ΔT kcal. If the heat loss factor that will occur during the process to be performed on the machine is accepted as 1.5, the actual total energy required will be 10 × ΔT kcal × 1.5 = 15 × ΔT kcal. As 1 kg steam gives 540 kcal energy, 15 × ΔT/540 = 0.028 × ΔT kg steam is needed. According to the need of 1900 kcal energy to produce 1 kg of steam; 0.028 × ΔT × 1900 = 53.2 × ΔT kcal energy is required. As 1 m3 of natural gas gives 9155 kcal energy; 53.2 × ΔT/9155 m3 = 0.0058 × ΔT m3 natural gas is required. If the price of natural gas is taken as 0.26 €/m3, natural gas cost is found as 0.11 × ΔT €.
The amount of water, precursor, chemical, electricity, natural gas and labor required for enzymatic coloring and reactive dyeing (including the washing processes) of 1 kg cotton knitted fabric and their costs are given in Table 29 and Table 30, respectively.
Consumables used in enzymatic coloring and their costs for coloring of 1 kg fabric
Consumables used in reactive dyeing and their costs for coloring of 1 kg fabric
The data given in Tables 29 and 30 are summarized in Table 31, allowing for comparison of enzymatic coloring and reactive dyeing costs for each color.
Cost items and their amounts in enzymatic coloring and reactive dyeing
When the cost items related to enzymatic and reactive dyeings are examined, the auxiliary cost is 50–70% higher than that of reactive dyeing, as enzyme should be used in enzymatic dyeings. In contrast, three rinsing steps are sufficient after dyeing in enzymatic dyeing, whereas at least four rinsing steps are required in reactive dyeing. Therefore, in enzymatic dyeing, water consumption and associated costs are 20% lower. Furthermore, total time in enzymatic coloring is significantly lower than reactive dyeing except yellow. This will lead to higher utilization efficiency per unit machine, and will increase the energy cost by 30–75% and labor cost by 35%. As a matter of fact, the cost of electricity and natural gas in enzymatic coloring is 75% lower and the energy cost is 60% lower than the reactive dyeing in red, where dyeing is done at room temperature. When the total costs of the dyeings are compared, it is seen that the cost of enzymatic coloring is 98% higher than reactive dyeing. However, this is because the prices of the precursors used in enzymatic coloring are 99% higher than the reactive dye prices. Because enzymatic coloring studies are still laboratory scale, chemicals of analytical purity are used. However, this disadvantage can be eliminated when those that are affordable for use in the textile field are also available in the market for use in enzymatic coloring in the future. Therefore, it will be more correct to compare the costs excluding dye in these conditions. When looking at the total costs excluding dye, it is remarkable that for yellow color reactive dyeing is close to enzymatic coloration, and for red and lilac colors, 55% lower dyeing costs were obtained compared with reactive dyeing. All these evaluations reveal that enzymatic coloring is an environmentally friendly process that allows less costly dyeing in shorter period of time by consuming less water and less energy.
Ecological comparison
To compare the environmental loads caused by the dyebath wastewater when the same color is obtained by enzymatic coloration and reactive dyeing, COD, BOD, TDS, Total Kjeldahl Nitrogen (TKN) and chloride analyses were carried for the waste waters of dyeings in yellow, red and lilac colors. Results are presented in Table 32.
Analysis results of enzymatic coloration and reactive dyeing wastewaters
Traditionally, organic matter is measured as COD and BOD in wastewater. 44 COD is the amount of oxygen required for chemical oxidation of oxidizable substances in water. One of the most important parameters used in determining the pollution degree of domestic and especially industrial wastewater is the need for chemical oxygen. Unlike BOD, it is based on oxidation of organic matter by redox reactions, not biochemical reactions. In the oxidation environment, carbon-containing organic substances transform into carbon dioxide and water; on the other hand, nitrogenous organic substances transform into ammonia. 44 BOD is the amount of oxygen consumed for organic substances that can be degraded by aerobic bacteria at a certain time (5 days) and at a certain temperature (20°C). 45 As COD contains some substances that are not biodegradable, unlike BOD, 44 the COD value is always greater than the BOD value in industrial or domestic wastewater. Therefore, the COD:BOD ratio is also greater than 1. The COD:BOD ratio indicates whether wastewater can be biologically treated or not. 46
Examining the values given in Table 32, it can be said that the amount of organic matter in the enzymatic coloration is much higher than in the reactive dyeing process. In fact, this can be understood more clearly when the amount of organic pollutants used in dyeing processes is calculated. For example, when enzymatic coloration is carried out for yellow color, 1 g/L laccase enzyme and 1.5505 g/L (188.18 g/mol × 8.10−3 mol/L = 1.550 g/L) 4-nitro-1-naphthylamine (A3) are used. On the other hand, when reactive dyeing is made for the same color, 0.11 g/L (0.11% means 0.0055 grams of dye for 5 g of fabric. When 5 g of fabric is dyed at the liquor ratio of 1:10, the volume of the liquor is 50 mL, so 0.0055 g of dye is put in 50 mL, which is equal to 0.11 g/L) C.I. Reactive Yellow 176, 0.16 g/L C.I. Reactive Yellow 160, 0.0035 g/L C.I. Reactive Blue 221 are used. In other words, while the total organic compound concentration used is 2.505 g/L in enzymatic coloration, it is 0.2735 g/L in reactive dyeing. Therefore, it is normal for COD and BOD values to be higher in enzymatic coloration. However, it is important to evaluate the COD:BOD ratio in terms of treatability of the wastewater. If the wastes do not contain toxic substances and only contain organic substances that can easily decompose, the COD value is approximately equal to the final BOD (carbonated) value. 44 A COD:BOD ratio <3 means that this wastewater can be biologically treated. 46 When the results are evaluated from this point of view, while the COD: BOD ratios obtained after reactive dyeing are between 4 and 4.5, this ratio is between 2 and 2.5 for enzymatic coloration. Therefore, although biological treatment is enough for wastewater of enzymatic coloration, chemical treatment is also needed in reactive dyeing wastewater. This is a very important issue in terms of both ecology and economy.
Another important parameter in textile wastewater is chloride content, in other words salinity. From this point of view, the normal wastewater of the reactive dyeing process in which sodium chloride salt is used is at a concentration of 30 g/L even in light colors. As salt usage is not necessary in enzymatic coloration, this will become an important advantage in the treatment and reuse of wastewater. In addition, removing salt from wastewater requires costly processes such as reverse osmosis. This has many ecological and economic disadvantages.
TDS indicates minerals, cations, anions, heavy metal ions, and small amounts of organic materials that are dissolved in water and cannot be trapped by simple filtration methods such as sand filters. The higher the TDS in water, the more foreign matter means. When evaluated from this perspective, it can be said that enzymatic coloration wastewater has an important advantage over reactive dyeing wastewater. The high amounts of TDS make it difficult to treat and reuse of wastewater. While the removal of organics is possible with biological treatment, advanced techniques such as membrane processes are required to remove TDS. This is an ecologically and economically undesirable situation.
TKN is the sum of organic nitrogen and ammonia nitrogen. When the TKN values are analyzed, it is seen that this value is much higher in enzymatic coloration than reactive dyeing. When looking at the source of N, it should be taken into account that there is also nitrogen in the structure of laccase enzyme consisting of amino acids as well as nitrogen in the molecular structure of the dye molecule. The use of a higher concentration of dye and laccase enzyme in enzymatic coloration explains the higher N content of enzymatic coloration wastewater compared with reactive dyeing. When the three enzymatic dyeings are compared among themselves, the highest value is obtained in lilac color. When the chemical structures of the yellow, red and lilac dyes are examined, it is understood that the highest N content in the dye chromophore is in the lilac dye (A7) in which 2-2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) precursor was used.
In addition, pH values of wastewater are between 5 and 5.5 in enzymatic colorations, whereas pH values of reactive dyeing wastewater are in the range of 10.5–11. This shows that the high alkalinity of the reactive dyeing wastewater disappears in the case of enzymatic dyeing. It is known that very high (greater than 9.5) or very low (less than 4.5) pH values are not suitable for most aquatic livings. Therefore, if the pH value of the wastewater is too acidic or very alkaline, pH balancing must be performed before discharging to the receiving waters or before biological treatment. 47 In addition, acidic and basic wastewaters need to be neutralized for biological treatment. This will cause excessive use of chemicals. From this point of view, it can be said that enzymatic coloring wastewater will be advantageous.
Conclusions
In recent years, environmentally friendly production has gained importance in all fields. In the textile industry, the most polluting process is dyeing. For this reason, eco-friendly alternatives to synthetic dyes are being sought. In this context, laccase-catalyzed simultaneous dye synthesis and dyeing is a promising process. In this study, brown, yellow, red and lilac colors could be obtained on 100% cotton knitted fabrics by using a wide variety of amine and phenol compounds as precursor. Yellow, red, lilac and brown colors with high color-yield values were successfully obtained. As only yellow and brown shades have been obtained in literature to date, it is believed that the findings of this study are original. It is important to note that from the view of textile coloration, yellow, red and blue colors are the primary colors. Although many precursors and their combinations have been examined in this study, blue color is still missing and it would be useful for further studies to concentrate on this issue.
After determining the colors, the effect of pH in enzymatic dyeing process was also investigated and pH 5 was found to be the most suitable for all precursors. Then the optimization of reaction conditions (enzyme concentration, precursor concentration, duration and temperature) was realized statistically for the precursors which gave the best results in terms of color. It was observed from the experimental results that the effect of enzyme concentration, precursor concentration, time and temperature on enzymatic dyeing was statistically significant (p<0.05) for all precursors. The optimum conditions were found to be 1 g/L enzyme, 8 mmol/L precursor, 80°C, 4 h; 1 g/L enzyme, 4 mmol/L precursor, 25°C, 1 h; 0.1 g/L enzyme, 8 mmol/L precursor, 60°C, 1 h; and 1 g/L enzyme, 8 mmol/L precursor, 60°C, 3 h for yellow, red, lilac and brown colors, respectively.
With the aim of achieving further improvements in color-yield values obtained in enzymatic dyeings, the effect of the pretreatment process and the use of ultrasound were also investigated. It was found that higher color-yield values could be obtained if fabrics are dyed without a bleaching process. Also, in the presence of ultrasound energy, higher color yields are achieved.
It was confirmed by means of FT-IR analysis that as a result of laccase-catalyzed oxidation of catechol, o-benzoquinone is formed. In addition, non-enzymatic coupling reactions occurred and dimers, which in further steps transformed into homomolecular polymers, formed. On the other hand, it was shown that 4-nitro-1-naphthylamine was subjected to oxidative coupling by laccase and a yellow dye containing C-N bonds was formed.
In the case of using phenol (4-methoxyphenol) and amine (3-methyl-2-benzothiazolinone hydrazone hydrochloride)-based dye precursors in combination, through the coupling of phenoxy radicals, which were formed as a result of laccase-catalyzed oxidation of phenol compound, with amine compounds by non-enzymatically 1,4-Michael-type reactions, a red dye containing new chemical bonds formed between the carbon and nitrogen atoms (C-N) was obtained. Furthermore, it was determined that 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) underwent reversible oxidation to form a stable and intensively colored dication ABTS2+.
With all these investigations, it was demonstrated that colorless compounds could be transformed into dye molecules having various colors by means of laccase-catalyzed oxidation. It was determined that the reaction pathways mainly depend on transformation of phenolic compounds to quinones and/or dimers, which may then react among themselves forming homopolymers or react with other compounds such as amines forming heteropolymers. Yellow, red, lilac and brown colors were successfully obtained by means of these enzymatic reactions.
According to the experimental results, it is possible to say that both enzymatic coloring and reactive dyeing have good reproducibility. In terms of the fastness levels achieved, it can be said that the enzymatic coloring is far behind the reactive dyeing, and it is necessary to work on developing the fastness values, especially the light fastness, in future studies.
When the UV protection values of the fabric samples are examined for reactive dyeing and enzymatic coloration of the same color, it is seen that there is no critical difference between them, and their UV protection values range from good to excellent.
When comparing the cost of dyeing 1 kg cotton knitted fabric of the same color (yellow, red or lilac) with the enzymatic and reactive dyeings, all cost items except dye were lower in enzymatic coloring. However, as enzymatic coloration studies are still laboratory-scale studies and chemicals with analytical purity are used, the costs seem higher than in reactive dyeing. It is thought that this disadvantage can be eliminated when affordable materials for use in the textile field are also available in the market.
Beyond these points, although wastewater of enzymatic coloring can be biologically treated, it is determined that chemical treatment will also be needed for reactive dyeing wastewater. Therefore, it can be said that environmentally friendly enzymatic dyeing is more advantageous than reactive dyeing. In addition, the lack of salt usage in enzymatic coloring will become an important advantage, especially when it comes to treating and reusing wastewater.
Footnotes
Acknowledgements
We would like to thank TUBITAK for supporting this study within 5170069 coded project.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors received financial support from TUBITAK within 5170069 coded project for the research.
