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Screening of supports and inducers for laccase production by Trametes versicolor in semi-solid-state conditions
Process Biochemistry 38 (2002) 249 /255 www.elsevier.com/locate/procbio
Screening of supports and inducers for laccase production by Trametes versicolor in semi-solid-state conditions ´ ngeles Sanroma´n * Susana Rodrı´guez Couto, Marı´a Gundı´n, Miriam Lorenzo, M. A Department of Chemical Engineering, University of Vigo, 36200 Vigo, Spain Received 29 January 2002; received in revised form 12 March 2002; accepted 12 March 2002
Abstract The production of laccase by Trametes versicolor under semi-solid-state conditions has been studied. Several supports (polyurethane foam, wheat straw, barley straw, wood shavings and barley bran) have been tested in order to determine the most suitable for laccase production by the above-mentioned microorganism. Barley bran led to the highest activity levels, reaching maximum values of about 1200 U/l. In this stage of research, several factors affecting laccase production (veratryl alcohol, xylidine, fresh support and the initial C/N ratio) were investigated. Xylidine was shown to be the best inducer of laccase activity, attaining values of about 1700 U/l. Moreover, the addition of fresh support not only prolonged culture lifetime but also enhanced activity levels, reaching in all the cases values higher than 2000 U/l. In addition, the decolourisation of three structurally different dyes by cultures grown on the best operating conditions determined in the present study, was monitored in order to assess the degrading capability of the ligninolytic complex secreted by such cultures. The decolourisation of all the dyes tested was almost total (85 /96%) after 6 days of incubation. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Laccase; Semi-solid-state cultures; Support; Trametes versicolor ; Veratryl alcohol; Xylidine
1. Introduction The white-rot fungus Trametes versicolor secretes, when cultured on appropriate conditions, an extracellular oxidase named laccase. This enzyme is able to catalyse the oxidation of phenolic and non-phenolic compounds [1,2], as well as to oxidise different synthetic dyes [3 /5]. Laccase finds commercial applications in oxidation of dyes, the polymerisation of lignin and lignosulphonates, preparation of musts and wines and in fruit juice stabilisation [6,7]. The former is becoming subject to scientific scrutiny due to the increasingly environmental problems caused by dyes released in textile industry wastewater. Wastewater from the textile industry is a complex mixture of several polluting substances ranging from organochlorine-based pesticides to heavy metals associated with dyes and the dyeing process [8]. During
* Corresponding author. Tel.: /34-986-812383; fax: /34-986812382. ´ . Sanroma´n). E-mail address: [email protected] (M.A
textile processing, inefficiencies in dyeing result in large amounts of the dyestuff being directly lost to the wastewater. The amount of dye lost depends on the class of dye used, varying from only 2% loss when using basic dyes to 50% loss when certain reactive dyes are utilised. Due to increasingly stringent environmental legislation, the textile industry is seeking to develop effective wastewater remediation technologies, especially those that allow colour removal that is largely unaffected by conventional treatment systems [9]. The development of processes based on laccase enzyme seems an attractive solution due to the potential of this enzyme in degrading dyes of diverse chemical structure [10]. However, despite of this, most studies on laccase enzyme deals with its purification and characterisation instead of with its production. Therefore, considering the importance of laccases in the removal of dyes from industrial effluents, studies to find the optimal production conditions are required. Most studies on lignin biodegradation by white-rot fungi have been performed in liquid cultures, which do
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S.R. Couto et al. / Process Biochemistry 38 (2002) 249 /255
not reflect the natural living conditions of these microorganisms, since they are wood inhabiting fungi [11]. Solid-state cultivation is generally defined as a culture in which a microorganism grows on a moist waterinsoluble solid material in the absence or near absence of free liquid, resulting in semi-solid or solid cultivation systems [12]. Two main types of solid materials can be employed in such cultures: inert materials (i.e. plastic foams) and non-inert materials (agricultural residues). The former acts as an attachment place for the microorganism, whereas the latter also functions as source of nutrients. The utilisation of a solid support by the microorganism is affected by several physical and chemical factors. Among them, particle size, porosity and chemical composition are outstanding. The cost and availability are other important factors to take into account. Therefore, the selection of an adequate solid material plays an important role in the development of an efficient solid state cultivation system. The main advantages that this type of cultivation offers over liquid cultures are the reduction in production cost, since they are usually carried out using agricultural by-products, like straw and bran [2,13], and they reproduce the natural living conditions of the white-rot fungi. Production of laccase is affected by diverse typical fermentation factors such as medium composition, C and N concentration, pH, temperature, aeration rate, etc. Moreover, different aromatic compounds (gallic acid, ferulic acid, xylidine, guaiacol, syringaldazine, veratryl alcohol) have also been widely used to stimulate laccase production [14 /17]. The aim of the present work was to select an adequate support for the production of laccase by T. versicolor in semi-solid-state conditions as well as to determine the best operation conditions in order to obtain high activities of the above-mentioned enzyme. In addition, the decolourisation of three dyes belonging to different groups, by the cultures grown on the best operating conditions determined in the present study, has been monitored to assess the degrading ability of the ligninolytic fluid secreted.
2. Materials and methods 2.1. Microorganism T. versicolor (CBS100.29) was maintained at 4 8C on 2% malt agar slants and plates. 2.2. Supports 2.2.1. Inert support Cubes of polyurethane foam (edge size 0.5 cm; characteristics: density, 20 kg/m3; surface area, 4149/
10 m2/m3) were used as an inert support. Prior to use, the cubes were washed once in methanol and three times in distilled water, they were then dried at room temperature overnight. 2.2.2. Non-inert supports Barley bran, wheat straw, barley straw and wood shavings (particle length about 3/7 mm) were employed as non-inert supports. Prior to use, all supports were autoclaved at 121 8C for 20 min. 2.3. Culture conditions 2.3.1. Inert support cultures The composition of the production medium was prepared according to Tien and Kirk [18] with 10 g/l glucose as carbon source, and 20 mM acetate buffer (pH 4.5) instead of dimethylsuccinate [19]. Tween 80 (0.5% v/ v) was added at the time of inoculation to stimulate the secretion of extracellular enzymes. 2.3.2. Non-inert support cultures The composition of the production medium was identical to the polyurethane foam cultures, except that glucose was at 2 g/l, since non-inert supports provide some nutrients to the fungus. Cultivation was carried out in 250 ml Erlenmeyer flasks, under semi-solid-state conditions. The volume of medium and the amount of support employed were the same that those utilised in a previous paper on Phanerochaete chrysosporium cultures [20]. The inoculation was carried out directly in the Erlenmeyer flasks. Three agar plugs (diameter, 6 mm), from a fungal colony growing on a glucose-malt extract plate, per Erlenmeyer were used as inoculum. The culture flasks were loosely capped with cellulose stoppers, which permitted a passive aeration and static incubation at an air temperature of 30 8C and 90% humidity, to avoid evaporation, was carried out in complete darkness. 2.3.3. Decolourisation of several synthetic dyes In vivo decolourisation of several synthetic dyes (Table 1), by the cultures grown under the best operating conditions established in this study, was assessed. The dyes were aseptically added to 3 day-old cultures as an aqueous solution at a final concentration of 0.02% (w/v). Two control tests were conducted in parallel: a biotic control (without dye) and an abiotic control (without fungus). In the former ligninolytic activities were analysed and in the latter the physical adsorption of the dye to the carrier was evaluated. Experiments were done in duplicate and samples were analysed three times.
S.R. Couto et al. / Process Biochemistry 38 (2002) 249 /255
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Table 1 Percentage of dye decolourisation obtained by T. versicolor grown in the best operating conditions after 2, 4 and 6 days of dye incubation Dye
Acid fuchsine Congo red Indigo carmine
Class
Triarylmethane Azo Anthraquinonic
lmax (nm)
529 498 611
Decolourisation (%) 2 days
4 days
6 days
32 68 53
46 75 65
85 90 96
The experiments were performed twice and the samples of each one were analysed three times. The values in the figures correspond to mean values, being the standard deviation less than 15%. Decolourisation percentage was determined as indicated in a previous work [24].
2.4. Analytical methods 2.4.1. Reducing sugars These were measured by a dinitrosalicylic acid method using D-glucose as a standard, according to Ghose [21].
3. Results and discussion
3.1. Selection of the support
2.4.3. Laccase activity This was determined spectrophotometrically by the method of Niku-Paavola et al. [23] with 2,2?-azino-di-[3ethyl-benzothiazoline-(6)-sulphonic acid] (ABTS, Boehringer) as a substrate. The laccase reaction mixture (in a total volume of 3 ml) contained 2.3 ml enzyme diluted to buffer (0.025 M succinic acid, pH 4.5) and 0.7 ml 0.02 M ABTS. The reaction was monitored at room temperature by measuring the change in A436 for 2 min. One unit was defined as the amount of enzyme that oxidised 1 mmol of ABTS per minute and the activities were expressed in U/l.
When polyurethane foam was employed as an inert support laccase activity first appeared on the 7th day (28 U/l) and then increased reaching a maximum value of 204 U/l on the 17th day (Fig. 1). When wood shavings were used as a non-inert support laccase activity began on the 3rd day (184 U/ l), and then increased progressively attaining a maximum value of 451 U/l on the 18th day. This value is 2fold higher than that obtained employing polyurethane foam as a support. When wheat straw was used as a non-inert support laccase activity started on the 3rd day (195 U/l), and then increased gradually achieving a maximum value of 490 U/l on the 17th day (Fig. 1). This value is also 2-fold higher than that obtained employing polyurethane foam as a support. When barley straw was used as a non-inert support laccase activity first appeared on the 3rd day (279 U/l), and then increased progressively reaching a maximum value of 591 U/l on the 18th day (Fig. 1). This value is
2.4.4. Estimation of decolourising ability Culture broth was collected immediately after dye addition and every day thereafter. It was centrifuged (8000 /g , 10 min) to eliminate suspended particles and the residual dye concentration was measured spectrophotometrically at the maximum visible absorbance of each dye (Table 1). Decolourisation percentage was determined as indicated in a previous work [24]. The physical adsorption of the dye to the carrier was evaluated, detecting that it was insignificant. This is due to the fungus grows not only on the surface of the carrier but also inside the carrier, decolourising the dye adsorbed onto the support. Dye adsorption to the mycelia was also tested. Spectrophotometric examinations of methanol extracts of the fungal mats showed that the dye bound to the mycelium of T. versicolor was insignificant.
Fig. 1. Laccase production in semi-solid-state cultures of T. versicolor grown on different supports: \ Polyurethane foam; % Barley bran; " Wood shavings; j Wheat straw; m Barley straw. The data shown are the mean of duplicate experiments.
2.4.2. Nitrogen ammonium content It was assayed by the phenol-hypochlorite method described by Weatherburn [22], using NH4Cl as a standard.
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almost 3-fold higher than that obtained employing polyurethane foam as a support. When barley bran was used as a non-inert support laccase began on the 4th day (130 U/l), increased up to 11th day and then increased sharply attaining a maximum value of 1155 U/l on the 18th day (Fig. 1). This value is almost 6-fold higher than that attained in the polyurethane foam cultures. The results attained clearly showed that non-inert supports led to higher activities than polyurethane foam, especially employing barley bran as a support. This could be due to the latter providing to the fungus an environment closer to its natural habitat (wood), with which maybe the fungus is more stimulated for the secretion of ligninolytic enzymes. Moreover, the cellulose content of these types of support could act as an activator of laccase activity [25]. This is an important result, since the non-inert supports employed are agricultural residues abundant in most countries. Moreover, these supports provide some nutrients to the fungus, due to which the initial amount of glucose in the culture medium was reduced in 80%, involving a great reduction in process costs. Therefore, in view of these results, the following experiments were performed employing barley bran as a support. In the subsequent sections, several operational variables affecting laccase production were investigated.
In all cases, a control culture (without additives) was also performed for comparison.
4.1. Effect of inducers At the beginning of the cultivation, there was an initial increase in reducing sugars from 2 (initial value) to 10/12 g/l on the 2nd day, which is due to a release of the sugars contained in the barley bran. From 2nd to 8th day, glucose was depleted more or less progressively, but
4. Effect of several variables on laccase activity After determining the best support, several operational variables were studied in order to select the most suitable conditions for laccase production. The factors studied were the addition of inducers of laccase activity, the addition of fresh support and the initial concentration of both carbon and ammonium in the culture medium. At the beginning of the cultivation some culture flasks were supplemented with veratryl alcohol (2 mM) and others with xylidine (1 mM) in order to study their influence on laccase production. The concentration of veratryl alcohol employed was selected according to Tonon and Odier [26]. As regards xylidine concentration, it was chosen taking into account the investigations done by Collins and Dobson [27] and Lee et al. [28]. In addition to this, after 12 days of operation fresh support (barley bran) was added to the cultures to study the possibility of enhancing culture lifetime, which would suppose an important reduction in process costs. Furthermore, a parallel cultivation with 5 g/l of glucose and 0.1 g/l of ammonium tartrate was performed to investigate the influence of the initial concentration of both carbon and ammonium on laccase production.
Fig. 2. Glucose and ammonium consumption, and laccase production in semi-solid-state cultures of T. versicolor grown on barley bran, supplement with different inducers: m xylidine-supplemented cultures; O veratryl alcohol-supplemented cultures; % control cultures. The data shown are the mean of duplicate experiments.
S.R. Couto et al. / Process Biochemistry 38 (2002) 249 /255
from 8th day onwards a residual value around 1 g/l remained in the culture (Fig. 2). Ammonium nitrogen showed a similar profile to reducing sugars, exhibiting an increase from 20 /25 (initial value) to 35/40 mg/l on the 2nd day. This is also due to the release of some compounds from barley bran. Ammonium nitrogen was totally consumed on 6th day, coinciding with the onset of the enzymatic activity. This suggests that laccase is mainly triggered by nitrogen limitation. Xylidine and control cultures exhibited two peaks of ammonium nitrogen at the end of the cultivation with values around 15 mg/l (Fig. 2). This could indicate that autolysis of the fungus is occurring, which would mean the end of the cultivation. As it can be observed in Fig. 2, supplementing the culture medium with veratryl alcohol or xylidine improved laccase activity levels. The latter gave the best results, showing activity values about 1700 U/l. Therefore, xylidine seems to be the best inducer of laccase activities under the conditions developed in the present study. This is in agreement with Kwang et al. [26], who found that the production of laccase increased 7-fold when 2,5-xylidine was added as an inducer to cultures of Trametes sp. CJ-105. From the two inducers tested in the present work, xylidine was shown to be the most effective, presenting values around 1700 U/l, which implies an increase of about 13% in relation to veratryl alcohol cultures and around 50% compared with control ones. In addition, the results also showed the superiority of xylidine as a laccase inducer over veratryl alcohol.
4.2. Feed-batch support The addition of more support (the same amount as the initial one) to the above cultures after 12 days of cultivation considerably increased laccase activities compared with cultures without addition, attaining values higher than 2000 U/l in all cases (Fig. 3). This indicates that it is possible not only to restart the process by adding a new amount of support to the cultures, but also to reactivate the enzyme production. This is a very interesting finding, since it would result in enzyme production for long periods at a very low cost. The addition of a new amount of support after 12 days of cultivation led to very high activity levels for several days, indicating that by adding fresh support to the cultures it is possible not only to prolong the cultivation but also to enhance activity levels. It implies that the fungus is able to metabolise the nutrients contained in the support. To metabolise the celluloses and hemicelluloses contained in the support lignin has to be, at least, partly destroyed. Therefore, the fungus has to secrete lignin-degrading enzymes.
253
Fig. 3. Laccase production in semi-solid-state cultures of T. versicolor grown on barley bran, supplement with different inducers after adding new support: m xylidine-supplemented cultures; k veratryl alcoholsupplemented cultures; % control cultures; --------cultures with new support; */ */ */cultures without new support. The data shown are the mean of duplicate experiments.
4.3. Effect of the initial concentration of both carbon and ammonium In this case the initial amount of glucose and ammonium tartrate added at the culture medium at the beginning of the cultivation were of 5 and 0.1 g/l, respectively. This implies a C/N ratio 5-fold higher than that employed in previous cultures. As shown in Fig. 4, glucose, measured as reducing sugars, increased from 5 to 12/14 g/l on the 2nd day of cultivation as it occurred in the cultures started with 2 g/ l. Therefore, at the beginning of cultivation the initial amount of glucose available for the microorganism was similar in both cases. In fact, the profiles obtained were analogous to those attained in the cultures with 2 g/l of glucose. As for ammonium nitrogen, an initial increase from 10 mg/l at day zero to 20/25 mg/l on 1st day was observed. These concentrations are about half those employed in the previous cultures. On the other hand, the profiles obtained were quite similar to those attained in the cultures with 0.2 g/l of ammonium tartrate. In view of the above results the C/N ratio employed was about twice higher than that employed in the previous cultures, since the initial glucose levels were almost identical. Laccase activity began very early, specially in xylidine cultures, reaching maximum values of 505, 796 and 1307 U/l for control, veratryl alcohol and xylidine cultures, respectively. These values are considerably lower than those attained in cultures started with 0.2 g/l of ammonium tartrate. So, a low initial ammonium concentration led to lower laccase levels. Maybe, a lower initial amount of ammonium nitrogen would provoke a decrease in the fungal growth, producing a lower
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different, indicating the non-specificity of the ligninolytic complex secreted by this fungus. This makes this organism very suitable for the application to the decolourisation of effluents from the textile industry. The enzymic complex produced by T. versicolor grown under the best conditions determined in the present study was able to decolourise three structurally different dyes. This suggests its possible application to the decolourisation of effluents containing dye mixtures, which would mean a great advance in wastewater treatment.
Acknowledgements This research was financed by XUNTA (PGIDT00PXI30118PR).
References
Fig. 4. Laccase production in semi-solid-state cultures of T. versicolor grown on barley bran, supplement with different inducers, with 0.1 g/l of ammonium tartrate: m xylidine-supplemented cultures; k veratryl alcohol-supplemented cultures; % control cultures. The data shown are the mean of duplicate experiments.
enzyme secretion. Therefore, an initial concentration of 2g/l of carbon and 0.2 g/l of ammonium (C/N ratio of 10) was optimal for laccase production under the conditions assayed in this work.
5. Decolourisation of different dyes The decolourisation reached by the different dyes studied is indicated in Table 1. Decolourisation of all the dyes was almost total after 6 days of incubation. This shows that the enzymic complex produced by T. versicolor is able to decolourise three dyes structurally
[1] Bourbonnais R, Paice MG. Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of ABTS. Appl Environ Microbiol 1997;36:823 /7. [2] Krastanov A. Removal of phenols from mixtures by co-immobilized laccase/tyrosinase and Polyclar adsorption. J Ind Microbiol Biotechnol 2000;24:383 /8. [3] Rodrı´guez E, Pickard MA, Vazquez-Duhalt R. Industrial dye decolorization by laccases from ligninolytic fungi. Curr Microbiol 1999;38:27 /32. [4] Swamy J, Ramsay JA. Effects of Mn 2 and NH4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor . Appl Microbiol Biotechnol 1999;51:391 /6. [5] Wong Y, Yu J. Laccase-catalysed decolorization of synthetic dyes. Water Res 1999;33:3512 /20. [6] Cantarelli C, Brenna O, Giovanelli G, Rossi M. Beer stabilization through enzymatic removal of phenolics. Food Biotechnol 1989;3:203 /13. [7] Giovanelli G, Ravasini G. Apple juice stabilization by combined enzyme membrane filtration process. Lebebsm-Wissu-Tes 1993;26:1 /7. [8] Correia VM, Stephenson T, Judd SJ. Characterisation of textilewastewaters */a review. Environ Technol 1994;15:917 /29. [9] O’Neill C, Lopez A, Esteves S, Hawkes FR, Hawkes DL, Wilcox S. Azo-dye degradation in an anaerobic-aerobic treatment system operating on simulated textile effluent. Appl Microbiol Biotechnol 2000;53:249 /54. [10] Thurston CF. The structure and function of fungal laccases. Microbiology 1994;140:19 /26. [11] Vares T, Ligninolytic enzymes and lignin-degrading activity of taxonomically different white-rot fungi. M.Sc. Thesis, University of Helsinki, Finland, 1996. [12] Hesseltine CW. Solid state fermentation: an overview. Int Biodeter 1987;23:75 /89. [13] Pandey A. Recent process developments in solid-state fermentation. Process Biochem 1992;27:109 /17. [14] Eggert C, Temp U, Eriksson KEL. The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus : purification and characterization of the laccase. Appl Environ Microbiol 1996;62:1151 /8.
S.R. Couto et al. / Process Biochemistry 38 (2002) 249 /255 [15] Fortina MG, Acquati A, Rossi P, Manachini PL, Di Genaro C. Production of laccase by Botrytis cinerea and fermentation studies with strain F226. J Ind Microbiol 1996;17:69 /72. [16] Rescigno A, Sollai F, Curreli N, Rinaldi A, Sanjust E. An extracellular laccase from Pleurotus sajor-caju . Ital J Biochem 1993;42:227A /8A. [17] Skorobogatko OV, Stepanova EV, Cavrilova VP, Yaropolov AI. Effects of inducers on the synthesis of extracellular laccase by Coriolus hirsutus , a basidal fungus. Prikl Biokhim Mikrobiol 1996;32:545 /8. [18] Tien M, Kirk TK. Lignin peroxidase of Phanerochate chrysosporium . Meth Enzymol 1988;161:238 /49. [19] Dosoretz CG, Chen HC, Grethlein ME. Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium . Appl Microbiol Biotechnol 1990;34:131 /7. ´. [20] Rodrı´guez Couto S, Longo MA, Cameselle C, Sanroma´n MA Ligninolytic enzymes from corncob cultures of Phanerochaete chrysosporium in semi-solid state conditions. Acta Biotechnol 1999;19:17 /25. [21] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59:257 /68.
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[22] Weatherburn MW. Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 1967;28:971 /4. [23] Niku-Paavola M-L, Raaska L, Ita¨vaara M. Detection of whiterot fungi by a non-toxic stain. Mycol Res 1990;94:27 /31. ´ . In vivo [24] Rodrı´guez Cout S, Taylor S, Rivela I, Sanroma´n MA decolourisation of the polymeric dye Poly R-478 by corncob cultures of Phanerochaete chrysosporium . Acta Biotechnol 2000;20:31 /8. [25] Srinivasan C, D’Souza TM, Boominathan K, Reddy CA. Demonstration of laccase in the white rot basidiomycete Phanerochaete chrysosporium BKM-F-1767. Appl Environ Microbiol 1995;61:4274 /7. [26] Tonon F, Odier E. Influence of veratryl alcohol and hydrogen peroxide on ligninase activity and ligninase production by Phanerochaete chrysosporium . Appl Environ Microbiol 1988;54:466 /72. [27] Collins PJ, Dobson ADW. Regulation of laccase gene transcription in Trametes versicolor . Appl Environ Microbiol 1997;63:3444 /50. [28] Lee I-Y, Jung K-H, Lee C, Park Y-H. Enhanced production of laccase in Trametes versicolor by the addition of ethanol. Biotechnol Lett 1999;21:965 /8.
Screening of supports and inducers for laccase production by Trametes versicolor in semi-solid-state conditions ´ ngeles Sanroma´n * Susana Rodrı´guez Couto, Marı´a Gundı´n, Miriam Lorenzo, M. A Department of Chemical Engineering, University of Vigo, 36200 Vigo, Spain Received 29 January 2002; received in revised form 12 March 2002; accepted 12 March 2002
Abstract The production of laccase by Trametes versicolor under semi-solid-state conditions has been studied. Several supports (polyurethane foam, wheat straw, barley straw, wood shavings and barley bran) have been tested in order to determine the most suitable for laccase production by the above-mentioned microorganism. Barley bran led to the highest activity levels, reaching maximum values of about 1200 U/l. In this stage of research, several factors affecting laccase production (veratryl alcohol, xylidine, fresh support and the initial C/N ratio) were investigated. Xylidine was shown to be the best inducer of laccase activity, attaining values of about 1700 U/l. Moreover, the addition of fresh support not only prolonged culture lifetime but also enhanced activity levels, reaching in all the cases values higher than 2000 U/l. In addition, the decolourisation of three structurally different dyes by cultures grown on the best operating conditions determined in the present study, was monitored in order to assess the degrading capability of the ligninolytic complex secreted by such cultures. The decolourisation of all the dyes tested was almost total (85 /96%) after 6 days of incubation. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Laccase; Semi-solid-state cultures; Support; Trametes versicolor ; Veratryl alcohol; Xylidine
1. Introduction The white-rot fungus Trametes versicolor secretes, when cultured on appropriate conditions, an extracellular oxidase named laccase. This enzyme is able to catalyse the oxidation of phenolic and non-phenolic compounds [1,2], as well as to oxidise different synthetic dyes [3 /5]. Laccase finds commercial applications in oxidation of dyes, the polymerisation of lignin and lignosulphonates, preparation of musts and wines and in fruit juice stabilisation [6,7]. The former is becoming subject to scientific scrutiny due to the increasingly environmental problems caused by dyes released in textile industry wastewater. Wastewater from the textile industry is a complex mixture of several polluting substances ranging from organochlorine-based pesticides to heavy metals associated with dyes and the dyeing process [8]. During
* Corresponding author. Tel.: /34-986-812383; fax: /34-986812382. ´ . Sanroma´n). E-mail address: [email protected] (M.A
textile processing, inefficiencies in dyeing result in large amounts of the dyestuff being directly lost to the wastewater. The amount of dye lost depends on the class of dye used, varying from only 2% loss when using basic dyes to 50% loss when certain reactive dyes are utilised. Due to increasingly stringent environmental legislation, the textile industry is seeking to develop effective wastewater remediation technologies, especially those that allow colour removal that is largely unaffected by conventional treatment systems [9]. The development of processes based on laccase enzyme seems an attractive solution due to the potential of this enzyme in degrading dyes of diverse chemical structure [10]. However, despite of this, most studies on laccase enzyme deals with its purification and characterisation instead of with its production. Therefore, considering the importance of laccases in the removal of dyes from industrial effluents, studies to find the optimal production conditions are required. Most studies on lignin biodegradation by white-rot fungi have been performed in liquid cultures, which do
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 8 7 - 0
250
S.R. Couto et al. / Process Biochemistry 38 (2002) 249 /255
not reflect the natural living conditions of these microorganisms, since they are wood inhabiting fungi [11]. Solid-state cultivation is generally defined as a culture in which a microorganism grows on a moist waterinsoluble solid material in the absence or near absence of free liquid, resulting in semi-solid or solid cultivation systems [12]. Two main types of solid materials can be employed in such cultures: inert materials (i.e. plastic foams) and non-inert materials (agricultural residues). The former acts as an attachment place for the microorganism, whereas the latter also functions as source of nutrients. The utilisation of a solid support by the microorganism is affected by several physical and chemical factors. Among them, particle size, porosity and chemical composition are outstanding. The cost and availability are other important factors to take into account. Therefore, the selection of an adequate solid material plays an important role in the development of an efficient solid state cultivation system. The main advantages that this type of cultivation offers over liquid cultures are the reduction in production cost, since they are usually carried out using agricultural by-products, like straw and bran [2,13], and they reproduce the natural living conditions of the white-rot fungi. Production of laccase is affected by diverse typical fermentation factors such as medium composition, C and N concentration, pH, temperature, aeration rate, etc. Moreover, different aromatic compounds (gallic acid, ferulic acid, xylidine, guaiacol, syringaldazine, veratryl alcohol) have also been widely used to stimulate laccase production [14 /17]. The aim of the present work was to select an adequate support for the production of laccase by T. versicolor in semi-solid-state conditions as well as to determine the best operation conditions in order to obtain high activities of the above-mentioned enzyme. In addition, the decolourisation of three dyes belonging to different groups, by the cultures grown on the best operating conditions determined in the present study, has been monitored to assess the degrading ability of the ligninolytic fluid secreted.
2. Materials and methods 2.1. Microorganism T. versicolor (CBS100.29) was maintained at 4 8C on 2% malt agar slants and plates. 2.2. Supports 2.2.1. Inert support Cubes of polyurethane foam (edge size 0.5 cm; characteristics: density, 20 kg/m3; surface area, 4149/
10 m2/m3) were used as an inert support. Prior to use, the cubes were washed once in methanol and three times in distilled water, they were then dried at room temperature overnight. 2.2.2. Non-inert supports Barley bran, wheat straw, barley straw and wood shavings (particle length about 3/7 mm) were employed as non-inert supports. Prior to use, all supports were autoclaved at 121 8C for 20 min. 2.3. Culture conditions 2.3.1. Inert support cultures The composition of the production medium was prepared according to Tien and Kirk [18] with 10 g/l glucose as carbon source, and 20 mM acetate buffer (pH 4.5) instead of dimethylsuccinate [19]. Tween 80 (0.5% v/ v) was added at the time of inoculation to stimulate the secretion of extracellular enzymes. 2.3.2. Non-inert support cultures The composition of the production medium was identical to the polyurethane foam cultures, except that glucose was at 2 g/l, since non-inert supports provide some nutrients to the fungus. Cultivation was carried out in 250 ml Erlenmeyer flasks, under semi-solid-state conditions. The volume of medium and the amount of support employed were the same that those utilised in a previous paper on Phanerochaete chrysosporium cultures [20]. The inoculation was carried out directly in the Erlenmeyer flasks. Three agar plugs (diameter, 6 mm), from a fungal colony growing on a glucose-malt extract plate, per Erlenmeyer were used as inoculum. The culture flasks were loosely capped with cellulose stoppers, which permitted a passive aeration and static incubation at an air temperature of 30 8C and 90% humidity, to avoid evaporation, was carried out in complete darkness. 2.3.3. Decolourisation of several synthetic dyes In vivo decolourisation of several synthetic dyes (Table 1), by the cultures grown under the best operating conditions established in this study, was assessed. The dyes were aseptically added to 3 day-old cultures as an aqueous solution at a final concentration of 0.02% (w/v). Two control tests were conducted in parallel: a biotic control (without dye) and an abiotic control (without fungus). In the former ligninolytic activities were analysed and in the latter the physical adsorption of the dye to the carrier was evaluated. Experiments were done in duplicate and samples were analysed three times.
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Table 1 Percentage of dye decolourisation obtained by T. versicolor grown in the best operating conditions after 2, 4 and 6 days of dye incubation Dye
Acid fuchsine Congo red Indigo carmine
Class
Triarylmethane Azo Anthraquinonic
lmax (nm)
529 498 611
Decolourisation (%) 2 days
4 days
6 days
32 68 53
46 75 65
85 90 96
The experiments were performed twice and the samples of each one were analysed three times. The values in the figures correspond to mean values, being the standard deviation less than 15%. Decolourisation percentage was determined as indicated in a previous work [24].
2.4. Analytical methods 2.4.1. Reducing sugars These were measured by a dinitrosalicylic acid method using D-glucose as a standard, according to Ghose [21].
3. Results and discussion
3.1. Selection of the support
2.4.3. Laccase activity This was determined spectrophotometrically by the method of Niku-Paavola et al. [23] with 2,2?-azino-di-[3ethyl-benzothiazoline-(6)-sulphonic acid] (ABTS, Boehringer) as a substrate. The laccase reaction mixture (in a total volume of 3 ml) contained 2.3 ml enzyme diluted to buffer (0.025 M succinic acid, pH 4.5) and 0.7 ml 0.02 M ABTS. The reaction was monitored at room temperature by measuring the change in A436 for 2 min. One unit was defined as the amount of enzyme that oxidised 1 mmol of ABTS per minute and the activities were expressed in U/l.
When polyurethane foam was employed as an inert support laccase activity first appeared on the 7th day (28 U/l) and then increased reaching a maximum value of 204 U/l on the 17th day (Fig. 1). When wood shavings were used as a non-inert support laccase activity began on the 3rd day (184 U/ l), and then increased progressively attaining a maximum value of 451 U/l on the 18th day. This value is 2fold higher than that obtained employing polyurethane foam as a support. When wheat straw was used as a non-inert support laccase activity started on the 3rd day (195 U/l), and then increased gradually achieving a maximum value of 490 U/l on the 17th day (Fig. 1). This value is also 2-fold higher than that obtained employing polyurethane foam as a support. When barley straw was used as a non-inert support laccase activity first appeared on the 3rd day (279 U/l), and then increased progressively reaching a maximum value of 591 U/l on the 18th day (Fig. 1). This value is
2.4.4. Estimation of decolourising ability Culture broth was collected immediately after dye addition and every day thereafter. It was centrifuged (8000 /g , 10 min) to eliminate suspended particles and the residual dye concentration was measured spectrophotometrically at the maximum visible absorbance of each dye (Table 1). Decolourisation percentage was determined as indicated in a previous work [24]. The physical adsorption of the dye to the carrier was evaluated, detecting that it was insignificant. This is due to the fungus grows not only on the surface of the carrier but also inside the carrier, decolourising the dye adsorbed onto the support. Dye adsorption to the mycelia was also tested. Spectrophotometric examinations of methanol extracts of the fungal mats showed that the dye bound to the mycelium of T. versicolor was insignificant.
Fig. 1. Laccase production in semi-solid-state cultures of T. versicolor grown on different supports: \ Polyurethane foam; % Barley bran; " Wood shavings; j Wheat straw; m Barley straw. The data shown are the mean of duplicate experiments.
2.4.2. Nitrogen ammonium content It was assayed by the phenol-hypochlorite method described by Weatherburn [22], using NH4Cl as a standard.
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almost 3-fold higher than that obtained employing polyurethane foam as a support. When barley bran was used as a non-inert support laccase began on the 4th day (130 U/l), increased up to 11th day and then increased sharply attaining a maximum value of 1155 U/l on the 18th day (Fig. 1). This value is almost 6-fold higher than that attained in the polyurethane foam cultures. The results attained clearly showed that non-inert supports led to higher activities than polyurethane foam, especially employing barley bran as a support. This could be due to the latter providing to the fungus an environment closer to its natural habitat (wood), with which maybe the fungus is more stimulated for the secretion of ligninolytic enzymes. Moreover, the cellulose content of these types of support could act as an activator of laccase activity [25]. This is an important result, since the non-inert supports employed are agricultural residues abundant in most countries. Moreover, these supports provide some nutrients to the fungus, due to which the initial amount of glucose in the culture medium was reduced in 80%, involving a great reduction in process costs. Therefore, in view of these results, the following experiments were performed employing barley bran as a support. In the subsequent sections, several operational variables affecting laccase production were investigated.
In all cases, a control culture (without additives) was also performed for comparison.
4.1. Effect of inducers At the beginning of the cultivation, there was an initial increase in reducing sugars from 2 (initial value) to 10/12 g/l on the 2nd day, which is due to a release of the sugars contained in the barley bran. From 2nd to 8th day, glucose was depleted more or less progressively, but
4. Effect of several variables on laccase activity After determining the best support, several operational variables were studied in order to select the most suitable conditions for laccase production. The factors studied were the addition of inducers of laccase activity, the addition of fresh support and the initial concentration of both carbon and ammonium in the culture medium. At the beginning of the cultivation some culture flasks were supplemented with veratryl alcohol (2 mM) and others with xylidine (1 mM) in order to study their influence on laccase production. The concentration of veratryl alcohol employed was selected according to Tonon and Odier [26]. As regards xylidine concentration, it was chosen taking into account the investigations done by Collins and Dobson [27] and Lee et al. [28]. In addition to this, after 12 days of operation fresh support (barley bran) was added to the cultures to study the possibility of enhancing culture lifetime, which would suppose an important reduction in process costs. Furthermore, a parallel cultivation with 5 g/l of glucose and 0.1 g/l of ammonium tartrate was performed to investigate the influence of the initial concentration of both carbon and ammonium on laccase production.
Fig. 2. Glucose and ammonium consumption, and laccase production in semi-solid-state cultures of T. versicolor grown on barley bran, supplement with different inducers: m xylidine-supplemented cultures; O veratryl alcohol-supplemented cultures; % control cultures. The data shown are the mean of duplicate experiments.
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from 8th day onwards a residual value around 1 g/l remained in the culture (Fig. 2). Ammonium nitrogen showed a similar profile to reducing sugars, exhibiting an increase from 20 /25 (initial value) to 35/40 mg/l on the 2nd day. This is also due to the release of some compounds from barley bran. Ammonium nitrogen was totally consumed on 6th day, coinciding with the onset of the enzymatic activity. This suggests that laccase is mainly triggered by nitrogen limitation. Xylidine and control cultures exhibited two peaks of ammonium nitrogen at the end of the cultivation with values around 15 mg/l (Fig. 2). This could indicate that autolysis of the fungus is occurring, which would mean the end of the cultivation. As it can be observed in Fig. 2, supplementing the culture medium with veratryl alcohol or xylidine improved laccase activity levels. The latter gave the best results, showing activity values about 1700 U/l. Therefore, xylidine seems to be the best inducer of laccase activities under the conditions developed in the present study. This is in agreement with Kwang et al. [26], who found that the production of laccase increased 7-fold when 2,5-xylidine was added as an inducer to cultures of Trametes sp. CJ-105. From the two inducers tested in the present work, xylidine was shown to be the most effective, presenting values around 1700 U/l, which implies an increase of about 13% in relation to veratryl alcohol cultures and around 50% compared with control ones. In addition, the results also showed the superiority of xylidine as a laccase inducer over veratryl alcohol.
4.2. Feed-batch support The addition of more support (the same amount as the initial one) to the above cultures after 12 days of cultivation considerably increased laccase activities compared with cultures without addition, attaining values higher than 2000 U/l in all cases (Fig. 3). This indicates that it is possible not only to restart the process by adding a new amount of support to the cultures, but also to reactivate the enzyme production. This is a very interesting finding, since it would result in enzyme production for long periods at a very low cost. The addition of a new amount of support after 12 days of cultivation led to very high activity levels for several days, indicating that by adding fresh support to the cultures it is possible not only to prolong the cultivation but also to enhance activity levels. It implies that the fungus is able to metabolise the nutrients contained in the support. To metabolise the celluloses and hemicelluloses contained in the support lignin has to be, at least, partly destroyed. Therefore, the fungus has to secrete lignin-degrading enzymes.
253
Fig. 3. Laccase production in semi-solid-state cultures of T. versicolor grown on barley bran, supplement with different inducers after adding new support: m xylidine-supplemented cultures; k veratryl alcoholsupplemented cultures; % control cultures; --------cultures with new support; */ */ */cultures without new support. The data shown are the mean of duplicate experiments.
4.3. Effect of the initial concentration of both carbon and ammonium In this case the initial amount of glucose and ammonium tartrate added at the culture medium at the beginning of the cultivation were of 5 and 0.1 g/l, respectively. This implies a C/N ratio 5-fold higher than that employed in previous cultures. As shown in Fig. 4, glucose, measured as reducing sugars, increased from 5 to 12/14 g/l on the 2nd day of cultivation as it occurred in the cultures started with 2 g/ l. Therefore, at the beginning of cultivation the initial amount of glucose available for the microorganism was similar in both cases. In fact, the profiles obtained were analogous to those attained in the cultures with 2 g/l of glucose. As for ammonium nitrogen, an initial increase from 10 mg/l at day zero to 20/25 mg/l on 1st day was observed. These concentrations are about half those employed in the previous cultures. On the other hand, the profiles obtained were quite similar to those attained in the cultures with 0.2 g/l of ammonium tartrate. In view of the above results the C/N ratio employed was about twice higher than that employed in the previous cultures, since the initial glucose levels were almost identical. Laccase activity began very early, specially in xylidine cultures, reaching maximum values of 505, 796 and 1307 U/l for control, veratryl alcohol and xylidine cultures, respectively. These values are considerably lower than those attained in cultures started with 0.2 g/l of ammonium tartrate. So, a low initial ammonium concentration led to lower laccase levels. Maybe, a lower initial amount of ammonium nitrogen would provoke a decrease in the fungal growth, producing a lower
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different, indicating the non-specificity of the ligninolytic complex secreted by this fungus. This makes this organism very suitable for the application to the decolourisation of effluents from the textile industry. The enzymic complex produced by T. versicolor grown under the best conditions determined in the present study was able to decolourise three structurally different dyes. This suggests its possible application to the decolourisation of effluents containing dye mixtures, which would mean a great advance in wastewater treatment.
Acknowledgements This research was financed by XUNTA (PGIDT00PXI30118PR).
References
Fig. 4. Laccase production in semi-solid-state cultures of T. versicolor grown on barley bran, supplement with different inducers, with 0.1 g/l of ammonium tartrate: m xylidine-supplemented cultures; k veratryl alcohol-supplemented cultures; % control cultures. The data shown are the mean of duplicate experiments.
enzyme secretion. Therefore, an initial concentration of 2g/l of carbon and 0.2 g/l of ammonium (C/N ratio of 10) was optimal for laccase production under the conditions assayed in this work.
5. Decolourisation of different dyes The decolourisation reached by the different dyes studied is indicated in Table 1. Decolourisation of all the dyes was almost total after 6 days of incubation. This shows that the enzymic complex produced by T. versicolor is able to decolourise three dyes structurally
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