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Increased laccase production by Trametes hirsuta grown on ground orange peelings
Enzyme and Microbial Technology 40 (2007) 1286–1290
Increased laccase production by Trametes hirsuta grown on ground orange peelings Emilio Rosales a , Susana Rodr´ıguez Couto b , Ma Angeles Sanrom´an a,∗ a
b
Department of Chemical Engineering, University of Vigo, 36200 Vigo, Spain Department of Chemical Engineering, Chemical Engineering School, Rovira i Virgili University, 43007 Tarragona, Spain Received 18 September 2006; accepted 25 September 2006
Abstract The present work focuses on the obtaining of high laccase activity levels by optimising several variables affecting laccase production under solid-state conditions. Thus, the effect of the addition of laccase-inducing compounds (syringaldazine, copper sulphate) and the amount of support utilised on laccase activity was studied. The highest activities (31,786 U/L) were obtained operating at the following conditions: 2.5 g ground orange peelings and 5 mM copper sulphate. The process was also scaled to 250 mL fixed-bed and 1.8 L tray reactors. The former produced maximal activities of about 3000 U/L, whereas the latter exhibited much higher values of around 12,000 U/L. To our knowledge, these activities are higher than those reported to date at bioreactor scale operating with a wild organism. © 2006 Elsevier Inc. All rights reserved. Keywords: Bioreactor; Laccase; Orange peelings; Solid-state fermentation; Trametes hirsuta
1. Introduction Laccases (benzenodiol:oxygen oxidoreductases; EC 1.10.3.2) have been subject of continuous study since the end of the 19th century. The genus Trametes, belonging to the white-rot fungi, is assumed to be the one of the main producing organisms. Among them, Trametes hirsuta has recently been described as a promising laccase producer [1]. The biotechnological importance of this enzyme lies in its ability to oxidise both phenolic and non-phenolic lignin related compounds [2,3] as well as highly recalcitrant environmental pollutants [4,5]. To apply this biocatalyst to large-scale biotechnological processes research focusing on obtaining high production is required. For this, several studies aimed at the promotion of enzyme production have been carried out. They are mainly based in the addition of diverse laccase-inducing substances to the culture medium, among which syringaldazine and copper sulphate are outstanding [6–9]. The selection of a substrate for SSF process depends upon several factors mainly related with cost and availability. Thus, recently there has been an increasing trend towards the utilisation of agro-industrial wastes as raw materials to
∗
Corresponding author. Fax: +34 986 812382. E-mail address: [email protected] (M.A. Sanrom´an).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.09.015
produce value-added products by this technique. The wastes generated by food processing industries like fruit peelings are a good example of this kind of materials. Thus, the production of orange and other citrus juices generates vast quantities of processing by-products, mainly peels, cores and segment membranes [10]. These by-products are not very useful as cattle feed, since their content in protein is rather low [11]. However, they are rich in both soluble and insoluble carbohydrates [10], making them an attractive raw material for the production of value-added products. In addition, the utilisation of this kind of supports helps in solving both the economic and the environmental problems caused by their disposal. However, despite the recent resurgence of SSF as a potential technology for the obtaining of value-added products, there are few designs available in the literature for bioreactors operating in solid-state conditions. This is principally due to several problems encountered in the control of different parameters such as pH, temperature, aeration and oxygen transfer and agitation [12]. The latter is particularly important when dealing with filamentous fungi. In the present work, orange peelings have been employed as a support-substrate for laccase production by T. hirsuta under SSF, since they have been previously shown to have potential as a support-substrate for laccase production [13]. Cultivation was performed in Erlenmeyer flasks and at laboratory scale
E. Rosales et al. / Enzyme and Microbial Technology 40 (2007) 1286–1290
bioreactors. For the former, the optimal culture conditions have been determined and for the latter, the effect of aeration has been analysed. 2. Materials and methods 2.1. Microorganism T. hirsuta (BT 2566), obtained from Dr. G.M. G¨ubitz (Institute for Environmental Biotechnology, Graz University of Technology, Graz, Austria) was maintained on potato dextrose agar (PDA) plates at 4 ◦ C and sub-cultured every 3 months.
2.2. Support Ground orange peelings were employed as support-substrates (size 5 mm). Orange peelings are mainly composed of both soluble and insoluble carbohydrates. The soluble sugars in orange peel are glucose, fructose and sucrose. The insoluble polysaccharides in cell walls of orange peel are composed of pectin, cellulose and hemicellulose. They also contain minor amounts of organic acids, mainly citric, malic, malonic and oxalic, proteins, mineral ions, phenolic compounds and polyols [14]. Prior to use, the peelings were pre-treated as follows: they were first soaked for 1 h in 30 mL of KOH 83.17 mM (10 g of fresh peelings) to neutralise organic acids [15]. Then, they were thoroughly washed with distilled water and dried at 30 ◦ C. The peelings were autoclaved at 121 ◦ C for 20 min.
2.3. Culture conditions 2.3.1. Cultivation in Erlenmeyer flasks The composition of the culture medium was according to Moldes et al. [16]. The basal medium containing per litre: 4 g glucose, 15 g yeast extract, 0.75 g NH4 Cl, 2 g KH2 PO4 , 0.5 g MgSO4 ·7H2 O, 0.1 g CaCl2 ·2H2 O, 0.5 g KCl and 20 mM acetate buffer (pH 4.5). The vitamin solution contained per litre 0.2 g thiamine. Basal medium was sterilised at 121 ◦ C for 20 min. After cooling, the vitamin previously sterilised by microfiltration (0.22 m) was added to the basal medium. The cultures were performed in cotton-plugged Erlenmeyer flasks (250 mL) containing 2.5 g or 5 g of ground orange peelings depending on the experiment and 15 mL of culture medium. Some cultures were supplemented with laccase-inducing compounds at the beginning of cultivation depending on the experiment. Inoculation was carried out directly in the Erlenmeyer flasks. Three agar plugs (diameter, 3 mm), from an actively growing fungus on PDA, per Erlenmeyer were used as inoculum. The Erlenmeyer flasks were incubated statically under an air atmosphere at 30 ◦ C and 90% humidity, to avoid evaporation, in complete darkness. 2.3.2. Bioreactor cultivation The composition of the culture medium was the optimal determined in the experiments at stationary flask scale. The following bioreactor configurations were considered: Fixed-bed tubular bioreactor: It consisted on a jacketed glass column with dimensions of 20 cm height and 4.5 cm in internal diameter (working volume of 200 mL). The temperature was maintained at 30 ◦ C by the circulation of temperature-controlled water. Two Erlenmeyer flasks containing T. hirsuta grown on ground orange peelings under solid-state conditions were used as inoculum, which were transferred to the bioreactor when maximal laccase activity was detected. The amount of orange peelings and the volume of medium employed were 77 g and 200 mL, respectively. Tray bioreactor: The static tray bioreactor, also known as koji bioreactor, is the generally used bioreactor for SSF. The configuration employed in this work consisted of a glass culture flask Fernbach type, conical shape (1.8 L), where the ground orange peelings were placed, forming a layer of about 1 cm of thickness (150 g orange peelings/200 mL medium). Inoculation was carried out directly in the bioreactor. Three agar plugs (diameter, 3 mm), from an actively growing
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fungus on PDA, were used as inoculum. The bioreactor was kept in a chamber at 30 ◦ C, 90% humidity and passive aeration, in complete darkness. Samples were collected once a day, centrifuged at 8000 × g for 10 min and analysed. Duplicate experiments were run for comparison and samples were analysed twice. The values in the figures correspond to mean values with a standard deviation lower than 15%.
2.4. Analytical determinations Reducing sugars were measured by the dinitrosalicylic acid method using d-glucose as a standard according to Ghose [17]. Protein concentration measurements were carried out using Bio-Rad reagent [18] with bovine serum albumin (Sigma) as a standard. pH evolution was measured by using a micro pH electrode (Sentron instruments). Laccase activity was determined spectrophotometrically as described by Niku-Paavola et al. [19] with ABTS (2,2 -azino-di-[3-ethyl-benzo-thiazolinsulphonate]) as a substrate. One activity unit was defined as the amount of enzyme that oxidised 1 mol ABTS/min. The activities were expressed in U/L.
3. Results and discussion 3.1. Flask cultivation 3.1.1. Effect of inducers on laccase activity In this section, the addition of inducers of laccase activity such as syringaldazine (0.11 M) [6] and the well-known laccase inducer copper sulphate (1 mM) [8] has been investigated. A control culture with no inducers has also been performed for comparison. The analysis of carbohydrate composition revealed that glucose, fructose and saccharose were the only carbohydrates present in the medium. Organic acids were not detected, indicating that peeling pre-treatment was effective. In all cultures, the ammonium nitrogen was depleted in 3 days, which coincided with the onset of secondary metabolism. Reducing sugars sharply increased reaching values three-fold higher than the initial one on the 2nd day and then, they abruptly decreased attaining values around 1.5 g/L during the last stage of cultivation (data not shown). This initial increase in reducing sugars could be due to the release of some hydrolysis products from the peelings. So, maybe these supports could be employed without adding any initial amount of glucose in the culture medium, which would imply an important advantage from the economical point of view. As it can be observed in Fig. 1, when the culture medium was supplemented with syringaldazine (0.11 M) laccase activity increased nearly 32% in comparison to control cultures. The addition of copper sulphate (1 mM) to the culture medium had an acute effect on laccase activity. Maximum laccase activities of nearly 20,000 U/L were obtained, which are about 3.5-fold higher than those attained in the reference cultures (Fig. 1). In addition, these activities are almost six-fold higher than those obtained in a recent paper by our research group with no copper addition and operating with a mixture of barley bran and orange peelings as a support-substrate [13]. These results clearly show the positive effect of copper sulphate as an inducer of laccase activity. They agree with those by Galhaup et al. [7], who reported that the addition of 2 mM
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Fig. 1. Laccase activities obtained by Trametes hirsuta grown in 250 mL Erlenmeyer flask on 2.5 g ground orange peelings in SSF under different conditions: (䊉) control cultures; () 1 mM copper-supplemented cultures; () 0.11 M syringaldazine-supplemented cultures.
copper sulphate considerably stimulated laccase production by Trametes pubescens in batch cultivation. These results are also in agreement with the investigations performed by Baldrian and Gabriel [8], who reported an increase in laccase activity of eight-fold by adding 1 mM copper sulphate to static cultures of Pleurotus ostreatus. To our knowledge the values of laccase activity obtained in the present study are higher than those found in the literature for Trametes species even for the newly isolated specie Trametes modesta, which produced a maximum value of 10,680 U/L in an optimised medium [20]. The high values of laccase detected are likely due to the pectin and cellulose content of the orange peelings [14], which may stimulate laccase production. In addition, they are also due to orange peelings allowed maintaining high glucose values along cultivation, which is an essential factor to keep high laccase levels by this fungus as it was shown in a recent paper by our research group [16]. This point out the enormous potential of orange peelings as a support-substrate for laccase production. 3.1.2. Effect of both copper concentration and amount of support on laccase activity Firstly, the joint effect of copper sulphate and syringaldazine on laccase activity was studied. As shown in Figs. 2 and 3 this effect was not very significant and similar activities were obtained by cultures supplemented only with 1 mM copper sulphate. So, this effect was not considered in the subsequent experiments. Finally, the amount of support employed as well as the copper sulphate concentration has been analysed in order to find the optimal ones for laccase production under solid-state conditions. For this, experiments at different copper sulphate concentrations (1 mM, 2 mM, 3.5 mM and 5 mM) and with different amounts of support (2.5 g and 5 g) were carried out. The cultures performed with 2.5 g of support, showed highest laccase activities of about 30,000 U/L when the medium was supplemented with 5 mM copper sulphate (Fig. 2). This supposed an increase higher than five-fold in relation to control cultures and nearly 60% higher
Fig. 2. Laccase activities obtained by T. hirsuta grown in 250 mL Erlenmeyer flask on 2.5 g ground orange peelings in SSF under different conditions: () 1 mM copper; () 2 mM copper; () 3.5 mM copper; () 5 mM copper; (♦) 1 mM copper + 0.11 M syringaldazine.
than those obtained in the cultures with copper sulphate at 1 mM. Moreover, the specific laccase activities, they were also higher in the 5 mM copper-supplemented cultures, indicating that the extracellular levels of protein were low. This is likely due to the activity of specific proteases produced in the high copper containing medium [21]. In the cultures with 5 g of support, the highest laccase activities (around 19,000 U/L) were also attained by the cultures supplemented with 5 mM of copper sulphate (Fig. 3). However, these values are similar to those attained previously with 1 mM copper sulphate but operating with 2.5 g of support. Therefore, operating with 2.5 g of support produced higher activities (around 58% higher). This could likely be due to the cultures are better aerated and clogging problems are avoided.
Fig. 3. Laccase activities obtained by T. hirsuta grown in 250 mL Erlenmeyer flask on 5 g ground orange peelings in SSF under different conditions: () 1 mM copper; () 2 mM copper; () 3.5 mM copper; () 5 mM copper; (♦) 1 mM copper + 0.11 M syringaldazine.
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3.2. Bioreactor cultivation As it has already been commented in the introduction section, agro-industrial residues have an enormous potential as supports for solid-state processes. However, they present several problems, which are not found operating with inert supports, such as support degradation and/or support accretion may occur along the fermentation process. This would cause mass and oxygen restrictions into the reactor bed hampering its proper performance. Nonetheless, due to the economical advantages that these materials present, it is worthy to investigate a bioreactor configuration that allows operating with them [22]. 3.2.1. Fixed-bed bioreactor Initially, laccase activity diminished as a consequence of the dilution by the addition of fresh medium. Then, from 11th day onwards the activity increased reaching values around 3000 U/L in the last stage of cultivation. As for pH, it was maintained around 4–4.5 along cultivation (Fig. 4). It was observed that the fungus grew well attached to the orange peelings, the liquid phase remaining clear along the whole cultivation. In addition, the ratio liquid volume/solid volume, allowed the moving of air bubbles throughout the bed, enabling a good oxygenation of the fungus and avoiding clogging problems. Moreover, orange peeling degradation was not detected along cultivation, indicating the suitability of such a support for performing this type of processes. 3.2.2. Tray bioreactor Laccase began on the 8th day and it increased peaking on the 23rd day (12,660 U/L). From there up to the end of cultivation laccase remained around 11,000 U/L (Fig. 4). These values are three-fold higher than those obtained by the fixed-bed bioreactor. This is likely due to the agitation produced by the fixed-bed reactor caused mechanical stress to the fungus, lessening laccase production [23]. Therefore, agitation is a key factor to take into account to design an adequate bioreactor
Fig. 4. Laccase activities obtained and pH evolution (dotted line) by T. hirsuta grown on ground orange peelings under solid-state conditions at bioreactor scale: (䊉) fixed-bed reactor; () tray reactor.
for this type of process. The results obtained agree with those reported in a recent paper by our research group [24], in which highest laccase activities by T. versicolor were attained in a tray bioreactor among several configurations tested. As regards pH, it was maintained around 4.5–5.5 along cultivation (Fig. 4). These pH values are slightly higher than those attained in the fixed-bed configuration, which indicates that a pH around 5.0 favours laccase production. This agrees with several papers, which reported that a pH value around 5.0–5.5 was very suitable for laccase production by the genus Trametes [25–28]. In view of the results obtained, it can be concluded that the tray bioreactor is the most suitable configuration for the production of laccase enzymes by T. hirsuta in solid-state conditions. This is mainly due to its shape permits a filamentous growing of the fungus, the fungus is not subjected to mechanical stress and it provides a low aeration to the fungus, avoiding microorganism stress as well as fungus overgrowth. In addition, the support is placed in a thin layer, which avoids support agglomeration.
Table 1 Maximum laccase activities obtained by several white-rot fungus at bioreactor scale cultivated under different conditions Fungus
Type of reactor
Max. laccase activity (U/L)
Ref.
Pycnoporus cinnabarinus Lentinus edodes Trametes multicolora Panus tigrinus P. tigrinus Irpex lacteus I. lacteus Trametes versicolorb T. versicolor T. versicolor T. versicolor Trametes hirsutaa T. hirsutac T. hirsutac
10 L packed-bed (nylon cubes) Column bioreactor (malt extract broth) Stirred tank 3 L stirred tank (olive mill) 3 L bubble column air-lift (olive mill) 27 mL packed-bed (polyurethane foam) 27 mL packed-bed (pine wood) 2 L airlift 2.5 L immersion (barley bran) 300 mL expanded-bed (barley bran) 1 L tray (barley bran) 1 L fixed-bed (stainless steel sponges) 200 mL fixed-bed reactor (orange peelings) 0.1 L tray reactor (orange peelings)
270 – – 4600 ± 98 410 ± 22 – – 1676 600 500–600 3000–3500 2206 4897 12,260
Schliephake et al. [29] Hatvani and M´ecs [30] Hess et al. [9] Fenice et al. [31] Fenice et al. [31] Kasinath et al. [32] Kasinath et al. [32] Ranca˜no et al. [33] Rodr´ıguez Couto et al. [24] Rodr´ıguez Couto et al. [24] Rodr´ıguez Couto et al. [24] Rodr´ıguez Couto et al. [34] This work This work
a b c
Supplemented with 1 mM copper sulphate. Supplemented with 1 mM xylidine. Supplemented with 5 mM copper sulphate.
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In summary, the results obtained in the present work are very novel, since to date most attempts performed to produce laccase at bioreactor scale have been unsuccessful or lower activities than reported here were obtained. Moreover, laccase activities were improved by far in relation to those obtained in previous papers by our research group (Table 1). 4. Conclusions In view of the results obtained, it can be concluded that orange peelings are an excellent support-substrate for laccase production by T. hirsuta under solid-state conditions due to their high content in sugars, cellulose and pectin. Operating with the appropriate amount of support-substrate and supplementing the medium with copper sulphate at a concentration of 5 mM allowed obtaining high activities at flask scale. The system was scaled to tray and fixed-bed laboratory reactors. Acknowledgements This research was financed by the Spanish Ministry of Science and Technology and European FEDER (Project CTM2004-01539/TECNO) and the University of Vigo. References [1] Vares T, Hatakka A. Lignin-degrading activity and ligninolytic enzymes of different white-rot fungi: effects of manganese and malonate. Can J Botany 1997;75:61–71. [2] Bourbonnais R, Paice MG. Oxidation of non-phenolic substrates: an expanded role of laccase in lignin biodegradation. FEBS Lett 1990;267:99–102. [3] Kadhim H, Graham C, Barrat P, Evans CS, Rastall RA. Removal of phenolic compounds in water using Coriolus versicolor grown on wheat bran. Enzyme Microb Technol 1999;24:303–7. [4] Field JA, de Jong E, Feijoo-Costa G, de Bont JAM. Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. Trends Biotechnol 1993;11:44–9. [5] Pointing SB. Feasibility of bioremediation by white-rot fungi. Appl Microbiol Biotechnol 2001;57:20–33. [6] Koroljova-Skorobogat’ko OV, Stepanova EV, Gavrilova VP, Morozova OV, Lubimova NV, Dzchafarova AN, et al. Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis. Biotechnol Appl Biochem 1998;28:47–54. [7] Galhaup C, Wagner H, Hintertoisser B, Haltrich D. Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 2002;30:529–36. [8] Baldrian P, Gabriel J. Copper and cadmium increase laccase activity in Pleurotus ostreatus. FEMS Microbiol Lett 2002;206:69–74. [9] Hess J, Leitner C, Galhaup C, Kulbe KD, Hinterstoisser B, Steinwender M, et al. Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor. Appl Biochem Biotechnol 2002;98–100:229–41. [10] Kesterson JW, Braddock RJ. Byproducts and speciality products of Florida Citrus, Bulletin 784. Gainesville, FL: Agricultural Experiment Stations, Institute of Food and Agricultural Science, University of Florida; 1976. [11] Wing JM. Citrus feedstuffs for dairy cattle. Gainesville, FL: Agricultural Experiment Stations, Institute of Food and Agricultural Sciences University of Florida; 1982.
[12] Lonsane BK, Ghildyal NP, Budiatman S, Ramakrishna SV. Engineering aspects of solid state fermentation. Enzyme Microb Technol 1985;7:258–65. [13] Rosales E, Rodr´ıguez Couto S, Sanrom´an Ma A. New uses of food waste: application to laccase production by Trametes hirsuta. Biotechnol Lett 2002;24:701–4. [14] Grohmann K, Cameron RG, Buslig BS. Fractionation and pretreatment of orange peel by dilute acid hydrolysis. Bioresour Technol 1995;54:129–41. [15] Stredansky M, Conti E. Xanthan production by solid state fermentation. Process Biochem 1999;34:581–7. [16] Moldes D, Gallego PP, Rodr´ıguez Couto S, Sanrom´an Ma A. Grape seeds: the best lignocellulosic waste to produce laccase by solid state cultures of Trametes hirsuta. Biotechnol Lett 2003;25:491–5. [17] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59:257–68. [18] Bradford MM. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Chem 1976;72:248–54. [19] Niku-Paavola ML, Raaska L, It¨avaara M. Detection of white-rot fungi by a non-toxic stain. Mycol Res 1990;94:27–31. [20] Nyanhongo GS, Gomes J, G¨ubitz G, Zvauya R, Read JS, Steiner W. Production of laccase by a newly isolated strain of Trametes modesta. Bioresour Technol 2002;84:259–63. [21] Palmieri G, Giardina P, Bianco C, Fontanella B, Sannia G. Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Appl Environ Microbiol 2000;66:920–4. [22] Pandey A, Selvakumar P, Ashakumary L. Performance of a column bioreactor for glucoamylase synthesis by Aspergillus niger in SSF. Process Biochem 1996;31:43–6. [23] Vasdev K, Kuhad RC. Induction of laccase production in Cyathus bulleri under shaking and static culture conditions. Folia Microbiol 1994;39:326–30. [24] Rodr´ıguez Couto S, Moldes D, Li´ebanas A, Sanrom´an Ma A. Investigation of several bioreactor configurations for laccase production by Trametes versicolor operating in solid-state conditions. Biochem Eng J 2003;15:21–6. [25] Swamy J, Ramsay JA. The evaluation of white rot fungi in the decoloration of textile dyes. Enzyme Microb Technol 1999;24:130–7. [26] Galhaup C, Haltrich D. Enhanced formation of laccase activity by the whiterot fungus Trametes pubescens in the presence of copper. Appl Microbiol Biotechnol 2001;56:225–32. [27] Jang MY, Ryu MM, Cho MH. Laccase production from repeated batch cultures using free mycelia of Trametes sp. Enzyme Microb Technol 2002;30:741–6. [28] Koroleva OV, Stepanova EV, Gavrilova VP, Yakovleva NS, Landesman EO, Yavmetdinov IS, et al. Laccase and Mn-peroxidase production by Coriolus hirsutus Strain 075 in a Jar Fermentor. J Biosci Bioeng 2002;93:449–55. [29] Schliephake K, Mainwaring DE, Lonergan GT, Jones IK, Baker WL. Transformation and degradation of the disazo dye Chicago Sky Blue by a purified laccase from Pycnoporus cinnabarinus. Enzyme Microb Technol 2000;27:100–7. [30] Hatvani N, M´ecs I. Production of laccase and manganese peroxidase by Lentinus edodes on malt-containing by-product of the brewing process. Process Biochem 2001;37:491–6. [31] Fenice M, Sermanni GG, Federici F. Submerged and solid-state production of laccase and Mn-peroxidase by Panus tigrinus on olive mill wastewaterbased media. J Biotechnol 2003;100:77–85. [32] Kasinath A, Novotn´y C, Svobodov´a K, Patel KC, Sasek V. Decolorization of synthetic dyes by Irpex lacteus in liquid cultures and packed-bed bioreactor. Enzyme Microb Technol 2003;32:167–73. [33] Ranca˜no G, Lorenzo M, Molares N, Rodr´ıguez Couto S, Sanrom´an Ma A. Production of laccase by Trametes versicolor in an airlift fermentor. Process Biochem 2003;39:467–73. [34] Rodr´ıguez Couto S, Sanrom´an Ma A, Hofer D, G¨ubitz GM. Stainless steel sponge: a novel carrier for the immobilisation of the white-rot fungus Trametes hirsuta for decolourisation of textile dyes. Bioresour Technol 2004;95:67–72.
Increased laccase production by Trametes hirsuta grown on ground orange peelings Emilio Rosales a , Susana Rodr´ıguez Couto b , Ma Angeles Sanrom´an a,∗ a
b
Department of Chemical Engineering, University of Vigo, 36200 Vigo, Spain Department of Chemical Engineering, Chemical Engineering School, Rovira i Virgili University, 43007 Tarragona, Spain Received 18 September 2006; accepted 25 September 2006
Abstract The present work focuses on the obtaining of high laccase activity levels by optimising several variables affecting laccase production under solid-state conditions. Thus, the effect of the addition of laccase-inducing compounds (syringaldazine, copper sulphate) and the amount of support utilised on laccase activity was studied. The highest activities (31,786 U/L) were obtained operating at the following conditions: 2.5 g ground orange peelings and 5 mM copper sulphate. The process was also scaled to 250 mL fixed-bed and 1.8 L tray reactors. The former produced maximal activities of about 3000 U/L, whereas the latter exhibited much higher values of around 12,000 U/L. To our knowledge, these activities are higher than those reported to date at bioreactor scale operating with a wild organism. © 2006 Elsevier Inc. All rights reserved. Keywords: Bioreactor; Laccase; Orange peelings; Solid-state fermentation; Trametes hirsuta
1. Introduction Laccases (benzenodiol:oxygen oxidoreductases; EC 1.10.3.2) have been subject of continuous study since the end of the 19th century. The genus Trametes, belonging to the white-rot fungi, is assumed to be the one of the main producing organisms. Among them, Trametes hirsuta has recently been described as a promising laccase producer [1]. The biotechnological importance of this enzyme lies in its ability to oxidise both phenolic and non-phenolic lignin related compounds [2,3] as well as highly recalcitrant environmental pollutants [4,5]. To apply this biocatalyst to large-scale biotechnological processes research focusing on obtaining high production is required. For this, several studies aimed at the promotion of enzyme production have been carried out. They are mainly based in the addition of diverse laccase-inducing substances to the culture medium, among which syringaldazine and copper sulphate are outstanding [6–9]. The selection of a substrate for SSF process depends upon several factors mainly related with cost and availability. Thus, recently there has been an increasing trend towards the utilisation of agro-industrial wastes as raw materials to
∗
Corresponding author. Fax: +34 986 812382. E-mail address: [email protected] (M.A. Sanrom´an).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.09.015
produce value-added products by this technique. The wastes generated by food processing industries like fruit peelings are a good example of this kind of materials. Thus, the production of orange and other citrus juices generates vast quantities of processing by-products, mainly peels, cores and segment membranes [10]. These by-products are not very useful as cattle feed, since their content in protein is rather low [11]. However, they are rich in both soluble and insoluble carbohydrates [10], making them an attractive raw material for the production of value-added products. In addition, the utilisation of this kind of supports helps in solving both the economic and the environmental problems caused by their disposal. However, despite the recent resurgence of SSF as a potential technology for the obtaining of value-added products, there are few designs available in the literature for bioreactors operating in solid-state conditions. This is principally due to several problems encountered in the control of different parameters such as pH, temperature, aeration and oxygen transfer and agitation [12]. The latter is particularly important when dealing with filamentous fungi. In the present work, orange peelings have been employed as a support-substrate for laccase production by T. hirsuta under SSF, since they have been previously shown to have potential as a support-substrate for laccase production [13]. Cultivation was performed in Erlenmeyer flasks and at laboratory scale
E. Rosales et al. / Enzyme and Microbial Technology 40 (2007) 1286–1290
bioreactors. For the former, the optimal culture conditions have been determined and for the latter, the effect of aeration has been analysed. 2. Materials and methods 2.1. Microorganism T. hirsuta (BT 2566), obtained from Dr. G.M. G¨ubitz (Institute for Environmental Biotechnology, Graz University of Technology, Graz, Austria) was maintained on potato dextrose agar (PDA) plates at 4 ◦ C and sub-cultured every 3 months.
2.2. Support Ground orange peelings were employed as support-substrates (size 5 mm). Orange peelings are mainly composed of both soluble and insoluble carbohydrates. The soluble sugars in orange peel are glucose, fructose and sucrose. The insoluble polysaccharides in cell walls of orange peel are composed of pectin, cellulose and hemicellulose. They also contain minor amounts of organic acids, mainly citric, malic, malonic and oxalic, proteins, mineral ions, phenolic compounds and polyols [14]. Prior to use, the peelings were pre-treated as follows: they were first soaked for 1 h in 30 mL of KOH 83.17 mM (10 g of fresh peelings) to neutralise organic acids [15]. Then, they were thoroughly washed with distilled water and dried at 30 ◦ C. The peelings were autoclaved at 121 ◦ C for 20 min.
2.3. Culture conditions 2.3.1. Cultivation in Erlenmeyer flasks The composition of the culture medium was according to Moldes et al. [16]. The basal medium containing per litre: 4 g glucose, 15 g yeast extract, 0.75 g NH4 Cl, 2 g KH2 PO4 , 0.5 g MgSO4 ·7H2 O, 0.1 g CaCl2 ·2H2 O, 0.5 g KCl and 20 mM acetate buffer (pH 4.5). The vitamin solution contained per litre 0.2 g thiamine. Basal medium was sterilised at 121 ◦ C for 20 min. After cooling, the vitamin previously sterilised by microfiltration (0.22 m) was added to the basal medium. The cultures were performed in cotton-plugged Erlenmeyer flasks (250 mL) containing 2.5 g or 5 g of ground orange peelings depending on the experiment and 15 mL of culture medium. Some cultures were supplemented with laccase-inducing compounds at the beginning of cultivation depending on the experiment. Inoculation was carried out directly in the Erlenmeyer flasks. Three agar plugs (diameter, 3 mm), from an actively growing fungus on PDA, per Erlenmeyer were used as inoculum. The Erlenmeyer flasks were incubated statically under an air atmosphere at 30 ◦ C and 90% humidity, to avoid evaporation, in complete darkness. 2.3.2. Bioreactor cultivation The composition of the culture medium was the optimal determined in the experiments at stationary flask scale. The following bioreactor configurations were considered: Fixed-bed tubular bioreactor: It consisted on a jacketed glass column with dimensions of 20 cm height and 4.5 cm in internal diameter (working volume of 200 mL). The temperature was maintained at 30 ◦ C by the circulation of temperature-controlled water. Two Erlenmeyer flasks containing T. hirsuta grown on ground orange peelings under solid-state conditions were used as inoculum, which were transferred to the bioreactor when maximal laccase activity was detected. The amount of orange peelings and the volume of medium employed were 77 g and 200 mL, respectively. Tray bioreactor: The static tray bioreactor, also known as koji bioreactor, is the generally used bioreactor for SSF. The configuration employed in this work consisted of a glass culture flask Fernbach type, conical shape (1.8 L), where the ground orange peelings were placed, forming a layer of about 1 cm of thickness (150 g orange peelings/200 mL medium). Inoculation was carried out directly in the bioreactor. Three agar plugs (diameter, 3 mm), from an actively growing
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fungus on PDA, were used as inoculum. The bioreactor was kept in a chamber at 30 ◦ C, 90% humidity and passive aeration, in complete darkness. Samples were collected once a day, centrifuged at 8000 × g for 10 min and analysed. Duplicate experiments were run for comparison and samples were analysed twice. The values in the figures correspond to mean values with a standard deviation lower than 15%.
2.4. Analytical determinations Reducing sugars were measured by the dinitrosalicylic acid method using d-glucose as a standard according to Ghose [17]. Protein concentration measurements were carried out using Bio-Rad reagent [18] with bovine serum albumin (Sigma) as a standard. pH evolution was measured by using a micro pH electrode (Sentron instruments). Laccase activity was determined spectrophotometrically as described by Niku-Paavola et al. [19] with ABTS (2,2 -azino-di-[3-ethyl-benzo-thiazolinsulphonate]) as a substrate. One activity unit was defined as the amount of enzyme that oxidised 1 mol ABTS/min. The activities were expressed in U/L.
3. Results and discussion 3.1. Flask cultivation 3.1.1. Effect of inducers on laccase activity In this section, the addition of inducers of laccase activity such as syringaldazine (0.11 M) [6] and the well-known laccase inducer copper sulphate (1 mM) [8] has been investigated. A control culture with no inducers has also been performed for comparison. The analysis of carbohydrate composition revealed that glucose, fructose and saccharose were the only carbohydrates present in the medium. Organic acids were not detected, indicating that peeling pre-treatment was effective. In all cultures, the ammonium nitrogen was depleted in 3 days, which coincided with the onset of secondary metabolism. Reducing sugars sharply increased reaching values three-fold higher than the initial one on the 2nd day and then, they abruptly decreased attaining values around 1.5 g/L during the last stage of cultivation (data not shown). This initial increase in reducing sugars could be due to the release of some hydrolysis products from the peelings. So, maybe these supports could be employed without adding any initial amount of glucose in the culture medium, which would imply an important advantage from the economical point of view. As it can be observed in Fig. 1, when the culture medium was supplemented with syringaldazine (0.11 M) laccase activity increased nearly 32% in comparison to control cultures. The addition of copper sulphate (1 mM) to the culture medium had an acute effect on laccase activity. Maximum laccase activities of nearly 20,000 U/L were obtained, which are about 3.5-fold higher than those attained in the reference cultures (Fig. 1). In addition, these activities are almost six-fold higher than those obtained in a recent paper by our research group with no copper addition and operating with a mixture of barley bran and orange peelings as a support-substrate [13]. These results clearly show the positive effect of copper sulphate as an inducer of laccase activity. They agree with those by Galhaup et al. [7], who reported that the addition of 2 mM
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Fig. 1. Laccase activities obtained by Trametes hirsuta grown in 250 mL Erlenmeyer flask on 2.5 g ground orange peelings in SSF under different conditions: (䊉) control cultures; () 1 mM copper-supplemented cultures; () 0.11 M syringaldazine-supplemented cultures.
copper sulphate considerably stimulated laccase production by Trametes pubescens in batch cultivation. These results are also in agreement with the investigations performed by Baldrian and Gabriel [8], who reported an increase in laccase activity of eight-fold by adding 1 mM copper sulphate to static cultures of Pleurotus ostreatus. To our knowledge the values of laccase activity obtained in the present study are higher than those found in the literature for Trametes species even for the newly isolated specie Trametes modesta, which produced a maximum value of 10,680 U/L in an optimised medium [20]. The high values of laccase detected are likely due to the pectin and cellulose content of the orange peelings [14], which may stimulate laccase production. In addition, they are also due to orange peelings allowed maintaining high glucose values along cultivation, which is an essential factor to keep high laccase levels by this fungus as it was shown in a recent paper by our research group [16]. This point out the enormous potential of orange peelings as a support-substrate for laccase production. 3.1.2. Effect of both copper concentration and amount of support on laccase activity Firstly, the joint effect of copper sulphate and syringaldazine on laccase activity was studied. As shown in Figs. 2 and 3 this effect was not very significant and similar activities were obtained by cultures supplemented only with 1 mM copper sulphate. So, this effect was not considered in the subsequent experiments. Finally, the amount of support employed as well as the copper sulphate concentration has been analysed in order to find the optimal ones for laccase production under solid-state conditions. For this, experiments at different copper sulphate concentrations (1 mM, 2 mM, 3.5 mM and 5 mM) and with different amounts of support (2.5 g and 5 g) were carried out. The cultures performed with 2.5 g of support, showed highest laccase activities of about 30,000 U/L when the medium was supplemented with 5 mM copper sulphate (Fig. 2). This supposed an increase higher than five-fold in relation to control cultures and nearly 60% higher
Fig. 2. Laccase activities obtained by T. hirsuta grown in 250 mL Erlenmeyer flask on 2.5 g ground orange peelings in SSF under different conditions: () 1 mM copper; () 2 mM copper; () 3.5 mM copper; () 5 mM copper; (♦) 1 mM copper + 0.11 M syringaldazine.
than those obtained in the cultures with copper sulphate at 1 mM. Moreover, the specific laccase activities, they were also higher in the 5 mM copper-supplemented cultures, indicating that the extracellular levels of protein were low. This is likely due to the activity of specific proteases produced in the high copper containing medium [21]. In the cultures with 5 g of support, the highest laccase activities (around 19,000 U/L) were also attained by the cultures supplemented with 5 mM of copper sulphate (Fig. 3). However, these values are similar to those attained previously with 1 mM copper sulphate but operating with 2.5 g of support. Therefore, operating with 2.5 g of support produced higher activities (around 58% higher). This could likely be due to the cultures are better aerated and clogging problems are avoided.
Fig. 3. Laccase activities obtained by T. hirsuta grown in 250 mL Erlenmeyer flask on 5 g ground orange peelings in SSF under different conditions: () 1 mM copper; () 2 mM copper; () 3.5 mM copper; () 5 mM copper; (♦) 1 mM copper + 0.11 M syringaldazine.
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3.2. Bioreactor cultivation As it has already been commented in the introduction section, agro-industrial residues have an enormous potential as supports for solid-state processes. However, they present several problems, which are not found operating with inert supports, such as support degradation and/or support accretion may occur along the fermentation process. This would cause mass and oxygen restrictions into the reactor bed hampering its proper performance. Nonetheless, due to the economical advantages that these materials present, it is worthy to investigate a bioreactor configuration that allows operating with them [22]. 3.2.1. Fixed-bed bioreactor Initially, laccase activity diminished as a consequence of the dilution by the addition of fresh medium. Then, from 11th day onwards the activity increased reaching values around 3000 U/L in the last stage of cultivation. As for pH, it was maintained around 4–4.5 along cultivation (Fig. 4). It was observed that the fungus grew well attached to the orange peelings, the liquid phase remaining clear along the whole cultivation. In addition, the ratio liquid volume/solid volume, allowed the moving of air bubbles throughout the bed, enabling a good oxygenation of the fungus and avoiding clogging problems. Moreover, orange peeling degradation was not detected along cultivation, indicating the suitability of such a support for performing this type of processes. 3.2.2. Tray bioreactor Laccase began on the 8th day and it increased peaking on the 23rd day (12,660 U/L). From there up to the end of cultivation laccase remained around 11,000 U/L (Fig. 4). These values are three-fold higher than those obtained by the fixed-bed bioreactor. This is likely due to the agitation produced by the fixed-bed reactor caused mechanical stress to the fungus, lessening laccase production [23]. Therefore, agitation is a key factor to take into account to design an adequate bioreactor
Fig. 4. Laccase activities obtained and pH evolution (dotted line) by T. hirsuta grown on ground orange peelings under solid-state conditions at bioreactor scale: (䊉) fixed-bed reactor; () tray reactor.
for this type of process. The results obtained agree with those reported in a recent paper by our research group [24], in which highest laccase activities by T. versicolor were attained in a tray bioreactor among several configurations tested. As regards pH, it was maintained around 4.5–5.5 along cultivation (Fig. 4). These pH values are slightly higher than those attained in the fixed-bed configuration, which indicates that a pH around 5.0 favours laccase production. This agrees with several papers, which reported that a pH value around 5.0–5.5 was very suitable for laccase production by the genus Trametes [25–28]. In view of the results obtained, it can be concluded that the tray bioreactor is the most suitable configuration for the production of laccase enzymes by T. hirsuta in solid-state conditions. This is mainly due to its shape permits a filamentous growing of the fungus, the fungus is not subjected to mechanical stress and it provides a low aeration to the fungus, avoiding microorganism stress as well as fungus overgrowth. In addition, the support is placed in a thin layer, which avoids support agglomeration.
Table 1 Maximum laccase activities obtained by several white-rot fungus at bioreactor scale cultivated under different conditions Fungus
Type of reactor
Max. laccase activity (U/L)
Ref.
Pycnoporus cinnabarinus Lentinus edodes Trametes multicolora Panus tigrinus P. tigrinus Irpex lacteus I. lacteus Trametes versicolorb T. versicolor T. versicolor T. versicolor Trametes hirsutaa T. hirsutac T. hirsutac
10 L packed-bed (nylon cubes) Column bioreactor (malt extract broth) Stirred tank 3 L stirred tank (olive mill) 3 L bubble column air-lift (olive mill) 27 mL packed-bed (polyurethane foam) 27 mL packed-bed (pine wood) 2 L airlift 2.5 L immersion (barley bran) 300 mL expanded-bed (barley bran) 1 L tray (barley bran) 1 L fixed-bed (stainless steel sponges) 200 mL fixed-bed reactor (orange peelings) 0.1 L tray reactor (orange peelings)
270 – – 4600 ± 98 410 ± 22 – – 1676 600 500–600 3000–3500 2206 4897 12,260
Schliephake et al. [29] Hatvani and M´ecs [30] Hess et al. [9] Fenice et al. [31] Fenice et al. [31] Kasinath et al. [32] Kasinath et al. [32] Ranca˜no et al. [33] Rodr´ıguez Couto et al. [24] Rodr´ıguez Couto et al. [24] Rodr´ıguez Couto et al. [24] Rodr´ıguez Couto et al. [34] This work This work
a b c
Supplemented with 1 mM copper sulphate. Supplemented with 1 mM xylidine. Supplemented with 5 mM copper sulphate.
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In summary, the results obtained in the present work are very novel, since to date most attempts performed to produce laccase at bioreactor scale have been unsuccessful or lower activities than reported here were obtained. Moreover, laccase activities were improved by far in relation to those obtained in previous papers by our research group (Table 1). 4. Conclusions In view of the results obtained, it can be concluded that orange peelings are an excellent support-substrate for laccase production by T. hirsuta under solid-state conditions due to their high content in sugars, cellulose and pectin. Operating with the appropriate amount of support-substrate and supplementing the medium with copper sulphate at a concentration of 5 mM allowed obtaining high activities at flask scale. The system was scaled to tray and fixed-bed laboratory reactors. Acknowledgements This research was financed by the Spanish Ministry of Science and Technology and European FEDER (Project CTM2004-01539/TECNO) and the University of Vigo. References [1] Vares T, Hatakka A. Lignin-degrading activity and ligninolytic enzymes of different white-rot fungi: effects of manganese and malonate. Can J Botany 1997;75:61–71. [2] Bourbonnais R, Paice MG. Oxidation of non-phenolic substrates: an expanded role of laccase in lignin biodegradation. FEBS Lett 1990;267:99–102. [3] Kadhim H, Graham C, Barrat P, Evans CS, Rastall RA. Removal of phenolic compounds in water using Coriolus versicolor grown on wheat bran. Enzyme Microb Technol 1999;24:303–7. [4] Field JA, de Jong E, Feijoo-Costa G, de Bont JAM. Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. Trends Biotechnol 1993;11:44–9. [5] Pointing SB. Feasibility of bioremediation by white-rot fungi. Appl Microbiol Biotechnol 2001;57:20–33. [6] Koroljova-Skorobogat’ko OV, Stepanova EV, Gavrilova VP, Morozova OV, Lubimova NV, Dzchafarova AN, et al. Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis. Biotechnol Appl Biochem 1998;28:47–54. [7] Galhaup C, Wagner H, Hintertoisser B, Haltrich D. Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 2002;30:529–36. [8] Baldrian P, Gabriel J. Copper and cadmium increase laccase activity in Pleurotus ostreatus. FEMS Microbiol Lett 2002;206:69–74. [9] Hess J, Leitner C, Galhaup C, Kulbe KD, Hinterstoisser B, Steinwender M, et al. Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor. Appl Biochem Biotechnol 2002;98–100:229–41. [10] Kesterson JW, Braddock RJ. Byproducts and speciality products of Florida Citrus, Bulletin 784. Gainesville, FL: Agricultural Experiment Stations, Institute of Food and Agricultural Science, University of Florida; 1976. [11] Wing JM. Citrus feedstuffs for dairy cattle. Gainesville, FL: Agricultural Experiment Stations, Institute of Food and Agricultural Sciences University of Florida; 1982.
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