Journal of Biotechnology 99 (2002) 249 /257

Enzymatic membrane reactors for biodegradation of
recalcitrant compounds. Application to dye decolourisation
C. Lo´pez, I. Mielgo, M.T. Moreira, G. Feijoo, J.M. Lema *
Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, Avda das Ciencias s/n,
E-15706 Santiago de Compostela, Spain
Received 6 July 2001; received in revised form 20 February 2002; accepted 27 March 2002

Membrane bioreactors are being increasingly used in enzymatic catalysed transformations. However, the application
of enzymatic-based treatment systems in the environmental field is rather unusual. The aim of this paper is to overview
the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation.
Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages
associated to their utilisation are revised as well as the application of this technology to wastewater treatment. Secondly,
dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in
vivo and in vitro systems. Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in
continuous operation is presented.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Enzymatic membrane reactors; Dye decolourisation; Manganese peroxidase; White-rot fungi

1. Enzyme membrane reactor
1.1. Definition and configuration
The fundamental characteristic of an enzymatic
membrane reactor is the separation of biocatalysts
from products and/or substrates by a semipermeable membrane that creates a selective
physical/chemical barrier. Permeable substrates
and products can be selectively separated from
* Corresponding author. Tel.: /34-981-563100, ext.14231;
fax: 34-981-595012
E-mail address: (J.M. Lema).

the reaction mixture by the action of a driving
force across the membrane (chemical potential,
pressure, electric field) that causes the movement
(diffusion, convection, electrophoretic migration)
of solutes. The biocatalyst is retained within the
system by the membrane, allowing the establishment of a continuous operation with substrate feed
and product withdrawal (Giorno and Drioli, 2000;
Prazeres and Cabral, 2001).
Enzymes can be used: (a) suspended in solution
in a reactor coupled with membrane unit; (b)
immobilised within the membrane matrix itself or
(c) entrapped in gels or microcapsules (Fig. 1). The
first configuration system comprises a stirred tank
reactor combined with a membrane-separation

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Livingston. high flow rates. Some selected examples of the different types are shown in Table 1. When working with immobilised enzymes. Enzyme membrane reactors (EMR) present advantages such as high enzyme loads. / Journal of Biotechnology 99 (2002) 249 /257 Fig. fibres or microcapsules (Klibanov. 1993). Lo´pez et al. EMR may result in a sustainable technology. where the enzyme is linked by either adsorption or covalent attachment to the membrane itself or by entrapment in gels.250 C. have a key role (Giorno and Drioli. therefore. mass transfer phenomena. 1. It is also interesting for reusing contaminated process water (Brindle and Stephenson. Products have to diffuse from the reaction site to the other side of the membrane.2. straightforward scale-up to large systems and high yields of pure material (Katchalski-Katzir. energy and waste products by recycling. The combination of membrane technology with enzymatic reactors for wastewater treatment has led to the development of three generic systems: Immobilised enzyme membrane reactor (IEMR). prolonged enzyme activity. 1. unit. Application to wastewater treatment Membrane bioreactors constitute an interesting possibility to be applied in wastewater treatment. contrarily to the immobilisation approach. Though commercial size membrane bioreactors exist. (b) stirred tank with enzyme immobilised within the membrane matrix itself and (c) stirred tank with enzyme entrapped in gels. Extractive membrane bioreactor (EMB) and Direct contact membrane reactor (DCMR). reductions in costs. it still remains a need to assess the durability and long-term integrity of the membrane material during the operation. 1983). Configurations of continuous membrane reactors: (a) stirred tank reactor combined with separation-membrane unit. substrate must be transported across the membrane to the biocatalyst. fibres or microcapsules. easy reactor operation and control. where it is recovered as a permeate. . 1996. This technology makes it possible not only degradation but also the recuperation of valuable components from effluent streams. 1994). According to these positive aspects. 2000).

.. the rate-limiting step is the permeation of the substrates across the membrane. Lo´pez et al.Bjerkandera sp.2) Laccase Source Reactor Pseudomonas sp. 1995) (Liese et al.Polyethersulphone and actor and hollow Hydrophobic membrane fiber DCMR Soybean peroxidase Ground soybean seed-hulls Pectolyase Y23 Aspergillus japonicus Manganese peroxi.10.14. 1983). 1997). which explains an inferior kinetic behaviour of enzymes in EMB compared with a free enzyme. Frame plate reactor module Agaricus bispor. 2000). Jolivalt et al. 1986. 1994. they can Synthetic industrial wastewater (glucose) Etanol oxidation (Kojima et al.. Protein immobilisation on membrane by covalent attachment involves interaction between an ‘activated’ group on the membrane and a functional group on the protein.1) Laccase (EC 1. the use of hollow-fibre bioreactors or capillary membrane reactors considerably increases the surface area / volume ratio and. . 2001.. 2000) (Jolivalt et al. 2000) Polyethersulphone Durapore hydrophilic EMB Glucose oxidase Aspergillus niger Hollow fiber Glycerol dehydrogenase Enterobacter aerogenes Stirred tank re.. dase Stirred tank reactor Stirred tank reactor Stirred tank reactor Polyethersulphone / Polyethersulphone Polyethersulphone The main characteristic of the IEMR is that the enzyme is immobilised onto the membrane.. The enzymes can be also entrapped within the polymeric matrix during the preparation of the membrane by phase-inversion (Chen et al. 1994..Capillary reactor um Pyricularia ory. most commonly b-amino groups of basic residues (Guisan et al. 1997. therefore. 2001). 2001) This work be encapsulated by filtration within the porous layer of the hydrophilic membrane (Bohdziewicz.. 2000).18. 1998) (Edwards et al. 1999) (Gallifuoco et al. The driving force for the extraction is the concentration gradient of the organic compounds (Livingston. (b) adsorption and (c) entrapment.Spiral-cel wound zae module Trametes Frame plate reversicolor actor module Membrane Application Reference Polyacrylonitrile Phenolic effluent Polysulphone Coal-gas conversion plant effluents (phenols) Synthetic industrial wastewater (phenols) Phenylurea pesticide in wastewater (Bohdziewicz. Prazeres and Cabral.. In both systems. The major disadvantages presented by this type of reactors are related to the fact that diffusion is the dominant transport mechanism. Roy et al..C... Ulbricht and Papra. Normally.. 2000). 1998). The direct immobilisation onto the surface and/or within the matrix of the membrane can be achieved by different techniques: (a) covalent attachment. 1999a) (Lante et al.. the enzymes are adhered on the membrane via van der Waals forces or electrostatic interactions (Arica et al. 1999a. 2000. / Journal of Biotechnology 99 (2002) 249 /257 251 Table 1 Summary of EMRs for the treatment of wastewaters Enzyme IEMR Enzyme crude extract Polyphenol oxidase (EC 1. the treatment capacity of the system (Edwards et al. Isono and Nakajima.3. 1996) Synthetic industrial wastewater (phenols) Depolymerization polygalacturonic acid Dye decolourisation (Flock et al. The principle of operation of the EMB is based on the selective extraction of pollutants from the wastewaters by means of specific membranes. Sisak et al.. Lante et al. In the adsorption process. IEMR and EMB. Alternatively. performing the catalytic reaction simultaneously with the separation of products (Gekas.

g. 2001. Zhang et al. which. The release of dyes into the environment constitutes only a small proportion of water pollution. wastewaters are characterised by a complex mixture of many polluting substances ranging from organic compounds to heavy metals associated to dyes. by the use of standardised commercial reactors and membranes. chrysosporium remains the most widely studied white-rot fungi..1. 2. anthraquinone. are prone to cause product inhibition by their binding within the active sites of the enzyme. bacterial treatment) in an integrated wastewater treatment plant. Fungal decolourisation The decolourisation of dyes by white-rot fungi was first reported by Glenn and Gold (1983). The operation was conducted continuously for 10/20 days and the decolourisation efficiency attained values higher than 80%. which have to be secluded and stored. Prazeres and Cabral. 1987). Zollinger. 1993). coupled to a bioreactor.. (1999) also considered the continuous treatment of an azo dye. Besides. as those of dyes (Banat et al. 2001). / Journal of Biotechnology 99 (2002) 249 /257 Direct contact membrane system consists on a solid/liquid membrane separation. Moreira et al.. 1987). Conventional biological treatment. in a packed-bed reactor. 2001). Nowadays. 1998b. 1996.. phthalocyanine etc. Bjerkandera and Pleurotus and many more are increasingly being studied to degrade different kinds of dyes: azo. Yang and Yu (1996) reported the treatment of a disperse dye (Red-553) in a fixed-film bioreactor. the system would complement more established technology (e. 1988). Whilst P. 1997.. Moreover. these units can be operated in a continuous mode and present an easy scale-up. Unfortunately. Orange II. 1986. 1999. most of the reports available which consider effluent decolourisation by whiterot fungi are restricted to batch or semi-continuous operation. 2. white-rot fungi appear as a valuable alternative because they are capable of oxidising compounds of complex structure such as lignin (Kirk and Farrell. 1999). Among the different configurations of DCMR. 2001).. For that purpose.. other species such as Trametes . This feature is based on the extracellular secretion of highly oxidative oxidases and peroxidases. which employs ultra or microfiltration modules for the retention of biocatalysts. with a high efficiency (90%). The main problem in the application of white-rot fungi to the continuous removal of colour from effluents is their remarkable predisposition for branching and ex- . There are few reports specifically on dye decolourisation in continuous bioreactors. 2001) or the in vitro decolourisation (Heinfling et al. costly physico-chemical processes are applied as the only treatment available to reduce their direct impact on the environment (Robinson et al. (Heinfling et al.252 C. Swamy and Ramsay. the most outstanding enzymes being lignin peroxidase (LiP) and manganese peroxidase (MnP) (Glenn et al. 1996. The proposed system permits to reuse the biocatalyst with the simultaneous removal of low molecular weight compounds. some of them generate hazardous by-products and sludges. who developed a method to measure ligninolytic activity of Phanerochaete chrysosporium based upon the decolourisation of a number of polymeric dyes.. Robinson et al. one of the most adequate for its utilisation as an enzymatic reactor for wastewater treatment is the recycled membrane reactor. Tien and Kirk.. where the reaction takes place (Brindle and Stephenson.. but dyes are visible in small quantities due to their brilliance (O’Neill et al. Moreira et al.. Ollikka et al. which comprises a stirred vessel coupled to an ultrafiltration module in a semi-loop configuration. Two different approaches could be considered: the in vivo decolourisation by fungal cultures (McMullan et al. This system would be useful for a stagewise operation. where only highly polluted streams would be degraded and partly converted in an in vitro process. in some cases. Then. Mielgo et al. However. 2000.. Lo´pez et al. exemplified in activated sludge or trickling filter plants is a suitable process for the removal of the bulk of the organic load but it is not effective in dealing with wastewaters generated in organic synthesis operations. Fungal and enzymatic dye decolourisation A main problem when treating industrial wastewater is the removal of dyes. 2001.

limited extension of mycelia and prevention of preferential paths were attained by the pneumatic pulsation. 2) (Kishi et al... the biological process is diverted to a biochemical one. we found that the strategy of H2O2 addition at a predetermined flow allowed improving both the efficiency and the rate of the process (data not shown). / Journal of Biotechnology 99 (2002) 249 /257 tending of the hyphae to the formation of a thick mycelial mat.. 1995.2. In a previous work. In order to maintain the culture alive and active but controlling grow. 1998a. Catalytic cycle of manganese peroxidase. 2001).. Results were very satisfactory. Almost a total decolourisation of a quite concentrated solution (100 mg l1) was possible at very low reaction times (10 min). We particularly studied the use of MnP for dye decolourisation. 2000) produced by white-rot fungi have been considered for developing colour removal in an in vitro system. we developed a continuous fungal decolourisation process for a long term considering two main factors: an adequate bioreactor configuration and proper nutrients (C and N) feeding rates.. 2001)... to which a pneumatic pulsation system was coupled in the aeration line. 1997. 1997. 2. good oxygen transfer. 1994. Accordingly. Kuan et al. absence of delays associated with the lag-phase of biomass. taking into account the fact that dye loads of 0. was proposed (Mielgo et al.. and the oxidase Laccase (Mansur et al. Perhaps the main limitation of the work was associated to the hydraulic retention time required for decolourisation (12 /24 h). with the advantages inherent to this system. which influence the action of the enzyme (Fig. The bioreactor was operated treating three different kinds of dyes: anthraquinone type (Poly R-478). Lo´pez et al. High dye decolourisation efficiencies (80 /98%) in parallel to continuous MnP production were maintained for long periods of time (more than 100 days) (Mielgo et al. 1985. Therefore. which would imply a great size bioreactor so as to treat large volumes of wastewater. Two main peroxidases. LiP (Chivukula et al. 1993).. In this case. 1996). azo type (Orange II) and the phtalocyanine type (Reactive Blue 98)... Mn2 and organic acids).2 g dye m 3 per day were effectively treated for prolonged periods of time (several months) with active decolourising capacity (higher than 90%) (Mielgo et al. 1994.. we initially investigated the factors (H2O2. 1997). The assays reported were performed on a very small scale and only a limited decolourisation yield was achieved. the ease and simplicity of controlling the process (Karam and Nicell.. operation at high and low contaminant concentrations. Among all the factors studied. Palma et al. Schliephake et al. reduction in sludge volume (no biomass generated) and 253 Fig. Moreira et al. 1994) and with fungi (Moreira et al.. Successful application of MnP requires the comprehensive knowledge of the interactions between the enzyme and the different reagents and cofactors for the catalytic action to proceed. Ollikka et al. Collins et al.. 1999)... This modification had already been proved to increase mass transfer rate and to enhance overall productivity of processes working with yeast (Roca et al. which rapidly disrupts proper operational conditions (Moreira et al. with a minimum consumption of enzyme. 2001). 2. an adequate flow of nutrients was supplied. 1997). 2002. . 1993) and MnP (Glenn and Gold. Heinfling et al. Sanroma´n et al.C. 2001. Enzyme decolourisation The potential advantages of enzymatic treatment as compared with fungal or bacterial treatment are mainly associated to several factors: shorter treatment periods. A fixed-bed bioreactor.

as just by assembling and interconnecting vessels and commercial membrane modules (Prep/ScaleTM-TFF Millipore). which had been treated with a chitosan solution with the objective to coat the entire outer surface. they can provide higher efficiencies. on the contrary. defined as the decrease in colour of the treated dye compared with the control and (ii) MnP activity. This system permitted the diffusion of phenolic components in the stream across the membrane. / Journal of Biotechnology 99 (2002) 249 /257 2. the next step was the development of a continuous enzymatic reactor. Edwards et al. The configuration of this system is quite simple. As can be observed. In this case. operating at extremely high dye loading rate of 2. Two parameters were followed: (i) decolourisation. Once MnP was again added in a new pulse to maintain a sufficient level (around 150 / 200 U l 1). especially when considering treatment capacity. corresponding to the steady state. Our proposal corresponds to a continuous stirred tank reactor with enforced flow where the retention of the soluble enzyme in the reaction vessel is accomplished by means of an ultrafiltration membrane. and the product was removed by adsorption on the chitosan coating. the enzyme was deactivated in Fig. the system was efficient to cope with dye decolourisation (95%). the membrane does not act as a support for immobilisation but as a separation module to recover the enzyme and to reprocess the unconverted dye. Lo´pez et al.254 C. the decolourisation proceeded optimally. Immobilisation or retention of the enzyme in the reactor are two good possible alternatives to maintain an effective operation. a fairly constant value was reached after 40 min. Decolourisation of the dye Orange II (m) and MnP activity (I) during the operation of DCMR with fed-batch enzyme addition. with efficiency higher than 90%. Thereafter. the system was effective to decolourise the dye.400 g m3 per day. 4. taking into account the results from the first set of experiments. H2O2 and dye. 3 illustrates a first experiment in which fresh MnP was added in pulses in fed-batch operation to maintain the highest degree of decolourisation. There are in the literature some available reports on continuous decolourisation by enzymatic reactors. Continuous flow reactors are more complex and costly than batch ones but. 4. Fig. parallel. The system allowed a very fast decolourisation in an initial period (20 min). on which enzyme was immobilised. The system combined characteristics coming from both physical and biochemical treatment.3. As can be observed in Fig. Decolourisation of the dye Orange II (m) and MnP activity (I) during the continuous operation of DCMR. 3. The enzyme (polyphenol oxidase) was immobilised on polysulfone capillary membranes. Fig. The continuous decolourisation of the dye was then evaluated in experiments carried out with continuous addition of MnP. to determine destabilisation of the enzyme and the threshold activity for addition of new pulses of MnP. however. Continuous enzymatic treatment Considering the success of the enzymatic treatment. . (1999b) have proposed a new EMR for decolourisation of coal-gas conversion plant effluents.

1997. M. Alfani. J. / Journal of Biotechnology 99 (2002) 249 /257 3. The application of membrane biological reactors for the treatment of wastewaters.. W. Enzyme Microb. J. 18. R. 47.. Biotechnol. the increase of the specific capacity.C. Biochem. Ferna´ndez-Lafuente. Nigam. FEMS Microbiol.. Giorno. A. J. 1997. 63. Microbiol.D.. NJ. Kacar. Transport and hydrolysis of urea in a reactor-separator combining an anion-exchange membrane and immobilised urease. A. 61. Removal of aqueous phenol and 2-chlorophenol with purified soybean peroxidase and raw soybean hylls. P. 8.J. 1999. Banat. immobilised onto ultrafiltration membranes. P. Purification and properties of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete. Reduction of steric problems. J. and hopefully the establishment of processes at industrial scale. Heinfling. Glenn. 261 /275. W. Enzymatic membrane reactors can be proposed as a promising technology.. Environ. 1983. Acknowledgements This work was funded by the Spanish Commission of Science and Technology (CICYT. 2001. P. Dobson. Artificial membranes as carriers for the immobilisation of biocatalysts. modules and materials commercially available. M. 1986.. 251. 1985.P. M. Bioresour.. A. 2543 /2548. Heinfling. 2000.H.. Gallifuoco.. Marchant. 1986. J. Process Biochem.. M.D.. Bohdziewicz.. 1999b. Bergbauer. Martı´nez. Biodegradation of phenol by enzymes from Pseudomnas sp.. Burton.. Technol. Technol. 261 /266. R. J.). 351 /357. Gekas.K. Phanerochaete chrysosporium . Glenn.. Cantarella.. Garcı´a-Junceda.. A. 2001. P. D. 1741 /1747.. the selection of new possibilities for the retention of the enzyme and its application for detoxification of other recalcitrant compounds. Lett. V.H. Akileswaran. Brindle. A. R.D. Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta . Gold. Leukes.. S.. Martı´nez. E. T. L. M. Jacobs. Process Biochem.. 33. Edwards.C.. Biocatalytic membrane reactors: applications and perspectives. 601 /610. 24. Ind. Project PPQ2001/3063). pp.T. 1996. Blanco. A capillary membrane bioreactor using immobilised polyphenol oxidase for the removal of phenols from industrial effluents. Eng. 303 /309. 58..M. Lignin peroxidase-catalyzed oxidation of sulfonated azo dyes generates novel sulfophenyl hydroperoxides. Denizli. Chem. P.. M. Viparelli. L. Trends Biotechnol. 74.P.. Biodegradation of azo and phthalocyanine dyes by Trametes versicolor and Bjerkandera adusta .D. 329 /341. 339 / 349. Among them. In: Bickerstaff.M. 1995.. Chen. Chem.. DCMR present several advantages for their short-term utilisation due to the wide variety of membrane shapes. Immobilisation of Enzymes and Cells.K. M..F.. 25. J. T... 165. 1999a. Spadaro. Appl. Microbial decolourisation of textile-dye-containing effluents: a review... A.. Enzyme Microb. Totowa. 217 /227. Stephenson. Renganathan. Rose. Teunissen. Biotechnol. Arch. 36. ongoing efforts are now focused to investigate the longest operational stability of the process. U. Bassi..K.. 40. R. Microbiol. 1997.D.G. 769 / 773. Bownes. S. K. A.C. Decolorization of several polymeric dyes by the lignin-degrading basidiomycete Phanerochaete chrysosporium . 255 Chivukula.. 1994. Appl. Microbiol.H. Biotechnol. 7765 /7772.. Currently. Bergbauer. R. 1998. I. Mn(II) oxidation is the principal function of the extracellular Mnperoxidase from Phanerochaete chrysosporium . W. D. E. 811 /818. Technol. V. Biochemistry 34. Biophys.. E.. M... J... Immobilization of enzymes acting on macromolecular substrates. M. 43 /50.C.. W. References Arica. Chem.. Leu. Huang. Bastida. A. Biotechnol. Gijzen.. 49. Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation. Environ. Reversible immobilisation of lipase on phenylalanine containing hydrogen membranes. 450 /461. A.. Field... Drioli. Studying enzyme-catalyzed depolymerization in continuous reactors.W.G. J. Edwards... Singh. Bioeng. Flock. Biophys. Res. (Ed... Gold. P. Gold. Burton. Humana Press Inc. Enzyme Microb.H. Glenn... 1996. Ergene. 242.. Methods in Biotechnology. C. M. Appl. Collins. The future will probably bring the development of even more complex enzymatic systems and elaborated configurations based on these types of reactors.. Szewzyk. Y..Y. 209 /217. U. Technol. . 1998a. Technol. Stabilization of lignin peroxidases in white-rot fungi by tryptophan. Arch. F.M. Lo´pez et al. Leukes. Conclusions and perspectives Dye decolourisation remains as an environmental problem still satisfactorily unsolved by conventional physicochemical and biological processes. 45. 5184 /5190. 688 /696. Technol. Guisan. J.. Sanderson. Rose. Biochem.. G.. 845 / 854.A. Szewzyk.

B.. Microbial hollowfibre bioreactors. Potential applications of enzymes in waste treatment. A pulsing device for packed-bed bioreactors. Biochemistry 33. 1994. Biotechnol.M. Kamphuis. Bioprocess Eng... Schliephake. Mielgo. C.S. J. C. M. Lema. 2001... A. Raijola. Conneely. Wilke. 268. II. 131 /136. Alhonma¨ki.. Pontie´. Kirby.M. Jpn.. Microbiol. Moreira. 75. Feijoo. I. Spettoli.H... Technol. O’Neill.. R. 75 /81. Kirk. N. Sci. 465 /505. 135 /139. R.. 103 /113. Rev. A.M. Jones. Biol. A. M...M. Bergbauer..B. Effect of organic acid chelators and pH. 77. 2002. Control of pellet morphology of filamentous fungi in fluidized bed bioreactors by means of a pulsing flow. Brenon. Feijoo. Science 219. Water Res.. Use of fungal bioreactor as a pretreatment or posttreatment step for continuous decolorisation of dyes.. heterocyclic. Yamada. W.T... Glumoff. M.. 69. Pinheiro.. Multiphase Bioreactor Design. M... Nagy. 61 /73. Biotechnol. Lema. H. V. Technol. Feijoo.). M. M.. J..G. J.. Transformation and degradation of the diazo dye Chicago Sky Blue by a purified laccase from Pycnoporus cinnabarinus . 28. Biotechnol. Nu´n˜ez. J. Moreira.. H.... I. 1. G..T.M. Biotechnol. C. In vitro degradation of a polymeric dye (Poly R-478) by manganese peroxidase. Martı´nez. Moreira. Nigam. A pulsing device for packed-bed bioreactors.M. I. 1896 /1901. Tramper.T. Process Biochem. Nu´n˜ez.S. Dunford. 1993... Lema. 27. J. Karam. M. R.. Meehan. 135 /180. Biotechnol. Leppa¨nen.M. J. Environ. S. I. Burfeind. Johnson. T. Lema. 2000. Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction.. J. Farrell..2-ethanediol by enantioselective oxidation: over coming product inhibition by continuous extraction. Smyth.A. L. Moreira. Szewzyk. 180. E.. 36. Wariishi. 41. A.L.. Palma.. 2000.. I. Eng. A. Microbiol.D. Biotechnol. Dele´e. 2001. C. 2000.K. Kragl. Lo´pez et al. 261 /266. 2788 /2793. E. Kinetic analysis of manganese peroxidase.. Nicell. Technol. Membr.A... A. Mechanism of manganese peroxidase compound II reduction. 1999.. J. J.. Mansur. J. K. 2001. 56.. 1499 /1503... Kuan. Kojima. Chem.. 74. G. measurement. Sisak. J.K.. D. (Eds. Mielgo. J.. 1996. Moreira. P. Baker. G. F.T. C. G. Technical aspects of separation and simultaneous enzymatic reaction . 544 /550.. Environ. 1983. 4010 /4016. Banat. Enzymatic ‘combustion’: the microbial degradation of lignin.... J. Blanch... Trends Biotechnol. J.. Bioeng... 51. G.... M. Bioeng. M. 247 /255.R. M.. Wandrey. Chem. 2637 /2647. Ollikka. In: Cabral. Taylor & Francis. G. Biodegradation of a polymeric dye in a pulsed bed bioreactor by immobilised Phanerochaete chrysosporium . Prog.. K. Hasunuma. C.. McMullan. Mielgo. 362 /368.. R. 117 /124. Bioeng. T. Klibanov. U. K. Lema. Lett. G. Appl... Colour in textile effluentssources.M. J. Suominen. A packed-bed fungal bioreactor for the continuous decolourisation of azo-dyes (Orange II). Microbiol.. J. 64. E. 2001. J. Moreira.B. E. Roca. Chem.. Appl.. Feijoo. Membrane phase separation of aqueous/alcohol biphase mixture and its application for enzyme bioreactor.. R. 89. H. Mota. R. Lema. Y. Caminade.. N.M. W. Moreira. P.. Marquez. Environ. 1997.. 10. I. Evaluation of different fungal strains in the decolourisation of synthetic dyes. Identification of a laccase gene family in the new lignin degrading basidiomycete CECT 20197. G. Microbiol. Feijoo.R.. 130 /137. Technol. Sua´rez. 2000. Hawkes. Biotechnol.. Tien. C. 1998b.F.M.C. H. Application to alcoholic fermentation. A. Ogawa. A. 59.. 471 /478.M. and polymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysosporium . 20064 /20070.M. Bioprocess Eng.. P.. 22. I.. 60.. Prazeres. Application to Aspergillus niger and Phanerochaete chrysosporium . Mainnaring.. Lonergan. 1994.. T. M. Gonza´lez. Enzyme Microb. Hydrodynamic behaviour. 1994. D..E. Kishi. Bioresour.. Mielgo.W.L. London. Technol. Marchant.T. Lema. 1999. Technol... J. I. 10. M.. K. Feijoo. M.. H. M.. Technol. C. 1997. Feijoo.T. M. 1009 /1018. Brizuela. M. I. Nigam.. Martı´nez. Krastanov. Roca. Immobilized enzymes-learning from past successes and failures.T. M. Roy. G. Enzymatic membrane reactors. Chem. I. Ferna´ndez-Larrea.. Biodegradation of phenols by laccase immobilised in a membrane reactor.J. 51 /58.J. Chem. Karutz. 1994..... 1987.. M. Biotechnol. / Journal of Biotechnology 99 (2002) 249 /257 Heinfling. Nakajima. D. Ann. K. 1993. 1993. 623 /626. Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. T. Schu¨gerl. Livingston.. J. Enzymatic resolution of 1-phenyl-1.. 2000.. Sanroma´n. Sanroma´n.E. M.M. U. 2001. Jolivalt.. Water Sci. J. Extractive membrane bioreactors: a new process technology for detoxifying chemical industry wastewaters.J. Mielgo. J. Liese. Katchalski-Katzir. 63 /68.256 C. E. McMullan. Microbial decolourisation and degradation of textile dyes. 1996.F. triphenyl methane. A. Immobilization of laccase from Trametes versicolor on a modified PVDF microfiltration membrane: characterization of the grafted support and application in removing a phenylurea pesticide in wastewater. M. 36.. A... 100 /107. C. T. Continuous production of manganese peroxidase by Phanerochaete chrysosporium immobilised on polyurethane foam in a pulsed packed-bed bioreactor. 81 /87.M.A. Sanroma´n. Decolorization of azo. Lante.T.. Hawkes. Isono. Biotechnol. Appl.M. G. S. 141 / 153. Enzyme Microb. 99 /106.. Lourenc¸o. 2000. Crapisi. 56.. P.. J. 298 /304. 63.... W.. Marchant... 19. 1983. Gold. Oxidation of glucose by glucose oxidase entrapped in hollow fiber membrane.L. Robinson. 1997. Cabral. Palma.M.T.. Appl. M. J. Palma. Lema.. Robinson. T. Lema. M. C. 722 /727. pp. discharge consents and simulation: a review. 40. J. 1995... Immobilized enzymes and cells as practical catalysts... Mougin.. 2000. Trends Biotechnol. Microbiol.. Tamai. T. 11.. Biotechnol.

24.. 24.. Swamy.. Development of bioreactor systems for decolourisation of Orange II using white-rot fungus. F. Germany. Methods Enzymol.. 61 /68. 1999. M. Technol. T. Bioprocess Eng. Development of a bioreactor system using an immobilised white-rot fungus for decolorization. Lignin peroxidase of Phanerochaete chrysosporium .. J. Bioprocess Eng. 48 /53. 20.. A. Technol..T. Part II: continuous decolorization tests.. Color Chemistry: Syntheses. 1999.. Weinheim. Enzyme Microb.K. 1988. Knapp. Yu. 161. J. Kirk. 1987. M.C. Lo´pez et al. 257 Yang.. / Journal of Biotechnology 99 (2002) 249 /257 in multiphase enzyme membrane reactors. Ramsay. H. 503 /512. J. 1997. F. VCH Publishers. Polyacrylonitrile enzyme ultrafiltration membranes prepared by adsorption.S. Ulbricht. J. 1996. Enzyme Microb. Tien. . Papra. 238 /249..N. 130 /137.. Zollinger.C. Enzyme Microb. cross-linking and covalent binding. Properties and Applications of Organic Dyes and Pigments.A. Technol. Zhang. 16. Tapley. The evaluation of white-rot fungi in the decolorization of textile dyes. K. 23. 9 /11.