· PRODUCTION OF CRUDE CELLULASE FROM Aspergillus versicolor UNDER SOLID STATE FERMENTATION (SSF) USING DIFFERENT

AGRO WASTE AS SUBSTRATE

Noorashikin binti Johari

Bachelor of Science with Honoun QK (Resource Biotechnology) 896 2015 N818 1015

Pusat Khidmat MakJumat Ak::t(k,.,.:· UNlVtRSm MAL4YSIA SARAWM·

PRODUCTION OF CRUDE CELLULASE FROM Aspergillus versicolor UNDER SOLID STATE FERMENTATION (SSF) USING DIFFERENT AGRO WASTE AS

SUBSTRATE

NOORASHIKIN BINT! lOHAR!

This project is submitted in partial fulfilment of the Final Year Project 2

(STF 3015)

Supervisor: Ms Rosmawati binti Saat

Co-supervisor: Assoc. Prof. Dr. Awang Ahmad Sallehin bin Awang Husaini

Bachelor of Science with Honours

(Resource Biotechnology)

2015

ACKNOWLEDGEMENTS

Alhamdulillah, all praises to Allah for His blessing and strength He gave to me in

completing this thesis. Special appreciation goes to my supervisor, Miss Rosmawati Binti Saat

and my co-supervisor Dr. Awang Ahmad Sallehin Bin Awang Husaini for their supervision

and extremely supportive towards me. Their valuable help, suggestions, and constructive

comments throughout the proposal, experimental and thesis work have significantly

contributed to the success of this research.

I would like to express my appreciation to all lecturers and staff members of Faculty of

Resource Science and Technology, University Malaysia Sarawak for their help and support

towards my research project.

Sincere appreciations to all my friends, course mates, laboratory mates and also .

postgraduate seniors for their helps, kindness and moral support they given to me along the

journey. My deepest gratitude goes to my parent, Mr. 10hari Bin Mohamed Noor, Mrs.

Rozanah Binti Mohd Tahir and also to my siblings for their endless love, prayers and

encouragement.

Lastly, it is impossible to list all the individuals that have been with me throughout this

research, thus, I dedicate my outmost gratitude to all of them. Thank you very much .

..

I

DECLARATION

I hereby declare that this thesis entitled "Production of Crude Cellulase from Aspergillus

versicolor under Solid State Fermentation (SSF) Using Different Agro Wastes as Substrates"

is my own work and all sources have been quoted and referred to have been acknowledged by

means of complete references. It has been submitted and shall not be submitted to other

university or institute of higher learning.

(NOORASHIKIN BINTi JOHAR!)

Date: J.'~ Junt ~OJ5

Bachelor of Science with Honours

(Resource Biotechnology)

II

I

Pusat 'hidmat M klum:Jl Akadcm" lJI';lVERSITI MALAYSIA SARAW.\ I

TABLE OF CONTENTS

Acknowledgement .......................................................................................................... I

Declaration.................................................................................................................... II

Table of contents ......................................................................................................... III

List of Abbreviations ................................................................................................... IV

List of Tables ............................................................................................................... VI

List of Figures....................................... · ...................................................................... VII

Abstract .......................................................................................................................... 1

1.0 Introduction........................................................................................................ 2

2.0 Literature Review .............................................................................................. 4

2.1 Aspergillus spp. and Aspergillus versicolor ................................................ 4

2.2 Cellulase and Cellulose ............................................................................... 5

2.3 Solid State Fermentation (SSF) ................................................................... 7

2.4 Substrate ...................................................................................................... 8

2.4.1 Sago hampas ..................................................................................... 8

2.4.2 Empty fruit bunch ............................................................................ 9

2.4.3 Rice Husk ....................................................................................... 10

3.0 Materials and Method ...................................................................................... 11 . . 3.1 Pre-treatment of Substrate ......................................................................... II

3.2 Preparation Petri Dish and PDA Medium ................................................. 11

3.3 Cultivation of Aspergillus . versicolor ........................................................ 11

3.4 Solid State Fermentation Using Three Different Types of Agriculture Waste As a Substrate ................................................................................. 12

3.5 Extraction of Crude Enzyme ..................................................................... 12

3.6 Enzyme Assay ........................................................................................... 13

3.6.1 Measurement of Cellulase Activity (FPase) ................................... 13

3.6.2 Protein Determination .................................................................... 14

3.7 Optimization of Production of Cellulase Enzyme ..................................... 14

3.7.1 Incubation Time ............................................................................ 15

3.7.2 Initial Moisture content. ................................................................. 15

3.7.3 Temperature ......................................~ ........................................... 15

4.0 Results.............................................................................................................. 16

4.1 Production of Aspergillus versicolor on the PDA and their morphological structure ..................................................................................................... 16

III

4.2 Enzyme Assay ........................................................................................... 17

4.2.1 Cellulase Activity Assay ................................................................ 17

4.2.2 Protein Determination .................................................................... 18

4.3 Optimization of Production of Cellulase................................................... 18

4.3.1 Production of Cellulase Enzyme by Different Substrate at Different Incubation Time .......................................................................... 19

4.3.2 Production of Cellulase Enzyme by Different Substrate at Different Moisture Content ........................................................................ 20

4.3.3 Production of Cellulase Enzyme by Different Substrate at Different Temperature (0 C) ........................... ............................................ 21

4.4 Production of Crudes Cellulase by Different Substrate for Optimum Condition ................................................................................................... 22

4.5 Specific Cellulase Activity (U/mg) Against Types of Substrates ............. 23

5.0 Discussion ........................................................................................................ 24

5.1 Measuring Cellulase Enzyme Activity Using Filter Paparase Assay and DNS Method ............................................................................................. 24

5.2 Protein Determination ............................................................................... 24

5.3 Effect of Different Fermentation Conditions and Types of Substrate

on Production of Cellulase Enzyme ......................................................... 25

5.3.1 Effect of Incubation Period on Enzyme Production Using Different Types of Substrate ...... : .............................................................. '26

5.3.2 Effect ofInitial Moisture Content (%) on Enzyme Production Using Different Types of Substrate ...................................................... 26

5.3.3 Effect of Temperature (OC) on Enzyme Production Using Different Types of Substrate ........................................................................ 28

5.4 Effect of Cellulase Production across Different Composition of Substrate under Optimum Condition ........................................................................ 29

5.5 Specific Cellulase Activity (U/mg) Against Types of Substrates ............. 31

6.0 Conclusion & Recommendations .................................................................... 32 ~'

7.0 References ........................................................ : ............................................... 33

8.0 Appendixes ...................................................................................................... 36

Appendix A: Calculation Moisture Content Dry Basis and Dry Matter Exist 36

Appendix B: Calculation for 70% ofInitial Moisture Content in EFB ........... 37

Appendix C: Standard Curve for reducing Sug~r Concentration .................... 38

Appendix D: Enzyme Activity Calculations ................................................... 39

Appendix E: Standard Curve for Total Number of Protein (BSA) .................. 41

IV

LIST OF ABBREVIATIONS

SSF

SmF

A. versicolor

EFB

Spp.

UNIMAS

FPase

Solid State F ennentation

Solid Submerged Fennentation

Aspergillus versicolor

Empty Fruit Brunch

Species

University Malaysia Sarawak

Filter-paperase

v

I

LIST OF TABLES

Table 1 Different intensity of colour from each substrates using Page 18 Bradford Method (B: blank; SH: sago hampas; EFB; empty fruit bunch; RH: rice husk)

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VI

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LIST OF FIGURES

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Complex component of cellulase that act synergistically for degradation of cellulose (Source: Singhania, 2009)

Aspergillus versicolor appeared to dark green with the white border after 14 days of cultivation

Colour fonnation of mixture of sample before (left) and after (right) boiling (B: blank; SH: sago hampas; EFB; empty fruit bunch; RH: rice husk)

Cellulase activities (U/ml) against days of incubation time using three different types of agro waste

Cellulase activities (D/ml) against moisture content (%) using three different types of agro waste from solid state fennentation

Cellulase activities (U/ml) against temperature (0 C) using three different types of agro waste from solid state fennentation

Cellulase' activities (U/mf) against different types of agro waste under optimum condition of incubation days, moisture content (%) and temperature (0 C)

Both cellulase activity (U/ml) and specific cellulase activity (D/mg) are different in number

Illustrated figure of plant cell showing the cellulose is surrounded by the hemicellulose and lignin (Sources: Annie, 2013)

Page 6

Page 16

Page 17

Page 19

Page 20

Page 21

Page 22

Page 23

Page 29

VII

Production of Crude Cellulase from Aspergillus versicolor under Solid State Fermentation (SSF) Using Different Agro Wastes as Substrates

Noorashikin Binti Johari

Resource Biotechnology Faculty of Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Malaysia produced abundant amount of plant waste residues from manufacturing and plantation industry. Sago hampas, rice husk and empty fruit bunch are the examples of agro wastes and they have many beneficial uses in bio based productions. The purpose of this study is to produce cellulase from Aspergillus versicolor (A. versicolor) under solid state fermentation by using three different agro-wastes as substrates which are sago hampas, rice husk and oil palm empty fruit bunch. Fungi are progressively used by the researchers to produce beneficial enzymes like cellulase, pectinase as well as xylanase. Activity of crude cellulase produced was determined by using Filter-paperase activity test (FPase) and DNS method by Mi ller (1959). Protein determination also was done using Bradford's method to determine the specific cellulase activity. SSF parameters such as initial moisture content, time of incubation, and temperature were optimised in order to produce high enzyme activity of cellulase. Optimum condition for the sago hampas to produce highest cellulase activity (U/ml) is on fourth day of incubation time, 70% of initial moisture content and 30°C of temperature; whereas, both cellulase activities (Ulml) in empty fruit bunch and rice husk substrates was optimum in sixth day of incubation time, with equal 70% of initial moisture content and also 30°C temperature. Hence, based on this study, sago hampas was found to be the best substrate for cellulase production from Aspergillus versicolor.

Key words: Aspergillus versicolor, empty fruit bunc"h, sago ham pas, dee husk

ABSTRAK

Malaysia mengeluarkan jumlah sisa buangan yang banyak di dalam pembuatan dan industri perladangan. Hampas sagu, sekam padi dan buah tandan kosong daripada kelapa sawit adalah contoh sisa buangan pertanian dan mempunyai banyak kegunaan yang baik dalam pengeluaran berasaskan bio. Tujuan kajian ini adalah untuk menghasilkan selulase dari Aspergillus versicolor CA. versicolor) di bawah penapaian keadaan pepejal dengan menggunakan tiga berbeza agro-bahan buangan sebagai substrat iaitu hampas sagu, sekam padi dan kelapa sawit taOOan buah kosong. Banyak spesies kulat yang telah digunakan secara progresif untuk menghasilkan enzim yang bermanfaat seperti selulase, pectinase serta xylanase. Aktivili selulase mentah yang dihasilkan telah ditentukan dengan menggunakan ujian aktiviti Penapis­paperase (FPase) dan ktledah DNS oleh Miller (1959). Penentuan Protein juga telah dilakukan dengan menggtlllakan kaedah Bradford untuk menentukan aktiviti !Jelulase. Parameter SSF seperti kaOOungan kelembapan awal, masa pengerarnan, dan suhu telah dioptimumkan untuk menghasilkan aktiviti enzim seluJase yang tinggi. Keadaan optimum untuk hampas sagu untuk menghasilkan aktiviti selulase tertillggi (Ulml) adalah pada hari keempat masa inkubasi, 70% kandungan kelembapan awal dan pada suhu 30°C; sedangkan, kedua-dl/a aktiviti selulase (U/ml) dalam tandan kosollg dan sekam padi adalah optimum pada hari keenam masa inkubasi, dengan sarna rata 70% kaOOungan kelembapan awal dan juga pada suhu 30 °e. O/eil itu, berdasarkan kajian ini, hampas sagu telah diken'tIl pasti sebagai substrat yang terbaik untuk pengeillarall selulase daripada Aspergillus versicolor.

Kata kunci: Aspergillus versicolor, buah tandan kosong kelapa sawit, hampas sagu, sekam padi

1

1.0 INTRODUCTION

In Malaysia, there are large proportion of waste products such as food waste, dump

waste, and solid waste every year (Samson et al., 2004). This situation also happened in other

countries such as Europe, India, and Thailand. Indeed, people use much money to get rid of

this waste and subsequently, the dumping cost is increasing. Although many efforts have been

done to reduce this waste, but it is still not enough to reduce this abundant of waste. Thus,

other alternative such as using these wastes to produce important products such as enzymes

may be done as to solve the problem.

Cellulase production is important in this research as the cost for producing cellulase by

using readily substrate such as glucose are getting higher in industrial cellulase production.

The current research was focused more on the green based technology by utilising agro wastes

as the substrate. · In this study, agro wastes such as sago hampas, rice husk and empty fruit

bunch were used in order to produce cellulosic enzyme under solid state fermentation by using

Aspergillus versicolor as the fungi.

Agro waste can be defined by any left-over of agriculture's activity. This kind of

product could be contributed as a carbon sources and it can be utilized by the microorganism

such as fungi to complete their living cycle; then the fungi will produce enzymes during their .. metabolism in the living system (Samson et al., 2004). The condition must be optimised, so

that the fungi are able to fully use their living system and produce their beneficial product

(Samson et al., 2004).

2

Solid state fennentation (SSF) is much more efficient compared to other type of

fermentations such as submerged fermentation (SmF) (Masutti et al., 2012). The production of

prnducts from SSF is higher with better characteristic compared to submerged fennentation

(SmF) (Masutti et al., 2012). Application of the three types of agro waste mentioned above as

the substrate in this SSF technique may reduce the process cost and also may solve the

pollution problems.

The aim of this study is to produce the crude cellulase from the Aspergillus versicolor

(A. versicolor) under SSF by using different types of agro waste as the substrate. Thus, the

objectives of this study are as follows:

1. To investigate the potential of A. versicolor to produce cellulase from the selected agro

wastes.

2. To detennine the best substrate for the production of cellulase among the three substrates

used.

3. To detennine the optimal SSF conditions for A. versicolor to produce cellulase.

~'

3

2.0 LITERATURE REVIEW

2.1 Aspergillus spp. and Aspergillus versicolor

Microorganism has been used by the scientist in industrial field to obtain the new

products. Aspergillus versicolor (A. versicolor) is one of the microorganisms that have been

applied by the researcher in bioprocessing technology (Silva et al., 2011). Moreover, the

national agencies of the United States Department of Health and Human Services, "Food and

Drug Administration" (FDA) have considered Aspergillus spp. as harmless. However, some of

these species are famous with the mycotoxin production which poisons to human (Silva et al.,

2011). For example, the aflatoxin that mostly caused by the Aspergillus flavus and Aspergillus

parasiticus is poisonous and carcinogenic substances (Richard & Payne, 2003). This aflatoxin

can cause severe hepatitis, immunosuppression, and hepatocellular carcinoma (Richard &

Payne, 2003).

According to Shahlaei and Pourhossein (2013), each strain of the microorganism such

as fungi, bacteria or yeast will produce different type of enzymes. Enzymes such as cellulase,

glucoamylase, protease and lipase have been produced by using the Apergillus spp. (Pandey et

al., 1999). Hence, this species of fungi are capable to yield the beneficial industrial enzyme.

Pandey et al. (1999) stated that common fungi strain that can be utilised to produce hydrolytic ~'

enzymes such as cellulase, pectinase and xylanase are Trichoderma spp. and Aspergillus spp.

(Pandey et al., 1999).

A. versicolor originated from the kingdom of Ftfngi, family of Trichocomaceae and

genus of Aspergillus (Samson et at., 2004). In 1903, it was first defined by lean-Paul

4

Pusat Khidm~t l\'lakJumar Akadrr. . UN] ~ In MALAYSIA SARA\\~

Vuillemin and also called with the name of Sterigmatocystis versicolor before being converted

to Aspergillus versicolor in 1908 by Carlo Tiraboschi. It is related with sterigmatocystin and

nidulotoxin toxic metabolites (Samson et al., 2004). This species of fungi can be widely found

in extensive range of places in nature as long as the substrates are provided. However, the

species of A. versicolor is a kind of mould that stays in an enclosed environments and it

becomes the main reason of why it can frequently being discovered in a places like grain

warehouse. Typically, it can be found on cheese, cereals, spices, dry meat products as well as

fresh or alive matter such as ground nuts (Samson et al., 2004).

A. versicolor uses their spores for asexually replicating in organic materials and soil.

This fungus is the type of fungi that can grow at or small level availability of water it could

propagate at high or low of 60% of the relative humidity (RH) (Samson et al., 2004). It will be

noticeable by its earthy volatiles scent and the existence of this odour always correlated with

this fungus (Bjurman & Kristensson, 1992). Even though their pattern of colony is varies but

their morphological feature is frequently alike (Samson et al., 2004). In a semi-synthetic solid

medium like Czapek Agar (contains a sucrose as carbon sources and nitrate as a nitrogen

sources), it can be identified firstly by its white in colour, then changing to yellow, orange-

yellow to green-yellow and sometimes mixing with meat to pink shades (Samson et al., 2004).

2.2 Crude CellulaS'e and Cellulose

The cellulose is made of linear polysaccharide glucose with ~-1, 4 linkages and it is a

non-cross-linked like chitin's structure (Spano et al., 1975). Native cellulose is unsolvable in oil

water and occurs as fibres of densely packed with hydrogen bond. Without the mechanical and

5

• (CBH)

" Cellobiose

• ..

chemical degradation, it is very resistant to hydrolysis. Hence, cellulase is needed in cellulose

decomposition (Spano et at., 1975).

Cellulase is a hydrolytic enzyme of the cellulose. It compnses of three malO

components which are Endo-~-glucanase, Exo-~-glucanase, 'and also ~-glucosidase (Spano et

ai., 1975). These three component of cellulase act synergistically for degradation of cellulosic

construction in a houseplant (refer to Figure 1). Cellulase hydrolyzes the cellulose to produce

glucose, cellobiose and other oligo-saccharides as primary products which can be used in

generating other bio-based products (Singhania, 2009), Degradation of cellulose firstly start by

Endo-~-glucanase which it acts on cellulose fibers and reacts in between the chain randomly,

thus releasing small fibers with free reducing and non-reducing ends. Next, the Exo-~-

glucanase acts on free ends to release the cellobiose, and finally help ~-glucosidase to produce

the end product of glucose (Singhania, 2009).

Glucoae • Celloblohydrolase

Figure 1. Complex component of cellulase that act synergistically for degradation of cellulose (Source: Singhania, 2009).

6

The use of enzymes cellulase can be applied in isolation of plant protoplast (Pandey et

al., 1999). On top of that, the cellulase has been broadly applied in textile and pulp industry

plus food processing. For drinking production like coffee, the cellulase is crucial in removing

cellulose during the coffee bean seed's ventilation (Pandey et ai., 1999). The attribute of

cellulase enzyme is different depending on their provenance. However, the main property of

the microbial cellulase has displayed proteins with pH of less than 7; this acidic property is

crucial for their degradation of cellulose substrates. Besides, this enzyme also can be repressed

completely by Mercury (Hg) and slightly inhibited by Manganese (Mn), Silver (Ag), Copper

(eu) and Zinc (Zn) ion (Pandey et ai., 1999).

2.3 Solid State Fermentation (SSF)

Nigam and Pandey (2009) stated that fermentation without consuming water or

approximately no water can be called as solid state fermentation. Nevertheless, the condition

of the substrate must be humid enough so that it can help in development and digestion of

microbe (Nigam & Pandey, 2009). Commonly, it has been chosen in the bioprocessing

industry because the method is suitable for development and growth of the microorganism

(Pandey et ai., 1999). According to Pandey et al. (2000), SSF is the one type of bioconversion

technique that can change a raw material to another type of value products such as chemical,

primary and also secondary metabolite which are beneficial in industrial. For example, SSF is

used to yield numerous microbial products such as biofuel, food, pharmaceutical and also

chemical products in the industrial field (Pandey et al., 2000).

SSF has ready moisture content in the substrate (Nigam & Pandey, 2009). Therefore, it

can reduce the usage of water in this method. Since a less water is used, little claim of sterility

7

\

is required. There are some benefits by using this technique over solid submerged

fennentation (SmF) or other fermentation technique; which is less energy consuming and high

concentration of product is generated during the SSF process (Nigam & Pandey, 2009). Next,

SSF method only needs a small fermenter size compared to SmF and this can reduce the whole

downstream processes. In overall, it can reduce operational cost. However, the process needs a

better knowledge or expert in choosing the substrate and material uses since it will affect the

cultivation of the microorganism later during fermentation (Nigam & Pandey, 2009).

2.4 Substrates

Substrate is the molecule that can be utilised to produce another product (Pandey et ai.,

1999). Three types of agro waste substrate were used in this propose experiment which are

empty fruit bunch from palm oil, sago hampas and also rice husk. Each substrate contains

different composition of nutrients and this can help for the development of Aspergillus

versicolor (A. versicoior) (Pandey et ai., 1999). By using agro-waste as a substrate in

bioprocess, the waste in our country can be reduced and the cost of the bioprocess may be

reduced.

2.4.1 Sago Hampas

Sago hampas is. a starchy lignocellulose agro-residue substrate from sago palm starch

production (Awg-Adeni et ai., 2010). This sago hampas was collected from the pith of

Metroxylon sagu after the processing of the starch. About 70 to 90 % of starch can be

extracted out of 100 kg of sago pith while the remaining' 10 to 30 % of the starch still inside

the sago hampas. Besides, around 45359.2 Kg to 99790.3 Kg of sago hampas is generated

8

2.4.3

daily in Sarawak (Awg-Adeni et al., 2010). It contains roughly 0.66% of starch and 0.14%

fiber on its dry mass; sago hampas consists of high carbon, nutrients and moisture (Chew &

Shim, 1993). Hence, sago hampas is the best choice as substrate in solid state fermentation

process since it can provide enough carbon sources for the cultivation of the fungi.

2.4.2 Oil Palm Empty Fruit Bunches (EF8)

Oil palm empty fruit bunch is the by product from the oil palm plantation. Kerdsuwan

and Laohalidanond (2011) stated that this fruit bunches grow in the middle of trunk and base

of the novel palm leaf part. Previously, it will be left aside after process of oil extraction at

industrial site of oil palm and become useless. Nowadays, it has been used widely to generate

bio-energy such as fuel (Kerdsuwan & Laohalidanond, 2011). The studied shown by Deraman

(1993) showed that 45% to 50% of EFB composed of cellulose and 25% to 35% of it consists

of hemicellulose and lignin. Hence, this is the huge opportunity to use it as the agro-waste

substrate in this study since the A. versicolor needs a carbon sources for their metabolism to

create enzymes.

Rice Husk

In Malaysia, Sarawak is in the fourth position for rice producer after Kelantan, Kedah,

and Perak (Lee et al., 2Q14). Yield of rice is increasing by the year due to high demands for

food supply. The higher the production of rice, the higher the amount of the by-products will

be generated, such as rich husk. Based on the research made by Lee et al. (2014), rice husk

consist of lignin, cellulose and hemicellulose with diverseolfunctional groups such as hydroxyl,

carbonyl and carboxylic. Every year, about 562 600 tons of rice husk are produced (Lee et al.,

9

2014). This is a huge amount of waste and they must be handled efficiently in order to sustain

the cleanness of our environment. Normally, the rice husk is used by the farmers to maintain

aeration, water holding capacities and sometimes use as a mulch or compost (Muchena &

Hilhorst, 2000). However, certain of the farmers convert this rice husk to ash and used it as a

fertilizer for improving the growth and condition of the plant (Muchena & Hilhorst, 2000).

The conversion of the rice husk into the ash used high amount of heat for burning and

somehow this activity can lead to air pollution. So, by using it as the substrate in this study,

hopefully this can indirectly maintain our environmental system.

10

3.0 MATERIALS AND METHOD

3.1 Pre-treatment of Substrate

All the three substrates were obtained from the Molecular Genomic Laboratory,

Faculty Resources Science and Technology (FRST), UNIMAS. The sago hampas and EFB

was ground separately using electrical blender. As for the rice husk, it was already pre-treated

mechanically by grinding. 1 gram of each substrate in a crucible was dried separately in an

oven at 70 DC temperature and the moisture content was determined by using moisture dry

basis calculation (refer to Appendix A and B).

32 Preparation Petri Dish and PDA medium.

Petri dish was sterilised under the UV light in the laminar floor and the PDA was

prepared by using the commercial Potato Dextrose Agar (PDA). Approximately, 100 ml of

medium agar was needed to prepare a three plate of medium. The PDA solution was prepared

in the glass bottle by using 3.9 g of commercial Potato Dextrose Agar (PDA) powder and 100

ml ofultrapure water. The PDA solution was mixed using the stirrer without applying the heat

and was autoclaved in 121°C, 15 psi for 15 min. After that, the autoclaved bottle containing

PDA solution was run under the tap water to decrease the temperature. 50 mg/IlL of ampicillin

was added after the temperature of the PDA solution was cooled for making 100 ml of

medium agar.

11

3.5

3.3 Cultivation of Aspergillus versicolor

The strain of Aspergillus versicolor was provided by the Molecular Genomic

Laboratory in Faculty Resources Science and Technology, UNlMAS and re-cultivated on the

Potato Dextrose Agar (PDA) media. The plates were incubated at room temperature for 10 to

14 days and stored at 4 °c in the refrigerator after the maturation of fungi before use.

3.4 Solid State Fermentation Using Three Different Types of Agricluture Waste as a

Substrate

An amount of 5 g for each substrate (rice husk and empty fruit bunch) was weighted

and put separately into a 250 ml conical flask with doubled distilled water to achieve 70% of

initial moisture content (refer to Appendix A and B for calculation). Then, 3 identical plugs of

Aspergillus versicolor grown on PDA were inoculated into the flask containing substrate. The

entire flasks were incubated for 6 days and at'30 °c with static condition. Each sample was

done in a duplicate with the same variable and whole step was repeated using 2.5 g of sago

hampas (Masutti et al., 2012).

Extraction of Crude Enzyme

The flasks were taken out from the incubator and carried to room temperature after r '

fementation. For the extraction, 20 ml of sodium acetate buffer O.OlM pH 5.8 was added into

the whole SSF component in the Erlenmeyer flask and the flask was shaken at 120 rpm for 30

minutes. Suspension in the flask was filtered in the ice box using muslin cloth into the 50 ml

falcon tube. Next, the filtrate was centrifuged at 6000 rpm and 4 DC for 20 minutes and the

remaining solid fraction was removed (Conti et al., 2001). After that, the supernatant (crude

12

enzyme) was filtered using qualitative filter paper twice into another 50 ml falcon tube and

kept at 4°C for analysis of cellulase (cellulase assay and protein determination).

3.6 Enzyme Assay

3.6.1 Measurement of Cellulase Activity (FPase)

Cellulase activity was analysed using Filter Paperase Assay and the liberated glucose

was estimated using 3, 5-dinitrosalicyclic acid (DNS) method by Miller (1959). Firstly, 50 mg

(1 x 6 cm) of rolled What man filter paper strips and 1 ml of 0.05 M sodium citrate buffer (pH

4.8) was added into the test tube. Then, 1 ml of crude enzyme (supernatant from each SSF)

were added into the mixture and incubated for exactly 1 hour at 50°C. At the same time, the

control without the crude enzyme was done by replacing the crude enzyme with the 0.01 M of

sodium acetate buffer pH 5.8 (extraction buffer).

After 1 hour of incubation, 2 ml of Dinitrosalicylic acids (DNS) was pipetted to each

of the sample and were mixed by vortex. After that, all the test tube was boiled for 15 minutes

and 1 ml of 40 % sodium potassium tartrate (Rochelle salt) was added immediately after 15

minutes of boiling. The mixtures in the test tubes were mixed again by vortex and 1 ml of each

sample from the test tube were pipetted out into the 1.5 ml cuvette after the samples were

cooled and the absorbance were read by using spectrophotometer at 540 nm. The glucose

concentration for each samples was determined by referring to the standard glucose graph

constructed (refer to Appendix C). Next, the cellulase enzyme activity was calculated using

the enzyme activity formula (refer to Appendix D). T~ cellulase activity was taken as units

13

3.7

per millilitre (U/ml). One unit of cellulase enzyme activity is defined as the amount of the

enzyme required to release 1 mg of reducing sugar per ml.

3.6.2 Measurement of Total Protein (Protein Determination)

The concentration of the protein was indicated by Bradford Protein Assay using BIO­

PROTOCOL (Lo et at., 2009). 0.1 % of Bradford reagent was prepared using Coomassie

Brilliant Blue G-250, 85% phosphoric acid, 95% ethanol and ultrapure water. The Coomassie

Brilliant Blue G-250 was first dissolved with 95% ethanol, and 85% phosphoric acid. Next,

the acid solution was poured slowly into the water and the dye was dissolved completely. The

precipitate was filtered before used by using the qualitative filter paper and stored in the dark

bottle at 4°C. The standard curve of absorbance against concentration of bovine serum

albumin (BSA) was generated (refer to Appendix E). The absorbance was identified by using

spectrophotometer at 595 nm after 20 minutes of incubation at the room temperature (Lo et

at., 2009). The total number of protein was determined by referring to the BSA standard graph

constructed (refer to Appendix E). The specific activity of cellulase was calculated using the

fonnula of specific enzyme activity (refer to Appendix F).

Optimisation of Production of Cellulase Enzyme

Initial moisture content of substrate, time of incubation, as well as temperature were

vital parameters for optimising the production of cellulase enzyme. The optimisation was done

by changing only one variable at one time and other parameters were maintained. SSF was

perfonned as described in section 3.4 with the three different substrates under the optimised

SSF parameters.

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3.7.1 Incubation Time

The solid state fermentation was set in duplication for 2, 4, 6, 8, and 10 days of

incubation time in the incubator and the others parameter such as 70 % (v/w) for initial

moisture content and 30°C temperature were maintained.

3.7.2 Initial Moisture Content

Each substrate (sago hampas, empty fruit bunch, rice husk) were duplicated and

supplied with variables of initial moisture content of 60%, 70%, and 80% (v/w). The other

parameters of incubation time of 6 days and temperature of 30°C were maintained.

3.7.3 Temperature

Substrates were duplicated and carried out at various temperatures of 25°C, 30 °C, and

35 ·C. The others parameter of incubation time of 6 days and 70% (v/w) initial moisture

content of substrate were maintained throughout the experiments.

r '

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