Proceedings of the 2003 Georgia Water Resources Conference, held April 23-24, 2003, at the University of Georgia.A very promising area for removing unwanted color from textile wastewater is biotreatment that is targeted at breaking down the dye molecules to basic elements (mineralizing them), and doing so in a way that has much less potential for environmental impact than conventional methods. Previous work in our laboratories demonstrated the success of using white rot fungi for this purpose.  The nature of the degradation fragments produced is of great interest because of concern that these may increase the aquatic toxicity of the overall effluent.  We report the development of method to identify the degradation products using thin layer chromatography, HPLC, capillary electrophoresis and mass spectrometry

Akin, Danny E.

Hardin, Ian R.

Zhao, Xueheng

Scholarly Materials And Research @ Georgia Tech

BIOTREATMENT APPROACH TO DECOLORIZINGTEXTILE WASTE EFFLUENTSIan R. Hardin1, Xueheng Zhao2 and Danny E. Akin3AUTHORS:  1Professor and 2Graduate Student, Textile Science Program, Department of Textiles, Merchandising and Interiors, Dawson Hall,The University of Georgia, Athens, Georgia 30602; and 3Research Scientist, Russell Research Laboratory, ARS-USDA, Athens, Georgia.REFERENCE:  Proceedings of the 2003 Georgia Water Resources Conference, held April 23-24, 2003, at The University of Georgia. KathrynJ. Hatcher, editor, Institute of Ecology, The University of Georgia, Athens, Georgia.    Abstract.  A very promising area for removingunwanted color from textile wastewater is biotreatmentthat is targeted at breaking down the dye molecules tobasic elements (mineralizing them), and doing so in a waythat has much less potential for environmental impact thanconventional methods.  Previous work in our laboratoriesdemonstrated the success of using white rot fungi for thispurpose.  The nature of the degradation fragmentsproduced is of great interest because of concern that thesemay increase the aquatic toxicity of the overall effluent.We report the development of method to identify thedegradation products using thin layer chromatography,HPLC, capillary electrophoresis and mass spectrometry.INTRODUCTION    Azo dyes, which account for approximately one-half ofall known dyes, are commonly used as coloring agents inthe food, pharmaceutical, and textile industries.  As aresult, they are the most common synthetic colorantsreleased into the environment (Bumpus 1995).  Becausethey are highly colored, azo dyes are readily visible ineffluent water and can be the focus of significantenvironmental complaints centered around that visibility.These compounds are also of concern because some of thedyes, dye precursors or their biotransformation products,such as aromatic amines, have been shown to becarcinogenic (Razo-Flores et al. 1997). The nonspecificnature of the lignin-degrading systems of fungi are ofincreasing interest in the study of textile effluenttreatment.  Although their function in nature is to degradelignin, they have the capability to degrade other types ofcompounds. Various investigations have shown that whiterot fungi can efficiently degrade various organic pollutantsother than lignin.    Examples include polycyclic aromatichydrocarbons, persistent environmental pollutants such asDDT (Bumpus et al. 1985, 1989), alkyl halide insecticides(Kennedy et al. 1990), xenobiotic compounds(Paszczynski et al. 1995), and dinitrotoluene (Valli et al.1992).   The similarities of the molecular structures of the abovecompounds to those of dyes was obvious to the earlyinvestigators.  This initiated interest in using the fungi fordecolorization of dye wastewater.  This application to thedecolorization of azo dyes using the white rot fungusPhanerochaete chrysosporium was first described byCripps et al. (1990).  They showed that three azo dyes,Orange II, Tropaeolin O, and Congo Red, could bedecolorized by Phanerochaete chrysosporium.  Otherwhite rot fungi, such as B. adusta and T. versicolor, alsoshowed the ability to degrade azo dyes efficiently(Heinfling et al. 1997).  In our own laboratory, variouswhite rot fungi including P. chrysosporium, P.cinnabarinus, T. versicolor, C. subvermispora, C.stercoreus, P. ostreatus, P. tremellia, P. oxysponum, andP. pini have been screened for their capability indegradation of commercially used dyes (Cao 2000).   Thisand other work showed that Pleurotus ostreatus, had highpotential for decolorizing a variety of  azo dyes (Cao2000, Shin et al. 1998).  Despite the known ability of thefungi to break down dyes and render them colorless, therehave been few attempts to comprehensively examine thedye effluent systems after biotreatment by white rot fungiand determine the molecular nature of the process.Decolorization, by itself, demonstrates only thetransformation of the chromophoric group of a dye, butdoes not reveal much about the mode of degradation of thedye molecules.  The objective of this paper is to examinethe decolorization of two widely used disperse azo dyes incultures of Pleurotus ostreatus and to analyze thedegradation products generated in the biotreatmentsystem. The results gained by treating these two dyes withrelatively simple structures will be used to startdevelopment of a comprehensive analytical method forinvestigating the degradation of dyes by white rot fungi.MATERIALS AND METHODSChemicals   Two disperse dyes of relatively simple structures (seeFigure 1) were chosen for this work.  Disperse Orange 3(20 percent purity) and Disperse Yellow 3 (30 percentpurity) were obtained from Aldrich Chemical Company(Milwaukee, Wis.), as was Disperse Orange 3 in a 90percent pure form.  The dyes were converted to pure formby recrystallization. In order to determine positively thedegradation products from these dyes, probablecompounds were were postulated.  These were:4-aminophenol 4-nitrophenol1,4-benzoquinone4-nitroanilineaniline1,4-phenylenediamine acetanilide4'-aminoacetanilide4-acetamidophenol4'-nitroacetanilide6-amino-m-cresolThese degradation “model” compounds were purchasedfrom Aldrich Chemical.  The organic solvents employedin the experiments were at least reagent grade and used aspurchased.N NH2NO2Na.Disperse Orange 3 (C.I. 11005)b.Disperse Yellow 3 (C.I. 11855)Figure 1. Structures of the disperse Dyes.Microorganism.   Previous work done in our lab hadinvolved screening a number of white rot fungi for theirability to decolorize a variety of dye molecular structures(Cao, 2000). The most successful candidate was thePleurotus ostreatus fungus (strain Florida) used in thework discussed herein.  This fungus was isolated at theUniversity of Georgia (Eriksson, 1987).  The culture wasmaintained on malt agar plates at 30 degrees  C, withsubcultures routinely made every month.Culture Conditions.   Nutrient nitrogen-limited culturesof P. ostreatus made with Kirk’s medium (Kirk et al.1978) were incubated at 30 degrees C in a liquid culturemedium contained in a 250 ml Erlenmeyer flask at pH 5.0.Decolorization of Commercial Dyes.   Cultures wereestablished in the incubator, shaken at 200 rpm, andallowed to grow for three days.  On day three, the twocommercial azo dyes were dissolved in methanol at 1.0mg/l concentration.  One disperse dye (includingdispersing agents) was added to each culture for final dyeconcentrations of 60 ppm.  Controls were run with thesame reaction conditions but excluding fungus.  Theseexperiments were replicated three times.Decolorization Determination.   Aliquots of 1-2 milliliters of clear dye solution weretaken from the each reaction flask at regular time intervalsand measured immediately using a UV- Vis recordingdouble beam spectrophotometer (Shimadzu).  Care wastaken not to draw out portions of the fungus in the aliquot.All samples from the dyebaths had to be diluted prior tomeasurement in order to keep the change in absorbancevalues measured below 2.5 absorbance units percentimeter of pathlength.  Because of the low watersolubility of these dyes, an equal volume of methanol wasmixed with the analytical solution to ensure completesolubilization prior to measurement.  Decolorization wasassessed in two ways: one way was by monitoringspectrophotometrically the absorbance at the wavelengthmaximum for each dye; secondly, by the reduction of themajor peak area in the visible region for each dye.  Bothapproaches were used because color change may occurduring biodegradation with a concommitant wavelengthshift.   In that case, just using the wavelength maximum ofthe dye would not be sufficient to accurately reflect thefull degree of decolorization.  To obtain additionalinformation regarding the changes, the area under thecurve in the visible regions (400-800 nm) was integrated.Dye Purification.  In order to analyze degradationproducts without interference from extraneous materials,the disperse dyes were purified before using in the experi-ments.   This was done by recrystallization in acetonitrilefor Disperse Orange 3 and in ethyl alcohol for DisperseYellow 3.  The purity of both dyes was checked by highperformance liquid chromatography (HPLC) and differ-ential scanning calorimetry (DSC).   Methanol solutions ofthe purified dyes were added to the fungal cultures toachieve a concentration of 60 ppm of the dye in theculture solution.Degradation of Purified Disperse Dyes.   All dyes weredissolved in methanol at 1.0 mg/mL concentration andwere added to the flask containing the fungus system togive a final concentration of 60 ppm.  After three daystreatment, the degradation samples were prepared for thinlayer chromatography analysis.Thin Layer Chromatography (TLC)  Sample preparation.  The mixture of the dye solution andthe dispersed fungus constituted a quantity of about 400milliliters (from three flasks).  After 3 days, the solutionwas filtered and concentrated by rotary vacuumevaporator. The concentrated solution was extracted bymethylene chloride of equal volume three times. Theorganic phases were combined and concentrated to about10 ml. A salting out procedure was used to facilitate theextraction. The samples were kept at a low temperatureO HN = N N H C O C H 3C H 3until further analysis. The fungal spores are also extractedby methylene chloride.Thin  Layer ChromatographySelecto Silica Gel 60, F-254, 5 × 20 cm plates (SelectoScientific, GA) were used in the analysis.  The specimensinvestigated were applied on the starting line with avolume of 4-5 microliters.  The mobile phase for analysisof the Disperse Orange 3 degradation components was anethyl acetate - hexane (2:1) mixture.  An ethyl acetate -hexane (4:1) mixture was used for analysis of the DisperseYellow 3 degradation solution.  The chromatograms wererun until the solvent front traveled 80 millimeters.  The Rfvalue of each degradation product was compared to thoseof the standards for identification of the structure.  Thesamples were run on three different plates and the averageRf value of spots of three runs was used for comparison.The standard compounds were run on the same plate.RESULTS AND DISCUSSIONThe absorptivities are intrinsic properties of a colorant.Measuring the spectral absorption curves of dyes duringbiodegradation provided a means to determine thereaction rate and gave evidence of the structural changeof dyes during biodegradation.  Previous experienceshowed that fading of the original color of the dyes issometimes accompanied by a color change (wavelengthshift) in the solution.  Because of this, both the ultra-violet and visible regions were monitored by a UV-Visspectrophotometer to provide more information aboutthe biodegradation. The decrease of the maximumabsorbance of disperse dyes occurred primarily in thefirst several days of treatment, with no substantialadditional decrease in absorbance as the treatment timeincreased.   This is shown in both Figure 2 and Table 1.   Disperse Orange 3 (Figure 1a) contains two substitutedaromatic rings, one with an amino substituent and theother with a nitro substituent, typical of many monoazodisperse dyes. The visible portion of the spectrum ofDisperse Orange 3 showed a major peak at 415 nm.  Afterfive days of biotreatment, this peak shifted to 388 nm anddecreased by 57 percent.  The reduction of the area of themajor peak in the visible region was 65 percent, whichindicated substantial decolorization.    A second dispersedye examined was Disperse Yellow 3 (Figure 1b), widelyused in the United States.   It is also a potential carcinogen(Spadaro et al. 1992). Decolorization with P. ostreatuscaused a 57 percent decrease in absorption after 5 days(Figure 3 and Table 2), This was indicated by the decreaseof absorption at the maximum wavelength and thedecrease in the area under the absorption curve in visibleregion.  No new peaks appeared in the UV region afterdecolorization.Figure 2. UV-Vis spectra of Disperse Orange 3before and after biodegradation by P. ostreatus. Thecurves represent 1 day intervals.Table 1. Decrease in Absoption of DisperseOrange 3 After Degradation (%)Day Absoption decrease(@ λmax) (%)Decrease in peak areain visible region (%)0 0 01 36 352 39 403 53 574 59 655 57 65Figure 3. . UV-Vis Spectra of Disperse Yellow 3before and after biodegradation by P. ostreatus. Thecurves represent 1 day intervals.Table 2. Decrease in Absoption of Disperseyellow 3 after Degradation (%)Day Absoption decrease(@ λmax) (%)Decrease in peak areain visible region (%)0 0 01 26 222 32 293 44 324 50 405 57 57The monitoring of the UV- Vis spectra suppliedinformation on the nature of the decolorization, particular-ly with the shifts in the absorption maxima.  These couldonly be the result of  chemical change in the conjugateddouble bond structure, rather than just removal of theunchanged dye molecules.  However, complexdegradation  products produced by the actions of theenzymes from the white rot fungi could only be identifiedby separation, followed by an identification scheme.  Tothis purpose, thin layer chromatography was employed toidentify the degradation products. A number of solvents,including benzene, methanol, acetone, chloroform,tetrahydrofuran, toluene, n-hexane, and ethyl acetate, werescreened for effectiveness in the thin layer chromat-ography approach. The combination of n-hexane and ethylacetate proved to be the best system for separation.     The ratio of ethyl acetate and n-hexane was changedfrom 8:1 to 1:2 at intervals of 2:1.  Satisfactory separationof the degradation products of Disperse Orange 3 wasachieved with ethyl acetate and n-hexane in a ratio of 2:1.A mixture of ethyl acetate and n-hexane in a ratio of 4:1was very successful in the separation of the degradationproducts of Disperse Yellow 3.After treatment with the white rot fungi for the threedays, two degradation products were detected fromtreatment of Disperse Orange 3 and one degradationproduct from Disperse Yellow 3.  The nine compoundswhich were postulated as degradation products -- 4-aminophenol, 4-nitrophenol, 1,4-benzoquinone, 4-nitroaniline, aniline, 1,4-phenylenediamine, acetanilide,4'-aminoacetanilide, 4-acetamidophenol, 4'-nitroaceta-nilide, and 6-amino-m-cresol -- were run under the sameconditions as the treated dye solution samples for thequalitative identification of the separated spots on theplates.  Among these compounds, 4-nitroaniline showedits possibility to be one of degradation products fromDisperse Orange 3.  After running both samples andstandard on the same plate and changing mobile phase toethyl acetate-benzene (4:1), it showed that 4-nitroanilinewas likely to be one of major products of Disperse Orange3 after degradation (Data is not shown here).  Because thedisperse dyes were added into the fungal culture in themethanol solution, the effect of methanol on the degrad-ation of dyes is investigating because the mechanism ofbiodegradation of azo dyes has not been elucidated yet.CONCLUSIONSUV-Vis spectra showed that P. ostreatus degradedwater unsoluble azo dyes in the decolorization.  Theabsorption of new compounds was indicated from thespectra, which indicated the degradation productsgenerated in the decolorization.  At least two majordegradation compounds of Disperse Orange 3 and onedegradation products of Disperse Yellow 3 had beenconfirmed by the results from thin layer chromatography.REFERENCESBumpus, J., Microbial Degradation of Azo Dyes.  Biotrans-formations: microbial degradation of health-riskcompounds. New York. 1995Bumpus, J., Biodegradation of oplycyclic aromatic hydro-carbons by Phanerochaete chrysosporium. Applied andEnvironmental Microbiology Vol. 55, pp. 154-158, 1989Bumpus, J.; Tien, M.; Wright, D.; Aust, S.       Oxidation ofpersistent environmental pollutants by a white-rot fungus.Science Vol. 228, pp. 1434-1436, 1985Cao, H. Decolorization of Textile Dyes by White Rot Fungi.Ph. D. dissertation, University of Georgia, 2000Cripps, C.; Bumpus, J.; and Aust, S.    Biodegradation of azoand heterocyclic dyes by Phanerochaete chrys-sporium.Applied & Envir. Microbiology  (56, pp. 1114-1118) 1990Eriksson, K.-E., and Kirk, T.K. Biopulping,    biobleachingand treatment of kraft    bleaching effluents with white-rotfungi. Madison, WI : Forest Products Laboratory, 1987Heinfling, A.; Bergbauer, M.; Szewzyk, U.  Biodegradationof azo and phthalocyanine dyes by Trametes versiclorand Bjerkandera adusta .  Applied Microbiology andBiotechnology. Vol. 48, pp. 261-266, 1997Kennedy, D.; Aust, S.; and Bumpus, J.  Comparative bio-degradation of alkyl halide    insecticides by white rotfungus, Phanerochaete chrysosporium  (BKM-F-1767).Applied & Envir. Microbiology (56, pp. 2347-2353) 1990Kirk, T. K.; Schultz, E.; Cornors, W. J.; Lorenz, L. F.; andZeikus, J. G. Influence of culture parameters on ligninmetabolism by PhanerochaetechrysosporiumArchives ofMicrobiology., Vol. 117, pp.3144-3149, 1978Paszcaynski, A. and Crawford, R. Potential for bioremedia-tion of xenobiotic compounds by the white-rot fungus                 Phanerochaete chrysosporium . Biotechnology ProgressVol. 11, pp. 368-379, 1995Razo-Flores, E.; Luijten, M.; Donlon, B. Lettinga, G.; andField, J. Biodegradation of Selected Azo Dyes underMethanogenic    Conditions. Water Science andTechnology. Vol. 36, pp. 65-72, 1997.Shin, KS; and Kim, CJ. Decolorisation of        artificial dyesby peroxidase from the white-rot fungus, Pleurotusostreatus.   Biotechnology letters. (20, pp. 569-572) 1998Spadaro, J. T.; Gold, M. H.; and Renganathan, V. Degrada-tion of Azo dyes by the lignin-degrading fungusPhaneochaete chrysosporium. Applied EnvironmentalMicrobiology. Vol. 58, pp.      2397-2401, 1992Stock, Naomi L.; Peller, Julie.; Vinodgopal      K.Combinative sonolysis and photocatalysis for textile dyedegradation.   Environmental Science & Technology Vol.34, pp. 1747-50, 2000Valli, K.; Brock, B.; Joshi, D.; and Gold, M.    Degradationof 2,4-dinitrotoluene by the lignin-degrading fungusPhanerochaete        chrysosporium. Applied andEnvironmental Microbiology Vol. 58, pp. 221-228, 1992

Biotreatment Approach to Decolorizing Textile Waste Effluents

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Biotreatment Approach to Decolorizing Textile Waste Effluents

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