Peptides Fixing Industrial Textile Dyes: A New Biochemical Method in Wastewater Treatment
The aim of the present work was the development of a new biological method for the treatment of textile industry effluents, which is cheaper, more profitable, and eco-friendly. This method is essentially based on the synthesis of dye-fixing peptides. The use of peptides synthesized via a solid-phase synthesis to fix a reference textile dye like “Cibacron blue” (CB) and the performance analysis of binding assays were the main objectives of this study. For this reason, two peptides P1 (NH2-C-G-G-W-R-S-Q-N-Q-G-NH2) and P2 (NH2-C-G-G-R-R-Y-Q-P-D-S-NH2) binding with the CB dye were synthesized by the solid-phase peptide synthesis (SPPS) technique. The obtained results showed significant fixation yields of CB-peptides of 91.5% and 45.9%, respectively, and consequently, their interesting potential as a tool for a new biochemical method in the pollution prevention of textile wastewater.
Expansion of the dye industry is explained by the fact that various industrial products can be colored, mainly  pigments (plastics industry); ink and paper; food dyestuff; pigments of paints, building materials, and ceramics (building industry); hair dyes (cosmetics industry); dyes and preservatives (pharmaceutical industry); fuels and oils (automotive industry, etc.); and textile dyes for clothing, decoration, building, transport, etc.
The toxicity of wastewater industry is one of the most serious problems facing humanity and other life forms on our planet today. This is a very serious environmental problem . Due to their large-scale production and widespread application, synthetic dyes cause considerable environmental pollution and constitute a serious risk factor to public health [6, 7].
The textile industry is one of the most polluting industries that produce large quantities of water polluted by various chemicals . In fact, world production of textile dyes is estimated to be around more than 10,000 tons per year and about 100 tons/year of dyes are released into water due to a lack of affinity of surfaces to be dyed . These dyes are stable, very toxic, and weakly biodegradable, which make their elimination a real challenge.
Indeed, eutrophication phosphates used as a detergent during the finishing process in textile industries  and nitrate released by microorganisms on dyes  are discharged into the external environment in large quantities. These products endanger marine species and alter the production of drinking water. Their consumption by aquatic plants accelerates their anarchic proliferation. The organic compounds are brought into the ecosystem in large quantities and disrupt the natural processes. Indeed, a study by Manahan  estimates that the degradation of 7 to 8 mg of organic compounds by microorganisms is sufficient to consume the oxygen contained in one liter of water.
It is worthy to note that synthetic dyes are compounds that are highly resistant to natural biological degradation . This persistence is related to their chemical reactivity. Generally, the treatment of textile rejects is carried out, thanks to a treatment chain ensuring the elimination of different pollutants in successive stages. The first step consists in eliminating insoluble pollutants by means of pretreatments (screening, grit removal, deoiling, etc.) and/or physical or physicochemical treatments ensuring solid-liquid separation.
Membrane filtration used as physical treatment of effluents, controlled by hydraulic pressure, is available in microfiltration, ultrafiltration, nanofiltration, and reverse osmosis, which is essentially done on the cutoff point of a membrane. The effluent passes through a semipermeable membrane that retains contaminants larger than the pore diameter upstream to produce a purified permeate and a concentrate that receives the organic impurities . Among the four types of processes, nanofiltration and reverse osmosis are most suitable for the partial retention of color and small organic molecules . However, reverse osmosis remains the most common . In fact, despite applications, these processes require significant investments . In addition, this filtration method is only suitable for water with low concentrations of dyes and it is rather ineffective in reducing dissolved solids .
Adsorption is an efficient treatment for removing organic compounds, especially when the molecular weight of the effluent is high and the polarity is low. It is a process where a solid is utilized to receive the dye of water, activated carbon being the most commonly used solid. This technique is effective only on certain categories of dyes such as cationic, mordant, dispersed, vat, and reagents [14, 18, 19]. In addition, such a technique requires subsequent operations of regeneration of activated carbon, which leads to losing about 10 to 15% of its efficiency , and posttreatment of expensive accumulated sludge.
Coagulation/flocculation is the aggregation or agglomeration of colloidal particles or fine suspended solids as flocs under the action of flocculants. An additional step of settling or flotation helps to eliminate flocs. It is a method of treating textile effluents by discoloration of wastewater. This method is inexpensive and uses coagulants such as alum, ferrous sulphate, and ferrous chloride. However, it is inefficient as it eliminates only 50% of reactive, azo, acid, and basic dyes [14, 18]. In addition, coagulation/flocculation cannot be used for highly water-soluble dyes . However, the sludge generated by these processes in huge quantities is the major disadvantage of such a method. These products are difficult to move and pose a real danger to the environment.
Since their application is easy, chemical oxidation methods are commonly used and become necessary when biological processes are ineffective. These are treatments that aim at the total mineralization of pollutants in CO2, H2O, and inorganic compounds . In the literature, this type of treatment is cited as a pretreatment for biological processes to increase biodegradability, as a treatment of hazardous organic compounds present in low concentration, and finally as a posttreatment to reduce aqueous toxicity. Chlorine (Cl2), chlorine dioxide (Cl2O2), and ozone (O3) are among the reagents used in conventional oxidation. And for the advanced chemical oxidation, other strongly oxidizing species such as the hydroxyl radical (OH) are used. Advanced oxidation processes are grouped into several categories . The efficiency of this process depends on the concentration of oxygen. The main drawback is the lack of knowledge concerning the degradation products generated and the final products that may be more toxic than the starting dyes [21, 22]. In addition, these processes require sophisticated and highly costly equipment [14, 18].
Although many microorganisms are able to cleave chromophores and auxochromes of certain dyes (hence, discoloration), some can mineralize the dyes in CO2 and H2O . Therefore, the use of microorganisms for the biodegradation of synthetic dyes is a simple and interesting method. Biological processes can be divided into three categories: aerobic, anaerobic, and mixed aerobic/anaerobic .
Aerobic treatment is the most applied , where aerobic bacteria and other microorganisms are placed in a biological unit consisting of an activated sludge basin. In the presence of CO2 and an adequate pH, these microorganisms break down pollutants into sludge that sediments. Organic pollutants can oxidize to carbon dioxide. After purification, the sludge is separated from the wastewater by sedimentation in a decanter, a part is recycled, and the surplus is removed after pressing or centrifugation.
For a high concentration of organic pollutants, these techniques are not sufficiently effective for the treatment of textile discharges. Many classes of dyes such as azo dyes, acids (because of sulphonated groups), and reactive dyes are recalcitrant , and the decrease in color is mainly due to adsorption on sludge rather than the degradation of the dye. Only weakly substituted dyes with a simple chemical structure and low molecular weight have significant discoloration rates .
Sheng and Chi  have shown that conventional biological treatment methods are ineffective for the decolorization and degradation of synthetic dyes since these dye molecules contain a very high degree of aromatic rings conferring them a very high stability and resistance to microbial attack. Moreover, the toxicity of the dyes inhibits the growth of bacteria, making the treatment of effluents containing dyes in the treatment plant difficult or impossible . In addition, the degradation of azo dyes by certain bacteria generates mutagenic products [21, 22].
An effluent treatment technique adapted to the dyes must eliminate them completely in order to avoid the formation of more dangerous by-products than the initial compounds and more particularly to prevent the formation of carcinogenic products. Conventional methods of treatment do not meet this expectation.
The objective of this study is the development of a new biological approach which is more suitable in terms of cost-effectiveness, pollutant removal efficiency, and recyclability and inexpensive, effective, and eco-friendly (biodegradable) for the treatment of effluents of the textile industry. To our knowledge, this is the first report on the textile effluent treatments using peptides. This technology will be based on the best binding affinity of textile dyes on peptides synthesized via a solid-phase peptide synthesis (SPPS) technique.
The peptide-dye interaction is governed by low-energy type links (electrostatic, van der Walls, hydrophobic, Pi, and ionic). This mechanism is the same as the mechanism of interaction of antibodies with antigens or the interaction of a receptor with a ligand or even the interaction of a substrate with an enzyme. This interaction is very specific, that is, a peptide may interact with a dye or a dye family. The affinity between the peptide and the dye is very important, and it can approach the affinity of a substrate to an enzyme.
2. Materials and Methods
A manual peptide synthesis was realized. It consists in using a cylindrical glass reactor made of a container with a sintered disc and a tap that controls the filtration of the solvents. A vacuum pump was used to create vacuum to ensure better filtration of the vial and the removal of reagents in excess.
2.1. Peptide Sequence Synthesis
Three amino acids (C-G-G) were added in the N-terminal sequence of peptides P1 and P2, selected by Iannolo et al. , and having the capacity to fix “Cibacron blue” using the phage display technique sequences, in order to be fixed to the support later. P1: NH2-C-G-G-W-R-S-Q-N-Q-G-NH2 P2: NH2-C-G-G-R-R-Y-Q-P-D-S-NH2
2.2. Dye Used: “Cibacron Blue”
“Cibacron blue” (2-anthracenesulfonic acid, 1-amino-4-[[4-[[4-chloro-6-[(2-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]-9,10-di/C29H20ClN7O11S3) with a molecular weight of 774.16 g/mol, a size of 25 × 10 Å, CAS-No. 84166-13-2 was provided by a textile industry “Huntsman.” The chemical structure of Cibacron blue (CB) is shown in Figure 1 .
2.3. Solid-Phase Peptide Synthesis
For the achievement of the solid-phase peptide synthesis, various components were used. They are listed below.
2.3.1. Fmoc-Rink-Amide Resin
The resin used for the synthesis gives a C-terminus amide. Its molecular formula is C31H29NO5 (8-[(E)-3-(9-ethylcarbazol-3-yl)prop-2-enoyl]-5,7-dimethoxy-4-propylchromen-2-one), it has a molecular weight of 495.575 g/mol, and its extent of labeling is 0.5 mmoles/g. The chemical structure of the used rink-amide resin is shown in Figure 2.
2.3.2. Base and Coupling Agent
The deprotection of the Fmoc protecting group is performed via the strong base, the piperidine (20%), premixed in N,N-dimethylformamide (DMF) (80%). The amino acid-coupling step requires a basic medium that is favored by the addition of diisopropylethylamine (DIPEA) as well as catalysts (coupling agents) such as PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HCTU O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, and EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbonate).
2.3.3. Cleavage Agent
The elimination of the protective groups of the side chains of the amino acids as well as the solid polymer is carried out with a cocktail mixture of trifluoroacetic acid (TFA), 0.5% water, and 0.5% triisopropyl silane (TIS) as scavengers.
2.3.4. Washing Solvent
To get rid of excess amino acids or coupling agents, a simple washing with dichloromethane (DCM) or DMF is required.
2.4. Kaiser Test
To ensure that the amino acid coupling is done, the “Kaiser test”  must be performed. This test requires two solutions A and B whose compositions are as follows: Solution A: a mixture of pyridine (49 mL) + 20 mL ethanol + 0.8 g phenol + 1 mL of KCN (1 mM) Solution B: a mixture of 1 g ninhydrin + 20 mL ethanol
2.4.1. Method of Treatment with Dye-Fixing Peptides
Water loaded with the dye passes through the column prefilled with a solid support-bound dye-fixing peptide, the dye binds to the peptide, and the exiting water is recovered.
2.4.2. Establishment of “Cibacron Blue” Dye Fixation Test
(1) Support Preparation. Peptides are water-soluble compounds. In order to avoid losses, they were fixed on a solid support by a disulfide bond. Like the first test, the same SPPS resin (rink amide resin) coupled to unprotected cysteine was used. The fixation of cysteine on the resin is done by following the same protocol of solid-phase peptide synthesis:(1)Loading the resin(2)Deprotection of the resin(3)Coupling of the unprotected cysteine because one needs the free thiol group.
(2) Peptide Fixation on the Solid Support. The peptide was solubilized in a buffer phosphate (20 mM) solution at pH = 5 and added to the support. Fifteen minutes later, the pH was adjusted to 8.5 for 24 hours. The next step consisted in recovering the supernatant by adjusting the pH to 5, and DTT (dithiothreitol) was added to cut the unwanted disulfide bridges formed between the peptides. To precipitate the peptides, 2 volumes of acetone were added, and the precipitate was recovered by centrifugation at 4000 rpm for 15 min. The precipitate was subsequently solubilized in buffer (pH = 5) and reprecipitated, to be added to the support. The pH of the mixture was adjusted to 8.5 again and the whole mixture was incubated for 24 h. Finally, the supernatant was recovered, the peptides were precipitated twice with acetone, and the precipitate was stored at −20°C.
(3) Dye Fixation on the Peptide. In order to calculate the dye fixation yield, 2 mL of Cibacron blue solution (10 mg/L) was mixed with the peptide bound to the support. The pH was adjusted to 8, and stirring for 3 h was carried out, favoring the connection between the dye and the peptide by the formation of no covalent interaction. Filtration and measurement of the optical density of the filtrate at 616 nm (absorbance of Cibacron blue) were performed.
3.1. Peptide Synthesis
The synthesis of peptides was confirmed manually by the detection of the correct mass of the two peptides in the mass spectrum (MS) analysis. Figure 3(a) shows a mass peak of 1376.10 g/mol which has a difference with the theoretical mass of 61.326 g/mol. The latter value corresponds to the sum of the masses of a potassium molecule and a sodium molecule coupled to the peptide during the passage in the LC-MS ionization column.
The spectrum of peptide 2 presented in Figure 3(b) shows a majority peak of mass 1399.53 g/mol which corresponds to the theoretical mass of the peptide associated with a potassium molecule. These results proved conformity between the theoretical and practical masses of peptides and therefore the success of our synthesis. As a result, our synthesized peptides are ready for the dye fixation test.
3.2. Preparation of the Support
A free cysteine was fixed to the resin by coupling its carboxylic group with the amine group of resin. The peptides P1 or P2 were added to this cysteyl resin in an equimolar concentration to the resin at pH = 5, which was adjusted carefully to 8.3 in order to link the peptide to the cysteyl resin.
3.3. Dye Fixation Test
The test is based on measuring the optical density of the dye solution before and after contact with the peptide. However, the optimal absorbance wavelength of the “Cibacron blue” is determined according to the calibration curve. By analyzing this curve, it can be concluded that the dye absorbs particularly at 616 nm.
To evaluate the amount of the dye fixed by the peptide, a range of dye “Cibacron blue” (774.16 g/mol) standards was prepared with increasing concentrations ranging from 0.5 mg·L−1 to 12 mg·L−1, and then the OD was monitored at 616 nm (Figure 4).
3.4. Dye-Peptide Binding Test
A solution of “Cibacron blue” at a concentration of 10 mg/L was contacted with the support-bound peptides (P1 or P2) for 48 h. The absorbance of the filtrate is measured. The obtained result shows that P1 and P2 made it possible to calculate a concentration of 0.85 mg/L and 5.41 mg/L of the remaining dye after its passage through the peptide, respectively. This result shows that P1 has fixed almost all dye, and the remaining half of “Cibacron blue” is fixed by P2 (Figure 5) with an efficacy yield of 91.5% and 45.9%, respectively. Thanks to the calibration curve OD = f (concentration of the dye) already drawn, we were able to determine the amount of dye fixed by the peptide. Since the concentration of the peptide is known, and knowing that each molecule of peptide can fix a dye molecule, we have been able to determine the ratio (expressed as a percentage of efficiency) of the number of moles of the dye and the number of moles of the peptide.
Dyes are an important class of synthetic organic compounds used in many industries, especially textiles. Consequently, they have become common industrial environmental pollutants during their synthesis and later during fiber dyeing. Textile industries are facing a challenge in the field of quality and productivity due to the globalization of the world market. The large-scale production and extensive application of synthetic textile dyes can cause considerable environmental pollution , making it a serious public concern. However, due to the toxic nature and adverse effect of synthetic textile dyes on all forms of life [32, 33], it is imperative to treat these effluents before rejecting them in the natural environment. Traditional physicochemical treatments (adsorption, coagulation/flocculation, etc.) as well as biological methods commonly used for the depollution of these effluents generally are proved to be inefficient, nonspecific, and very expensive . It seems interesting to develop new alternative treatments free from these drawbacks.
The present study aims at developing a new biological approach for the treatment of textile industry effluents. This method is essentially based on the synthesis of dye-fixing peptides. The chemical approach of this research focuses on the peptide synthesis strategy described for the first time by Merrifield  using the Fmoc-tert-butyl technique, to synthesize the peptides in question with a good yield. This technique is very well described in the literature [35–37]. The use of two peptides synthesized and selected by Iannolo et al.  (NH2-W-R-S-Q-N-Q-G-NH2 and NH2-R-R-Y-Q-P-D-S-NH2) via a solid-phase peptide synthesis (SPPS) technique and fixing a reference textile dye like “Cibacron blue” (CB) and the performance of binding assays have been realized by adding 3 amino acids (C-G-G) in the P1 or P2 N-terminal sequence. The main results showed significant fixation yields of CB-peptides of 91.5% and 45.9%, respectively, hence their remarkable potential as a tool for the new biochemical method in the pollution prevention of textile wastewater.
These results are consistent with the work of Iannolo et al.  who discovered that “Cibacron blue” fixed two peptides bound to the major proteins of the filamentous phage membrane. In pursuit of their work, the same authors selected new phage clones that can specifically bind the dye 1000 times more than wild phages. The results also revealed that a concentration of 1 mM of “Cibacron blue” was sufficient to stain all phages. Indeed, according to these researchers, the strong interaction between phage peptides and dye can be explained by their chemical properties. In fact, the peptides selected in this work are characterized by (i) one or two positively charged residues flanked by an aromatic end on the amine end and (ii) the “Cibacron blue” that contains three negatively charged sulphonic acid groups which are attached to the backbone of the molecule by several aromatic groups.
This new biochemical method, based on the fixation of textile dyes by peptides, offers a great contribution to the industry. This method allows the reuse of the depolluted water, the dyes (after the release of the peptides), and the synthesized peptides. On the contrary, it is free from disadvantages that characterize conventional methods (physical, chemical, and biological). Such methods are not effective for certain dyes, suitable only for waters containing low concentrations of these toxic substances, and they generate degradation products that may themselves be more toxic than the starting dyes [14, 18, 21, 22, 24].
This new approach goes beyond these limits, and it is adaptable to large concentrations of dyes given the high affinity of peptides to them and given their high yield. It is also effective in a wide range of dyes by the use of various peptides having affinities to different dyes or peptides with high affinity to a family of dyes. Also, by the “phage display” technique, one can select more than one peptide for a single dye or one peptide is able to fix more than one dye or a family of dye, as reported by Iannolo et al. . These researchers identified 11 sequences with high affinity (more than 90%) for the “Cibacron blue” textile dye. In our laboratory, we are currently extending our study to other dyestuff classes and more specifically to diazo and monoazo dyes, which are highly hazardous, and to dyes used in the cooking industry, which can be currently eliminated.
It should be pointed that, to better exploit this new approach, some assays are in progress for other dyestuff classes, in particular monoazo and diazo dyes, as well as for dyes used in the cooking industry.
This study allows the setting up of a new biochemical method in textile wastewater treatment. It is based on the passing of the textile wastewater through a column which contains in advance a fixing peptide of the dye or a fixing peptide of a family of dyes bound to a support. The dye binds to the peptide, and the water passing through the column will be recovered. Thanks to this eco-friendly as well as a cost-saving method, the water and the colorant can be reused. To begin this project and before embarking on the “phage display” technique to identify a whole range of dye-fixing peptides, an attempt was made to validate the methodology with peptides that bind the “Cibacron blue.”
Thanks to this new approach, significant fixation yields of CB-peptides were obtained (91.5% and 45.9%). These results reveal the effectiveness of the peptides synthesized (by SPPS or by cell expression in multimer) for the fixation of the “Cibacron blue” dye and to get rid of the depollution of textile effluents. As a perspective, it is interesting to adapt and extrapolate this simple, fast, inexpensive, and eco-friendly technique on an industrial scale.
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
The authors are grateful to the ISBST, Biotechpole, Manouba University, Sidi Thabet, Tunisia, for providing infrastructural facilities and assistance.
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