ASEAN Review of Biodiversity and Environmental Conservation (ARBEC)

July 1998

ISOLATION OF CELLULOLYTIC FUNGI FROM SAYAP-KINABALU PARK, SABAH

Abdul Jalil Kader and Othman Omar Faculty of Life Sciences, Universiti Kebangsaan Malaysia, Bangi, Malaysia

A total of 16 cellulolytic fungi was isolated from various locations within Sayap substation, Kinabalu Park. Ten were selected based on the ratio clearing gone on cellulose medium and colony size for further studies. Except for cellobiohydrolase activity which was demonstrated highest in the Control, five isolates showed high hydrolytic Filter paper activity, carboxymethylcellulase and B-glucosidase enzymatic activities. The five isolates identified are three Aspergillus species (C2, Dl and EI) and two are Trichoderma (D3 and E2). These isolates will be used for further studies into the enzyme production and their ability to degrade cellulose.

INTRODUCTION Cellulolytic enzymes are a group of hydrolytic enzymes (cellulases) capable of hydrolysing cellulose to glucose. The glucose produced can be used for human and animal food or for production of chemicals. These enzymes are used to perform a multitude of functions including to remove cell walls or crude fibre to release valuable components (flavours, enzymes, polysaccharides and other proteins) from plants cells to improve nutritional value of animal feeds or to prepare plant protoplasts for genetic research (Mandels, 1985). There are at least three major types of cellulolytic enzymes produced by fungi: endoglucanases, cellobiohydrolases and cellobiases (Klyosov, 1990). Cellulolytic enzymes are produced by a large number of microorganisms which include fungi and bacteria (Enari, 1983). In microorganisms the enzymes are either cell-bound or extra cellular. The ability to produce extra cellular cellulolytic enzymes is widespread in fungi and these enzyme systems have been most extensively studied (Enari, 1983; Wood, 1985).

METHODS Soil samples were taken along the Sungai Wario, Sungai Lumutuk Besar, Sungai Lumutuk Kecil, Gua Melayu and the entrance to Sayap substation.

Isolation of cellulolytic fungi. The samples was first inoculated on potato dextrose agar (PDA) and later transfered into Mandels agar to screen for cellulolytic fungi (Mandels & Reese, 1957).

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Fungal growth The fungal spores (1x107) were inoculated into 250ml modified Mandels medium (Mandels & Reese, 1957) in a 5OOml Erlenmeyer flask and incubated at 30C for 7 days shaken at 150rpm.

Sample preparation Samples of 10ml were collected daily for a total of seven and centrifuged at 8000g for 10min (at 4C). Each sample was monitored for pH, protein and cellulolytic activities.

Enzymatic assays. Carboxymethylcellulase was determined in a 20min assay at 37C with the method of Wood & McCrae(1977). Cellobiohydrolase was also determined by the method of Wood & McCrae(1977) in a 1-hour assay at 37C. Filter paper activity was determined by the method of Mandels et al,, (1976). The unit of activity (U) is defined as the amount of enzyme liberating l umol glucose per minute in a standard assay. B-glucosidase was determined in a 1-hour assay at 37C using the method of Theodorou et al., ( 1980). The unit of activity (U) for B-glucosidase is defined as the amount of enzyme liberating 1 umol p-nitrophenol per minute.

Table 1 Numbers of fungus isolated from different locations. Sampling location A. B. C. D. E. Sungai Wario Sungai Lumutuk Besar Gua Melayu Sungai Lumutuk Kecil Sayap substation (entrance) No. of fungus isolated 2 7 2 3 2

RESULTS AND DISCUSSION Sixteen different fungus was isolated from the soil samples taken as shown in Table 1. The highest number isolated was from Sungai Lumutuk Besar with 7 isolates. Fifteen out of the total number of isolates has been identified. They are Trichoderma (10) and Aspergillus (5). One of the isolate from Sungai Lumutuk Besar has not been identified (Table 2).

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Table 2 The fungus isolated from Sayap substation, the alphabets used indicates the location where they were isolate as in Table 1. Fungus A1 A2 B1 B2 B3 B4 B5 B6 Genus Trichoderma Trichoderma Trichoderma Asperigillus Trichoderma Trichoderma Trichoderma Not identified Fungus B7 Cl C2 D1 D2 D3 E1 E2 Genus Trichoderma Asperigilius Asperigillus Asperigillus Trichoderma Trichoderma Asperigillus Trichoderma

All isolates were able to grow on Mandels's medium. Judging from the ratio between the clearing zone diameter and colony diameter where only 10 isolates were chosen for enzyme production studies (Table 3). The profile of the average enzyme production over a period of 7 days of the selected 10 organisms is as in Figure 1. As seen from Figure 1 isolates BI, DI, D2 and EI produces a higher Filter paper activity as compared to the control while EI produces the highest carboxymethylcellulase activity. Only 2 isolates; C2 and EI have higher activity of B-glucosidase than the Control but the Control has the highest production of cellobiohydrolase.

Table 3 The ratio between the diameter of the clearing zone and that of colony diameter grown on Mandels's medium to select cellulolytic fungi. The control is Aspergillus terreus isolated from Palm oil mill effluent (Zainal Abidin, 1990). Fungus A1 A2 B1 B2 B3 B4 B5 B6 B7 Clearing zone/diameter 2.50/2.25= 1.11 2.30/2.10= 1.10 5.50/4.15= 1.33 3.20/2.65= 1.21 2.65/2.45= 1.10 1.90/1.80= 1.06 2.90/2.40= 1.20 1.70/1.50= 1.13 4.00/3.20= 1.25 Fungus C1 C2 D1 D2 D3 E1 E2 Control Clearing zone/diameter 2.90/2.75 = 1.05 3.05/2.50= 1.22 5.00/2.70= 1.85 6.00/3.50= 1.71 2.75/2.50= 1.10 4.20/2.40= 1.75 4.50/3.50= 1.29 5.00/4.00 =1.32

The result from indicates that the 5 isolates; C2, D], D2, EI and E2 have the ability to introduce celulolytic enzymes and degrade cellulose comparable to control. These 5 isolates will be used for further studies to determine their potential as a cellulolytic enzymes producers.

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July 1998

Figure 1 Average cellulolytic enzymes activity over a period of 7 days.

ACKNOWLEDGEMENT We are grateful to the Organizing Commitee for allowing us to be part of the Sayap-Kinabalu Scientific Expedition. This study was supported under the IRPA program No. 4-07-03-008.

REFERENCES Enari, T.M. [1983] Microbial cellulases. In: Microbial enzymes and biotechnology. W.F. Forgaty (ed). Applied Sciences publishers, London. p. I 83-223. Klyosov,A.A. [1990] Trends in biochemistry and enzmology. Biochemistry. 29:10577-10585. Theodorou, M.K., MJ. Bazin and A.P.J. Trinci [1980] Cellulose degradation in a structured ecosystem which is analogous to soil. Transaction of the British Mycological Society. 75:432-454. Mandels, M. and E.T. Reese [1957] Induction of cellulase in fungi by cellobiose. J. Bacteriology. 73:816- 826.

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Mandels, M., R. Andreotti and C. Roche [1976] Measurement of saccharifying cellulase. Biotechnology & Bioengineering symposium. 6:2 1-33. Mandels, M. [1985] Application of cellulases. Biochemical Society Transactions. 13:414- 416. Wood, T.M. and S.I. McCrae [1977] Cellulase from Fusarium solani. Purification and properties of C1 component. Carbohydrate Research. 57:117-133. Wood, T.M. [1985] Properties of cellulolytic enzyme system. Biochemical Society Transactions. 13:407-410.

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Zainal Abidin, M.Y. [1990] Characterisation and production of cellulolytic enzymes of Aspergillus terreus IMI 282743. PhD thesis. Department of Microbiology, Faculty of Life Sciences, Universiti Kebangsaan Malaysia.

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