Biotechnology Advances 25 (2007) 558 569 www.elsevier.

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Research review paper

Laccase production at reactor scale by filamentous fungi

Susana Rodrguez Couto a,, Jos L. Toca-Herrera b,
a

Department of Chemical Engineering, Rovira i Virgili University. Av. Pasos Catalans 26, 43007 Tarragona, Spain b Biosurfaces Unit. CIC BiomaGUNE. Paseo Miramn 182, 20009 San Sebastin, Spain Received 15 February 2007; received in revised form 5 July 2007; accepted 11 July 2007 Available online 18 July 2007

Abstract Laccases have received much attention from researchers during the past decades due to their broad substrate specificity and to the fact that they use molecular oxygen as the final electron acceptor instead of hydrogen peroxide as used by peroxidases. This makes laccases highly interesting for a wide variety of processes, such as textile dye decolouration, pulp bleaching, effluent detoxification, biosensors and bioremediation. The successful application of laccases to the above-mentioned processes requires the production of large quantities of enzyme at low cost. Filamentous fungi are able to produce laccases in high amounts, however, an efficient production system at bioreactor scale is still lacking. This is mainly due to the fact that laccase production by wild-type strains of filamentous fungi is linked to secondary metabolism, which implies that the following drawbacks must be overcome: uncontrolled fungal growth, the formation of polysaccharides around mycelia and the secretion of certain compounds (i.e. proteases) that inactivate laccases. This review summarizes the current status of laccase production by wild-type strains of filamentous fungi at the bioreactor scale. 2007 Elsevier Inc. All rights reserved.
Keywords: Filamentous fungi; Laccase; Reactor; Solid-state fermentation; Submerged fermentation; Trickle-film processing

Contents 1. 2. Introduction . . . . . . . . . . . Laccase production at bioreactor 2.1. Submerged fermentation . 2.2. Solid-state fermentation . 3. Trickle-film processing . . . . . 4. Conclusions and outlook . . . . Acknowledgments . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . scale by filamentous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 559 560 564 565 566 566 566

Abbreviations: ABTS, 2-2-azinobis (3-ethylbenzthiazoline-6-sulfonate); ALLR, air-lift loop reactor; ALR, air-lift reactor; DCP, dichlorophenol; EC, European Commission; HBT, N-hydroxybenzotriazole; OMW, olive mill wastewater; PCP, pentachlorophenol; PUF, polyurethane foam; PW, pine wood; RDR, rotary drum reactor; RITA, Rcipient Immersion Temporaire Automatique; SEM, scanning electron microscope; SmF, submerged fermentation; SSF, solid-state fermentation; STR, stirred tank reactor; VA, veratryl alcohol. Corresponding authors. S.R. Couto is to be contacted at tel.: +34 977 55 9617/8661; fax: +34 977 55 9667. J.L. Toca-Herrera, tel.: +34 943 00 5313; fax: +34 943 00 5301. E-mail addresses: susana.rodriguez@urv.cat (S.R. Couto), jltocaherrera@cicbiomagune.es (J.L. Toca-Herrera). 0734-9750/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2007.07.002

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1. Introduction Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are multicopper enzymes capable of oxidising phenols and aromatic amines, reducing molecular oxygen to water, with the involvement of three types of copper centres which have different functions: type 1 (blue copper) catalyses the electron transfer from the substrate while type 2 and type 3 form a three-member cluster that collectively activates molecular oxygen. Among physiological groups of fungi, laccases are typical of the wood-rotting basidiomycetes causing white-rot and a related group of litter-decomposing fungi, i.e. the species causing lignin degradation. Almost all species of white-rot fungi were reported to produce laccases to varying degrees (Hatakka, 2001) and laccases from many species have been purified (Baldrian, 2006). Laccases are remarkably non-specific regarding their reducing substrate. This range can even be widened in the presence of aromatic electron transfer or radicalforming mediators (Knutson and Ragauskas, 2004; Camarero et al., 2005; Rodrguez Couto et al., 2005). Thus, in the presence of redox mediators such as 2-2azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) (Cantarella et al., 2003), N-hydroxybenzotriazole (HBT) (Kleen et al., 2003) and 3-hydroxyanthranilic acid (Li et al., 2001), laccases are capable to oxidise non-phenolic aromatic compounds. This makes laccases highly interesting for different industrial applications such as pulp bleaching, textile dye decolouration, wastewater treatment, bioremediation, etc. Recently, the potential applications of laccases within different industrial fields have been reviewed (Rodrguez Couto and Toca Herrera, 2006). The successful application of laccases to the above-mentioned processes requires the production of high amounts at low cost. Reducing the costs of laccase production by optimising the fermentation medium is the basic research for industrial applications. Another approach is the overproduction of laccase in a suitable host. However, ligninolytic enzymes are difficult to overexpress heterologously in an active form (Jnsson et al., 1997) contrary to other oxidoreductases such as glucose oxidase which are industrially produced by recombinant strains of filamentous fungi. Fungal laccases are glycosylated enzymes whose sugar moieties are involved in the stabilisation process against proteolysis (Yoshitake et al., 1993). Consequently, it is necessary to express these enzymes in eukaryotic microorganisms able to perform such post-translational modifications. Several fungal

laccase genes have been cloned and heterologously expressed in the filamentous fungi Aspergillus niger (Record et al., 2002; Larrondo et al., 2003), Aspergillus oryzae (Yaver et al., 1996; Ducros et al., 1997; Hoshida et al., 2005) and Trichoderma reesei (Kiiskinen et al., 2004; Bailey et al., 2007). The latter gave high laccase production levels. Yeasts are suitable as hosts for heterologous protein production because they combine a high capacity for growth, the easy manipulation of unicellular organisms and a eukaryotic organisation enabling post-translational modifications. Laccase genes have been heterologously expressed in the yeasts Saccharomyces cerevisiae (Kojima et al., 1990; Hoshida et al., 2001; Larsson et al., 2001; Bulter et al., 2003;Kiiskinen and Saloheimo, 2004; Necochea et al., 2005; Piscitelli et al., 2005), Pichia pastoris (Jnsson et al., 1997; Otterbein et al., 2000; Hoshida et al., 2001; Brown et al., 2002; O'Callaghan et al., 2002; Soden et al., 2002; Liu et al., 2003; Colao et al., 2006), Pichia methanolica (Guo et al., 2005, 2006), Kluyveromyces lactis (Piscitelli et al., 2005) and Yarrowia lipolytica (Jolivalt et al., 2005; Madzak et al., 2005). The latter led to very promising results opening the way to the use of directed mutagenesis or in vitro evolution for improvement of laccase for industrial applications. Also, recently Hong et al. (2007) expressed a new laccase gene isolated from a novel laccase-producing fungus Trametes sp. 420 in P. pastoris obtaining a high laccase yield (8.3 10 4 U/L). The main technological applications of laccases are in the textile, dye or printing industries in processes related to decolouration of dyes (Claus et al., 2002) and in the pulp and paper industries for the delignification of woody fibres, particularly during the bleaching process (Bajpai, 1999; Leonowicz et al., 2001). In most of these applications, laccases are used together with a redox mediator. At the end of 2005 three industrial processes were using laccases: dye bleaching, lignin bleaching and bleaching of cork for bottled wine (Riva, 2006). This review provides a current overview of laccase production by wild-type strains of filamentous fungi at bioreactor scale. 2. Laccase production at bioreactor scale by filamentous fungi Fungal laccases are secreted into the medium by the mycelium of filamentous fungi (Bollag and Leonowicz 1984, Agematu et al., 1993). The highest amounts of laccases are produced by white-rot fungi (Leonowicz

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et al., 1997), which are the only organisms able to mineralise all components of lignin to carbon dioxide and water. One of the factors limiting the large-scale application of fungal laccases is the lack of an efficient production system at bioreactor scale. The different morphological growth forms of the filamentous fungi have a significant effect on the rheology of the fermentation broth and, thus, in the performance of the bioreactor. The effects of broth rheology on mass, momentum and heat transfer within a bioreactor have been well studied (Charles, 1985; Moo-Young et al., 1987; Funahashi et al., 1988). Thus, different morphological forms result in different types of broth rheology. Cultures with filamentous growth usually exhibit a high apparent viscosity and non-newtonian rheology. At moderate to high biomass levels, these broths display shear thinning or pseudoplasticity (Harvey and McNeil, 1994). These effects can have a number of undesirable results such as poor mass transfer, which would decrease the overall productivity. In addition, the control of hyphal extension is of great importance to operate the bioreactor in a continuous mode. In the last years different cultivation techniques and strategies have been used to produce laccase at bioreactor scale by wild-type strains of filamentous fungi (Table 1). 2.1. Submerged fermentation Submerged fermentation (SmF) involves the growth of microorganisms in a liquid medium rich in nutrients and with a high oxygen concentration (aerobic conditions). The industrial production of enzymes is mainly performed by SmF. Growth patterns in submerged cultures usually result into uncontrolled growth of mycelium. The extension of the fungal biomass has profound effects on mass transfer, metabolic rate and product secretion. Fungal mycelia can wrap around impellers, cause blockages and spread into sampling and nutrient feed lines as well as increase broth viscosity, which results in mass and oxygen transfer limitations. These drawbacks limit the time of operation in bioreactors. Different strategies have been used to control fungal growth in bioreactors. Thus, Lema et al. (2001) developed a pulsed system that allows controlling pellet growth, making possible a long-term operation. Recently, this system has been applied to decolouration of a synthetic dye by the white-rot fungus Trametes versicolor (Blnquez et al., 2004; Blnquez et al., 2006; Romero et al., 2006; Blnquez et al., 2007) making possible to operate the bioreactor in a continuous mode for prolonged times with high efficiency.

Other authors immobilised the fungi on different materials to control the growth rate. Filamentous fungi have a natural inclination to adhere to surfaces and, in this way, become immobilised. The immobilisation of microorganisms can be defined as any technique that limits the free migration of cells. Immobilised fungal cells have several advantages over dispersed cells. Thus, immobilised cell systems make easy to separate cells from the liquid medium, which makes repeated batch culture possible and simplifies the operation of both the continuous culture and the subsequent downstream processes. Cell immobilisation also decreases the apparent broth viscosity and makes the rheological features more favourable for oxygen supply and mass transfer. (Thongchul and Yang, 2003). In addition, immobilised cultures tend to have a higher level of activity and are more resilient to environmental perturbations such as pH, or exposure to toxic chemical concentrations than suspension cultures (Shin et al., 2002) Moreover, immobilisation protects the cells from shear damage (Abraham et al., 1991; Fiedurek and Ilczuk, 1991; Vassilev and Vassileva, 1992). When applied to recombinant strains, cell immobilisation can also alleviate strain genetic stability problems (Caunt et al., 1988; Dincbas et al., 1993). Basically, there are two types of cell immobilisation: entrapment and attachment. In the former, the microorganisms are entrapped in the interstices of fibrous or porous materials or are physically restrained within or by a solid or porous matrix such as a stabilised gel or a membrane. In the latter, the microorganisms adhere to surfaces by self-adhesion or chemical bonding. Natural polymers such as alginate, chitosan, chitin and cellulose derivatives have been mostly used as the matrix for the immobilisation of cells via the entrapment technique (Arica et al., 2001). This procedure is mainly used in the decolouration of wastewater. The materials commonly employed for the attachment procedure are synthetic foams like polyurethane foam (PUF) (Nakamura et al., 1997) and nylon sponge (Haapala and Linko, 1993) (Fig. 1). Both types of carrier materials were reported to be very appropriate for the immobilisation of the whiterot fungus Phanerochaete chrysosporium in the treatment of a sugar refinery effluent (Guimaraes et al., 2002). Also, Schliephake et al. (2000) produced laccase by Pycnoporus cinnabarinus immobilised on cubes of nylon sponge in a 10-L packed-bed bioreactor operated in batch. Luke and Burton (2001) reported that the immobilisation of the fungus Neurospora crassa on membrane supports allowed the continuous production of laccases for a 4-month period without enzyme deactivation.

S.R. Couto, J.L. Toca-Herrera / Biotechnology Advances 25 (2007) 558569 Table 1 Maximum laccase activities obtained by different filamentous fungi at bioreactor scale in the last years Fungus Type of reactor Type of cultivation SmF, immobilised on nylon cubes SmF, free cells SmF, immobilised on membrane supports SmF, free cells SmF, free cells SmF, free cells fed-batch SmF SmF, free cells, semi-continuous Inducer 10 mM VA Max. laccase activity (U/L) 280 Reference Schliephake et al. (2000) Galhaup and Haltrich (2001) Luke and Burton (2001) Blnquez et al. (2002) Galhaup et al. (2002) Galhaup et al. (2002) Hess et al. (2002) Koroleva et al. (2002)

561

Pycnoporus 10-L packed-bed cinnabarinus Trametes pubescens 20-L STR (150 rpm) Neurospora crassa Capillary membrane Phanerochaete flavido-alba T. pubescens T. pubescens Trametes multicolor Coriolus hirsutus Bioflo III (975 mL a; 70 rpm) 20-L STR (100 rpm) 20-L STR (100 rpm) STR 10-L jar fementor (160 rpm)

2 mM Cu+2 61,900 1 M 10,000 cycloheximide OMW 72 2 mM Cu+2 2 mM Cu+2 0.25 g/L Cu+2 333,000 740,000 83,830 (1st fermentation) 80,730 (2nd fermentation) 460 8131 65 4600 4300 1309 1187 16,000 1670 229 600 126 600 343 3500 5.3 5.3 1685 3500 4 2206 4892 2800 2700

15-L Biostat C (8 L; 250 rpm) Pleurotus ostreatus Benchtop fermenter (3 L; 200 rpm) Panus tigrinus 3-L STR (2 L; 250 rpm) P. tigrinus 3-L ALR (2.5 L) P. tigrinus 20-L RDR Trametes versicolor Biostat Q (4 reactors of 330 mL each) Trametes versicolor 0.5-L fluidised-bed T. versicolor 0.5-L pulsed-bed Irpex lacteus Packed-bed (27 mL) I. lacteus T. versicolor T. versicolor T. versicolor T. versicolor T. versicolor T. versicolor T. versicolor T. versicolor T. versicolor T. versicolor P. ostreatus Packed-bed (27 mL) ALR (2 L) Immersion (2.5 L) Immersion (2.5 L) Expanded-bed (300 mL) Expanded-bed (300 mL) Tray (1 L) Tray (1 L) 2-L STR (1.5 L) 2-L STR (1.5 L) Fluidised (1.5 L) Solid-substrate

Pycnoporus sanguineus P. sanguineus

2-L Biostat C

SmF SmF, free cells SmF, free cells SmF, free cells SmF, free cells SSF (maize stalks) SmF (pellets) SmF (pellets) SmF (pellets) SmF (immobilised on PUF) SmF (immobilised on PW) SmF free cells SSF (nylon sponge) SSF (barley bran) SSF (nylon sponge) SSF (barley bran) SSF (nylon sponge) SSF (barley bran) SmF free cells SmF (immobilised on nylon mesh) SmF (pellets) Trickle-film processing (sugarcane bagasse) SmF (immobilised on plastic net) SmF (immobilised on stainless steel sponges) SSF SmF SmF pellets

16 mM VA 16 mM VA OMW OMW OMW OMW Tween 80 Tween 80 Tween 80 Tween 80 Tween 80 Tween 80 Tween 80 Sugarcane bagasse Cu+2 Cu+2

Van der Merwe (2002) Van der Merwe (2002) Aggelis et al. (2003) Fenice et al. (2003) Fenice et al. (2003) Fenice et al. (2003) Font et al. (2003) Font et al. (2003) Font et al. (2003) Kasinath et al. (2003) Kasinath et al. (2003) Rancao et al. (2003) Rodrguez Couto et al. (2003) Rodrguez Couto et al. (2003) Rodrguez Couto et al. (2003) Rodrguez Couto et al. (2003) Rodrguez Couto et al. (2003) Rodrguez Couto et al. (2003) Sedarati et al. (2003) Sedarati et al. (2003) Blnquez et al. (2004) Lenz and Hlker (2004) Mohori et al. (2004) Rodrguez Couto et al. (2004a) Rodrguez Couto et al. (2004b) Sigoillot et al. (2004) Blnquez (2005) (continued on next page)

Bjerkandera adusta STR (5 L) Trametes hirsuta T. hirsuta P. cinabarinus ss3 T. versicolor 1-L fixed-bed 0.5-L immersion STR (12 L), 150 rpm Fluidised-bed with air pulses (10 L)

562 Table 1 (continued ) Fungus T. hirsuta P. ostreatus T. pubescens T. versicolor

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Type of reactor ALR (2 L) Packed-bed (280 mL) ALLR (3.5 L) Fluidised-bed with air pulses (1.5 mL) RITA System RITA System Bench scale bioreactor (800 mL)

Type of cultivation SmF (immobilised in alginate beads) SmF (immobilised on PUF) Pellets Pellets SSF (palm oil fibre) SSF (pine wood chips) Batch fermentation

Inducer

Max. laccase activity (U/L)

Reference Domnguez et al. (2005) Prasad et al. (2005) Ryan et al. (2005) Blnquez et al. (2006) Bhmer et al. (2006) Bhmer et al. (2006) Gnanamani et al. (2006)

4 Mm veratryl 1043 alcohol 1403 Cu+2 Phenolic effluent 30 mM Cu+2 11,800 1160 138.6 54 30.2

T. versicolor T. versicolor Phanerochaete chrysosporium NCIM 1197 P. cinnabarinus ss3 Vapour phase bioreactor (300 mL; 18 L) P. ostreatus ALLR (5 L) Funalia trogii 2-L STR T. hirsuta T. hirsuta T. hirsuta T. hirsuta T. versicolor T. versicolor T. versicolor T. hirsuta T. hirsuta T. versicolor

SSF (sugarcane bagasse)

Ethanol vapour OMW Cu+2, glycerol 30 M xyldine 5 mM Cu+2 5 mM Cu+2

10,000

Meza et al. (2006)

Tray (0.2 L) Tray (0.2 L) Immersion (0.5 L) ALLR (6 L) Pulsed fluidised-bed (0.5 L) STR (1 L) Fluidised-bed with air pulses (1.5 mL) 250-mL fluidised-bed (200 mL) 1.8-L tray (200 mL) 5-L STR (1.25 L)

SmF pellets Immobilised on Na-alginate beads SSF (nylon songe) SSF (grape seeds) SSF (grape seeds) SmF free cells Pellets SmF Pellets SSF (orange peels) SSF (orange peels) SmF (pellets)

1200 1000 6898 18,715 12,877 19,400 N1500 11,403 2123 3000 12,000 1385

Olivieri et al. (2006) Park et al. (2006) Rodrguez Couto et al. (2006a) Rodrguez Couto et al. (2006a) Rodrguez Couto et al. (2006a) Rodrguez Couto et al. (2006b) Romero et al. (2006) Tavares et al. (2006) Blnquez et al. (2007) Rosales et al. (2007) Rosales et al. (2007) Thiruchelvam and Ramsay (2007)

STR: Stirred tank reactor; ALR: air-lift reactor; ALLR: air-lift loop reactor; RDR: rotatory drum reactor. SSF: Solid-state fermentation; SmF: submerged fermentation; PUF: polyurethane foam; VA: veratryl alcohol; OMW: olive mill wastewater. a The volume in brackets refers to the working volume.

Sedarati et al. (2003) compared free-cell cultures of T. versicolor with cultures immobilised on nylon mesh in a 2-L stirred tank reactor (STR) for the transformation of pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP). They found that the immobilised cultures led to a more efficient removal. Also, Rodriguez Couto et al. (2004a,b) investigated different synthetic materials as carriers for the immobilisation of the white-rot fungus Trametes hirsuta in fixed-bed bioreactors operated in batch. They found that, among the different materials tested, stainless steel sponge led to the highest laccase activities (Table 1; Fig. 2). Prasad et al. (2005) reported an increase yield on laccase activity due to immobilisation of the fungus Pleurotus ostreatus on PUF cubes in flask cultures. They scaled the system to a 280-mL packed-bed bioreactor and found that the laccase yield was comparatively higher than those of the shake flask

experiments. More recently, Park et al. (2006) have found that the immobilisation of the white-rot fungus Funalia trogii in Na-alginate beads allowed the decolouration of the dye Acid Black 52 at a high concentration in a STR operated in batch. Also, ula et al. (2007) found that the white-rot fungus Dichomitus squalens immobilised on pine wood cubes in a fixedbed reactor operated in batch was able to decolourise the synthetic dyes Remazol Brilliant Blue R and Reactive Orange 16. In industrial operations immobilised microbial cell systems provide additional advantages over freely suspended cells such as simpler reuse of the biomass, easier liquidsolid separation and minimal clogging in continuous-flow systems (Arica et al., 1993; Tieng and Sun, 2000). The selection of the immobilisation technique as well as the immobilisation material is

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Fig. 1. Scanning electronic microscope images (taken at Scientific and Technical Services of the Rovira i Virgili University, Tarragona, Spain) of the white-rot fungus Trametes pubescens immobilised on cubes of nylon sponge, A: without fungus; B: with fungus.

essential to design an effective system for each particular purpose. Thus, Shin et al. (2002) pointed that immobilisation by encapsulation in a matrix such as alginate may be too costly for wastewater treatment while surface immobilisation on an inexpensive material such as woodchips was cheaper. They tested several natural (wheat straw, jute, hemp and maple woodchips) and synthetic materials (nylon and polyethylene teraphthalate fibres) for T. versicolor immobilisation in flask cultures and found that jute was the best support material. Successful applications of immobilised fungal cells in industry remain limited due to several disadvantages. The process of immobilisation may be uneconomical. Mass transfer limitations have an important effect in

altering the physiology and kinetics of cells. The product of interest has to be secreted from the cell. The immobilising matrix might be disrupted because of cell growth and gas evolution due to cell metabolism. Oxygen mass transfer limitations are more severe in an immobilised cell culture than in a suspended cell culture. Due to the oxygen supply problems, immobilisation techniques have been mainly confined to anaerobic processes (Anderson, 1975). In conclusion, immobilised cell culture systems offer a great potential for industrial applications, but with certain unsolved technical difficulties. Therefore, additional research investigating their proper application is required. Other factor affecting laccase production is agitation. Several studies showed that laccase production can be repressed by mechanical stress on the fungi. Thus, Hess et al. (2002) found that laccase production by Trametes multicolor decreased considerably when the fungus was grown in a STR, presumably because of the damage to the mycelia caused by shear stress. Fenice et al. (2003) also confirmed the repressing effect of agitation on laccase production. Thus, they found that laccase activity was strongly affected by the impeller speed. In addition, they also compared the efficiencies and productivities of three different bioreactor configurations, a STR, an air-lift reactor (ALR) and a rotary drum reactor (RDR), for the production of laccase. They confirmed the negative effect exerted by agitation (mechanical stress) on the production of fungal laccases. Mohori et al. (2004) found that it was possible to cultivate the white-rot fungus Bjerkandera adusta in a STR after its immobilisation on a plastic net, although very low laccase activities were attained. On the contrary, Tavares et al. (2006) reported that agitation did not play an important role in laccase production by T. versicolor. Ryan et al. (2005) developed an air-lift loop reactor (ALLR), which allowed the production of high laccase activities by the white-rot fungus Trametes pubescens. Thus, they obtained a maximum laccase activity of 2540 U/L after 7 days without induction and of 11,800 U/L by adding a phenolic effluent as laccase inducer. Also, Rodrguez Couto et al. (2006a,b) found the configuration of an ALLR very suitable for the production of laccase from the white-rot fungus T. hirsuta, obtaining a maximum copper-induced laccase activity of 19,400 U/L. Moreover, Olivieri et al. (2006) reported that the ALLR was fully adequate for olive mill wastewater (OMW) remediation by the extracellular laccase secreted by the white-rot fungus P. ostreatus. This is likely due to ALR provides a low shear environment for laccase production (Bonnarme

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Fig. 2. Scheme of a fixed-bed bioreactor with stainless steel sponges as supports for the fungus.

and Jeffries, 1990). Moreover, they are simple, reliable and of low cost (Kiese et al., 1980). On the other hand, operating in fed-batch has shown to be very effective for the production of laccases by white-rot fungi in bioreactors. This strategy allows controlling the growth rate by regulating substrate concentration to avoid catabolite repression and sugaroverflow metabolism. In addition, it avoids protease synthesis, which rapidly inactivates laccases. The main advantage of the fed-batch operation is the possibility to control both reaction rate and metabolic reactions by substrate feeding rate, avoiding, thus, the limitations caused by oxygen transfer and cooling. Thus, Galhaup et al. (2002) found that operating in fed-batch increased the laccase production of T. pubescens by two fold and obtained the remarkable high laccase activity of 740,000 U/L. 2.2. Solid-state fermentation Solid-state fermentation (SSF) is defined as any fermentation process occurring in absence or near absence of free liquid, employing an inert substrate (synthetic materials) or a natural substrate (organic materials) as a solid support (Pandey et al., 1999a). In recent years, SSF has received more and more interest from researchers, since several studies have shown superior product yields and simpler downstream processing than SmF (Robinson and Nigam, 2003). SSF processes have shown to be particularly suitable for the production of enzymes by filamentous fungi (Moo-Young et al., 1983; Pandey et al., 1999a), since they reproduce the conditions under which these fungi grow in nature (Pandey et al., 1999b).

Moreover, Tllez-Jurado et al. (2006) have shown recently that the expression of a heterologous laccase in A. niger substantially improved using the SSF technique. The use of natural solid substrates, especially lignocellulosic agricultural residues, as growth substrates for fungi has been studied for laccase production in recent years (Rodrguez Couto and Sanromn, 2005 and references therein). Furthermore, such residues contain lignin or/and cellulose and hemicellulose, which act as inducers of laccase. Moreover, most of them are rich in sugars, which due to their organic nature are easily metabolised by the microorganisms. This makes the whole process much more economical. Nevertheless, the use of lignocellulosic materials presents several problems such as support degradation and support accretion, causing mass and oxygen restrictions (Rodrguez Couto et al., 2002a). All this hampers the proper performance of the reactor. Despite the numerous both processing and biological advantages that SSF offers over SmF (Hlker et al., 2004), there are few designs available in the literature for bioreactors operating in SSF conditions. This is principally due to several problems encountered in the control of different parameters such as pH, temperature, aeration and oxygen transfer, moisture and agitation (Lonsane et al., 1985). The latter is particularly important when dealing with filamentous fungi. The different disadvantages detected in the conventional bioreactor designs to perform SSF processes have promoted the necessity of developing new bioreactor configurations or modifying the already existing designs. Thus, Rivela et al. (2000) developed a new bioreactor design for the production

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of ligninolytic enzymes under SSF conditions named immersion bioreactor (Fig. 3). They attained high ligninolytic activities and, in addition to this, the bioreactor was able to operate in continuous mode (Rodrguez Couto et al., 2002b). Also, Bhmer et al. (2006) reported the advantages of adapting the temporary immersion RITA-System (Rcipient Immersion Temporaire Automatique) as a bioreactor for laccase production by white-rot fungi and its application to synthetic dye decolouration. On the other hand, Rodrguez Couto et al. (2003) tested three bioreactor configurations (immersion, expanded-bed and tray) with different agitation systems (mechanical, pneumatic and static, respectively) for laccase production by T. versicolor under SSF conditions using an inert (nylon sponge) and a non-inert (barley bran) support. They found that the tray configuration led to the highest laccase activities (Table 1). More recently (Rodrguez Couto et al., 2006a), they compared two bioreactor configurations (immersion and tray) for laccase production by T. hirsuta using grape seeds as supports and reported much higher laccase activities in the tray bioreactor (Table 1). Also, they reported much higher laccase activities in a tray bioreactor than in a fixed-bed one for T. hirsuta grown on ground orange peelings (Rosales et al., 2007). 3. Trickle-film processing Trickle-film processing is mainly known from wastewater and waste air treatment and from heterogeneous

catalysis so far. However, this technique, where a fluid is continuously or periodically trickled through a fixedbed of solid supports covered by the growing microorganisms, has also turned out to be of special interest for producing extracellular fungal enzymes. Thus, Lenz and Hlker (2004) applied trickle-film processing to the production of laccase at lab-scale by the white-rot fungus P. ostreatus grown on sugarcane bagasse. They obtained a productivity of 600 U/L per day for 9 weeks. In contrast, when they cultivated P. ostreatus under SmF conditions only 20 U/L per day of laccase were produced. On the other hand, trickle-film processing allowed semi-continuous harvesting of the enzymes by periodically substituting the liquid growth medium. Therefore, the potential of this technique for industrial enzyme production should be evaluated on a large scale. Scale-up may be possible based on some modifications of the industrial bioreactors already applied as well as on using proper natural or synthetic carrier materials. While combining the advantages of common SmF (easy and standardised process control) and of classical SSF (growth on solid supports, large gas exchange interface), this technique helps to overcome some problems of both types of fermentation, which are e.g. the large amounts of water used in SmF and the difficulties in scaling-up of SSF (Lenz et al., 2004). Recently, Tavar et al. (2006) reported high decolouration efficiency for the synthetic dye Reactive Orange 16 by immobilised cultures of the white-rot fungus Irpex lacteus in a trickle bed reactor.

Fig. 3. Scheme of the immersion bioreactor developed by Rivela et al. (2000).

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S.R. Couto, J.L. Toca-Herrera / Biotechnology Advances 25 (2007) 558569 braneapplication to continuous flow reactors. J Chem Technol Biotechnol 1993;58:2815. Arica MY, Kacar Y, Gene O. Entrapment of whiterot fungus Trametes versicolor in Ca-alginate beads: preparation and biosorption kinetic analysis for cadmium removal from an aqueous solution. Bioresour Technol 2001;80:1219. Bailey MJ, Adamitsch B, Rautio J, von Weymarn N, Saloheimo M. Use of a growth-associated control algorithm for efficient production of a heterologous laccase in Trichoderma reesei in fed-batch and continuous cultivation. Enzyme Microb Tech 2007;41:48491. Bajpai P. Application of enzymes in the pulp and paper industry. Biotechnol Prog 1999;15:14757. Baldrian P. Fungal laccases-occurrence and properties. FEMS Microbiol Rev 2006;30:21542. Blnquez P, Caminal G, Sarr M, Vicent MT, Gabarrell X. Olive oil mill waste waters decoloration and detoxification in a bioreactor by the white rot fungus Phanerochaete flavido-alba. Biotechnol Prog 2002;18:6602. Blnquez P, Casas N, Font X, Gabarrell M, Sarr M, Caminal G, et al. Mechanism of textile metal dye biotransformation by Trametes versicolor. Water Res 2004;38:216672. Blnquez P, Sarr M, Vicent MT. Study of the cellular retention time and the partial biomass renovation in a fungal decolourisation continuous process. Water Res 2006;40:16506. Blnquez P, Caminal G, Sarr M, Vicent MT. The effect of HRT on the decolourisation of the Grey Lanaset G textile dye by Trametes versicolor. Chem Eng J 2007;126:1639. Blnquez P. Desenvolupament d'un procs a escala pilot per al tractament del colorant txtil Grey Lanaset G amb Trametes versicolor. PhD Thesis. Universitat Autnoma de Barcelona, Bellaterra, Spain, 2005. Bhmer U, Suhardi SH, Bley T. Decolorizing reactive textile dyes with white-rot fungi by temporary immersion cultivation. Eng Life Sci 2006;6:41720. Bollag JM, Leonowicz A. Comparative studies of extracellular fungal laccases. Appl Environ Microbiol 1984;48:84954. Bonnarme P, Jeffries TW. Mn(II) regulation of lignin peroxidases and manganese-dependent peroxidases from lignin-degrading white rot fungi. Appl Environ Microbiol 1990;56:2107. Brown MA, Zhao Z, Mauk AG. Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor. Inorg Chim Acta 2002;331:2328. Bulter T, Alcalde M, Sieber V, Meinhold P, Schlachtbauer C, Arnold FH. Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ Microbiol 2003;69:98795. Camarero S, Ibarra D, Martinez MJ, Martinez AT. Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl Environ Microbiol 2005;71:177584. Cantarella G, Galli C, Gentili P. Free radical versus electrontransfer routes of oxidation of hydrocarbons by laccase-mediator systems. Catalytic and stoichiometric procedures. J Mol Catal B-Enzym 2003;22:13544. Caunt P, Impoolsup A, Greenfield PF. Stability of recombinant plasmids in yeast. J Biotechnol 1988;8:17392. Charles M. Fermentation scale-up: problems and possibilities. Trends Biotechnol 1985;3:13449. Claus H, Faber G, Knig H. Redox-mediated decolorization of synthetic dyes by fungal laccasse. Appl Microbiol Biotechnol 2002;59:6728. Colao MC, Lupino S, Garzillo AM, Buonocore V, Ruzzi M. Heterologous expression of lcc1 gene from Trametes trogii in

4. Conclusions and outlook On the whole, it can be asserted that the combination of environmental conditions (suitable inducers, feeding medium composition, aeration, agitation system, etc.), culture technique and bioreactor design should be taken into account to produce high titres of laccase. From the data found in the literature it can be stated that a fed-batch strategy, mild agitation and the addition of copper to the culture medium favour laccase production at reactor scale. In any case, the selection of the optimal conditions will also depend on the strain and on each particular purpose. On the other hand, despite the advantages offered by SSF more studies are needed to design a system at industrial scale able to compete with SmF. In addition, SSF would contribute to the so-called green biotechnology. Also, trickle-film processing appears as a promising technology. Although not dealt with in this review, overexpression of recombinant laccases in heterologous systems has been actively studied to enhance the production of fungal laccases as well as to improve their catalytic activity and stability, during the last years. Recent results obtained in this field have opened the way for the use of directed mutagenesis or in vitro evolution for the improvement of laccase enzyme for industrial applications. Acknowledgments SRC and JLTH are Ramn y Cajal Senior Research Fellows. The authors thank the Spanish Ministry of Education and Science for promoting the Ramn y Cajal Programme. The authors thank MSc Vernica Saravia for taking the SEM photographs. References
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