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Faculty of Resource Science and Technology PRODUCTION AND OPTIMIZATION OF RAW STARCH DEGRADING AMYLASE AND CELLULASE IN SOLID STATE FERMENTATION (SSF...
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Faculty of Resource Science and Technology

PRODUCTION AND OPTIMIZATION OF RAW STARCH DEGRADING AMYLASE AND CELLULASE IN SOLID STATE FERMENTATION (SSF) OF AGRICULTURAL WASTE BY ASPERGILLUS sp.

NUR FATIN HUSNA BINTI ROSLAN

Bachelor of Science with Honours (Resource Biotechnology) 2012

PRODUCTION AND OPTIMIZATION OF RAW STARCH DEGRADING AMYLASE AND CELLULASE IN SOLID STATE FERMENTATION (SSF) OF AGRICULTURAL WASTE BY ASPERGILLUS NIGER

Front cover Nur Fatin Husna binti Roslan (24573)

A final project report submitted in partial fulfillment of the Final Year Project II (STF 3015) Resource Biotechnology

Supervisor: Assoc. Prof Dr. Awang Ahmad Sallehin Awang Husaini Co-supervisor: Assoc. Prof Dr. Cirilo Nolasco Hipolito

Resource Biotechnology Programme Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak 2012

Acknowledgement I would like to thank to my supervisor, Assoc. Prof. Dr. Awang Ahmad Sallehin Awang Husaini who has given me a chance to become one of his Final Year Project’s students. Endless thanks for his valuable guidance, advices and encouragements towards the completion of this project. Special thank to my co-supervisor, Assoc. Prof. Dr. Cirilo Nolasco Hipolito for his kindness to support and help me towards the completion of this project. I also would like to express my appreciation to the Department of Molecular Biology, Universiti Malaysia Sarawak for giving me this opportunity to fulfill my Final Year Project. I really appreciate all the materials, equipments, instruments and other facilities provided which are necessary during the progression of my project. Beside I would like to express my gratitude to the master student in Molecular Genetic Laboratory and lab assistants for their help, support and cooperation when this project was carried out. Lastly, thank you to all the colleagues for their ideas and advice while we were working together at the laboratory. Not forgetting, to my beloved family who has given me a lot of spiritual and financial supports.

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Table of Contents Acknowledgement ............................................................................................................................i Table of Contents ........................................................................................................................... ii List of Abbreviation .......................................................................................................................iv List of Tables ................................................................................................................................... v List of Figures .................................................................................................................................vi Abstract ............................................................................................................................................ 1 1.0 Introduction ............................................................................................................................... 2 2.0 Literature review........................................................................................................................ 4 2.1 Agricultural waste................................................................................................................. 4 2.2 Solid state fermentation ........................................................................................................ 5 2.3 Aspergillus sp. ...................................................................................................................... 6 2.4 Amylase ................................................................................................................................ 7 2.5 Cellulase ............................................................................................................................... 8 3.0 Materials and Methods .............................................................................................................. 9 3.1 Fungi culture and maintenance ............................................................................................. 9 3.2 Preparation of spore suspension ........................................................................................... 9 3.3 Amylase and cellulase enzyme screening .......................................................................... 10 3.4 Substrate preparation .......................................................................................................... 10 3.5 Solid-state fermentation (SSF) for enzyme production ...................................................... 11 3.6 Enzyme extraction .............................................................................................................. 11 3.7 Enzyme Assay .................................................................................................................... 12 3.7.1 Amylase activity measurement ................................................................................... 12 3.7.2 Cellulase activity measurement ................................................................................. 12 3.8 Production and optimization of amylase and cellulase in Solid State fermentation (SSF) 14

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3.8.1 Effect of temperature on SSF ..................................................................................... 14 3.8.2 Effect of inoculum size on SSF .................................................................................. 14 3.8.3 Effect of time of incubation on SSF ............................................................................ 14 3.8.4 Effect of pH on SSF ................................................................................................... 15 3.8.5 Effect on moisture content on SSF ............................................................................. 15 4.0 Results and Discussion ............................................................................................................ 16 4.1 Strain Selection ................................................................................................................... 16 4.1.1 Screening .................................................................................................................... 16 4.2 Preparation of Aspergillus niger culture............................................................................. 17 4.2.1 Fungal culture ............................................................................................................ 17 4.3 Production and Optimization on Solid State Fermentation ................................................ 18 4.3.1 Effect of time incubation ............................................................................................ 18 4.3 .2 Effect of pH ............................................................................................................... 21 4.3.3 Effect of temperature ................................................................................................... 24 4.3.4 Effect of inoculum size ............................................................................................... 27 4.3.5 Effect of moisture content .......................................................................................... 29 5.0 Conclusion and Recommendation ........................................................................................... 33 Reference ....................................................................................................................................... 35 Appendix ....................................................................................................................................... 38

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List of Abbreviation PDA

Potato dextrose agar

CMC

Carbomethylcellulose

SSF

Solid state fermentation

DSNA 3,5-dinitrosalicylic acid g

Gram

mL

milligram

µg

microgram

nm

nanometer

pH

A measurement of the acidity or alkalinity of solution [p stands for “potenz” which means the potential to be while H stands for Hydrogen]

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List of Tables Table 1: Diameter of halos around the isolate ........................................................................... 16 Table 2: Amylase enzyme activity produces at different time of incubation by SSF of agricultural was as substrate. ...................................................................................... 19 Table 3: Cellulase enzyme activity produces at different time of incubation by SSF of agricultural was as substrate. ...................................................................................... 20 Table 4: Amylase enzyme activity produced by SSF of different agricultural waste as substrate at different pH. ............................................................................................................ 21 Table 5: The cellulase enzyme activity produced by SSF of different agricultural waste as substrate at different pH. ............................................................................................ 22 Table 6: Amylase enzyme activity produces at different temperature by SSF of agricultural was as substrate. ......................................................................................................... 24 Table 7: Cellulase enzyme activity produces at different temperature by SSF of agricultural was as substrate. ......................................................................................................... 25 Table 8: Amylase enzyme activity by SSF at different inoculum size. ..................................... 27 Table 9: Cellulase enzyme activity produced by SSF at different inoculum size. .................... 29 Table 10: Amylase enzyme activity produces at different moisture content by SSF of agricultural was as substrate. ...................................................................................... 30 Table 11: Cellulase enzyme activity produces at different moisture content by SSF of agricultural was as substrate. ...................................................................................... 31

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List of Figures Figure 1: Growth of Aspergillus niger at day 7 on Potato Dextrose Agar (PDA). ................... 17 Figure 2: Effect enzymatic activity on time of incubation of amylase under SSF of different agricultural waste as substrate................................................................................... 19 Figure 3: Effect of time incubation on cellulase production under of different agricultural was as substrate. ............................................................................................................... 20 Figure 4: Effect of pH on amylase production. ......................................................................... 22 Figure 5: Effect of pH on production of cellulase. .................................................................... 23 Figure 6: Effect of temperature on amylase production. ........................................................... 25 Figure 7: Effect of temperature on cellulase production. .......................................................... 27 Figure 8: Effect of inoculum size on amylase production. ........................................................ 28 Figure 9: Effect of inoculum size on cellulase production. ....................................................... 29 Figure 10: Effect of moisture content on amylase production. ................................................. 30 Figure 11: Effect of moisture content on cellulase activity. ...................................................... 32

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Production and Optimization of Raw Starch Degrading Amylase and Cellulase in Solid State Fermentation (SSF) of Agricultural Waste by Aspergillus Niger Nur Fatin Husna Binti Roslan Resources Biotechnology Programme Department of Molecular Biology Faculty of Resource Science and Technology Universiti Malaysia Sarawak

Abstract

Agricultural waste such as sago hampas, rice husk, pineapple waste and cassava waste were used as solid substrate on solid state fermentation for the production of amylase and cellulase. The aims of this project were to identify the best strain in production of amylase and cellulase enzymes and to determine the optimum condition for the production on both enzymes on solid state fermentation system. There were five important fermentation parameters being studied; temperature, size of inoculum, time of incubation, moisture content and pH of the medium. In this project, Aspergillus niger PAN1 was selected as the best strain capable of producing both enzymes at the highest level. The optimum condition on solid state fermentation for the production of amylase and cellulase was recorded at day 3 for amylase and day 6 for cellulase of time of incubation, respectively. The pH suitable for high enzymatic activity was at pH 5.5 for amylase and pH 7.5 for cellulase activity, respectively. The optimum temperature of 30oC showed the highest yield of amylase activity and the highest production of cellulase activity was at the temperature of 40 oC, respectively. In addition, the optimum inoculates that produce highest production of both amylase and cellulase enzymes are at 10 7 of spore suspension/ml. The medium was further optimized with 70% of moisture content. The enzyme was extracted and assayed by using DNS method to determine the total amount of reducing sugar released. Key words: Amylase, Cellulase, Aspergillus niger, Solid Substrate Fermentation (SSF) Abstrak Sisa pertanian seperti hampas sagu, sekam padi, sisa nanas dan sisa ubi kayu digukan sebagai substrat pepejal dalam fermentasi pepejal untuk pengeluaran enzim amilase dan sellulase. Matlamat projek ini adalah untuk mengenal pasti fungus terbaik untuk pengeluaran amilase dan sellulase serta menentukan keadaan yang optimum untuk penghasilan kedua-dua enzim dalam sistem fermentasi pepejal. Terdapat lima parameter yang dikaji iaitu; suhu, saiz inoculum, masa fermentasi, kelembapan medium dan pH. Dalam projek ini Aspergillus niger telah dipilih sebagai fungus yang terbaik untuk menghasilkan kedua-dua enzim. Keadaan optimum dalam sistem fermentasi pepejal bagi masa pengeraman untuk pengeluaran enzim amilase dan selulase dicatatkan pada hari ke 3 bagi amylase dan hari ke 6 untuk enzim selulase, masing-masing. pH yang sesuai untuk aktiviti enzim yang tinggi adalah pada pH-5.5 untuk enzim amilase dan pH-7.5 untuk aktiviti enzim selulase. Selain daripada itu, pada suhu 30oC penghasilan enzim adalah tertinggi bagi aktiviti enzim amilase dan untuk aktiviti enzim selulase, suhu yang sesuai ialah pada 40 oC, masing-masing dalam medium yang optimum yang telah difermentasikan dengan 107inokula spora per ml bagi kedua-dua enzim yang terlibat. Medium juga ditambahbaik lagi dengan kandungan kelembapan sebanyak 70% juga bagi penghasilan kedua-dua enzim yang terlibat. Enzim yang dihasilkan diekstrak dan dianalisa dengan menggunakan kaedah DNS untuk menentukan jumlah penghasilan gula penurun yang terhasil. Kata kunci: amylase, selulase, Aspergillus niger, fermentasi substrat pepejal

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1.0 Introduction α-amylase enzymes is used for the hydrolysis of polysaccharides such as starch into simple sugar constituents and are important in the starch processing industries (Akpan et al., 1999; Fogarty and Kelly, 1980; Nigam and Singh, 1995) whereas cellulase was used for the hydrolysis of cellulose. The amylase enzyme received great attention because this enzyme can be used commercially in production of glucose and economically benefits. Nowadays, the study of extracellular source enzymatic activities in several microorganisms has inspired curiosity in the new potential of using microorganism as biotechnological source of industrially relevant enzymes (Suganthi et al., 2011). These enzymes can be found in saliva and pancreas of an animals, malt of plants, bacteria and molds (Abu et al., 2005). Many trials have been made to improve culture conditions and appropriate strains of fungi (Abu et al., 2005). On a commercial scale, production of amylase from fungal origin found to be more stable than bacterial enzyme. Molds are capable of producing great amounts of amylase. Aspergillus sp. usually used for commercial production of amylase and cellulose enzyme. Aspergillus sp. is found to give more yields of enzyme as compared to bacteria. Solid state fermentation holds remarkable capabilities for the production of enzyme. The water-free is crucial to the microorganism’s growth and is adsorbed on a solid substrate or complexes into the interior of a solid matrix. This process has cost-effective value for countries, as these methods used cheap raw material in large quantity biomass and agro industrial waste (Tunga and Tunga, 2003). In the present work, different agricultural waste substrates were used such as rice husk, sago hampas, pineapple waste, cassava waste. Different methods and conditions were studied to obtain maximum production of amylase and cellulase production, by employing several experimental designs. 2

Hence, the objectives of this study were: 1. To screen for the best strain from Aspergillus sp. that produces high production of amylase and cellulase enzymes. 2. To identify the enzymatic activity of amylase and cellulase produced from solid state fermentation using different agricultural waste as solid substrate. 3. To identify and characterize the optimum condition of fermentation on solid state fermentation for amylase and cellulase enzyme production.

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2.0 Literature review 2.1 Agricultural waste In developing countries, large quantities of waste and crops residues are made available every year causing severe environmental pollution problems. Agricultural waste such as sago waste is one of the major wastes in West Malaysia. Other than that, rice husk, pineapple waste and cassava waste are other examples of agricultural waste that disposed off during their processing in industries. Cassava waste is a fibrous material which contains about 30–50% starch on dry weight basis. According to Pandey (2000), due to its rich organic nature and low ash content, it can serve as an ideal substrate for microbial processes for the production of value added products. Attempts have been made to produce several products such as organic acids, flavour and aroma compounds, and mushrooms from cassava bagasse. Solid-statefermentation has been mostly employed for bioconversion processes. Pineapple is important foods which are good for human health. One whole pineapple is contains about 452 calories, 118.74 g of carbohydrate, 4.89 g of protein, 1.09 g of fat and 12.7 g of fiber. Processing pineapple in industries can leave a lot of waste which can cause serious problems. Pineapple waste is a by-product of the pineapple processing industry and it consists of residual pulp, peels and skin. These wastes can cause environmental pollution problems if not utilized. Recently there are investigations or studies have been carried out on how to utilize these wastes. It was found that pineapple waste may give a big help in the production of enzyme through fermentation. Pineapple peel is rich in cellulose, hemicellulose and other carbohydrates. Ensilaging of pineapple peels produces methane which can be used as a biogas. 4

Anaerobic digestion takes place and the digested slurry may find further application as animal, poultry and fish feeds. Sago hampas is another product waste from processing industries. The waste from sago starch industry is a complex material with starchy lignocellulosic, one of the major byproducts in the industry. Literally, the sago hampas contains about 69.82% of starch and 13.88% lingo cellulose materials on dry weight basic also made up 25% of lignin (Awg-Adeni et al., 2010). Sago hampas can serves as ideal substrate for microbial processes for the production of enzyme and sugar, due to its abundant availability. Rice husk is the outer part of grains of rice that protecting the rice during the growing season. This rice husk can be put to use as building material, fertilizer, insulation material or fuel. Around 20% of the paddy weight is husk in 2008 with 661 million tons of production and consequently 132 million tons of rice husk were produced. The composition of rice husk is about 66% of lignocellulosic material, 24% of xylose and arabinose and 10% of galactose (Chockalingam et al., 2005). The utility of rice husk can be used as a carbon source for the growth of microbe.

2.2 Solid state fermentation Solid state fermentation (SSF) is one of the famous methods for fungal sporulation. SSF with fungal strain results as a more effective way to produce higher product than submerged fermentation (Cannel et al., 1980; Losane et al., 1985). SSF is economical and have many advantages, including greater volumetric yield, use of simpler equipment, inexpensive substrate, simpler downstream processing and lower energy requirement (Cannel et al., 1989; 5

Lonsane et al., 1985). Furthermore, there is low wastewater output, less problems with waste treatment than are experienced with submerged fermentation (Barreto et al., 1989). Submerged fermentation is known as high cost intensively, have highly problematic and poorly understood its unit operations and also due to its low concentration in the product, have consequent handling, reduction and dispose large volume of water during down-stream processing (Ramesh et al., 1987).

2.3 Aspergillus sp. Aspergillus is a genus of moulds which have structure that bears asexual spore. These types of fungi possess important roles in natural ecosystem and the human economy. Aspergillus is said to have a continuing attraction with their biotechnological potential especially in producing various valuable extracellular enzyme and organic acids. These groups of fungi have notorious pathogens such as A.flavus which produces aflatoxin, one of the most potent, naturally occurring compounds. Oppositely, A.niger used for the production of citric acid and enzyme such as glucose oxidase and lysozyme. The color of the spores they bear are important in identifying characteristic of the fungus such as A.flavus group bear green spore, A.niger group bear black spores and A.versicolor bear green-white spores. Aspergillus sp. has varying morphological and growth response to different nutrients so it is important to standardize conditions. Species identification depends upon pure culture grown on known media. This fungi commonly involved in many industrial processes including enzymes (amylases), commodity chemicals (citric acid) and food stuff (soy sauce) (Bennett J. W., 2010). When the spore come into contact with a solid or liquid surface, they deposited and if moisture of the

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environment are right, they are able to grow (Kanaani et al., 2008) because the fungal growth are possible almost everywhere when suitable condition of food and water are available. Aspergillus sp. also plays important roles as they are adept at recycling starches, hemicelluloses, celluloses, pectinases and other sugar polymers. Their extracellular enzymes can be exploited for the production of enzyme used in the baking, beverage and brewing industries, in making animals feeds, and in the paper pulping industries. A.niger had been developed as an efficient host for the production of heterologous proteins using genetic engineering techniques (Archer and Turner, 20006).

2.4 Amylase Amylase originated from the fungal are found to be more stable on commercial scale compared to amylase from bacteria (Abu et al., 2005), thus few actions have been made to expose the controlling mechanism that take part in the formation and secretion of extra cellular enzymes. Starch degrading enzyme like amylase has great attention in commercial industries as they give huge significance to technologies and benefits to economies (Suganthi et al., 2011). Capable in producing high amounts of amylases, molds mainly Aspergillus sp. is used in the production of α-amylase commercially probably due to the ubiquitous nature and non fastidious nutritional requirement of these organisms (Abu et al., 2005). Among best operational

in

preparation

of

this

enzyme

contain

other

enzymes,

especially

amyloglucosidases and submerged methods will give narrow range of additional enzyme, so an efficient mechanism is worthwhile to be use in production of amylase such as by using solid state fermentation. This method can be of special interest in those processes as the crude

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fermented product maybe used directly as the enzyme source (Suganthi et al., 2011). The free water is replaceable to the microorganism’s growth and then is absorbed by interior of a solid matrix by solid support.

2.5 Cellulase Cellulase is a complex enzyme composed of cellobiohydrolases, endoglucanases and βglucosidases. These enzymes act effectively in the conversion of complex carbohydrate present in lignocellulosic biomass into glucose. This enzyme is also one of important enzyme that are needed in different industrial application and been sold in huge volumes. Cellulase is used for various industries such in starch processing, animal feed production, grain alcohol fermentation, malting and brewing, extraction of fruits and vegetable juices, pulp and paper industries and textile industries (Abo-State et al., 2010). Usually, the production of cellulase is produced from submerged fermentation (SmF) method but the production cost of cellulase and low yield of these enzyme are the major problems in the industrial applications (Kang et al., 2004). Therefore, in order to optimize the production of cellulase and also lowering the cost of production of cellulase, solid state fermentation (SSF) is being used. This method has been reported that has an attractive process which is economical due to its lower capital investment and lower operating expenses (Yang et al., 2004 and Singhania et al., 2009).

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3.0 Materials and Methods 3.1 Fungi culture and maintenance There are three different strains from Aspergillus sp. used and selected in this project; Aspergillus versicolor FP13, Aspergillus niger PAN1 and Aspergillus flavus NSH9. The stock culture was obtained from UNIMAS fungi collection at Molecular Genetic Laboratory. As for the fungal growth, Potato Dextrose Agar (PDA) was used to culture Aspergillus sp. Approximately, 9.75 g of Potato Dextrose Agar powder was prepared, then mixed with 250mL of distilled water. The medium was then stirred until completely dissolved and undergo sterilization by autoclaving at 121oC for 20 minutes. After that, the medium was cooled dowm to ambient before poured into petri dishes. The media was stored at 4oC for further used. The culture were grown and maintained on Potato Dextrose Agar (PDA) at 28oC for 7 days in order to allow sufficient spore formation.

3.2 Preparation of spore suspension

The cultures then repeatedly subcultured into fresh media. After several weeks, a pure culture was obtained from the subculturing. The spores were then harvested from the media by pouring sterile 0.1% Tween-80 on surface of media to wash off the spores. The spore concentration is measured by counting with a hemacytometer under the microscope. The spore suspension was used as inoculums in the subsequent fermentation experiments. Aspergillus sp. were routinely maintained on PDA at 4oC by regular sub-culturing (no longer then 3 months).

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3.3 Amylase and cellulase enzyme screening Preliminary screening for amylase and cellulase production from Aspergillus isolates were carried out on starch (amylase) and carbomethyl cellulose (CMC) (cellulase) agar plate assay on standard media (Abe, et al., 1988; Akpan et al., 1999 and Fogarty, 1983) with minor modification. The final constituents of media was consisted of the following in (g/L): 1.5g yeast extract, 2.0 g soluble starch, 0.5 g peptone, 1.5g NaCl and 15.0g agar dissolved in 1.0 liter double distilled water, and autoclaved. After 72 hours of incubation, the inoculated plates containing media supplemented with starch were stained with Gram’s iodine reagent. Plates were flooded by iodine solution (amylase) and Congo red (cellulase), respectively, for 15 minutes and washed with 1 M of NaCl to remove the excess color and subsequently photographed. The isolate that produces amylase and cellulase will be detected by the formation clear halo on soluble starch solution (amylase) and orange digestion halos (cellulase) when treated with iodine solution and Congo red, respectively (Sanghi et al., 2008).

3.4 Substrate preparation Agricultural waste such as rice husk, sago hampas, pineapple waste and cassava waste were used as the substrate for solid state fermentation. The wastes were cut into small pieces and washed under tap water, then boiled for at least one hour to remove any remaining reducing sugar residue, then oven dried. The oven dried wastes were ground into small particles using a mill and finally separated by sieves. The fraction that passes through the sieve was used for medium preparation in the SSF. (Gao et al., 2008).

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3.5 Solid-state fermentation (SSF) for enzyme production

Different agricultural waste such as sago hampas, rice husk, pineapple waste and cassava waste were obtained and prepared were used as solid substrate for comparison in solid state fermentation. Approximately, 5g of agricultural waste was mixed with 2 mL of medium salt solution in a 250 mL Erlenmeyer flask and autoclaved at 121oC for 20 minute (Francis et al., 2003). After cooled down to room temperature, the waste was inoculated with 0.5 mL of spore suspension (106 spores mL-1). After sterilization, the fermentation was inoculated with 1% of inoculum and fermentation was carried out at room temperature for six days under relatively humidity of 97%.

3.6 Enzyme extraction Approximately, 22mL of 0.1M phosphate buffer saline (pH 7) was added to each of inoculated substrate beds and vigorously shaken in rotary shaker for 55 minutes at 130rpm. The mixture was filtered through cheese cloth and centrifuged at 5000rpm at 4oC for 15 min. The supernatant was filtered through cheesecloth and filtrate is used as the crude enzyme preparation. Amylase and cellulose enzyme were assayed by 3, 5-dinitrosalicilic acid method (Miller, 1959).

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3.7 Enzyme Assay 3.7.1 Amylase activity measurement Amylase assay was determined by using a reaction mixture contained 0.8 ml of 1% (v/v) starch solution mixed with 1.0 mL of 0.5M phosphate buffer, pH 6.5 and 0.2 mL crude enzyme, and the mixture was incubated for 15 min at 30oC. After incubation, 1 ml DNS reagents was added and mixed well to detect enzyme activity and then the mixture was boiled for 10 minute. After boiling, 1.0 mL of chilled 40% (w/v) sodium potassium tartarate (Rochelle salt) was added to stabilize the color of reaction. The resulting color due to reaction of DSNA and reducing sugar is measured at 540 nm wavelength (Miller 1959) against blank. The blank was prepared with the same mixture but the crude enzyme used was boiled first for 5 minutes. One enzyme unit (U) was equivalent as the amount of enzyme released 1.0 µM of glucose per unit time per unit volume. A graph of absorbance at 540 nm versus glucose standards was prepared (Appendix A).

3.7.2 Cellulase activity measurement

The enzyme activity for endoglucanase, carboxymethyl cellulase (CMCase) was determined as reported by Wang et al., (1988). The enzyme activity was carried out in the total reaction mixture of 0.2 mL of the crude enzyme supernatant and 0.8 ml of 1% (w/v) CMC solution in sodium acetate buffer solution at pH 5. This mixture was incubated at 60oC for 30 min. The release of reducing sugars was determined by the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). After incubation, the mixture was mixed with 1 mL of DNS reagent was added 12

and mixed well. The mixture then was boiled for 10 min. After boiling, the reaction mixture was added with 1 mL of 40% (w/v) sodium potassium tartarate (Rochelle salt) to stabilize the color reaction. The reaction mixture was measured at absorbance of 540 nm against blank using spectrocytometer. The blank was prepared same as the mixture but the crude enzyme was boiled first for 5 minutes. One unit (U) of enzyme activity was defined as the amount of enzyme required to release 1.0 µM of glucose from the appropriate substrates per minute under the assay. A graph of absorbance at 548 nm versus glucose standards was prepared (Appendix A).

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3.8 Production and optimization of amylase and cellulase in Solid State fermentation (SSF) 3.8.1 Effect of temperature on SSF The effect of temperature on enzyme production was determined on SSF in different substrates and incubated at different temperature. In this experiment, the range of temperature used was 30oC, 35 oC, 40 oC, 45 oC, and 50 oC. at pH 7 for six days. The SSF was done according the description as described in Section 3.4, 3.5, and 3.6.

3.8.2 Effect of inoculum size on SSF The effect of inoculum size on production of enzyme on SSF was determined at different range of size of spore suspension. The range size of 105, 106, 107, and 108 spore per ml were used in this experiment. The substrate was fermented with different size of inoculum and assay was performed as described in Section 3.4, 3.5, and 3.6.

3.8.3 Effect of time of incubation on SSF The effect of time incubation on production of enzyme on SSF was determined at different time of incubation. The different range of time of incubation is done by incubating the fermentation at different period of time. The fermentation was incubated and harvested at 3 days interval; at 3days, 6days, 9days, 12days and 15days. The substrate was fermented with different time of incubation and assay was performed as described in Section 3.4, 3.5, and 3.6.

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3.8.4 Effect of pH on SSF In SSF, the effect of pH on production of amylase and cellulase in different substrate was determined by adjusting the pH of medium salt solution. The pH used in this fermentation areexperiments were 4.5, 5.5, 6.5, and 7.5. The different substrate was fermented with different pH and assay was performed as described in Section 3.4, 3.5, and 3.6.

3.8.5 Effect on moisture content on SSF

The effect of moisture content on production of enzyme on SSF in different substrate was determined by using variable moisture condition for fermentation. In this fermentation, the moisture conditions of the substrate were adjusted to 50%, 60%, 70% and 80% of moisture content. The different substrate was fermented with different moisture content and assay was performed as described in Section 3.4, 3.5, and 3.6.

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4.0 Results and Discussion 4.1 Strain Selection 4.1.1 Screening During the screening, the isolate were grown on soluble starch for amylase and carbomethylcellulose (CMC) for cellulase. The minimal agar plate for screening cellulase is consisting of the following in g/L: yeast extract (2g), KH2PO4 (1g), MGSO4.7H2O (5g), a soluble form of cellulose, carbomethylcellulose (CMC) (5g) and agar powder (17g). While for the preparation for screening for amylase consists of the following in g/L: yeast extract (2g), KH2PO4 (1g), MGSO4.7H2O (5g), a soluble starch (5g) and agar powder (17g) (Apun et al., 2000). The isolate plates are incubated in 30oC for 48 hours. After 2 days, the isolate were undergo screening steps. The isolate plates were flooded with an aqueous solution 1% of congo red in CMC’s plate and Iodine solution in soluble starch’s plates for 30 minutes. Iodine solution is prepared by mixing of 0.2% of Iodine and 0.4% of potassium iodide. Then plate is washed with 1M of NaCl as cited by Teather and Wood (1982). After washing with NaCl, the zone of clearance produced on the starch and CMC plate. The halos produce around the isolate is measured and used as indication of cellulolytic and amylase activity of the each strains. Based on the results obtained as shown in Table 1, Aspergillus niger PAN1was selected for further studies on amylase and cellulase production in solid substrate fermentation. Table 1: Diameter of halos around the isolate

CMC Soluble starch

A.flavus 1.2 cm ± 4.5 cm ±

A.niger 1.5 cm ± 5.5 cm ±

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A.versicolor 1.7 cm ± 3.5 cm ±

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