production and optimization of raw starch

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

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 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


Universiti Malaysia Sarawak

2012

i

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.

ii

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

iii

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

iv

List of Abbreviation

PDA

CMC

SSF

DSNA

g

mL

µg

nm

pH

Potato dextrose agar

Carbomethylcellulose

Solid state fermentation

3,5-dinitrosalicylic acid

Gram

milligram

microgram

nanometer

A measurement of the acidity or alkalinity of solution [p stands for “potenz” which

means the potential to be while H stands for Hydrogen]

v

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


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


Table 11: Cellulase enzyme activity produces at different moisture content by SSF of


vi

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

1

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


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 40oC, respectively. In addition, the optimum inoculates that produce

highest production of both amylase and cellulase enzymes are at 107 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 40oC, 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

2

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.

3

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.

4

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.

5

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 by-

products 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;

6

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

7

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

8

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 β-

glucosidases. 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).

9

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).

10

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).

11

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).

12

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

13

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).

14

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 10

8 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.

15

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.

16

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

A.flavus A.niger A.versicolor

CMC 1.2 cm ± 1.5 cm ± 1.7 cm ±

Soluble starch 4.5 cm ± 5.5 cm ± 3.5 cm ±

production and optimization of raw starch

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