1. Introduction
Dyeing clothes is a prehistoric process. This process involved the application of early natural dyes for furs and textiles of vegetable origin, though some dyes were of animal origin. More complex coloring materials were developed over thousands of years. The woad (natural indigo), for example, was obtained from the plant
Indigofera tinctoria, and the Tyrian purple was extracted from the gland of a purple snail and developed by the Phoenicians, whereas the Alizarine was taken out from madder Campeachi wood [
1]. By the end of the 19th and early 20th centuries, the synthetic dye industry was established in many countries, and thousands of dye molecules were synthesized and produced on a large scale [
2].
Despite the chemical diversity of dyes, dye molecules share a common chemical structure. In fact, each dye molecule has four components, namely a chromophore group, an auxochrome group, a solubilizing group, and a matrix. The chromophore groups are responsible for the absorption of the light energy and the creation of the dye’s color through the excitation of electrons. The auxochrome groups help with the dye fixation into the support, while the solubilizing ones ensure the solubility of the molecule in water or organic solvents. The remaining parts of the dye molecule form the matrix or the skeleton [
1].
Dyes are classified according to several parameters, including color, chemical structure, application, manufacturer, synthesis route, fastness, and date invented. However, based on their chemical structure and the chromophore groups, the following dye families were identified: azo, anthraquinone, nitroso, nitro, indigoid, cyanine, phthalocyanine, and triphenylmethane [
3].
Azo dyes are characterized by two aromatic groups linked to each other by an azo bond (-N=N-). They are classified based on the number of azo linkages, mono azo dyes, diazo dyes, etc. The number of azo groups varies from 1 to 4. Other than textile industries, this family of dyes is used in various fields such as pharmaceuticals, cosmetics, food, paint, paperwork, etc. Their success is mostly due to the stability of coloring, the ease of a coupling reaction between the dyes and the support, the high molar extinction coefficient (capacity to absorb light), the flexibility of the coloring structure, and their adaptability to a variety of applications [
4,
5,
6].
Approximately 70% of the dyes used in the textile industry are of the azo type. However, during the coloring process, non-adsorbed dyes are estimated to be between 15 and 20% and are discharged into the wastewater [
7,
8,
9]. Due to their toxicities, industries using this type of dye are currently attempting to minimize their negative impact on the environment. This includes improving their binding to the matrix or their degradation once discharged into industrial wastewater using biological or physicochemical processes.
Many studies have demonstrated that the released sewage contains, other than dyes, toxic molecules like heavy metals. Once released into the environment, the wastewater may affect both human health and ecosystem [
10,
11,
12]. Many health issues, including cancer, chronic diseases, and skin irritation, have been associated with exposure to azo dyes [
10]. Furthermore, the death of aquatic organisms and the stunting of plant growth were mentioned as a consequence of the release of untreated textile wastewater [
13]. To treat sewage from the textile industry, many attempts have been made, and many physicochemical methods have been developed (e.g., filtration, adsorption, coagulation/flocculation). Those treatments were mostly used at the outset. Nevertheless, their unwanted outcomes, like the formation of secondary mud, the limited efficacy, and the high cost, have prompted industries to look for alternative biological methods that are especially eco-friendly and low-cost and where plant microorganisms and/or their enzymes can be used. Yeast [
14], bacteria [
15,
16], algae [
17], and fungi [
18] have widely been used for this purpose. Several studies demonstrated the efficiency of white-rot fungi such as
Trametes versicolor [
19],
Trametes trogii [
20], and
Coriolopsis gallica [
7,
21], and other fungi like
Aspergillus niger [
22] in the removal of textile dyes using their enzymes or biomasses.
White-rot fungi secrete a number of oxidoreductases that are involved in lignin depolymerization [
23,
24]. These oxidoreductases encompass heme-containing peroxidases (manganese, lignin, and versatile peroxidases) and copper-dependent polyphenol oxidases named laccases (E.C. 1.10.3.2). Laccases from white-rot fungi exhibit a higher redox potential (0.720–0.790 V) compared to other fungal, bacterial, or plant laccases (0.400–0.700 V). High redox potential laccases do not oxidize lignin directly but through small aromatic compounds (laccase-mediators) that can attack lignin after their oxidation in the active site [
25,
26]. Although laccases are widely used, their efficiency in removing pollutants is sometimes limited; this has prompted the use of a laccase-mediator system to enhance the laccase activity. Mediators allowed the active center of the enzyme to interact with large molecules of substrates or substrates with high redox potential. Several laccase mediators have been studied, including mediators of natural molecules (e.g., 3-hydroxy-anthranilic acid, syringaldehyde, vanillin, etc.) or synthetic molecules (such as HBT, TEMPO, Violoric acid, etc.) [
27].
In this paper, we aim to study the biodegradation of the four azo-bond dye Sirius grey by the laccase mediator system and to optimize its decolorization conditions using a response surface methodology approach.
3. Discussion
Strain BS9 shares more than 99% similarity of its ITS1-ITS4 region of rDNA with members of the species
C. gallica. This fungus is known for its capacity to grow on several woods to produce ligninolytic enzymes [
7,
28]. Based on this fact,
C. gallica was also shown to degrade many pollutants, including dyes [
7,
21], hydrocarbons [
29], phenols, and bisphenol A [
30]. Recently,
C. gallica was shown to be able to degrade antibiotics [
31]. In the same work, proteomic analysis showed the presence of one major secreted laccase, although there were several laccase genes in the genome.
Decolorization of a wide range of synthetic and textile dyes using laccases from basidiomycetes has been investigated in recent years. For this reason, we used
C. gallica for the decolorization of Sirius grey; the latter belongs to the azo compounds that contain one or more azo groups (N=N), and most of them are xenobiotics [
32]. By using the culture filtrate from
C. gallica, 48% decolorization of Sirius grey was achieved. This decolorization yield was improved by adding 1 mM of HBT to the reaction mixture. Under these conditions, the decolorization yield increased to 81%. Therefore, the use of a mediator (such as HBT) is necessary, especially for certain laccases with low redox potential or in case the substrate is highly recalcitrant. Indeed, laccase mediators are low molecular weight molecules with a significant redox potential, enabling them to act as an electron messenger between the substrate and the enzyme [
27].
The experimental design that was performed used four variable factors, namely: initial enzyme concentration, initial dye concentration, initial HBT concentration, and pH. Ben Ayed et al. [
7] found that these factors had a significant effect on Reactive black 5 (RB5) decolorization using a laccase-like activity of cell-free supernatant from
C. gallica. The optimized conditions obtained for laccase concentration, HBT concentration, and pH were 1 U/mL, 50 mg/L, 1 mM, and pH 5, respectively, with a maximum decolorization yield of 87%.
In this study, the crude laccase of
C. gallica was used in the decolorization experiments, and the presence of four azo groups in the Sirius grey makes its treatment more challenging. The decolorization rate obtained here (87% after 4 h) is significant when compared to the results reported by Daâssi et al. [
21]. In their study, they used partially purified
C. gallica laccase for the treatment of three different groups of dyes. However, the RB5 and Bismarck brown R (BBR), which are diazoic dyes, did not show significant decolorization rate. Concerning BBR, the rate was approximately 47.1% over 24 h, whereas, for RB5, this rate did not exceed 70%, even after 24 h of incubation in the presence of 1 mM HBT.
To identify the influence of the interactions between studied factors on decolorization yield and rate, 3D-surface responses were designed (
Figure 3). Increasing the HBT concentration to its highest level (1 mM), followed by the increase of pH to 4–5, resulted in enhancing decolorization yield to a level of 80% (
Figure 3a). This effect was observed in the interactions between pH × dye concentration and pH × enzyme concentration (
Figure 3d,e, respectively). However, increasing the pH beyond 5 led to a reduction of decolorization yield, and this aligns with Forootanfar et al.’s [
33] observations. These findings were explained by the fact that hydroxide anions could bind to the enzyme at acid pH, and this negatively affects the electron transfer. In contrast, Aksu and Tezer [
34] considered the possibility of basic azo dyes becoming charged positively at higher pH, and this affects their interactions with the mediator and enzyme. The effect of this interaction on the decolorization rate showed that the highest rate was achieved at pH 3, independent of the concentration of the other factors (
Figure 5a,d,e). This can be explained by the fact that pH 3 matches the optimum pH for the used enzyme, allowing the decolorization to reach a high speed. This aligns with previous studies claiming that fungal laccases are active at the 3–5 pH range [
7,
35]. In addition, the stability of the dye can be affected at pH 3, increasing the rate of decolorization even more, as reported before by Yin et al. [
36] and Ben Ayed et al. [
7].
Figure 3b shows the interaction between HBT concentration × dye concentration at pH 4.5 and 0.6 U/mL of enzyme. Increasing the HBT concentration to its maximum level at different dye concentrations resulted in an increase in the decolorization rate from values less than 1.80%/min (for 0.2 mM of HBT and 150 mg/L of dye) to values more than 2.25% (for 1 mM of HBT and 50, 100 and 150 mg/L of dye). This means that higher HBT concentrations improved the dye oxidation by laccase. However, at 150 mg/L dye concentration (and 1 mM of HBT), the decolorization yield decreased slightly, and this was likely due to enzyme and HBT saturation. As a matter of fact, excessive dye concentration can lead to enzyme inhibition and/or unproductive reactions by intensifying competition for the enzyme’s active sites and substrate saturation. This hypothesis about enzyme-substrate concentration was discussed by Cifçi et al. [
37]. The effect of saturation can also be seen in the enzyme concentration × HBT concentration interaction (
Figure 3c). In fact, increasing HBT concentration up to 0.8 mM and that of the enzyme to 0.8 U/mL could lead to an increase in the decolorization yield reaching 85%, but a higher concentration of enzyme and HBT led to a slight decrease attaining 80%. Accordingly, the presence of the enzyme and the mediator facilitates an efficient dye cleavage.
The effect of the interaction of dye concentration × enzyme concentration on the decolorization yield is depicted in
Figure 3f. It can be seen that treating high dye concentrations at low enzyme levels (and at an HBT concentration of 0.6 mM) might cause a saturation effect of the enzyme and, therefore, of the decolorization yield. However, when the substrate concentration was decreased while increasing that of the enzyme, the decolorization yield was enhanced. This suggests that higher enzyme concentrations provide more active sites for the degradation of the dye.
With regard to the decolorization rate,
Figure 5 represents the effects of factor interactions.
Figure 5b illustrates the interaction between HBT and dye concentrations. The lowest decolorization rate (1.8%/min) was observed for a minimal HBT concentration (0.2 mM) and a maximum concentration of Sirius grey concentration (150 mg/L). Conversely, a high response (2.2%/min) was obtained with a maximum mediator level (1 mM of HBT) and at different dye concentrations or at low concentrations of both dye and HBT. These results were expected since they align with the mediator’s role in improving electron transfer between the enzyme and substrate and enhancing the treatment rate [
27,
38]. In fact, higher dye concentrations necessitate greater mediator concentrations to accelerate the decolorization rate. The need for a mediator was also shown in the interaction between HBT concentration × enzyme concentration (
Figure 5c). Indeed, this interaction showed that a higher response of approximately 3%/min was reached at higher concentrations of both factors (1 mM of HBT and 1 U/mL of enzyme). So, as the levels of the enzyme and mediator were increased, the rate of dye decolorization was accelerated. The dye concentration × enzyme concentration interaction presented in
Figure 5f exhibited linearity, showcasing a high response (2.8%/min) at 1 U/mL of laccase for various dye concentrations, maintaining a pH of 4.5 and 0.6 mM of HBT. Increasing enzyme concentration means increasing the active site number, which boosts the decolorization rate [
39].
As textile industry effluents could be used for the irrigation of some crops [
40,
41], it is necessary to evaluate their phytotoxicity. In the present study, the toxicity of the treated and untreated Sirius grey solution was evaluated by measuring the germination index of radish seeds. It was found that the Germination Index (%IG) was significantly increased after the treatment of Sirius grey by the supernatant of
C. gallica compared to the dye solution. This indicates that the treatment with laccase has effectively minimized the toxicity of the dye to lower levels compared to that of untreated dye.