AIA Alonso-Calderon1, E. Chavez-Bravo2, E. Vidal-Robles3,
S. Martinez-Galindo4, J.R. Omaire-Vargas4 and M.A. Marin-Castro5

1Chemical Engineering Faculty, Autonomous University of Puebla, Mexico.
University City, Avenue San Claudio and south 18, Colony San Manuel.
2Center for Research in Microbiological Sciences,
Institute of Sciences Autonomous University of Puebla, Mexico.
3Chemical Engineering Faculty, Autonomous University of Puebla, Mexico.
4Engineering Faculty, Autonomous University of Puebla, Mexico.
5Agricultural Sciences Research Department of the Sciences-Institute,
Autonomous University of Puebla, Mexico.


In this research, extracellular fungal extracts containing Pleurotusostreatus and Pleurotusdjamor were used for the removal of erionyl blue A-R (anthraquinone) and solophenyl black FR (azo) textile dyes in artificially contaminated water at 200 ppm. For erionyl blue A-R, removals of up to 93.6 and 42.85% were achieved, and for solophenyl black FR of 27 and 31.14%, using the enzymatic extracts of P. ostreatus and P. djamor respectively. Enzymatic activity values ​​of 888.41 IU and 152.22 IU were reached for the laccases obtained from the submerged culture extract of Pleurotusostreatus and Pleurotusdjamor respectively. The extract obtained from P. ostreatus was partially purified using dialysis and anion exchange chromatography (DEAE), by polyacrylamide gel electrophoresis. A molecular mass of 67 kDa was determined.

Keywords: Pleurotus, dye, laccase, enzyme.


The textile industry is one of the main sources of pollution, it discharges effluents containing a wide variety of dyes, the most important ones are the azoids and the anthraquinones and therefore constitute the largest group of all organic dyes on the market (1, 2).

The toxicity of azo acids is because they produce aromatic amines during natural processes of degradation (3-6). In addition, azo dyes affect water quality by preventing the passage of light and by increasing the demand of chemical oxygen. In contrast, there is little information on the biodegradation of anthraquinone dyes (7).

The “maquilas” or laundries, as they are called locally in the State of Puebla, are responsible for the discoloration, bleaching or dyeing of the denim. They are mainly located in community dwellings such as San Rafael Tenanyecac, Villa Alta, San Mateo Ayecac and Santa Ana Xalmimilulco,(8) which requires a large amount of water use, which at the end of the process contains, in addition to the dyes, chemical additives used to treat the garments and give them different finishes. Therefore, the wastewater they produce contains various chemical pollutants that are discharged to municipal drainage or irrigation canals most of the time, without treatment or with inadequate treatments, which eventually flow into the Atoyac River. In order to contribute to this problem, the use of ligninolytic fungi such as those of the genus Pleurotuscapable of synthesizing a complex of extracellular enzymes, mainly peroxidases andLaccases (p-diphenol: dioxygen: oxide-reductase) these are extracellular glycoproteins containing copper and a molecular weight between 60 and 80 kDa, have the capacity to reduce molecular oxygen to two molecules of water and simultaneously work in the oxidation of many aromatic substrates (9). The range of oxidizable substrates is broad and includes pentachlorophenol, 2,6-dimethoxyphenol aromatic amines and other easilyoxidizable aromatic compounds, as well as azodyes.{G. Davila, 2006 #171}(10, 11)


The production of laccase enzymes was carried out by submerged culture, using a mineral medium consisting of magnesium sulfate heptahydrate (J.T.Baker®) ferrous sulfate heptahydrate(J.T.Baker®) manganese sulfate(J.T.Baker®), sodium chloride(J.T.Baker®), ammonium sulfate(J.T.Baker®), potassium phosphate dibasic(J.T.Baker®) and as carbon source wheat bran at a pH of 6.2 and inoculated with 4 units of 1 cm2 of PDA (BD Bioxon®) with the strains of P.ostreatus and P. djamor of the ceparium  of the Department of Research in Agricultural Sciences of the Institute of Sciences of the Benemérita Universidad Autónoma de Puebla, it was incubated at 28 ° C and a stirring speed of 120 rpm. Monitoring of laccase activity was performed on the seventh day using 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich®)ABTS as an oxidizable substrate and measuring at a wavelength (ʎ) of 420 nm for 1 min. using a molar extinction coefficient (ɛ) 36000 M-1cm-1 (12). The enzymatic activity was expressed as international units as a function of volume (IU / ml).

Solutions of water artificially contaminated with the dyes were prepared at 200 ppm erionyl blue A-R (Ciba Specialty Chemicals Inc. ®) and solophenyl blackFR (Ciba Specialty Chemicals Inc. ®). To the water artificially contaminated with the dyes to be studied was gradually added the enzymatic extract of laccase, determining the percentage of removal by UV-Vis spectroscopy (Varian Cary  50) all the determinations were made by  triplicate-sample method, one of the extracts was subjected to a dialysis process for 15 h in 20 mM phosphate buffer, at a pH of 7.4 and at a temperature of 4 ° C, once the time was over, the dialysate was centrifuged at 7500 rpm for 20 min, then anion exchange chromatography was performed with the pre-equilibrated Diethylaminoethyl (DE-53 Whatman) resin at a pH of 7.4. The proteins obtained were eluted with 100 ml of 20 mM phosphate buffer – 1 M NaCl, at a pH of 7.4, for determination of the molecular mass, was performed by electrophoresis in 12% concentration polyacrylamide gel, where 15 μl of the extract were added and let it run at 120 V / 2.30 h.


The maximum enzymatic activity obtained for the laccase produced by P. ostreatus was 888.41 IU / ml and for P. djamor 153.22 IU / ml. With these enzymatic extracts, it was possible to remove the dyes studied in the percentages reported in Table 1.

In figure 1, the UV-Vis spectra of the erionyl blue dye A-R are presented before and after the reaction

In this research using the P.ostreatus extract, it was possible to remove 93.77% of the dyeerionylblue A-R (Figure 2), classified as acid blue 260 with CAS number 62168-86-9 and molecular formula C26H23ClN3NaO6S, molecular mass of 563.96 g / mol and belongs to the family ofanthraquinones.(13)

On the other hand, Sánchez-López(14) demonstrated the ability of the enzyme extract (laccase and manganese peroxidase) from Trametes maxima strain MUCL 44155 to remove acid blue anthraquinone dye 62 by 91% measured at 630 nm. Other fungi such as Thanatephoruscucumeris have been used to degrade anthraquinone dyes as the blue reactive dye 5 by DyP peroxidase having percentages above 90% measured at 630 nm (15). In Japan, Itohet. al.(16) have used the white rot fungus Coriolusversicolor to degrade violet dye 12 (4-dihydroxyanthraquinone).

Table 1. Percentage of removal of the studied dyes

P. ostreatus
P. djamor
Erionyl Blue A-R
Solophenyl Black FR


Fig. 1. Spectrum of UV-Vis absorption of water artificially contaminated with erionyl blue A-R dye at 200 ppm and treated with Pleurotusostreatus

The UV/Vis absorption spectra of the reaction products (Figure 1) showed an increase in absorbance from 520 nm and towards the ultraviolet region behavior very similar to those reported by Sánchez-López(14) and Sugano (15). With regard to the Pleurotusdjamor strain, lower removal values were observed with respect to P. ostreatus (Table 1).

Thesolophenyl black dye (direct black 22) is classified as Index 35435, indicating that it is a polyazodye. (17, 18) its structure is reported in figure 3.

With respect to the oxidation of azo dyes like the solophenyl black FR by laccases and peroxidases, several authors report the formation of naphthoquinones(19-22).In contrast, Mohana et al. (23) report that using a microbial consortium applied in the treatment of water contaminated with direct black 22 (solophenyl black), obtained as degradation products 1-naphthol and diphenylamine as a product of anaerobic reduction, similar results obtained by Chávez(24), but using microbial consortia isolated from the Alseseca River of the City of Puebla, Mexico, these products are toxic so it would be convenient to use oxidation mechanisms with laccases or peroxidases and not chemical reduction. Although the removal rates for this azo-type dye were low (Table 1), it is important to emphasize that in this case, the laccases of the strain Pleurotusdjamor were more efficient than those of Pleurotusostreatus. In addition, for wastewater treatment it is convenient to use a treatment train for the process to be efficient. It has been reported that ligninolytic enzymes produced by Basidiomycetes of the genus Trametes remove better the dyes of an anthraquinone nature than the azo(25, 26), as found in this research.

Fig. 2. Erionyl Blue A-R

Fig. 3. Solophenyl Black FR

The partially purified enzyme had a molecular mass of 67 kDa as determined by polyacrylamide gel electrophoresis. It has been reported that most of these enzymes range from 50-70 kDa(27, 28).


Thanks are due to Programapara el DesarrolloProfesionalDocente (PRODEP) for supporting the generation and application of knowledge: Folio BUAP-PTC-449 and  agreement number DSA/103-5/16/10420


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