Immobilization of Pleurotus ostreatus 1804 on PUF cubes: Influence of mycelial growth pattern on laccase yield

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1 Indian Journal of Biotechnology Vol 5, January 2006, pp Immobilization of Pleurotus ostreatus 1804 on PUF cubes: Influence of mycelial growth pattern on laccase yield K Krishna Prasad 1, S Venkata Mohan 1, B R Pati 2 and P N Sarma 1 * 1 Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology, Hyderabad , India 2 Department of Microbiology, Vidyasagar University, Midnapore , India Received 16 November 2004; revised 13 April 2005; accepted 25 April 2005 The influence of immobilized Pleurotus ostreatus 1804, on polyurethane foam (PUF) cubes, was investigated on laccase yield as a function of immobilized PUF cubes and their repeated application. Enhanced laccase expression after fermentation was observed with immobilized PUF cubes (3109 U in 168 h) compared to free mycelia (2583 U in 192 h). The expression of laccase during fermentation was monitored by SDS-PAGE electrophoresis, which showed a band with a molecular mass of 63 kda. Effective laccase yield was also observed with repeated application of immobilized PUF cubes consistently upto 8 batches of fermentation. Scanning electron microscope (SEM) images revealed the pattern of spiraling colonization and aggregation of the P. ostreatus mycelia on PUF cubes. During repeated application, the hyphal growth on the PUF cubes increased consistently, manifested by both the processes of tip extension and branching, permitting the fungal colonization. Keywords: laccase, immobilization, PUF cubes, mycelial aggregation, SEM imaging, SDS-PAGE electrophoresis IPC Code: Int. Cl. 7 A01N63/04; C12N9/00, 11/08; C12R1:645 Introduction Laccases (p-diphenol:dioxygen oxidoreductase, EC ) are multicopper-containing enzymes, having great biotechnological potential for their broad substrate specificity in the diverse fields of industry and environment, such as pulp delignification 1-5, textile dye bleaching 6, detoxification of wastewater 2,7, xenobiotics 8-10, detergent manufacturing, and transformation of antibiotics and steroids 4. It is reported that Pleurotus ostreatus showed strong laccase activity among the edible mushrooms and was relatively easy to culture in vitro 11. Due to a wide range of substrate utilization, P. ostreatus is considered as a novel producer of ligninolytic enzymes. Considering biosafety aspect, production of industrially important ligninolytic enzymes by using P. ostreatus is a potential alternative to Phanerochaete chrysosporium 4. In recent times, fungal fermentations were exploited extensively in the commercial production of a wide range of secondary metabolites, including enzymes 12. Fungi are considered to be morphologically complex organisms, which differ in *Author for correspondence: Tel & Fax: sarma1950@yahoo.com structure at different stages of their life cycle, in surface and submerged growth, and on the nature of the growth medium, physical environment and culture conditions 13,14. During submerged fungal fermentation, dispersed mycelial and filamentous (hyphal) growth was observed in densely interwoven mycelial masses 15. Fungal morphology plays a fundamental and crucial role in determining the overall process productivity, and its manipulation results in increased yield of metabolites 12,16. Enhanced secondary metabolite production (gluconic acid, vinegar and lignolytic enzymes) was reported because of immobilization on polyurethane foam (PUF) Application of hyphal aggregates to enhance the metabolite production by natural (aggregation) processes or by artificial immobilization methods is on the increase 9. Thus, immobilization is critical in the production of metabolites, especially because of changes in fungal morphology and its repeated applications. Studies in this direction presume importance. In the present study, the influence of P. ostreatus 1804 mycelia aggregation on PUF cubes due to immobilization was investigated with respect to laccase yield. Further, morphological changes were studied as a function of repeated applications. The

2 PRASAD et al.: IMMOBILIZATION OF P. OSTREATUS 1804 ON PUF CUBES 85 hyphal morphology on PUF cubes and its subsequent aggregation was also studied employing scanning electron microscope (SEM) images. Materials and Methods Chemicals All the chemicals used were of pure and reagent grade unless otherwise stated. ABTS (2,2 -azino-bis- 3-ethyl benzothiazoline-6-sulphonic acid) was purchased from Sigma, Germany. All other chemicals were purchased from Himedia and Qualigens, India. White Rot Fungi White rot fungus, Pleurotus ostreatus 1804 (hyper laccase yielding strain), used in the present study, was procured from Microbial Testing and Collection Center (MTCC), Institute of Microbial Technology (IMTech), Chandigarh, India. The fungus was maintained on PDA plates (4 C) and sub-cultured once in every 30 d. Cultivation The production of laccase by P. ostreatus 1804 submerged agitation cultures was studied in cotton plugged 250 ml Erlenmeyer flasks, containing 100 ml of production medium. Young culture of the fungus was grown for 8 d in potato dextrose broth and the homogenized culture was used as initial inoculum for the immobilization. The production medium consisted of (g/100 ml of distilled water) 1.0 g glucose; 0.5 g yeast extract, 0.5 g urea (90 ml) and 10 ml of salt solution. The salt solution consisted of KH 2 PO g, MgSO 4.7H 2 O g, CaCl g, KCl g and CuSO 4.5H 2 O - 1 mm dissolved in 100 ml of distilled water and ph was adjusted to 5.6, using 3 N HCl prior to sterilization (15 lbs, 15 min, 121 C). Laccase Production PUF cubes (1 1 1 cm 3 ) were used as supporting material for the immobilization of fungal aggregated mycelium. Prior to use, the PUF cubes were pretreated by boiling for 20 min at 80 o C and washed with distilled water. The cubes were then placed in methanol overnight and washed twice with distilled water and dried in an hot air oven at 50 o C till the moisture was completely removed from the cubes. For immobilizing P. ostreatus on PUF cubes, 5 g of pretreated PUF cubes were placed in 250 ml Erlenmeyer flask containing potato dextrose broth. After sterilization (121 o C, 15 lbs, 15 min), the flasks were inoculated with 0.6 ml of homogenized mycelia ( g/l dry wt. of cell) under sterile conditions. P. ostreatus was found to grow profusely on PUF cubes after 5 days of incubation. The growth medium was then removed and the immobilized cubes were thoroughly washed with autoclaved saline water under sterile conditions. Laccase production was studied by aseptically transferring the immobilized cubes into 10 flasks, containing the production medium. Each flask was inoculated with four PUF cubes. Of 10 flasks, one was used throughout the study for estimating laccase yield. The remaining 9 flasks were used to study the fungal aggregation on the PUF cube surface by removing one flask after every 168 h of incubation. Initially, experiments were carried out to find the optimum fermentation period for maximum laccase yield and to assess the capability of immobilized P. ostreatus on laccase yield compared to the free mycelia. The mycelial concentration was maintained constant in all the flasks (1.38 g/l wet wt of mycelia) and laccase yield and glucose utilization were monitored for every 24 h. Repeated Fermentation with Immobilized Cubes Repeated batch fermentations were conducted with immobilized P. ostreatus cubes for a period of 168 h. At the end of each cycle, the production medium was decanted and the immobilized cells were thoroughly washed with sterile saline water and fresh production medium was added to continue the fermentation. Analytical Assay Laccase activity was estimated by ABTS oxidation procedure 20. The assay mixture consisted of 5 mm ABTS, 0.1 M sodium acetate buffer (ph 5.0) and an enzyme solution at 25 o C. Enzyme activity was expressed in units (U/L). U denotes amount of enzyme that oxidizes one μm of ABTS per min. The residual glucose in the media during fermentation was measured colorimetrically (UV-Vis spectrophotometer-beckmann DU-7400, USA) by DNS (dinitrosalicylic acid) method using glucose as standard 21. Extracellular protein concentration in the fermentation broth was estimated by Lowry s method using bovine serum albumin (BSA) as standard 22. The wet wt of immobilized fungal mycelium on PUF was determined every time by using separate set of fermented flasks. From the fermentation broth, PUF cubes at predetermined time were removed and washed with distilled water. Scanning Electron Microscope (SEM) Imaging P. ostreatus mycelia formation on PUF cubes during repeated laccase fermentation was studied by

3 86 INDIAN J BIOTECHNOL, JANUARY 2006 SEM imaging. The PUF cubes were fixed with 5% (v/v) glutaraldehyde in 100 mm phosphate buffer for a period of 1 h at 4 o C. The cubes were subsequently washed with distilled water and dehydrated in acetone solution (by gradually increasing the concentration of acetone from 10 to 100%) and dried at 40 o C for 6 h. Further, the cubes were coated with a gold layer and observed under SEM (Hitachi-S520). SDS Gel Electrophoresis To determine the expression of laccase along with molecular weight, sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the procedure outlined by Chefetz et al 23 employing 10% of polyacrylamide gel containing 0.1% of SDS. Protein samples (10 μg) were treated with 0.5% SDS and 5% β- mercaptoethanol mixture and denatured by boiling at 95 C for 5 min before loading onto the gel. The protein was visualized by staining the gel with silver nitrate and comparing with the low range of molecular marker from 14.2 to 66 kda (Sigma). Results and Discussion Laccase Expression with Free and Immobilized Mycelia In order to know the influence of immobilization on laccase expression, fermentation experiments were carried out with both free and immobilized P. ostreatus mycelia under similar conditions (25 o C; 100 rpm). In case of free mycelia, the highest level of laccase activity was observed at 192 h of incubation time, whereas it was observed only after 168 h in immobilized mycelia (Fig. 1). Similarly, maximum laccase activity of only 2583 U was observed with the free mycelia, while an activity of 3109 U was evident with immobilized mycelia. Thus, enhanced laccase yield at relatively less incubation period was observed in the case of immobilized mycelia as compared to the free mycelia. This phenomenon may be attributed to the increased surface area of fungal mycelia due to their immobilization on PUF cubes. Immobilization enhanced the available surface area and reduced the mass transfer limitations, facilitating easy access to the substrate utilization. The release of laccase during fermentation was detected by SDS-PAGE electrophoresis (Fig. 2). The analysis was done in order to know the expression and also to estimate the molecular mass of laccase secreted by P. ostreatus It was observed that molecular mass of laccase was approximately 63 kda when compared to the standard protein marker. Laccase yield (U/L) Fig. 1 Comparative representation of laccase yield with the function of fermentation period for immobilized and free mycelia Fig. 2 SDS-PAGE showing the expression of laccase during fermentation (Lane M, standard molecular weight marker; Lane 1, fermentation broth) Repeated Batch Fermentation with Immobilized Mycelia Repeated application of immobilized mycelia was studied to understand the relative laccase expression. After each batch of fermentation, the extracellular culture fluid was replaced by fresh laccase production medium in order to remove residual laccase and the immobilized cubes were washed twice with sterile saline water (0.9% NaCl solution). The laccase yield obtained from the repeated batch fermentation experiments are shown in Fig. 3. An increase in the laccase yield from 2956 to 4012 U was observed upto eight batches of fermentation, which gradually dropped to 2783 U upto the 10 th batch. Generally, mycelial growth may be viewed not merely as mechanical conglomerates but rather as complex differentiated tissues. In mycelial aggregation, the cell-to-cell interaction and signaling that resulted from shoot differentiation was qualitatively dissimilar

4 PRASAD et al.: IMMOBILIZATION OF P. OSTREATUS 1804 ON PUF CUBES 87 Fig. 3 Laccase yield during repeated application of immobilized mycelia from that of free dispersed mycelia and facilitated more substrate and oxygen availability 15. The effective laccase yield achieved upto eight batches might be attributed to this effect. After exceeding eight batches of repeated application of immobilized cubes, the yield decreased due to excessive aggregation of mycelia in multiple layers around the PUF cube, which have negative influence on the mass transfer limitations of substrate. This restricted the contact between mycelia and growth media only near the peripheral zone of the aggregated mycelia, resulting in reduced surface (contact) area. Layers of aggregations resulted in retarding the internal mass transport imposing non-diffusion of oxygen and other nutrients, resulting in the loss of biosynthetic activity. Mycelial Growth During fermentation experiments with repeated application of immobilized PUF cubes, mycelial aggregation mass on wet wt basis was monitored and the results obtained are presented in Fig. 4. For each repeated application of immobilized cubes, a consistent increase in mycelial mass was observed (0.43 to 3.15 g/l). The immobilization pattern and morphological changes of mycelia aggregation on PUF cubes during repeated batch fermentation were observed by SEM imaging (Fig. 5). The SEM images provided information about the colonization pattern of the P. ostreatus mycelia (hyphae). Colonization on Fig. 4 Mycelia growth on PUF cubes during repeated application of immobilized mycelia Fig. 5 SEM images of PUF cubes with formation of mycelia aggregation with the function of repeated applications PUF cubes was observed in the form of spiraling during the process of repeated application. With each additional batch of fermentation, a consistent increase in the growth of hyphae on the surface of the cubes was observed. Also, the growth of hyphae led to the formation of densely formed aggregation on the PUF cube surfaces, with every additional batch. Consistent profuse growth of mycelia was observed up to 8 th batch of fermentation experiments by reducing the existing porous network. It is clearly evident from SEM images that the porous network was relatively

5 88 INDIAN J BIOTECHNOL, JANUARY 2006 better on zero day. With increase of each application, a gradual reduction was evident. Even though the laccase expression was suppressed after eighth batch of fermentation, the mycelia aggregation was not inhibited. In fungal mycelia, hyphae extend by a highly polarized process of cell extension, known as tip extension 24. It was clearly observed from the images that, as the tip extended, periodic branches also appeared in a polarized manner forming new tips. The processes of tip extension and branching permitted the fungi to colonize and efficiently utilize the substrate, which can be directly related to the process performance. Conclusion Immobilization of P. ostreatus 1804 on PUF cubes showed enhanced laccase expression in rapid fermentation time compared to free mycelia fermentation. Effective laccase yield was also observed with repeated application of immobilized PUF cubes. Scanning electron microscope (SEM) images revealed the aggregation of the P. ostreatus mycelia on PUF cubes. Effective laccase yield was achieved upto eight batches, which could be attributed to the availability of more substrate and oxygen due to more surface area. After exceeding eight batches of repeated application of immobilized cubes, the yield decreased due to excessive aggregation of mycelia in multiple layers around the PUF cube, which have negative influence on the mass transfer limitations of substrate. References 1 Higuchi T, Lignin biochemistry: Biosynthesis and biodegradation, Wood Sci Technol, 24 (1990) Eriksson K E L, Blanchette R A & Ander P, Microbial and enzymatic degradation of wood and wood components (Springer, Berlin-Heidelberg, New York) Duran N & Esposito E, Potential application of oxidative enzymes and phenoloxidases like compounds in wastewater and soil treatment: A review, Appl Catal B: Environ, 28 (2000) Cohen R, Persky L & Hadar Y, Biotechnological applications and potential of wood degrading mushrooms of the genus Pleurotus, Appl Microbiol Biotechnol, 58 (2002) Breen A & Singleton F L, Fungi in lignocellulose breakdown an biopulping, Curr Opin Biotechnol, 10 (1999) Bajpai P, Application of enzymes in the pulp and paper industry, Biotechnol Prog, 15 (1999) Shah V & Nerud F, Lignin degrading system of white rot fungi and its exploitation of dye decolourization, Can J Microbiol, 48 (2002) Karam J & Nicell J A, Potential applications of enzymes in waste treatment, J Chem Techol Biotechnol, 69 (1997) Paszczynski A & Crawford R L, Potential for bioremediation of xenobiotic compounds by the white rot fungus Phanerochaete chryososporium, Biotechnol Prog, 11 (1995) Reddy C A, The potential for white rot fungi in the treatment of pollutants, Curr Opin Biotechnol, 6 (1995) Okamoto K, Ito Y, Shigematsu I, Ochiai S & Yanase H, Cloning and characterization of a laccase gene from the white-rot basidiomycete Pleurotus ostreatus, Mycoscience, 44 (2003) Papagianni M, Morphology and citric acid production in Aspergillus niger submerged fermentation. Ph D Thesis, University of Strathclyde, Glasgow, Atkinson B & Daoud I, Microbial flocs and flocculation in fermentation process engineering, Adv Biochem Eng, 4 (1976) Kossen N W F, The morphology of filamentous fungi, Adv Biochem Eng Biotechnol, 70 (2000) Papagianni M, Fungal morphology and metabolite production in submerged mycelial processes, Biotechnol Adv, 22 (2004) Schugerl K, Gerlach S R & Siebenberg D, Influence of the process parameters on the morphology and enzyme production of Aspergilli, Adv Biochem Eng Biotechnol, 60 (1997) Mukhopadhyaya R, Chatterjee S, Chatterjee B P, Banerjee P C & Guha A K, Production of gluconic acid from whey by free and immobilized Aspergillus niger, Int Dairy J, 15 (2005) De Ory I, Romero L E & Cantero D, Optimization for immobilization conditions for vinegar production. Siran, wood chips and PUF as carriers for Acetobacter aceti, Process Biochem, 39 (2004) Nakamura Y, Sungusia M G, Sawada T & Kuwahara M. Lignin degrading enzyme production by Bjerkandera adusta immobilized on polyurethane foam, J Biosci Bioeng, 1 (1999) Krishna Prasad V, Venkata Mohan S, Srinivas R, Pati B R & Sarma P N, Optimization of culture conditions for laccase production by Taguchi orthogonal array experimental design, Biochem Eng J, 24 (2005) Ghose T K, Measurement of cellulase activities, Pure Appl Chem, 59 (1987) Lowery O H, Rosebrough N J, Farr A L & Randall R J, Protein measurement with the Folin-phenol reagent, J Biol Chem, 193 (1951) Chefetz B, Chen Y & Hadar Y, Purification and characterization of laccase from Chaetomium thermophilium and its role in humification, Appl Environ Microbiol, 64 (1998) Heath I B, Integration and regulation of hyphal tip growth, Can J Bot, 73 (1995)