Indian Journal o f Experimental Bi o logy Vol. 4 1, September 2003, pp. 1076- 1087

Synthetic dye decolourization by white rot fungi

K Murugesan & P T Kalaichelvan*

Centre for Advanced SllIdi es in Botany , Unive rs ity o f Madras, G uindy Campus, C hennai 600025 , Ind ia

Synthet ic dyes are integra l pa rt of many indus tri a l products. The e f'fluents gene rated from tex tile dyei ng un its c rea te major environme ntal proble ms and issues both in public and tex tile units. Industria l wastewate r treatment is one of the major prob lems in the present scenari o. Though, the phys ical and chemi ca l methods otTer some soluti ons to the probl ems. it is no t affordable by the unit opera tors. Bi o log ical degrada ti on is recognized as the most effec ti ve me thod for degrad ing the dye present in the waste. Research ove r a pe ri od of two decades had provided ins ight into the va ri ous aspects of bio logical deg radation o f dyes. It is obse rved th at the white rot fun g i have a non-spec ifi c enzy me sys tem, wh ich ox id izcs thc reca lc itrant dyes. De tai led and ex tcnsive sllId ies have bcen made and process devcloped for trcatmcnt o f dye con taining wastewate rs by white rot fun g i and the ir enzy me systems. An attempt is made to summari ze the detai led research contributions on these lines.

Keywords: Bioremed iation, Deeolouri za ti on, Lignolti c enzy mes, Reca lc itrant compounds, Synthe ti c dyes, White ro t fun gi

Wastewater from textile dye units is one of the major environmentall y undes irable pollutants. A wide variety of sy ntheti c dyes namely azo, anthraquinone, polycyc lic and triphenylmethane are being increas ingly used in tex tile dyeing and printing processes. The textile dyeing process requires a large volume of fresh water of fairl y high purity and di scharge equall y large volume of wastewater after the dyeing process. The wastewater contains dyes at concentrations ranging from of 10-200 mgll and about 10-20% of the dye present in effl uents along with other organic and inorganic accessory chemicals in volved in the dyeing process. Dye bearing efflu ents are compl ex , most often non-bi odegradable and ex hibit tox icity to both aquatic and non-aquatic biota. The untreated effl uents released from the dyeing units cause a maj or threat to the environment. Direct discharge of dye effluents causes formation of tox ic aromatic amtnes under anaerobic conditions 111

receiving media. Colour can be removed from wastewater by

physical and physicochemical methods namely, adsorption, coagulation , fl occulati on, ox idation, filtration and electrochemical methods 1.2. These methods are quite ex pensive and have operational prob lems. The physicochemi ca l process aims to transfer pollutants fro m one system to other system without degrad ing it. The complete breakdown of an orga nic molecule to inorganic component should be the des ired outcome to avoid the persistence of

"'Correspo ndent autho r : E- mail : botany@vs nl.colll

potenti ally hazardous compounds in the environment'. Although the chemical treatment process bring about 60-70% colour removal, the BOD removal does not exceed more than 30-40% in most cases studied. Eventhough chemical coagul ation appears to be effective in treating the dye bearing efflu ents the toxic sludge handling problem associated with the chemical cost retarded its usage4

.5. Thus the tex tile dyeing

industry is facing an increasing pressure from environmentall y concerned organi zations to replace the conventi onal treatment technolog ies with environmentall y fri end ly ones . In thi s contex t in recent years biological methods are rece ived more attention since they are considered as sustainable and ecofriendl y. Biological methods of remediation have potenti al to convert or degrade the pollutants into water, carbon dioxide and various salts of inorgan ic nature. Non-v iable biomateri als are also used for sorpti on of the dyes and many other pollutants6

-IO

.

The isolation of potent microb ial species and its use in degradati on are of main interest in biological aspect of effl uents treatment tt -t5. Many synthetic dyes

II I . b ' I d d ' 16- I X are genera y reca CItrant to actena egra auon . Under anaerobic conditi ons, bacteria reduce azo dyes leading to the formation of aryl amine deri va ti ves t9

which are carcinogen ic2o. Decolouri zati on generall y

occurs by adsorption of dyestuffs on bacteri a rather than oxidation in ae robic system. But more recent studies have demonstrated that many bacteria are ab le to degrade azo dyes aerobicall y as well as anaerobically.

MURUGESAN & KALAICHELVAN : SYNTHETIC DYE DECOLOURIZATION BY WHITE ROT FUNGI 1077

Lignolyti c fungi are well known , not only as decomposers of lignin but also for their ability to degrade a wide variety of organopollutants2

1.22.

Several studies showed that the degradation of azo, anthraquinone, heterocycli c, triphenylmethane and polymorphic dyes is by Phanerochaele chrysosporium, the most extensively studied white-rot fun gus23

.25 .

Decolourization as well as parti al minerali zation of azo dye have been . demonstrated' ~ ·26. Subsequently several other potential white rot fun gi namely Bjerkandera adusta. Irpex lacteus, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus ostreatus, Pycnoporus cinnabarinus, and Trametes versicolor, have been studied for decolouri zation of several groups of recalcitrant dyes and dye effluents27-35. In the present review paper accounts the literature and strategy of recent achievements in synthetic dye decolourization by the white rot fun gi.

Dye

A dye is a substance used to impart colour to fabrics, food and other objects fo r beauti ficati on. Based on the chemi cal compos ition, sy nthetic dyes are class ifi ed as azo dyes, nitro dyes, triphenylmethane dyes, phthalocyanin dyes, indigo id dyes and anthraquinone dyes. Based on the applicati on and usage, the dyes are class ified as ac id dyes, bas ic dyes, reac ti ve dyes, polyazo dyes, vat dyes, azoic or naphthol dyes and disperse dyes.

Synthetic dyes are used ex tensively for textil e dyeing, paper printing and colour photography and as additi ves in petroleum products. About 10000 di fferent dyes and pi gments ex ist, and over 7 x 105

tons of these dyes are produced annuall y36. About 50% of the industrial dyes produced in the world are azo dyes3

? These dyes are released mostl y into the aquatic environment primaril y fro m tex til e and dyestuff industries3R

. Syntheti c dyes share a common fea ture in th at they are not readily biodegradable; when di scharged into the environment and they are therefore persistent and many are also tox ic ' 6

Pollution problems caused by dyes

The major environmental prob lem of colourants is the removal of dyes from effluents39

. The untreated effluents of these industries may be hi ghl y co loured and thus particul arly dangerous when di scharged into open water bodies. The concentration of dye may be much less than 1 ppm but the dye is visible even at that lesser concentration.

The problems caused by them are - a) Sunlight penetration of the streams would be reduced, which is essential for photosynthesis and consequently, the ecosystem of the stream will be seriously affected4o

; b) Toxicity to fi sh and mammalian life; c) Inhibits acti vity and growth of microorganisms, particularl y in hi gh concentration. Some cationic spec ies (mostly tri pheny I methanes) affect the flora and fa una even at lesser concentrations41; d) Possible chronic ri sks of co lourants and their intermedi ates, which are carcinogenic and to a lesser extent sensitizing and allergic40

; e) Intestinal cancers are common in highl y industri ali zed societi es and poss ible connection between these tumors and the uses of azo dyes has been investigated; and f) Some dyes are reported to cause cerebral abnormality in foetuses and skeletal abnormalities.

Sulfonated azo compounds are widely used as dyes fo r textile and cosmetics. Both aromatic sul fonic ac id and azo groups are rare among natural products and thus confer a xenobioti c character to sulfonated azo dyes. Several amino substituted azo dyes are mutagenic as well as carcinogeni c and their tox ici ty have been ex tensively studied along with the ri sk of occupational cancer assoc iated with their use42.43 . The carcinogeni city of an azo dye may be due to the dye itself or to the aryl amine deri vati ves generated during the reducti ve biotransformation of the azo Iinkage4~.46 . Fujita and Peisach46 reported that azo dyes are reduced to the aryl amines by cytochrome p-4S0 and a flav in dependent cytosolic reductase in mammals. Gray el al. 47 indicated that in female and male mice ex posed to congo red (11), the utero gonadal development has been adversely affec ted in both the sexes . However, only females di spl ayed reduced fe rtility.

Although the commonl y used azo dyes are not mutageni c in the standard Ames pl ate assa/8 they are reduced by azo reductase from intestinal bacteri a and, to a lesser ex tent, by enzy me of the microsomal fraction of the Ii verI 9.49.5 1. The first catabolic step in the reduction of azo dyes is the reducti on of azo bridge to produce aromatic amines. Aromati c amines whi ch are known human carcinogens, have been fo und in urine of many dyes tuff workers and test animal fo ll owing the admini stration of azo dyesI 9.5o.52. Azo dye compou nds are linked to bl adder cancer in humans and hepatocarcinoma and nucl ear anomalies in intes tinal ep itheli al ce ll s in mice.

Tex til e dyeing, paper printing and leather finishing industry workers ex posed to benzidine based dyes

1078 INDIAN J EXP BIOL, SEPTEMBER 2003

have a higher than normal incidence of urinary bladder cancerS3. Benzidine based dyes when admjnistered to various experimental animal s, undergo reduction of the azo bands with the appearance in the urine of the human bladder carcinogen benzidine and benzidine metabolitess4.s7 .

Effluent treatment One of the more press ing envi ronmental problems

that has been facing the textile industry is the removal of colour from dye bath effl uent, which contai ns a variety of processed chemicals and dyes, prior to discharge into local sewage treatment facilities o r adjoining water courses. Brightly coloured water soluble reactive dyes and acid dyes are particularly problematic , because the dyes can exhibit low leve ls of fixation with the fibre and passing unaffected through conventional treatment systems at the sewage works and entering water courses . Thi s is particularly noticeable as the human eye can detect reacti ve dyes of concentration as low as 0.005 mg/l in clear river water58

.

The treatment of wastewater containing dyes and its decolouri zation involves serious problems. A wide range of several pH intervals, salt concentration and chemical s very often add to complications. Removal of colour from effluents is a major problem and tighter constraints on di scharges are forcing waste creators and managers to consider new options for effluent treatment and disposal 59. Methods of effluent treatment for dyes may be cl assified into three main categories: physical, chemical and biological as ind icated in Table 1.

Unfortunately, the physical and chemical methods of effluent treatment have high operating costs and of li mited applicabilitlo.6' . An effective and low cost treatment system would be of great value. Among the low cost viable alternatives available for effluent

Table I - Methods of effl uent lreatment

Physical

Adsorption Sedimentation Floalation Flocculation Coagulation Foam frac tion Polymer fl occu lation Reverse osmos is Ultrafiltra lion Ioni zation radiat ion

Chemical

Neutralization Reduction Oxidation Elec trolysis Ion exchange Wet air oxidation

Biological

Stabilization Aerated lagoons Trickling filter Activated sludge Anaerobic di gest ion Bioaugmentation

treatment and decolouri zation, the biological systems seem to be the best ones nowadays. Biological systems are recogni zed by their capac ity to reduce biological oxygen demand and chem ical oxygen demand by conventional aerobic biodegradati on. But there is a problem with its inability to remove colour62 . However, potential microbial decolouri zation sys tems have been developed wi th total colour removal, in some cases within few hours63.65 .

Biological degradation of dyes An organic chemical introduced in to a terrestri al or

aquatic ecosystem may be subjected to enzy mati c or non-enzymatic reactions brought about by inhabitants of the environment66. This method is most effecti ve and economical for the treatment of organic and certain ino rganic wastes. Biological removal of degradable organics invo lves a sequence of steps including mass transfer, adsorpti on, absorption and biochemical enzymatic reactions67.

Many synthetic dyes are recalcitrant to biologica l degradati on under conditions normally found in wastewater treatment plants'7.24.68.69 . Such

recalcitrance is desirable for a commercial textile dye under typical usage conditions70

. The metaboli sm of pure dyes has been extensively studied but little has been published on biolog ical treatments of industri al dye effluents71.74 .

Many microbes have been isolated which can catalyse anaerobic reductive fission of the azo linkage resulting in formation of colourless aromatic amines75 . These can be highly toxic and carcinogenic and anaerobic degradation of many of these aromatic intermediates have not been reported. Further, on exposure to air, some of these produc ts may be oxidized to highly coloured compounds76. These problems limit the full-scale application of bacteri al decolourization.

Degradation of synthetic dyes by white rot fungi White rot fungi are a heterogeneous group of

organisms but have in common the capacity to degrade lignin as well as other wood components and wide variety of recalcitrant compounds. The capability to degrade lignin is due to the extracellular non-specific and non-stereoselective enzyme system. The extracellular enzy me system involved in lignin degradation is composed of lignin peroxidase, laccases and manganese dependent peroxidases as well as H20 2 producing oxidases. T he same unique non-specific mechani sm th at gives these fungi the

MURUGESAN & KALAICHELVAN: SYNTHETIC DYE DECOLOURIZATION BY WHITE ROT FUNGI 1079

ability to degrade lignin also allow them to degrade a wide range of pollutants and they possess a number of advantage not associated with other bioremediation systems77.78. Because the key components of lignin degrading system are extracellular, the fungi can degrade very insoluble chemicals such as lignin and many of the hazardous environmental pollutants. Furthermore they do not require pre-conditioning to a particular pollutant79

.8o. This capability has extended

their use to a series of biotechnological applications, all of them related to the degradation of structurally diverse aromatic compounds.

Glenn and Gold82 first demonstrated that ligninolytic cultures of P. chrysosporium decolouri ze several pol ymeric dyes. Subsequent work has received the ab ility to decolouri ze a wide array of azo, triphenylmethane and heterocyclic dyes. Decolouri­zation of the azo dyes such as orange II , tropeolin 0 , congo red, acid red 114, acid red 88 , biebrich scariet, direct blue 15, chrysophenine, tetrazine, and yellow 924.83.84 triphenyl methane dyes, bas ic green 4 , crystal

violet, brilliant green , cresol red , bromonphenol blue and para rose anilines23 by various white rot fungi has been reported . The degradation of azo, anthraquinone, heterocyclic , triphenylmethane and po lymeric dyes by P I · . I d · d23-2585 . c 1rysosponum was most extensive y stu Ie ..

Cripps et al. 24 reported th at P. chrysosporium removed 87 to 93 % of orange n, tropeolin ° and congo red and the mycelial mats were visibly coloured after 5 days of inc ubatio n. Further they have reported that congo red was resistant to decolorization by P. chrysosporium in nitrogen limited cultures and remained tightly bound to the fungal mycelium after 12 days of incubation. N-limited ligninolytic cultures of Trametes versicolor have been reported to degrade

PCBs, anthracene, fluorene, phenanthrene, benzo (a) pyrene and dichloroaniline~6.88 . Ollika et al. 25 showed that 54% decolourization of congo red occurred in the presence of a crude preparatio n of lignin peroxidase and hydrogen perox ide. He infling el al.29 have screencd 18 fungal stra ins for the ir potenti a l to decolourize commercially used reactive textile dyes (reactive orange 96, reactive violet 5 and reacti ve black 5) and two phthalocyanine dyes (reactive blue 15 and reac tive blue 38). They reported that only Trameles versicolor, Bjerkandera adusla and Phan erochaete chrysosporium were ab le to decolori ze all the dyes. The dye HRB 8 was decolourised rapidly within 24 hr to below 40 % by Bjekandera adusta and Trametes versicolor and 95 % decolourization was achieved within 4 days.

The white rot fungi have been shown as superior dye decolourizers particularly in comparison to prokaryotes which are generally poor or non­decolourizer. Even the lignin transforming actinomycete Streptomyces chromouscus is a weak decolourizer compared to P. chrysosporiuml 8

.

The question whether fungal dye decolouri zation represents mineralization, has been addressed by relatively few studies. Mineralization rates of 23.1 -48.1 for a wide range of '4C_ring labeled azo dyes after a 12 day incubation with P. chrysosporium have been recorded26. Another study confirmed the ability of '4C-radiolabeled azo dyes, yet found to correlate between aromatic ring substitution pattern and mineralization rates l8

. Toxic intermediates probab ly do not accumulate during dye decolourization and mineralization although experimental evidence is lacking .

The majority of experiments on the degradation of dye-stuffs by fungi have been carried out with e ither whole cultures or crude enzyme preparations of ex tracellular enzymes of the lignolytic system of fungi. Eventhough the white-rot fungi are routinely screened for degradation of xenobiotics, many studies have been focused mainly on P. chrysosporium. However, the practical applications of this fungus in waste treatment and bioremediation do not always enable the culture conditions for lignolytic to be fulfilled . [t may therefore be beneficial to screen a variety of white rot fungi to understand their ability to degrade xenobiotics under a wide range of environmental conditions. Although most studies have been used the standard test organisms P. chrysosporium and T. versioclor dye-decolouri zi ng ability among diverse taxa and white rot fungi with very different LME-producing characteristics has a lso been recorded in many recent studies against wide variety of dyes32.34.s9.9 1. In addition to screening

established culture collection strains, new isolates can y ie ld stra ins that are able to degrade xenobiotics more rapidly. For that, a simple pl ate test92 for direct visualization of biological dye-decolouri zation with Iignolytic activities have been used in many recent studies in order to select potential fungal strains. [n our laboratory, thirteen white rot fungi were sc reened for decolourization of commercial tex tile dyes33 .

Mechanism of dye decolourization From various studies conducted so far it becomes

clear that a two-step mechani sm viz the physical adsorption and enzymatic degradation are invol ved in

1080 INDI AN J EXP BIOL, SEPTEM BER 2003

dye decolouri zation by the white rot fun gus. Knapp and Newb/ 6 observed that in many cases adsorption of dye is in the microbi a l ce ll surface with primary mechani sm of decolouri zation . Young and Yu93

suggested that the binding o f dyes to the fungal hyphae and physical adsorption and enzymatic degradation by ex tracellul ar and intracellular enzy mes as reasons for the colo ur removal. The dye-saturated myce lium can be regenerated and used for repeated dye adsorption . They have further stated that the dyes were not decolo urized by manganese dependent pe rox idase (MllP) while above 80% colo ur was removed by li gninase-catalyzed ox idation. Dyes with di fferent structures are decolouri zed at different intrinsic enzymatic rates and high dye concentratio n resu lts in s lower decolo uri zatio n rateD. The dye was adsorbed to the myce li a l pe ll ets in both li gnino lyti c and non-li gninolyti c cultures. W ang and Yu CJ4

reported the adsorption of ac id green 27, ac id vio let 7 and indigo carmine dyes on li ving and dead mycelium of T. versicolor. The phys ical desorptio n and enzy mati c degradation of the adsorbed dye mo lecules were also investigated and repo rted that the enzy matic degradati on o f adsorbed dyes was the major mechanism in which the regeneration o f dye adsorption capac ity of the mycelium was achieved.

The involvement of LM E in the dye decolouri zation process has been confirmed in several studies using puri fied cell free enzymes. Enzy mes such as lignin peroxidase (Lip), manganese dependent peroxidase (MnP) and laccase are in vo lved in lignin degradation which parti cipate in the decolouri zation of the dyes9s

.

Kim el al.96 reported the presence of H20 2 dependent Remazol brilliant blue R deco louri zing enzymatic acti vity in the culture filtrate of Pleurotus ostreatus in chemica ll y defined medium. Lignin perox idase of P. chrysosporium has been shown to decolouri ze azo, triphenylmethane and heterocyclic dyes in the presence of veratryl alcohol and H20/ 4

.2S

. It is interesting that the ability of enzyme preparation to decolouri ze identical dyes varied between the two studies.

Extracellular fluid from cultures of P. chrysosporiwn and purified lig nin peroxidase were able to degrade c rysta l vio let and six other triphenylmethane dyes by sequenti a l N-demethy latio n23 . The ro le of purified li gnin perox idase in the decolourizatio n of several azo dyes has been c learl y demo nstrated97 . The different isoenzy mes of lignin peroxidase produced by P. chrysosfJorium are able to deco louri ze severa l dyes with different chemical structures including azo, triphenylmethane, hete ro cyclic and polymeric dyes25

.

In P. chrysosporium cultures, dye decolouri zation is not one-step ox idation process, as several dyes are extensive ly minerali zed26

. Dyes such as Poly R98 and Azure B 99 have been proposed as a standard assay for dete rmination of lignin peroxidase acti vity .

The chemical steps invo lved in the degradati o n o f azo dyes by LiP and MnP has been el ucidated 100.101.

The mechani sm o f azo dye ox idati on by perox idases such as lignin perox idase probabl y in volves the ox idati on o f the phenolic groups to produce a radical at the carbon bea ri ng the azo linkage. Then water attacks this pheno li c carbo n to c leave the mo lecule producing pheny ldi az ine. The pheny l d iazine can be ox idi zed by a o ne-e lectron reactio n generating N 102.103

2 .

The ea rl y stages o f azo dye deco lo urizati on is the breaking of the azo bond , the ease of whi ch has been fo und to be depending o n the identity, number and positio n of func ti onal groups in the aro mati c region and the resulting interacti ons with the azo bond. Furthe r degradatio n of azo dyes in vo lves a romatic cleavage whi ch has a lso been fo und ro be dependen t o n the identity of the ring substituents with the presence o f pheno lic, amino acetamido, 2-methoxypheno l, o r othe r eas il y biodegradable functi onal groups resulting in a greate r ex tent of degradation 18.97.

Manganese perox idase fro m P. chrysosporium was also able to decolouri ze severa l azo dyes in vitra , and with both enzymes the decolo uri zati o n rate was dependent on the chemi cal struc ture o f the dye lO I

. A corre latio n has been observed between MnP acti vity and Po ly R-478 decolouri zatio n in P. chrysosporium grow n in corncob cultures by semi-solid state cultures, inc luding that MnP is mainly responsible fo r dye degradation.

The MnP's o f Bjerkandera adusla and Pleuralus eryngii have a lso been shown to cata lyse dye decol ouri za tion3o

. The enzymes fro m both fung i were unusual in that they did so in M n2

+ independent reaction (ie the Mn 3

+ lactate complex was not the oxidative agent created by the enzy me) .

Decolouri zatio n o f a number of phenol azo dyes has been examined us ing a co mmercia l crude laccase preparatio n of Pyricularia oryzae27 . Two laccase isoforms purified from Trametes hispida were able to cata lyze decolouri zation of severa l sy ntheti c dyes lD4

.

For laccase oxidation of phenolic dyes, a simil ar mechani sm has been proposed as li ke LiP27 . In the proposed mechani sm laccase ca ta lyses two sequentia l e lectron oxidatio n reactions to generate firstl y a

MURUGESAN & KALA ICHELVAN : SYNTHETIC DYE DECOLOURIZATION BY WHITE ROT FUNGI 1081

phenoxy radical and then a carbonium ion in the phenol ring nucleophilic attack of water then yields a di azinc derivati ve and a quinone. Partial decolori zation of two azo dyes (orange G and amaranth) and complete decolouri zati on of two triphenylmethane dyes (bromophenol blue and malachite green) was achieved by cultures in submerged liquid culture producing laccase as the sole phenol oxidase 105. Laccase based decolouri zation treatments are potenti ally advantageous to bioremedi ation technologies since the enzy me is produced in large amounts and often produced constitutively or requires less fas tidious induction conditions than either LiP or MnP3 1.77. los.los.

Effect of environmental conditiolls Oil dye decolouri­zatioll

Culture conditions affect fungal ph ysiology and the expression and acti vity of the li gnolytic enzy mes. Several studies have been focused on optimi zation of cultural conditions for dye decolouri zation. Knapp et al. I09 and Swamy and Ramsa/9 reported that pH was a critical factor fo r decolouri zation of dyes. They found that additi on of dye in the culture medium increased the pH, when a pH change with additi on of dye resulted in no decolouri zati on. Agitati on played a major ro le in dye decolouri zation. In stati stica lly grown cultures a decrease in dye adsorpti on was accompani ed by visibl e sorption of the dyes to fungal mat. The superi or and increased performance of the agitated cultures may be due to the ph ysiolog ical state of the fungi as pellets and increased mass and oxygen transfer between the cell s and the medium due to mi xing. In many studi es greater decolouri zation was observed in ag itated cultures th an in stati c cultures which, offers many advantages over stati c culti vati on for development of practi cal process32. IIO.

The formation of a mat at the surface in static cultures restricts O2 transfer to the cell s beneath the surface and in the medium. Oxygen limitati on is likely to in hibit the ox idati ve enzy mes and prevent deco lourizati on. Stati c cultures of P. chrysosfJorium have been reported to decolourize dyes, without sorption, thi s was with a large surface area to vol ume

. d . . fl I ' . h 0 7 1 . 7 (1 97 9X ratio an Intermlltent uSllng Wit pure 2-- -. - .

Glucose co ncentra ti ons have a strong effect on the decolourizati on of the dye stuff by the fung i. The decolourizat ion ab ili ty of myceli a decreased rap idl y in the absence of glucose, supporting tha t the fun gus req uires an additional carbon source to fuel the degrada ti on process in basidiomycete strain F29 109

Glucose concentrations, either at growth or decolouri zation phase, caused insufficient growth or loss of fungal activity leading to a decrease in colour removal efficienc/ II . In addition to carbon sources, both the nature and the quantity of available nitrogen sources exert a great influence on the extracellular lignolyti c enzyme production and dye decolouri zation actI vity of wood-rotting bas idiomycetes. The degradati on of Congo red by P. chrysosporium is also inhibited by a high concentrati on N,( 12 mM)11 2 and mineralization studies with several dyes have revealed that most of the dyes in vesti gated were degraded extensively onl y in a certain range of N concentra-. . PI' 76 d T . l 15 X9 11 0 tlons III . C 1rysosponum- . an . ve rSI.co or . . .

In the recent study, Hatvani and Mecs l1 3 also reported that low N concentration ( 1-5 mM) proved optimum fo r enzy me production and dye decolouri zation by L. edodes on a solid medium, regardtess of the N-source used . -.-- --------

The composition optimum fo r growth and that optimum for enzy me produc~n and enzy matic degradation were not the same even for a given spec ies and there were still greater di ffe rences between the requirements of the diffe rent spec ies. This must be taken into considerati on III

biotechnological applications of white rot fungi. The conditi ons must be optimized for each strain with regard to the aim of the applicati on.

Dye decolourizatioll ill immobilized system The most important extern al fac tors affecting the

acti vity of the organi sm are temperatures, p H, di sso lved oxygen concentrati on and fi xed nitrogen concentrati on. In order to be very effecti ve in degrading the dye compounds by white rot fungus, a practi cal treatment system must be developed, within which the fungal cell can grow well and maintain hi gh viability to excrete degrading enzy mes continuously fo r a long term operati on. A promi sing method to accompli sh thi s goal lies in the use of immobil izing white rot fungus. The immobili zation method using biomass support parti cles has several advantages over the other methods. It is easy to perfo rm under aseptic condi tion ill Si lU within the bioreactor and easy to scale up . Numerous carriers have been studied for the immobili zation of fungi with good resu lts being obta ined in man y cases with carriers such as sintered glass . nylon web. po lyurethane foam , porus polystyre ne and sili con tubing. The foam matrix is cheap and mechan ica ll y strong. Imlllob i I ized cultures of whi te rot fungi produced lignolytic enzymes over a

1082 INDI AN J EXP BIOl, SEPTEMBER 2003

longer peri od and effec tively decolouri zed the syntheti c dyes 114. Other materi als such as nylon sponge, corncob, pine wood, pelleted wheat bran and lignocellulosic fibres have been used fo r the immobili zati on of mycelium33.J5.94. 115. Recentl y, Kas inath el a /.35 observed that immobili zed while rot fungus, lrpex /acteus on polyurethane foam and pinewood were found to decolouri ze the dyes and effl uents effecti vely than free mycelium.

Dye decolourization by solid-state fermentation culture

Solid substrates offer additional advantages over liquid culture media for the bioremediati on of xenobioti cs pollution in that pollutants bind to organic matrices, which effects some removal from polluted sites/eftl uents I1 6. Production of lignolytic enzy mes on solid substrates is establi shed with hi gher activities of laccase being reported as compared with submerged culturesI 17. 119. Use of solid substrates for the remova l of xenobioti cs fro m aqueous effluent IS well establi shedl2o. Several industri al dyes and effluents decolouri zed biocatalyticall y by ex tracellul ar enzy mes from different strains of white rot fungi grown on li gnocellulos ic substrates in so lid-state fe rmentati on and proved its effi ciency in continuous useJ33.,.12 1.

Dye decolourization ill using bioreactors

Most of the ava ilable reports considering effl uent decolouri zation by white rot fungi are restricted to b h .. . 12? 123 Wh O atc or semi-contllluous operations -'. Ite rot fungi have been used for decompos ition of several reca lcitrant compounds in different types of bioreactors namely fi xed film bioreactors, packed bed bioreactors, rotating biological contactors and pulsed fl ow bioreators. There are few reports spec ifically on dye decolouri zati on in continuous bioreactors. Young and Yu9J reported the decolouri zati on of a di sperse dye (Red 553) in a fi xed-film bioreactor. The decolourizati on was conducted continuously for 10-20 days and the decolouri zation rate remained > 80%. Many authors have reported continuous deco louri zation of many dyes by white rot fungi in

d b d b· 15?4175 d' . packe e loreactors'- . - an III rotatlllg . I . I I I I 126 . h h' I d 1 . . blo og lca contactors . Wit Ig l eco OUrI zatlon

efficiency. Deco louri zati on effi ciency of tex ti Ic dyestu ff by white rot fungi in rotating biodi sc contractor va ri es depending on biofilm thickness,

. I I d b' III rotatl ona speec an car on source concentration .

Pulsed fl ow bioreactors system permitted the continuous production of enzy mes fo r greater than fi ve months by contro lling fungal grow th J27

·m . The

introducti on of oxygen in intermirtent pulsations and the adequate rate of nutri ents supply were the key fac tors responsible for such a long operation peri od.

However, a number of operational pro blems such as formation of myceli a, aggregates, electrode fouling and clogging emerged after a short time and made necessary the periodical removal of funga l biomass from the reactors.

Degradation metabolites Literature survey reveals that much work has been

carried out on the dye decolourizati on but onl y few studies have been focused on the elucidati on of possib k degradation path ways. The anaerobic degradati on of reacti ve dyes by bacteria has been shown to produce several aromati c and a ni o ni ~

byproducts 129. Atomic absorption spectrophotomett:;J has been used to fo llow the fa te of the copper moieq. during biodegradation in both culture supernatant an biomass samples I4. IJO. Polarography has also alreacl\) been utili zed to determine in what chemica l fo rm thl copper was present at the vari ous different stages of the deg'radati on process 14, 130. Recently, a study has been made by Conneely et a /.lJ I for eluc idation of a microbial degradati on pathway for a copper phthalocyanin dye by capill ary electrophoresis an,l liquid chromatography. This study has postul ated that the lignolyti c ex tracellul ar enzy me laccases ale involved in the early producti on of a metabolite whi ch involved break-up of the conjugated phthalocyanin ring structure l31. Another lignolytic ex trace llul ar enzy me manganese peroxidase is beli eved to be in vol ved in the release of Cu2

+ fro m the phthalocyan i n structure.

Thin layer chromatography of fungal culture extracts showed only one unknown metabolite of Rr= 0.6 as a result of dye degradation 132. The decolouri zation was expected due to high capacity of dye (i ndigo) absorption by mycelium of fungi as well as the reduction of dye intensity in solution because of changes caused by them. Dye degradation was also demonstrated by thin layer chromatography of supernatants and mycelium extract of decolouri zati on assay. Very few repoils are available on the biodegradation products or in termed iates of indigo dyes. The degradation of indigo by laccases produces isatin (indole2-3 dione) which was fLlli her degraded to anthranilic acid (2-aminobenzoic acid) by HPLC analysis.

MURUGESAN & KALAICHELVAN: SYNTHETIC DYE DECOLOURIZATION BY WHITE ROT FUNGI 1083

Dye decolourization by mycelial pellets A new approach has been developed for

decolourization of dyes by complex mycelial pellet133.

Complex mycelium pellets of a white rot fun gus , Trametes versicolor with activated carbon powder were prepared and investigated for decolourization of an azo dye, acid violet 7 133. The pellets had a bl ack core of activated carbon powder that was surrounded by a layer of white fungal mycelium. Compared to the activated carbon powder and the mycelial pellets, the complex pellets showed that highest and the most stable activity of dye decolourization in batch cultures. The enhanced decolouri zation ability of the complex pellets implies that the microenvironment . around the fungal cells was changed in the presence of acti vated carbon which became more favorable for dye degradation . The activated carbon can adsorb and concentrate the chemicals from the compounds for microbial or enzymatic attack. Activated carbon can -:1 lso retain the extracellular enzymes and mediators hat are necessary for the degradation .

~fjluent treatment by white rot fungi The successful applications of white rot fungi for

decolourization of textil e dye efflu ent have been achieved for several white rot fungi 26.72.134 Immobili zed laccase of Coriolopsis gallica efficiently decolourized the dye effluent J35

. Abadulla et al.73 ;'eported that the textile dye effluent treated with the partially purified laccase reused for dyeing process. Recently, Wesenberg et al.74 reported that the white rot fungu s Clitocybula dusenii partially decolori zed the wastewater containing dye and the fungus produced higher manganese peroxidase and laccase activities when grown iii effluent.

Dye degradation in soil Since high amount of lignolytic enzyme

production, robust growth, capability of so il colonization and relative resistance to the inhibitory action of soil bacteria and toxic compounds the white rot fungi have the potential to remediate the

. d .\ 136 B . d d' f contamInate SOl S. 10 egra atlOn 0

environmental pollutants by white-rot fungu s has been demonstrated for numerous pollutants in both liquid and soiI 34. 137. 141. Novotny et al .34. 142 have

reported that the white rot fungi Irpex lacteus and Pleuratus ostreatus are the potential candidates for removal of dye RBBR present in the soil under in vitro conditions and bioremediation of water and soil bioremediation. P. sajor-caju effectively colonize in

sterile and non-sterile dye contaminated soil and produced extracellular laccase which reduced the

.. 33 tOXICIty .

A huge amount of sludge is formed during dye effluent treatment process by chemical precipitation method. Thi s sludge is toxic and highly problematic to safe disposal. The detoxification and di sposal of the sludge is a bigger problem to the tex tile dye units. The treatment of sludge by white rot fungi is the best way for the detoxification. Recently in our laboratory studies , it is proved that the white rot ed ible mushrooms effectively grown on sludge, produced high amount of laccase and the toxicity of the sludge was reduced33. The results of the study with white rot fungi on dye decolourization are very much practical because thi s is the time for textile effluent retention in conventional stations of biological treatment.

Conclusion Critical analysis of the literature shows that

decolourization of dyes by white rot fungi offers several advantages over the conventional treatment system due to its potential lignolytic enzyme system. It is hoped that enzyme based treatment systems will be the technologies of the future. Genetically engineered strains, suitable for degradation process would play an important role in this . Though the white rot fungi are efficient decolourizer, fungal­based colour removal process in field level are still limited however, the scope of use of this method for large scale treatment in effluent treatment plant. Treatments employing white-rot fungi offer the possibility to expand the substrate range of exi sting treatments via biodegradation of pollutants that can not be removed by prokaryotes (or by chemical means) . White-rot fungal bioremediation treatments may be particularly appropriate for in silu remediation of soils, where recalcitrant compounds and bioavailability are problematic . A further application may lie in the operation of bioreactors for synthetic dyes in liquid waste, where near-lOO% degradation efficiencies have been achieved using white-rot fungi.

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