Biotechnology Letters 24: 701704, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

701

New uses of food waste: application to laccase production by Trametes hirsuta

E. Rosales, S. Rodrguez Couto & A. Sanrom n a
Department of Chemical Engineering, University of Vigo, 36200 Vigo, Spain Author for correspondence (Fax: +34 986 812382; E-mail: sanroman@uvigo.es)
Received 5 February 2002; Revisions requested 15 February 2002; Revisions received 26 February 2002; Accepted 26 February 2002

Key words: food industry wastes, laccase, solid state fermentation, Trametes hirsuta

Abstract Diverse food wastes, apple, orange and potato, were screened for laccase production, under solid-state fermentation conditions, by the white-rot fungus Trametes hirsuta. Potato peelings gave the highest activity, reaching about 5000 U l1 within 8 days. These values are higher than those reported to date.

Introduction Laccase (E.C. 1.10.3.2) is the most common ligninmodifying enzyme produced by the white-rot fungi belonging to the family Polyporaceae sensu lato (Pelaez et al. 1995). Among them, Trametes versicolor has extensively been used as the main experimental organism for laccase production studies. However, another fungus belonging to the same genus, Trametes hirsuta, has also been described as a very promising candidate for the production of laccase (Vares & Hatakka 1997). Moreover, laccase from T. hirsuta can efciently degrade a wide variety of synthetic dyes (Abadulla et al. 2000, Campos et al. 2001). This makes this biocatalyst very suitable for the treatment of wastewater from the textile industry. Most studies dealing with ligninolytic enzyme production by white-rot fungi have been carried out using liquid culture conditions, in spite of the fact that these organisms grow in nature in solid-state conditions. Recent reviews on solid-state fermentation (SSF) point out the enormous potential of this culture technique for the development of different bioprocesses (Pandey et al. 1999). The selection of a substrate for SSF processes depends upon several factors mainly related with cost and availability and thus may involve screening of several agro-industrial residues. Moreover, the utilisation of this type of supports helps to solve the pollution

problems caused by their disposal. Wheat bran has commonly been used for the cultivation of microorganisms in SSF processes. In the present work, in addition to barley bran, several other agro-industrial residues, traditionally employed in composting, such as apple, orange and potato peelings have been tested as supports for laccase production by T. hirsuta under solid-state conditions. In addition, cultivation employing an inert support like nylon sponge has been done for comparison. To our knowledge there are no reports dealing with laccase production by T. hirsuta in solid-state conditions.

Materials and methods Microorganism Trametes hirsuta (BT 2566), a kind gift of Dr G.M. Gbitz from the Department of Environmental Biotechnology, Graz University of Technology, was grown on potato/dextrose/agar (PDA) plates. Supports Inert support Cubes (edge size 0.5 cm) of nylon sponge (Scotch Brite, 3M Spain, S.A.) were used as inert supports. Prior to use, the nylon sponge was pre-treated by

702 boiling for 10 min, washing thoroughly three times with distilled water then was dried overnight at room temperature. Non-inert supports Barley bran (particle length about 37 mm) and barley bran mixed with apple, orange or potato, or peelings (cut in cubes of 0.5 cm of edge) were employed as non-inert supports. Prior to use the peelings, 10 g fresh wt, were pre-treated by soaking for 1 h in 30 ml 83 mM KOH to neutralise organic acids (Stredansky & Cont 1999) then, they were thoroughly washed with distilled water and dried. Prior to use, all the supports were autoclaved at 121 C for 20 min. Culture conditions Inert support cultures The composition of the culture medium was prepared according to Abadulla et al. (2000) but without wheat bran akes. Non-inert support cultures The composition of the culture medium was identical to the nylon sponge cultures, except that glucose was at 4 g l1 , since non-inert supports provide some nutritive substances to the fungus. The cultures were performed in cotton-plugged Erlenmeyer asks (250 ml) containing different supports depending on the experiment and 15 ml of culture medium. Inoculation was carried out directly in the Erlenmeyer asks. Three agar plugs (diameter, 3 mm), from an actively growing fungus on PDA, per ask were used as inoculum. The Erlenmeyer asks were incubated statically under air at 30 C and 90% humidity to avoid evaporation, in complete darkness. Two experiments for each support were conducted in parallel and triplicate samples were analysed. The values in the gures correspond to mean values with a standard deviation lower than 15%. Analytical determinations Reducing sugars They were measured by the dinitrosalicylic acid method using D-glucose as a standard.

Fig. 1. Production and utilization of reducing sugars in solid-state cultures of T. hirsuta grown on different supports: , nylon sponge (0.95 g); , barley bran (2.5 g); , barley bran and orange peelings; , barley bran and apple peelings; , barley bran and potato peelings (0.625 g bran plus 1.875 g peelings).

Laccase activity It was determined spectrophotometrically as described by Niku-Paavola et al. (1990) with ABTS (2,2 -azinodi-[3-ethyl-benzo-thiazolin-sulphonate]) as substrate. One activity unit was dened as the amount of enzyme that oxidised 1 mol ABTS per min. The activities were expressed in U l1 .

Results and discussion The potential of several food wastes, commonly used in composting, such as apple, orange and potato peelings, as substrates for laccase production by T. hirsuta under solid-state conditions, was investigated. These materials were selected due to their availability and low cost. Moreover, there are no reports dealing with these materials as supports for laccase production. Cultivation with an inert support (nylon sponge) and with a non-inert support usually used in SSF processes (barley bran) was also made for comparison. Glucose evolution When T. hirsuta was grown in cubes of nylon sponge, glucose, measured as reducing sugars, was totally consumed by the 3rd day (Figure 1). With barley bran cultures, glucose, measured as reducing sugars, dropped to 1.5 g l1 by the 3rd day, and from there onwards it remained around 2 g l1 . A similar prole was found when T. hirsuta was cultivated on potato peelings (Figure 1).

703 3437 U l1 on the 6th day (Figure 2). This value was almost 5-fold higher than that found in nylon sponge cultures and around 53% higher than that obtained in barley bran cultures. When T. hirsuta was grown on potato peelings, laccase activity peaked on the 8th day (5372 U l1 ) (Figure 2). This value was about 7-fold higher than that attained employing nylon sponge as a support and much higher than that found in the other cultures as well. The results show the potential of food wastes to promote laccase activity by T. hirsuta under solid-state conditions, since they led to much higher activities than growth on a nylon sponge. In addition to this, by employing these bio-wastes the initial amount of the added glucose was decreased to 60% of that used with the inert carrier (i.e., nylon sponge). Therefore, the utilisation of such wastes on the one hand promoted high enzymatic levels and for the other hand a considerable reduction in production costs.

Fig. 2. Laccase production in solid-state cultures of T. hirsuta grown on different supports: , nylon sponge (0.95 g); , barley bran (2.5 g); , barley bran and orange peelings; , barley bran and apple peelings; , barley bran and potato peelings (0.625 g bran plus 1.875 g peelings).

Operating with apple peelings as a support, reducing sugars were about 78-fold higher than the basal medium at the beginning of cultivation and then, they abruptly decreased attaining values around 2.5 g l1 during the last stage of fermentation. A similar prole was obtained when orange peelings were used as a support (Figure 1). This initial increase in reducing sugars is probably due to the release of some hydrolysis products from the peelings. These supports might be employed without adding any initial amount of glucose in the culture medium, which would imply an important economic advantage. Enzyme production When T. hirsuta was cultivated in cubes of nylon sponge, laccase rst appeared on the 2nd day (217 U l1 ), then it decreased up to the 5th day and from there onwards it increased peaking on the 8th day (779 U l1 ) (Figure 2). As regards barley bran cultures, laccase production began on the 4th day (230 U l1 ). Then, it increased peaking on the 8th day (2247 U l1 ) and from there onwards it decreased (Figure 2). This peak was almost 3-fold higher than that found operating with nylon sponge as a support. With apple peel cultures, laccase production reached a maximum value of 3148 U l1 on the 5th day of cultivation (Figure 2). This value was 4-fold higher than that found in nylon sponge cultures and about 40% higher than that obtained using barley bran as a support. With orange peelings as a support, laccase production reached a maximum value of

Conclusions According to the results obtained in the present work, food wastes, especially potato peelings, have an enormous potential as supports for laccase production by T. hirsuta in solid-state conditions. Furthermore, these supports make the process more economical due to their waste status and they provide some nutrients for the microorganism. Moreover, the utilisation of such materials also helps to solve pollution problems caused for their disposal. In view of these encouraging results more studies in order to determine the optimal operation conditions (addition of inducers, amount of support, initial concentration of carbon and nitrogen, etc.) are underway in our laboratory.

Acknowledgements This research was nanced by XUNTA (PGIDT00PXI 30118PR). The authors would like to thank Dr Georg M. Gbitz, Dr Stephanie Ryan and MSc. Luciana Pereira, Department of Environmental Biotechnology, Graz University of Technology, for the kind gift of Trametes hirsuta strain.

704 References
Abadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A, Gbitz G (2000) Decolorization and detoxication of textile dyes with a laccase from Trametes hirsuta. Appl. Environ. Microbiol. 66: 33573362. Campos R, Kandelbauer A, Robra KH, Cavaco-Paulo A, Gbitz GM (2001) Indigo degradation with puried laccases from Trametes hirsuta and Sclerotium rolfsii. J. Biotechnol. 89: 131139. Niku-Paavola ML, Raaska L, Itvaara M (1990) Detection of whiterot fungi by a non-toxic stain. Mycol. Res. 94: 2731. Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77: 149162. Pelaez F, Martnez MJ, Martnez AT (1995) Screening of 68 species basidiomycetes for enzymes involved in lignin degradation. Mycol. Res. 99: 3742. Stredansky M, Conti E (1999) Xanthan production by solid state fermentation. Process Biochem. 34: 581587. Vares T, Hatakka A (1997) Lignin-degrading activity and ligninolytic enzymes of different white-rot fungi: effects of manganese and malonate. Can. J. Botany 75: 6171.