Journal of Scientific & Industri al Research Vo1.59, April 2000, pp 286-293

Detoxification of Phenols and Aromatic Amines from Polluted Wastewater by Using Phenol Oxidases

Qayyum Husain * and Ulfat Jan

Department of Bioche mi stry, Faculty o f Life Sciences , Aligarh Mus lim U ni vers ity, Aligarh 202002, India

Received: 30 August 1999; accepted: 15 November 1999

The applications of treatment of industri al wastewater containing phenol s and aromat ic amines have received increased atten ti on in recent past. Classical chemical and physical methods arc not successfu l due to varying drawbacks in these methods. Biological treatment o f was tewater is also facing some serious limitations and is more expensive. It is expected that those methods based on oxido-reducti ve enzymes have the potential to detoxify the was tewater contai ning pheno ls and aromati c amines and can provide an a lternative procedure to all other known methods. An advant age of this procedure is that these enzymes ac t on a broad range of substrates and under the low concent ra tion of substrates . Nu merous phenol-axidases are de­scribed here such as perox idases, tyrosinases, and laccases. An attempt has been made to review the work carried out by using sol ubl e as well as immobiiized ox ido-reductive enzymes fo r the remo val o f phenols and aromati c ami nes from industrial waste­waters.

Introduction

Decades of industrial acti vity have resulted in the synthesis and introduction of a plethora of tox ic chemi­cals into the biosphere. However, "Management" of these wastes cannot be equated with safe di sposa l. Continua­tion of thi s trend may have catastrophic impact on hu­man health and environment. Therefore, effective means of solving thi s po llution problem must be developed to preserve the quality of life for future generations. Phenols and aromatic amines are the most important c lasses of synthetic industrial chemicals and are often present in the industri al effluents from various manufacturing op­erations I. Numerous chemical industries, sending these tox ic chemicals in the environment, are: coal conver­sion , petroleum refinin g, res in and plasti c, dyes and other organic chemicals, textile, timber, mining and dress ing, pulp and paper2-4. In addition, among the different classes of pollutants those that enter the environment , some of them are also deri ved from agricultural acti v i t i ~s . Large scale use of herbicides, insecti sides and pes ticides in agriculture contributes to the presence of these hazard­ous material s in the surface and in the ground water. Phenol s and aromatic amines are also released as inter­mediary products during the microbial degradation of pest icides or some other xenobiotics5.0 . The vast major­ity of phenol s have been classified as tox ic ity priority pollutants and some of them are even known suspected * Author for co rresponde nce

carc inogens 7- 111. The pollutants such as phenols and aro­matic amines play an important ro le in the ecolog ica l balance of some of the compartments of so il and wa­ter". The presence of these che mical in ground water or in drinking water pose a significant hea lth risk. There­fore, the decontamination of these molecules from an industrial aqueous effluent, is an important practical as­pect prior to the ir final di scharge. umerous methods have been tried in recent years to remove o r to trans­form these molecules .

Con ventional phys ical and chemical methods for the removal or transformation of phenols and aromatic amines are actually outdated due to their limitations, such as high cost, incompleteness of purification, formation of hazardous byproducts and due to app licability to onl y a limited range of phenol concentrati o 12-15 However, one of the most promis ing way to remove these sub­stances, is via bi olog ical treatment, us ing d ifferent types of micro-organi sms due to its potential it ies for complete mineralization 10-211 .

There are certain limitations of us ing microbes for treat ing these po llutants such as those h igh cost o f pro­duction of microbial culture, limited mobility and sur­vival of cells in the soil , alternate carbon ources, comple­tion of the indigenous population and metabo lic inhibi ­tion 2 1

,22 . Numerous organisms have been used for the complete degradation of phenols and other related chemi­cals but much success has not been achieved yet23-25.

HUSAIN & JAN : DETOXIFICATION OF PHENOLS AND AROMATIC AMINES 287

Enzymatic Treatment of Phenols and Aromatic Amines

In the recent past an enzymatic approach has at­tracted much interest, as an alternative method to the conventional, physical an-i chemical procedures for the removal of phenols and aromatic amines from wastewa­ters. In early I 980s, researchers first time developed an idea of using ox ido-reductive enzymes for treating waste­water containing these toxic chemicals26

.30. Some work­

ers suggested that enzymes could be used to oxidize phenol s to free radicals or to quinones and benzoquinoneimine30. These oxidation products can couple to each other resulting in the formation of water insoluble oligomers or polymers. These insoluble com­plexes are less toxic as compared to their so luble sub­strates and can be easily removed from the reaction mix­ture by simple filtration or sedimentation or centrifuga­tion26 .. 31• A wide spectmm of oxidoreductive enzy mes have been considered for these studies, such as perox i­dases26 .. 2Y , laccases6.32 and tyrosinases3o. It has been dem­onstrated that peroxidases from different sources can be used in the decontaminati on of phenols and aromatic am ines" .. 3Y .

An advantage of thi s enzymatic approach is that these enzymes can react with a broad range of phenols under the dilute condition and are less sensiti ve to op­erational upsets than the microbial populations30.

The work on the use of horseradish peroxidase in the detoxi ficati on of various phenols and other aromatic compounds has been in focus due to economical source

of enzyme, slow inhibition caused by product and the action of enzyme on a wide spectrum of substrates. Horseradish peroxidase catalyzes the precipitation of phenols and aromatic amines from aqueous solution and decolorization of phenolic industrial eftluents26 .. 2X.40. Vari­ous phenols and aromatic amines are oxidized and the corresponding free radicals by perox idases in the pres­ence of hydrogen peroxide as an oxidant are formed . Free radicals polymerize to form the products which are less soluble in water4 1

• Peroxidase catalyzes the poly­merization of phenols by three distinct reaction steps, initiation, radical-radical coupling and radical tran sf~r ' .

The major problem in the development of peroxi­dase based catalysis for industri al applications is the susceptibility of the enzyme to inactivation42

.. 44

, which is caused by adsorption of enzyme molecules on the final product of reaction . Some researchers have reported the add ition of certain adsorbants in the reaction mixture to prevent the inactivation of peroxidase during the cata­lytic cycle. These additives adsorb the product of reac­tion and thus prevent the loss of enzyme activity3~ 30 . Wu et a /4

-' have optimized the reaction conditions for enzy­matic removal of phenols from wastewater by using per­ox idase in the presence of polyethylene glycol. The pres­ence of thi s adsorbant helps in increasing the shelf-life of enzyme. In another study, Tatasumi et al.46 have shown that phenols could be effectively removed by treatment with horseradish peroxidase in the presence of a coagu­lant. Peroxidase when used alone gets rapidly inacti vated during the reaction while in the presence of the coagu­lant the amount of peroxidase required for the removal

Dr QayyulII Husain is presently working as a Reader in the Department of Biochemistry, Faculty of Life Sc iences. Aligarh Muslim University (AMU), Aligarh. He obtained his both Master 's deg ree and M.Phil in Biochemistn ' fro m AMU. SlIbseqllently he was also awarcled Ph.D f or his work on "The Imm obilization of Some Glycoenzyml's Using Concanavalin A As A Support " by AMU. He did his pos/-doctoral work on "The Role of Serine-threonine Protein­phosphatases Dllring the Development and Differentiation of Dictyostelillm-discoideum " at the FacilIty of Biolog \', Uni versity of Kons/(lnz, Germany. Dllring his post-doclOral work in German); he was awarded with the German Aca­demic !:.x c/wnge Service Fellowship (DAAD -F ellowship). He joined AM U as a Lecturer in 1988 and since then he has worked on variolls aspect of enzymes and cells immobilization. Recently, he is working as a co-investigator on {/ UGC sponsored project "Polyc/onal AllIibody Mediated Immobilization of Some Enzymes. " At the same time he is also involved in the sw dy of immobilized phenol oxidases fo r the decontamination of phenols and aromatic wllines from indllstrial eJJ7uents. He has 15 y of research experience and has mallY international and national publications 10 his credit. His cllrrent interest is in the area of enzyme- immobilizatioll and its applicatiolls in various fields, particlliarly in the area of en virollmen/(ll technology.

Miss Ulfat Jan is presently working as a Research Fellow in the Department of Biochemistry, Faculty of Life Sciences, Aligarh Mll slim University, Aligarh. She obtained her M.Sc degree in Biochemistry from Kashmir University, SrinagCll; India. Presen tly she is working on the removal of phenols and aromatic aminesfrom wastewaters by using immobilized phenol oxidases.

288 J SCI IND RES VOL 59 APRIL 2000

of phenols is reduced and enzyme remain s acti ve during the e treatment. Arsegue l and Baboulene47 have demon­strated the acti on of peroxidase in the presence of a min­erals and have shown that it could pro long the action of enzyme by adsorbing reaction products. Dec and Bollag4X

have used plant materi als for the detox ification of pol­luted water conta ining phenol s and chlorophenol s. This decontamination effect was due to the presence of per­oxidases in the plant ti ssues. An industri al water con­ta ining vari ous chl orophenols was success fully treated with minced horseradish, potato and white radish. Horse­radi sh medi ated re mova l of 2,4 dichlorophenol from mode l was te wate r was quite comparabl e w ith th at achieved by using purified horseradi sh perox idase. In a further study the selected removal of aromatic contami ­nants from water was observed by using a funga l per­ox idase from Coprinus moccrrohizus in batch reactors3X

•

The cost of the perox idase is compet it ive due to the use of expensi ve hydrogen perox ide in the trea tment of industri al effluents. The use of haemoglobin has been suggested as an alternati ve source, as it has perox idase acti vity and it is more abundant and cheaper pro te in ~~.

However use of hydrogen peroxide as an ox idant is found essentia l in e ither case.

Laccase and tyros inase require molecul ar oxygen for the formation of o ligomeric or po lymeric henols. Laccase can be eas ily isolated from the culture medium of Coriolus versicolor~()' ''' and it can act on a broad range of sub s titut ed ph e no ls 32 . Th e mec ha ni s m of dephenoli zation by laccase is the same as for perox i­dase. Thus, laccase ox idi zes phenols to the correspond­ing radicals which spontaneously polymeri ze to form insoluble compounds52. Moreover, laccase is also capable of cross-coupling toxic molecules with naturally occur­ring phenol s like syring ic ac id and vanillic ac id s"3"'~ and converts them into humic like polymers"""'6 . In an aer­ated liquid medium laccase can polymerize successfull y lignosulphonates from spent sulphite effluents.,7. Phe­nolic azodyes have been oxidi zed by using laccase from Pyricularia oryzae. It has been suggested that laccase oxidation can result in the detoxificati on of azo dyes"x.

The use of tyros inase was proposed as a cheaper alternative to horseradi sh peroxidase because it uses molecular oxygen as an oxidant instead of expens ive hydrogen peroxide. The cost of the laccase isolation from microbial sources is also a hurdle in the use of enzymes for the decontamination of various wastewaters. An easy ava ilability of ty ros inase and utili zation of free oxygen as an ox idant, will defini te ly reduce the cost of phenols

and aromatic a mines detoxificati on from indust ri a l wastewaters. An advantage of using tyrosinase as the dephenolizing enzy me was shown by some worke rs. Atlow and his co-workers have reported that the crude preparation of tyros i nase from mushroom was qui te com­parable to the pure e nzyme in c learing t e phenols from coke plant was tewater31l.

There are certa in drawbacks in the use of ty rosi­nase, such as the format ion of low molecular weight o li ­gomers which remain in soluti on, less fo rmati on of in­soluble prec ipitate and in acti vati on of enzy me by reac­tive in termedi ates during reacti o n 305~ . 1 order to over­come these limitations a two-step approach was sug­gested . The use of additives removes the reactive inter­mediate species by adsorbing from the olution and form­ing insol uble precipitate. Recentl y, ch itosan has been used as a suitabl e adso rbant during the ty rosi nase cata­lyzed reaction. Chitosan is a polysaccharide and obtained fro m waste material of she llfi sh indu try. This polymer contai ns amino groups whic h specifically binds with benzoquinone molecules and form in soluble aggregate which can be eas ily removed by centrifugation or fil tra­ti onOil

.

Wada et 0 1.61.62 have in vestigated the remova l of phenols from industrial wastewater by us ing tyros inase, no precipitate was observed but a colour change from co lourless to dark brown was seen. The coloured prod­uct was precipitated by adding chi tosan and the reduc­tion rate of phenols was reported to be enhanced in the presence of c hitosan. However, it has become necessary to add a large quantity of chitosan (1.4 mg/m l) is re­quired for the complete removal of coloured products62

.

Sun e l al.63 have also described the effec t of chitosan on the tyrosinase catalyzed reaction duting the removal of phenols from the reacti on mix ture. C resol was treated with tyrosinase and it was observed that the enzyme go t stabili zed in the presence of chitosan. These findi ngs further strengthen the significance of adso rption on the chitosan during the action of tyrosinase fo r the remova l of co loured products formed by enzymatic reaction. It was observed that almost whole p enol was converted into oxidized product in the presence of this adsorbant6.\

Payne et al.M have successfully explored the feas ibility of an enzymatic approach to separate phenols from non­phenols. They have reported two-step approach to re­move selectively phenol s from the mi xtures containing non-phenolic isomers. The procedure involves the li se of enzyme, tyros inase wh ich catalyzes the oxidation of phenols into a-qu inone products and adsorbant, chitosan

HUSAIN & JAN: DETOXIFICATION OF PHENOLS AND AROMATIC AMINES 289

Table I - Enzymes used in the detoxification of phenols and aromatic amines

Name of enzymes Source Ref.

Chloroperoxidase

Laccase

Caldariol1l),ces JlIlI1ago

Tral1letes versicolor

Coriolus versicolo r

36, 37, 39 94

6, 32, 50-57, 84

Lignin peroxidase Chrysollilica sitophila 37,85-87 Phanerochaele CI)'sosphorillll1 82

Manganese peroxidase Lentinllia edodes 90-92

Peroxidase A rl1loracia m sticana (Horseradish) Caprinus cinereas

26-31 , 46, 88, 89 38,44

Caprin liS macrrohiZlIs 31, 38 Raphanus sativlIs (White radish) Rrassica rapa (Turnip)'

48 95

Tyrosinase AgariCils bispora (M ushrooms) Sololllul/ tllberOSlI1I1 (Potato)

30, 60-64, 93, 94 48

which can specifically adsorb reaction product. In an­other study, the same effect of chitosan on the tyrosi­nase catalyzed reaction has been reported during the re­moval of phenol s from the reaction mixture. The treat­ment of cresol with tyrosinase in the presence of chitosan , results in the stabilization of enzyme. This enzyme prepa­ration appears to be quite effective in re moving the coloured product formed during the reactiono,.

Oxido-reductive enzymes have been shown to be able to remove various phenols and aromatic amines from an aqueous solution and to decolourize phenolic indus­trial waste (Table I ). It has been already demonstrated that phenols could be effectively removed by treatment with oxido-reductive enzymes in the presence of a co­agulant. Nume rous coagulants have been tried for thi s purpose. However, the enzymes used in these processes quickly get inactivated during the progress of reaction . Coagulant prevents their inactivation and reduces the amount of enzymes used for phenol detoxification. In a most recent study Lee et ai.o5 have described an enzy­matic method for the removal of phenols from wastewa­ter of the phenolic resin manufacturing. An enzyme used

was a thermostable ~-tyrosinase which catalyzed the synthesis of L-tyrosine from phenol , pyruvate and am­monia. This enzymic removal of phenol was effective at

pH values from 6.5-9.0 and below 70°C. Wastewater containing 10 mM phenol was successfully reduced to 8

mM in 24 h by this enzyme at 37°C.

In addition to mushroom tyrosinase, other plant sources should be evaluated for the decontaminati on potential because polyphenol oxidases do not require the addition of expensive H

20

2. The presence of such en­

zymes in plants is well documented by various workers. There is quite early report about the presence of laccase activity in Japanese lacquer tree Rhus verniciferaoo and in mango fruits67

. Various aspects of polyphenol oxidases in plants were extensively reviewed by Mayor and HareV>X and pesticide metabolism mediated by various oxido­reductive enzymes in higher plants has been described by Lamoureux and FrearOY. Oda et aL.70 have reported oxido-reductive enzyme activity in spinach leaves and SoderhalF' has discovered a latent polyphenol oxidase from carrot. The search is continuing to isolate various polyphenol oxidases from cheaper plant sources which can be exploited for the detoxification of industrial ef­fluents. The major reasons that enzymatic treatments have not been applied on an industrial scale are due to the huge volume of polluted environments demanding bio-remediation . For instance, hundreds of thousands of liters of wastewater is produced daily from an average

290 J SCI IND RES VOL 59 APRIL 2000

industrial site and hundreds of tonnes of soil may be contaminated through continuous industrial emission or through a single spill accident. Use of soluble enzyme is practically impossible for determination of such as an extensive contamination. The applications of soluble enzymes also suffers from certain other drawbacks such as thermal instability, susceptibility to attack by pro­teases, activity inhibition and the lack of know how for separating and reusing free catalyst at the end of the re­actionn . Another important disadvantage of using soluble enzymes in the detoxification of these hazardous mol­ecules is that the free enzyme cannot be used in continu­ous processes. To overcome all these limitations, enzyme immobilization has been suggested. Numerous matrices or supports have been employed for the immobilization of various enzymes73-75. Enzyme immobilization is ex­cellent due to its high storage, operational stability, and better control towards catalytic processes7(,.7X. In addi­tion , immobilization allows no contamination of the so­lution treated by enzymes because the immobili zed en­zymes can eas il y be separated from the reacti on mixture and can be used effi ciently in the reactors7X.

Immobilization of Phenol Oxidases for the Detoxification of Phenols

Phenol oxidases like other enzymes have been suc­cessfully immobilized on various natural and sy nthetic carriers like soils, clays, Sepharose 4B, AE-cellu lose, etc. 7'J.X3 Early immobilized preparations were not suc­cessfully used for the detoxification of phenols and aro­matic amines due to above mentioned draw-backs. Davis and BurnsX4 have reported the rol e of alginate entrapped phenol oxidases in the removal of colour fro m phenolic indu strial effluents. Entrapped enzyme exhibited higher decolourization as compared to the so luble enzyme. However, the beads were not suitable for continuolls lise in the reactors due to leakage of enzymes into the solu­tion . In an another study, Wada et al.ol have developed an immobilized preparation of tyrosin ase by using the amino groups of the enzymes onto magnetite. This im­mobilization method could improve storage and opera­tional stabilities. Para-substituted phenol s were found as a etter substrate than the 0- and m-substituted phenols in immobilized preparations. Immobilized lignin peroxi­dase from Chrysonilica sitophila decolorizes phenolic kraft effluent from pulp millsx5 . Lentinula edodes has been immobilized in a packed bed reactor and could ef­ficiently be used to degrade tox ic chlorophenols and deco lorize kraft effl uents. ~-glucosidase, laccase, lignin peroxidase and manganese peroxidase were acti vely in-

volved in this processXfi.X7 . Siddique et al. XH have described

the removal of p-chlorophenol from an aqueous so lu­tion by horseradish peroxidase. The enzyme was immo­bilized on three different reactors matrices . However, the immobilized preparation was not found successfu l for repeated use. Horseradish peroxidase was immobi­lized onto magnetite by physical adsorpti on and thi s method proved more successful as compared to cross­linking method because the enzyme retained nearl y com­plete activity on the matrix. Magnetite bound peroxi­dase was used for the treatment of variou ' chlorophenols and the preparation was 100 per cent efficient in clear­ing phenol s and their derivative from solutions. Al­though soluble enzyme are not effici ent for complete removal of each chlorophenols x~ .

In some recent studi es, Grabski el (!f.Y0.~1 have shown the degradation of various organopollutants by usi ng immobili zed manganese perox idase. In another study the same workers have used the immobilized man­ganese perox idase in a two-stage bioreactor for catal ytic generation of chelated Mn3+ and for ubsequent oxida­tion of chlorophenol. 78 per cent oxidation of 1.0 mM Mn 2

+ to Mn3+ was initially meas red under optimized conditions. After 24 h of contin ous operation under optimized reacti on conditions, the reactor still oxidized 1.0 mM Mn2

+ to Mn3+ with 69 per cent efficiency corre­

sponding to 88 per cent of initial Manganese peroxidase act ivit/1

.

Tyrosinase was immobilized on magnetite using amino groups of enzyme, for the purpose of removing phenols from wastewater. Immobilized preparation was superior in operational and storage stability as compared to the soluble enzyme. p-Substituted pheno l was removed more efficiently than the /11- and a-substitu ted pheno ls by immobilized enzyme preparationol . Wada ef al.62 have demonstrated the immobilization of tyros inase on the cation-exchange res in via amino groups and thi s prepa­ration was found to be effective in treating a large vol­ume of wastewater conta ining phenol s. A combination of immobilized enzyme and chitosan was espec ially ef­fective in removing toxic phenol from aqueous so lu­tion. Tyrosinase immobilized by this method was suc­cessful in removing 100 per cent pheno l in 2 h and a marginal reduction in activity was observed after ten repeated treatments. Immobilization of tyrosinase on magnetite gave a good retention of activity, nearly 80 per cent and storage stability. A marginal loss of 5 per cent activity after IS d of storage at ambient tempera­ture was observed. In the treatment of immobili zed ty-

HUSAIN & JAN : DETOXIFICATION OF PHENOLS AND AROMATIC AMINES 29 1

rosinase, coloured enzymatic reaction products were re­moved by less amount of coagulant as compared to the soluble enzyme. Synthetic cation polymers containing amino groups were more effective than chitosan in re­moving the coloured product formed during the tyrosi­nase reaction93 . Although enzyme was immobilized on magnetite and better results were shown by adsorbants synthetic cation polymers. This immobilized preparation could be reused for the removal of chlorophenols93

.

The addition of adsorbants becomes necessary for the rapid removal of coloured product formed during the oxidation of various phenols by oxido-reductive en­zymes. However, Crecchio el aU4 have developed a new approach for the immobilization of these enzymes. Organogels have been employed for the immobilization of lacease and tyrosinase. These gels were obtained by simple gelation of reverse micells. Organogels immobi­lized enzymes preparation exhibited high operational stability and resistant to heat and proteolytic denatur­ation . A column packed with gel immobilized tyrosinase was used to demon strate the reusability of the tech­nique94

. These columns were used several times without loosing much efficiency. An additional advantage of thi s method is that there is no add ition of the adsorbant dur­ing the catalytic process and it will simplify the removal of phenols and aromatic amines at large scale. More re­cently, some research workers have investigated, ror the first time the role of turnip peroxidases in the decon­tamination of various phenols. A partially puri fied prepa­ration of turnip peroxidases was entrapped into calcium alginate beads directly and after the cross-linking with glutaraldehyde. Both immobilized preparations and soluble enzyme preparation were used for the treatment of various phenols present in the model wastewater. The higher removal of phenols from the model wastewater was achieved by immobilized enzyme. Cross-linked en­trapped enzyme exhibits higher stabi lity and reusability as compared to the soluble and directly entrapped en­zyme preparation (Mushtapa S, Jan U & Husain Q, 1999, Unpublished Worlk) .

Conclusions

It is evident from the literature that the applica­tions of ox ido-reductive enzymes for the treatment of industrial effluents has been well recognized. However, the most of the work presented here is restricted only to the model wastewaters. This work indicates the poten­tial of various phenol oxidases in the treatment of indus-

trial effluents . It is mentioned that those methods based on oxido-reductive enzymes have significant advantages over the physico-chemical and biological processes, whereas the information regarding the use of these en­zymes at commercial level is still in infancy. Therefore, it is suggested that more detailed research, on the immo­bilized enzymes and their applications at the industrial level is necessary. However, the cost of enzymes and support for preparing the immobilized enzyme is al so a major drawback in using these enzymes for detoxifica­tion . For making these preparations more successful com­mercially, efforts should be made to search new cheaper sources of enzymes and supports. In order to prevent the inactivation of enzymes during reaction , some new adsorbants of the intermediate product should be searched for removal from the wastewaters.

Acknowledgements The authors are thankful to Dr Faheem Haleem

Khan and Dr Farah Khan , Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh for their valuable suggestions .

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