Research ArticlePeptides Fixing Industrial Textile Dyes: A New BiochemicalMethod in Wastewater Treatment

Amor Mosbah , Habib Chouchane, Soukaina Abdelwahed, Alaeddine Redissi,Manel Hamdi, Soumaya Kouidhi, Mohamed Neifar, Ahmed Slaheddine Masmoudi,Ameur Cherif , and Wissem Mnif

University of Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi #abet, 2020 Ariana, Tunisia

Correspondence should be addressed to Amor Mosbah; amor.mosbah@gmail.com and Wissem Mnif; w_mnif@yahoo.fr

Received 8 March 2019; Revised 28 June 2019; Accepted 10 July 2019; Published 30 July 2019

Academic Editor: Wenshan Guo

Copyright © 2019 AmorMosbah et al.)is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

)e aim of the present work was the development of a new biological method for the treatment of textile industry effluents, whichis cheaper, more profitable, and eco-friendly. )is method is essentially based on the synthesis of dye-fixing peptides. )e use ofpeptides synthesized via a solid-phase synthesis to fix a reference textile dye like “Cibacron blue” (CB) and the performanceanalysis 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. )e obtained results showed significant fixation yields of CB-peptides of 91.5% and 45.9%, respectively, andconsequently, their interesting potential as a tool for a new biochemical method in the pollution prevention of textile wastewater.

1. Introduction

)e protection of the environment is becoming a majorconcern for humankind [1, 2], particularly the protection ofwater resources against pollution by industrial dyes [3].

Expansion of the dye industry is explained by the factthat various industrial products can be colored, mainly [4]pigments (plastics industry); ink and paper; food dyestuff;pigments of paints, building materials, and ceramics(building industry); hair dyes (cosmetics industry); dyes andpreservatives (pharmaceutical industry); fuels and oils(automotive industry, etc.); and textile dyes for clothing,decoration, building, transport, etc.

)e toxicity of wastewater industry is one of the mostserious problems facing humanity and other life forms on ourplanet today.)is is a very serious environmental problem [5].Due to their large-scale production and widespread applica-tion, synthetic dyes cause considerable environmental pollu-tion and constitute a serious risk factor to public health [6, 7].

)e textile industry is one of the most polluting in-dustries that produce large quantities of water polluted byvarious chemicals [8]. In fact, world production of textile

dyes is estimated to be around more than 10,000 tons peryear and about 100 tons/year of dyes are released into waterdue to a lack of affinity of surfaces to be dyed [9]. )ese dyesare stable, very toxic, and weakly biodegradable, which maketheir elimination a real challenge.

Indeed, eutrophication phosphates used as a detergentduring the finishing process in textile industries [10] andnitrate released by microorganisms on dyes [11] are dis-charged into the external environment in large quantities.)ese products endanger marine species and alter theproduction of drinking water.)eir consumption by aquaticplants accelerates their anarchic proliferation. )e organiccompounds are brought into the ecosystem in large quan-tities and disrupt the natural processes. Indeed, a study byManahan [12] estimates that the degradation of 7 to 8mg oforganic compounds by microorganisms is sufficient toconsume the oxygen contained in one liter of water.

It is worthy to note that synthetic dyes are compoundsthat are highly resistant to natural biological degradation[13]. )is 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

HindawiJournal of ChemistryVolume 2019, Article ID 5081807, 7 pageshttps://doi.org/10.1155/2019/5081807

different pollutants in successive stages. )e first stepconsists in eliminating insoluble pollutants by means ofpretreatments (screening, grit removal, deoiling, etc.) and/orphysical or physicochemical treatments ensuring solid-liq-uid separation.

Membrane filtration used as physical treatment of ef-fluents, controlled by hydraulic pressure, is available inmicrofiltration, ultrafiltration, nanofiltration, and reverseosmosis, which is essentially done on the cutoff point of amembrane. )e effluent passes through a semipermeablemembrane that retains contaminants larger than the porediameter upstream to produce a purified permeate and aconcentrate that receives the organic impurities [14]. Amongthe four types of processes, nanofiltration and reverse os-mosis are most suitable for the partial retention of color andsmall organic molecules [15]. However, reverse osmosisremains the most common [16]. In fact, despite applications,these processes require significant investments [17]. Inaddition, this filtrationmethod is only suitable for water withlow concentrations of dyes and it is rather ineffective inreducing dissolved solids [14].

Adsorption is an efficient treatment for removing or-ganic compounds, especially when the molecular weight ofthe effluent is high and the polarity is low. It is a processwhere a solid is utilized to receive the dye of water, activatedcarbon being the most commonly used solid. )is techniqueis effective only on certain categories of dyes such as cationic,mordant, dispersed, vat, and reagents [14, 18, 19]. In ad-dition, such a technique requires subsequent operations ofregeneration of activated carbon, which leads to losing about10 to 15% of its efficiency [14], and posttreatment of ex-pensive accumulated sludge.

Coagulation/flocculation is the aggregation or agglom-eration of colloidal particles or fine suspended solids as flocsunder the action of flocculants. An additional step of settlingor flotation helps to eliminate flocs. It is a method of treatingtextile effluents by discoloration of wastewater. )is methodis inexpensive and uses coagulants such as alum, ferroussulphate, and ferrous chloride. However, it is inefficient as iteliminates only 50% of reactive, azo, acid, and basic dyes[14, 18]. In addition, coagulation/flocculation cannot beused for highly water-soluble dyes [18]. However, the sludgegenerated by these processes in huge quantities is the majordisadvantage of such a method. )ese products are difficultto move and pose a real danger to the environment.

Since their application is easy, chemical oxidationmethods are commonly used and become necessary whenbiological processes are ineffective. )ese are treatments thataim at the total mineralization of pollutants in CO2, H2O,and inorganic compounds [20]. In the literature, this type oftreatment is cited as a pretreatment for biological processesto increase biodegradability, as a treatment of hazardousorganic compounds present in low concentration, and fi-nally as a posttreatment to reduce aqueous toxicity. Chlorine(Cl2), chlorine dioxide (Cl2O2), and ozone (O3) are amongthe reagents used in conventional oxidation. And for theadvanced chemical oxidation, other strongly oxidizingspecies such as the hydroxyl radical (OH) are used. Ad-vanced oxidation processes are grouped into several

categories [20]. )e efficiency of this process depends on theconcentration of oxygen. )e main drawback is the lack ofknowledge concerning the degradation products generatedand the final products that may be more toxic than thestarting dyes [21, 22]. In addition, these processes requiresophisticated and highly costly equipment [14, 18].

Although many microorganisms are able to cleave chro-mophores and auxochromes of certain dyes (hence, discol-oration), some can mineralize the dyes in CO2 and H2O [18].)erefore, the use of microorganisms for the biodegradation ofsynthetic dyes is a simple and interesting method. Biologicalprocesses can be divided into three categories: aerobic, an-aerobic, and mixed aerobic/anaerobic [23].

Aerobic treatment is the most applied [24], where aer-obic bacteria and other microorganisms are placed in abiological unit consisting of an activated sludge basin. In thepresence of CO2 and an adequate pH, these microorganismsbreak down pollutants into sludge that sediments. Organicpollutants can oxidize to carbon dioxide. After purification,the sludge is separated from the wastewater by sedimen-tation in a decanter, a part is recycled, and the surplus isremoved after pressing or centrifugation.

For a high concentration of organic pollutants, thesetechniques are not sufficiently effective for the treatment oftextile discharges. Many classes of dyes such as azo dyes, acids(because of sulphonated groups), and reactive dyes are re-calcitrant [24], and the decrease in color is mainly due to ad-sorption on sludge rather than the degradation of the dye. Onlyweakly substituted dyes with a simple chemical structure andlow molecular weight have significant discoloration rates [25].

Sheng and Chi [26] have shown that conventional bi-ological treatment methods are ineffective for the de-colorization and degradation of synthetic dyes since thesedye molecules contain a very high degree of aromatic ringsconferring them a very high stability and resistance tomicrobial attack. Moreover, the toxicity of the dyes inhibitsthe growth of bacteria, making the treatment of effluentscontaining dyes in the treatment plant difficult or impossible[26]. In addition, the degradation of azo dyes by certainbacteria generates mutagenic products [21, 22].

An effluent treatment technique adapted to the dyesmust eliminate them completely in order to avoid the for-mation of more dangerous by-products than the initialcompounds and more particularly to prevent the formationof carcinogenic products. Conventional methods of treat-ment do not meet this expectation.

)e objective of this study is the development of a newbiological approach which is more suitable in terms of cost-effectiveness, pollutant removal efficiency, and recyclabilityand inexpensive, effective, and eco-friendly (biodegradable)for the treatment of effluents of the textile industry. To ourknowledge, this is the first report on the textile effluenttreatments using peptides. )is technology will be based onthe best binding affinity of textile dyes on peptides synthesizedvia a solid-phase peptide synthesis (SPPS) technique.

)e peptide-dye interaction is governed by low-energytype links (electrostatic, van der Walls, hydrophobic, Pi, andionic). )is mechanism is the same as the mechanism ofinteraction of antibodies with antigens or the interaction of a

2 Journal of Chemistry

receptor with a ligand or even the interaction of a substratewith an enzyme. )is interaction is very specific, that is, apeptide may interact with a dye or a dye family. )e affinitybetween the peptide and the dye is very important, and it canapproach the affinity of a substrate to an enzyme.

2. Materials and Methods

A manual peptide synthesis was realized. It consists in usinga cylindrical glass reactor made of a container with a sintereddisc and a tap that controls the filtration of the solvents. Avacuum pump was used to create vacuum to ensure betterfiltration of the vial and the removal of reagents in excess.

2.1. Peptide Sequence Synthesis. )ree amino acids (C-G-G)were added in the N-terminal sequence of peptides P1 andP2, selected by Iannolo et al. [27], and having the capacity tofix “Cibacron blue” using the phage display technique se-quences, 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-anthrace-nesulfonic 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 A, CAS-No. 84166-13-2 was provided bya textile industry “Huntsman.” )e chemical structure ofCibacron blue (CB) is shown in Figure 1 [28].

2.3. Solid-Phase Peptide Synthesis. For the achievement ofthe solid-phase peptide synthesis, various components wereused. )ey are listed below.

2.3.1. Fmoc-Rink-Amide Resin. )e resin used for thesynthesis gives a C-terminus amide. Its molecular formula isC31H29NO5 (8-[(E)-3-(9-ethylcarbazol-3-yl)prop-2-enoyl]-5,7-dimethoxy-4-propylchromen-2-one), it has a molecularweight of 495.575 g/mol, and its extent of labeling is0.5mmoles/g.)e chemical structure of the used rink-amideresin is shown in Figure 2.

2.3.2. Base and Coupling Agent. )e deprotection of theFmoc protecting group is performed via the strong base, thepiperidine (20%), premixed in N,N-dimethylformamide(DMF) (80%). )e amino acid-coupling step requires a basicmedium that is favored by the addition of diisopropyle-thylamine (DIPEA) as well as catalysts (coupling agents) suchas PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), HCTU O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, andEDC (N-(3-dimethylaminopropyl)-N′-ethylcarbonate).

2.3.3. Cleavage Agent. )e elimination of the protectivegroups of the side chains of the amino acids as well as the

solid polymer is carried out with a cocktail mixture of tri-fluoroacetic acid (TFA), 0.5% water, and 0.5% triisopropylsilane (TIS) as scavengers.

2.3.4. Washing Solvent. To get rid of excess amino acids orcoupling agents, a simple washing with dichloromethane(DCM) or DMF is required.

2.4. Kaiser Test. To ensure that the amino acid coupling isdone, the “Kaiser test” [30] must be performed. )is testrequires two solutions A and B whose compositions are asfollows:

Solution A: a mixture of pyridine (49mL) + 20mLethanol + 0.8 g phenol + 1mL of KCN (1mM)Solution B: a mixture of 1 g ninhydrin + 20mL ethanol

2.4.1. Method of Treatment with Dye-Fixing Peptides.Water loaded with the dye passes through the columnprefilled with a solid support-bound dye-fixing peptide, thedye 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 com-pounds. In order to avoid losses, they were fixed on a solid

O

O NH2

NHNH

SO3H

SO3H

N

NNSO3H

Cl

NH

Figure 1: Chemical structure of Cibacron blue [28].

O

O

NH

O

OO

Figure 2: Chemical structure of the rink-amide resin used in theSPPS [29].

Journal of Chemistry 3

support by a disulfide bond. Like the first test, the same SPPSresin (rink amide resin) coupled to unprotected cysteine wasused. )e fixation of cysteine on the resin is done by fol-lowing 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. )e peptide wassolubilized in a buffer phosphate (20mM) solution at pH� 5and added to the support. Fifteen minutes later, the pH wasadjusted to 8.5 for 24 hours. )e next step consisted inrecovering the supernatant by adjusting the pH to 5, andDTT (dithiothreitol) was added to cut the unwanteddisulfide bridges formed between the peptides. To precipitatethe peptides, 2 volumes of acetone were added, and theprecipitate was recovered by centrifugation at 4000 rpm for15min. )e precipitate was subsequently solubilized inbuffer (pH� 5) and reprecipitated, to be added to thesupport.)e pH of the mixture was adjusted to 8.5 again andthe whole mixture was incubated for 24 h. Finally, the su-pernatant was recovered, the peptides were precipitatedtwice with acetone, and the precipitate was stored at −20°C.

(3) Dye Fixation on the Peptide. In order to calculate the dyefixation yield, 2mL of Cibacron blue solution (10mg/L) wasmixed with the peptide bound to the support. )e pH wasadjusted to 8, and stirring for 3 h was carried out, favoringthe connection between the dye and the peptide by theformation of no covalent interaction. Filtration and mea-surement of the optical density of the filtrate at 616 nm(absorbance of Cibacron blue) were performed.

3. Result

3.1. Peptide Synthesis. )e synthesis of peptides was con-firmed manually by the detection of the correct mass of thetwo peptides in the mass spectrum (MS) analysis. Figure 3(a)shows a mass peak of 1376.10 g/mol which has a differencewith the theoretical mass of 61.326 g/mol. )e latter valuecorresponds to the sum of the masses of a potassiummolecule and a sodium molecule coupled to the peptideduring the passage in the LC-MS ionization column.

)e spectrum of peptide 2 presented in Figure 3(b)shows a majority peak of mass 1399.53 g/mol which cor-responds to the theoretical mass of the peptide associatedwith a potassium molecule. )ese results proved conformitybetween the theoretical and practical masses of peptides andtherefore the success of our synthesis. As a result, oursynthesized peptides are ready for the dye fixation test.

3.2. Preparation of the Support. A free cysteine was fixed tothe resin by coupling its carboxylic group with the aminegroup of resin. )e peptides P1 or P2 were added to thiscysteyl resin in an equimolar concentration to the resin at

pH� 5, which was adjusted carefully to 8.3 in order to linkthe peptide to the cysteyl resin.

3.3. Dye Fixation Test. )e test is based on measuring theoptical density of the dye solution before and after contactwith the peptide. However, the optimal absorbance wave-length of the “Cibacron blue” is determined according to thecalibration curve. By analyzing this curve, it can be con-cluded that the dye absorbs particularly at 616 nm.

To evaluate the amount of the dye fixed by the peptide, arange of dye “Cibacron blue” (774.16 g/mol) standards wasprepared with increasing concentrations ranging from0.5mg·L−1 to 12mg·L−1, and then the OD was monitored at616 nm (Figure 4).

3.4. Dye-Peptide Binding Test. A solution of “Cibacron blue”at a concentration of 10mg/L was contacted with thesupport-bound peptides (P1 or P2) for 48 h. )e absorbanceof the filtrate is measured. )e obtained result shows that P1and P2 made it possible to calculate a concentration of0.85mg/L and 5.41mg/L of the remaining dye after itspassage through the peptide, respectively. )is result showsthat P1 has fixed almost all dye, and the remaining half of“Cibacron blue” is fixed by P2 (Figure 5) with an efficacyyield of 91.5% and 45.9%, respectively. )anks to the cali-bration curve OD� f (concentration of the dye) alreadydrawn, we were able to determine the amount of dye fixed bythe peptide. Since the concentration of the peptide is known,and knowing that each molecule of peptide can fix a dyemolecule, we have been able to determine the ratio(expressed as a percentage of efficiency) of the number ofmoles of the dye and the number of moles of the peptide.

4. Discussion

Dyes are an important class of synthetic organic compoundsused in many industries, especially textiles. Consequently,they have become common industrial environmental pol-lutants during their synthesis and later during fiber dyeing.Textile industries are facing a challenge in the field of qualityand productivity due to the globalization of the worldmarket. )e large-scale production and extensive applica-tion of synthetic textile dyes can cause considerable envi-ronmental pollution [31], making it a serious public concern.However, due to the toxic nature and adverse effect ofsynthetic textile dyes on all forms of life [32, 33], it is im-perative to treat these effluents before rejecting them in thenatural environment. Traditional physicochemical treat-ments (adsorption, coagulation/flocculation, etc.) as well asbiological methods commonly used for the depollution ofthese effluents generally are proved to be inefficient, non-specific, and very expensive [34]. It seems interesting todevelop new alternative treatments free from thesedrawbacks.

)e present study aims at developing a new biologicalapproach for the treatment of textile industry effluents. )ismethod is essentially based on the synthesis of dye-fixingpeptides. )e chemical approach of this research focuses on

4 Journal of Chemistry

the peptide synthesis strategy described for the first time byMerrifield [35] using the Fmoc-tert-butyl technique, tosynthesize the peptides in question with a good yield. )istechnique is very well described in the literature [35–37].)e use of two peptides synthesized and selected by Iannoloet al. [27] (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 havebeen realized by adding 3 amino acids (C-G-G) in the P1 orP2 N-terminal sequence. )e main results showed signif-icant fixation yields of CB-peptides of 91.5% and 45.9%,respectively, hence their remarkable potential as a tool forthe new biochemical method in the pollution prevention oftextile wastewater.

)ese results are consistent with the work of Iannolo et al.[27] who discovered that “Cibacron blue” fixed two peptidesbound to the major proteins of the filamentous phagemembrane. In pursuit of their work, the same authors selectednew phage clones that can specifically bind the dye 1000 timesmore than wild phages. )e results also revealed that aconcentration of 1mM of “Cibacron blue” was sufficient tostain all phages. Indeed, according to these researchers, thestrong interaction between phage peptides and dye can beexplained by their chemical properties. In fact, the peptidesselected in this work are characterized by (i) one or twopositively charged residues flanked by an aromatic end on theamine end and (ii) the “Cibacron blue” that contains threenegatively charged sulphonic acid groups which are attachedto the backbone of the molecule by several aromatic groups.

)is new biochemical method, based on the fixation oftextile dyes by peptides, offers a great contribution to the

(a)

(b)

Figure 3: Mass spectrum analysis of peptide synthesis products by liquid chromatography coupled to mass spectrometry (LCMS). (a) Syntheticpeptide 1 (P1). (b) Synthetic peptide 2 (P2).

y = 0.0224x + 0.0068R2 = 0.9897

0

0.05

0.1

0.15

0.2

0.25

0.3

0 2 4 6 8 10 12 14

Abs

orba

nce (λ

=61

6nm

)

Concentration (mg/L)

Figure 4: Calibration curve (concentration vs. absorbance) for“Cibacron blue.”

Journal of Chemistry 5

industry. )is method allows the reuse of the depollutedwater, the dyes (after the release of the peptides), and thesynthesized peptides. On the contrary, it is free from dis-advantages that characterize conventional methods (physi-cal, chemical, and biological). Such methods are not effectivefor certain dyes, suitable only for waters containing lowconcentrations of these toxic substances, and they generatedegradation products that may themselves be more toxicthan the starting dyes [14, 18, 21, 22, 24].

)is new approach goes beyond these limits, and it isadaptable to large concentrations of dyes given the highaffinity of peptides to them and given their high yield. It isalso effective in a wide range of dyes by the use of variouspeptides having affinities to different dyes or peptides withhigh affinity to a family of dyes. Also, by the “phagedisplay” technique, one can select more than one peptidefor a single dye or one peptide is able to fix more than onedye or a family of dye, as reported by Iannolo et al. [27].)ese researchers identified 11 sequences with high af-finity (more than 90%) for the “Cibacron blue” textile dye.In our laboratory, we are currently extending our study toother dyestuff classes and more specifically to diazo andmonoazo dyes, which are highly hazardous, and to dyesused in the cooking industry, which can be currentlyeliminated.

It should be pointed that, to better exploit this newapproach, some assays are in progress for other dyestuff

classes, in particular monoazo and diazo dyes, as well as fordyes used in the cooking industry.

5. Conclusion

)is study allows the setting up of a new biochemicalmethod in textile wastewater treatment. It is based on thepassing of the textile wastewater through a column whichcontains in advance a fixing peptide of the dye or a fixingpeptide of a family of dyes bound to a support.)e dye bindsto the peptide, and the water passing through the columnwill be recovered. )anks to this eco-friendly as well as acost-saving method, the water and the colorant can bereused. 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 themethodology with peptides that bind the “Cibacron blue.”

)anks to this new approach, significant fixation yieldsof CB-peptides were obtained (91.5% and 45.9%). )eseresults reveal the effectiveness of the peptides synthesized(by SPPS or by cell expression in multimer) for the fixationof the “Cibacron blue” dye and to get rid of the depollutionof textile effluents. As a perspective, it is interesting to adaptand extrapolate this simple, fast, inexpensive, and eco-friendly technique on an industrial scale.

Data Availability

)e data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

)e authors declare that they have no conflicts of interest.

Acknowledgments

)e authors are grateful to the ISBST, Biotechpole, ManoubaUniversity, Sidi )abet, Tunisia, for providing infra-structural facilities and assistance.

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A B

Figure 5: Dye-peptide binding test: Once the support is ready, acontact with the peptide in the basic medium allows their bindingby means of disulfide bond formation between the cysteine of thesupport and the cysteine of the peptide. A solution of “Cibacronblue” at a concentration of 10mg/L for 48 h is then contacted withthe support-bound peptide, and finally, the OD of the filtrate ismeasured.

6 Journal of Chemistry

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