ISOLATION AND CHARACTERIZATION OF AMYLOLYTIC, PECTINOLYTIC AND CELLULOLYTIC MICROORGANISMS

FROM LOCAL HOTSPRING

Nurfarahin Binti Jainuddin

QR 84.8 N974 Bachelor of Science with Honours 2013 (Resource Biotechnology)

2013

Pusat Khidmat Maklumat Akademik tTNlV"',R~1TT M.\LAVSIA SARAWAK

Isolation and Characterization of Amylolytic, Pectinolytic and Cellulolytic

Microorganisms from Local Hotspring P.KHIDMAT MAKLUMAT AKADEMIK

UNIMAI

II U111111111111111111111 1000246609

Nurfarahin Binti Jainuddin (27737)

A thesis submitted in fulfillment of the Bachelor of Science with Honours Resource

Biotechnology

Supervisor: Assoc. Prof. Dr. Awang Ahmad Sallehin Awang Husaini

Co-supervisor: Dr. Azham Zulkharnain

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ii

ACKNOWLEDGEMENT

First of all, I would like to express my highest gratitude and deepest appreciation to

my supervisor, Assoc. Prof. Dr. Awang Ahmad Sallehin Awang Husaini, for all the advices,

encouragement and supports. Thank you so much for knowledge, sharing and time spent for

me in order to complete this research successfully. Without any of your criticisms and

advices, this thesis would not be completed.

Plus, I would like to express special thanks to my parents Mr. lainudin Mangeesha

and Mrs. Khamis Ando for all the love, support, encouragement and advices to keep moving

on and never give up.

Most of all, I would like to thank to all Master students in the Molecular Genetic

Laboratory especially to Mr. Simon Ngieng and Ms. Siti Nur Ratna for all the help and

knowledge-sharing during the lab work. Your kindness and dedication is very much

appreciated.

Last but not least, very special thanks to all my FVP colleagues who have helped me

with my lab work and the never-ending supports and encouragement. Not forgotten, all

course mates who have spent three years together.

iii

DECLARATION

I hereby declare that no portion of the work referred in this project has been submitted in

support of application for another degree qualification of this or any other universities or

institution of higher learning.

NURFARAHIN JAINUDDIN

(27737)

JUNE 2013

iv

Pusat Khidmat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS DECLARATION ................ .. ...... ............. ....................................... ... .......................................... ....... ........ iv

LIST OF TABLES .................. .. ...... .. ....... ........ .. .... ......................... .. ........................................................... vi

LIST OF FIGURES ................ .... ........................................... .......... ... ........ ............................. .. ... ............... vi

LIST OF ABBREVIATIONS ...................................................... ................................. ..... .............. ............. vii

ABSTRACT................... .................................................. .. ... ......................................... ...... .... ...... ........ .. viii

ABSTRAK....................... ....................................................... .......................................... ...... .. ................... .

INTRODUCTION ..... ..... .... .............................................. ... ............................................. ... ... ... ... ............... 1

LITERATURE REVIEW ......... ........... ..................................................................................... ..... ................. 2

2.1 Thermophilic fungi .................. .. .. ............ .. ..... .......... ........................................... ... ....................... 2

2.2 Amylase, pectinase and cellulase .............. ........... ....... .. ..................... ......... .. ... ....... ...................... 2

2.3 Importance of thermo stable enzymes ......................................................... .. ........ .. .. .................. 3

MATERIALS AND METHODS ............................................................................................ ... ... ................ .. 5

3.1 Enrichment .. ... .................................................. .............................................. ... .... ..... ... ........ ........ 5

3.2 Serial dilution and spread plating .......... .......... .................................................. ........................... 6

3.3. Isolation of amylolytic, pectinolytic and cellulolytic fungi .................................. ......................... 6

3.4 Screening test............ .. ...... .. .. ....... .... .. .............. ........................................... .... .............................. 6

3.5 Morphological characterization ............. ........ ...... ........ .. .. .. .. .... .. .. .... .. ..... ....... .. .. ......... .. ................ 8

3.6 Molecular characterization ....................................................... .. .. .. ... ..... ....... .. ........ ........ ............. 8

3.6.1 Genomic DNA extraction ................ .... ................................................ .. ......................... ........ 8

3.6.2 Agarose Gel Electrophoresis ......... ...... ................................................................................... 9

3.6.3 PCR amplification and purification of PCR product .......................... .. ................................. 10

RESULTS AND DiSCUSSiON.. .............................. ............................................. .. .... .. ............................... 11

4.1 Screening of extra cellular enzymes ...... .................................................... ................................. 11

4.1.1 Qualitative Screening ............. .. .. .. ................................ .. .. ...... .. ..... ... .................................... 11

4.1.2 QuanHtative Screening ............. .... ................................................................ ......... .. ............ 13

4.2 Morphological characterization ..... .... .................................................. ...................................... . 14

4.3 Molecular characterization ............ ... ..................................................... ..................................... 16

4.3.1 Genomic DNA extraction ... .. ........................................................ ........................................ 16

4.3.2 PCR Amplification ... .. .............. .. .......................................... .. ... ......... .................................... 18

CONCLUSION .............................. ... ............. ..... ....... ..... ...... .......... .......... .... ...... ...................................... 21

REFERENCES ............................................................... .... ... .. .. ........ ... ....... ...... .................................. ...... 22

APPENDIX ........................................... ..... .. ................................ ..... .... ........ ...... .... .. .... ....... ..... . ~ ............ 24

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r~· ~

LIST OF TABLES

LIST OF FIGURES

Description PageFigure no.

12Zone clearance of F02 on pectin supplemented agar1

14Culture a_nd morphological characteristics of strain FOI2

15Culture and morphological characteristics of strain F02 3 I

Genomic DNA of strain Fa1 gel photo 174 I

Genomic and PCR product of strain Fa1 gel photo 195

)

vi

Table no. Description Page

1 , Clear zone fonnation 12

2 Enzyme activity 13

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LIST OF ABBREVIATIONS

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vii

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Isolation and characterisation of amylolytic, pectinolytic and cellulolytic microorganisms from local hotspring

Nurfarahin binti Jainuddin

Resource Biotechnology Programme

Faculty of Resourse Science and Technology

University Malaysia Sarawak

ABSTRACT Thennophilic enzymes produces by microorganisms such as fungi has a very high potential in the

industry. The objectives of this research study are to isolate and characterize amylolytic, pectinolytic and cellulolytic indigenous microorganism (in particular fungi) from local hot spring. Sampling was done at Kampung Panchor Dayak Hotspring by which two thenno tolerant fungal strains were successfully isolated. The fungal strains were named as FO I and F02. Screening test employed shows that both of the fungal strains have the ability to degrade starch, pectin and cellulose. Qualitative screening was done by supplementing citrus pectin, soluble starch and also carboxymethy1cellulose (CMC) separately as a substrate and there were halo region observed in both fungal strains culture in the supplemented agar plates after the application of iodine solution. As for quantitative screening test, sago 'hampas' was utilized as the substrate for the fungal strains to produce crude enzyme by which to be used for enzyme activity test using DNS method. Both screening test indicates that strain F02 produce high amount of cellulose (0.1943 U/mL) and pectinase (0.2058 U/mL).

However, F02 identification was not done due to some limitation. Morphology characterisation shows that strain FOI was related to Apergillus sp. and identification through sequencing analysis showed that strain FOI was actually Aspergillus jitmigatus strain DF7 _ F 18S ribosomal RNA gene, 99% similarity.

Keywords: Thermophilic, fungi, amylolytic, pectinolytic, cellulolytic.

ABSTRAK Enzim thermophilic yang dihasilkan oleh mikroorganisma seperti fungus mempunyai potensi yang

sangat tinggi dalam industri. ObjektiJ penyelidikan ini adalah untuk mengasingkan dan mencirikan amylolytic, pectinolytic dan cellulolytic mikroorganisma asli (terutamanya fungus) dari kolam air panas tempatan. Persampelan telah dilakukan di Kampung Panchor Dayak Hotspring dan dua stren jimgus termo toleran telah berjaya dikllmpul. Fungus yang diperolehi telah dinamakan sebagai FO] dan F02. Ujian saringan yang digunakan menunjukkan bahawa kedua-dua jenis fungus mempunyai keupayaan untuk merendahkan kanji, pektin dan selulosa. Pemeriksaan kualitatiJ telah dilakukan dengan menggunakan pektin limau, kanji terlarut dan juga carboxymethylcellulose (CMC) secara berasingan sebagai substrat dan terdapat kawasan halo diperhatikan dalam kedua-dua stren fungus dalam Petri plat selepas peillmuran lanttan iodin. Bagi ujian saringan kuantitatif, sagu 'hampas'telah digunakan sebagai substrat untllk strain jimgus untuk menghasilkan enzim yang akan digunakan untuk ujian aktiviti enzim menggunakan kaedah DNS. Kedua-dua lIjian saringan menzmjukkan bahawa stren F02 mengeluarkan enzim cellulase (0.]943 U/mL) and pectinase (0.2058 U/mL). yang tinggi. Walaubagaimanapun, pengenalpastian identiti F02 tidak dapat dilakukan atas sebab-sebab tertentu. Pencirian morfologi menunjukkan bahawajimgus FO] adalah berkait-rapat dengan Apergillus sp. dan mengenal p(lsti melalui analisis unttan menunjukkan bahawa stren FO] sebenarnya Aspergillus fumigatus stren DF7_F ]8SRNA ribosom gen, dengan 99% persamaan.

Kata kunci: Termophilik, fungus, amylolytic, pectinolytic, cellulolytic

viii

INTRODUCTION

Thermophilic fungi are the fungi which are able to tolerate high temperature and have

the optimum temperature for growth at or above 50°C and minimum temperature of growth

at or above 20°C. Other than hot spring, thermophilic fungi can also be obtained from

various habitats which include composts, stored grain, piles of hay, wood chip piles, nesting

material of bird and other animals snuff, municipal waste and other accumulation of organic

matter by which favour the warm, humid and aerobic environment to provide the

physiological condition for the fungi development (Salar & Aneja, 2007). In this research

study, sampling in Kampung Panchor Dayak Hot spring in Serian were done and water

samples together with the sediments were taken to the laboratory for further analysis. The hot

spring where sampling located have the temperature ranging 42°C and above.

The problem statements for this research include there is no prevIOus study on

indigeneous amylolytic, pectinolytic and cellulolytic from Sarawak local hotspring. Plus, the

morphological and molecular biology of thermophiles from local hotspring need to be

investigated in order to highlight their application potential especially in the utilization as

potential inoculum for sago waste bioconversion. Lastly, there is high demand of thermo­

stable enzyme especially in the industry.

The objectives of this study are to isolate the amylolytic, pectinolytic and cellulolytic

fungi from local hot spring and characterize it morphologically and molecularly.

1

LITERATURE REVIEW

2.1 Thermophilic fungi

Thermophilic fungi are much more common in acid thermal habitats than those of

neutral to alkaline pH. It also constitute a heterogeneous physiological group of various

genera in the Zygomycetes, Ascomycetes, Deuteromycetes (Anamorphic fungi), and Mycelia

Sterilia (Salar & Aneja, 2007).

2.2 Amylase, pectinase and cellulase

Amylase degrades starch and associated polymers to yield products depending on the

characteristics of individual amylolytic enzymes. Amylase is not stable in aqueous solution

and retrogrades (precipitates spontaneously) due to the molecular shape and structure of the

amylase itself. This is because linear chains align themselves by hydrogen bonding and thus

forms aggregates which are irreversible (Aiyer, 2005). Some application of amylase involves

the process of liquefaction and can act as thinning agent. Starch by which usually used as

substrate include com, wheat, rice, potato and sago pith.

Pectin is present at the middle lamellae and pnmary cell wall between cells.

Pectinolytic enzymes are categorized according to their mode of attack on the galacturonan

part of the pectin molecule. They can be differentiated from pectin methylesterases (EC

3.1.11.1) that deesterify pectins to low methoxyl pectins or pectic acid, and form pectin

depolymerases, that split the glycosidic linkages between galacturonosyl (methyl esther)

residues (Silva et al., 2002). Silva et al. (2002) also reported that pectinolytic enzymes have

great importance in commercial industry such as improving fruit juice production and clarity,

paper an~ pulp industry, waste management, animal feed and also textile industry. Oranges

2

peel, wheat bran and also banana peel are usually used as a substrate to determine the starch­

degrading activity of the fungi.

Cellulase is group of hydrolytic enzyme capable of hydrolysing the most abundant organic

polymer, cellulose into smaller subunits. This enzyme system which shows excellent

degradative action towards crude natural cellulosic material is regularly found in most of

fungi and comprises three main components that are exoglucanase, endoglucanase and ~­

glucosidase (Gautam et at., 2010). Sago pith waste is one of the most suitable and popular

substrate used to study cellulolytic enzyme activity.

2.3 Importance of thermo stable enzymes.

The main focus of these thermophiles that had been gaining attention ever since is that

the enzyme produced by these thermophiles which are resistance to such a high temperature.

Enzyme produced by thermophiles has the potential which would denote many benefits that

definitely reduces the process cost (Johri et at., 1999). The screening of thermophilic fungi

and other thermophilic organisms for improved enzyme varieties may contribute to lowering

the costs of enzyme preparations (Morgenstern, 2012). In 1894, Dr. Thokichi Takamine

makes the history of industrial production of enzyme by the production of digestive enzyme

preparation by wheat bran. koji culture of Aspergi/us oryzae (Aiyer, 2005). Since then,

amylases are isolated for varieties of purposes. There has been a need of thermostable a­

amylase since a-amylase is required to be active in high temperature for the process of

gelatinization (100-11O°C) and liquefaction (80-90°C) so that the processes can be

economizes (Reddy, Nimmagadda & Rao, 2003).

3

Geetha et al. (2012) reported that pectinase have important roles in fruit juice and

mine making industry by which it is used for clarification and pulp formation in fruit juices.

Thus, thermostable pectinase is absolutely useful and important. Chuan Ii, Na Ii and

Papageorgious (2011) reported that thermophilic cellulases are the solution for efficient

biomass degradation due to the fact that cellulose swells at higher temperature, thus it became

easier to break down and speed up the reaction process.

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Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK

MATERIALS AND METHODS

Sampling was carried out at Kampung Panchor Dayak Hotspring. Six bottles of water

sample were collected from six different locations at the hot spring area. The samples were

kept in mobile incubator to maintain the original temperature of the sample before being

transferred into the incubator in the laboratory.

3.1 Enrichment

The samples were enriched in Potato Dextrose Broth (PDB) for seven days. A volume

of I ml of water from each 6 samples was inoculated into 6 universal bottles containing 9 ml

of media broth (PDB). All the inoculation techniques were applied aseptically and the sample

broths were incubated in a shaker incubator at 50°C with 200 rpm of agitation speed. After 7

days of enrichment in Potato Dextrose Broth, the sample broths were then inoculated into

Minimal Medium with the addition of sago 'hampas' as the sole carbon source. 2 ml of

culture broth were inoculated into 250 ml Erlenmeyer flasks containing 100 ml of Minimal

Medium with sago 'hampas'. Antibiotic (chloramphenicol) were added into the medium in

order to prevent the growth of bacteria and other unwanted organisms. The culture broths

were then incubated in the shaker incubator at 50°C with 200 rpm speed of agitation for 7

days.

5

3.2 Serial dilution and spread plating

After two weeks of enrichment, the culture broths were diluted and then plated using

spread plate method. The dilutions were made with factor of 10-1 to 10-4 for each sample.

Approximately 1 ml of culture broths were transferred into 4 test tube containing 9 ml of

distilled water. Then, 0.1 ml of the dilutions was transferred aseptically using spread plate

method into petri plate containing Potato Dextrose Agar. The plates were sealed, labelled

accordingly and incubated in the incubator at 50°C for 7 days.

3.3. Isolation of amylolytic, pectinolytic and cellulolytic fungi

Two fungal isolates were collected from the sample. The isolates were inoculated into

petri dish containing Potato Dextrose Agar and sub-cultured to obtain pure colony of the

fungal isolates.

3.4 Screening test

The isolated fungi were tested for their ability to produce extracellular enzyme that

degrade starch, pectin and also cellulose for the screening of the presence of enzymes

amylase, pectinase and cellulase respectively. Two screening test was done; qualitative and

quantitative screening.

3.4.1 Qualitative Screening Test

The qualitative screenmg was done by inoculating the selected fungus into Petri

dishes containing Mineral Salt Medium agar supplemented with substrates as the carbon

sources (Soluble starch, carboxymethyl cellulose and citrus pectin). Mineral Salt Medium agar

was prepared with some modification. The Minimal Salt Medium agar were prepared in 300

6

mL by mixing 0.639 g of Na2HP04, 0.39 g of K2HP04, 0.15 g of NH4CI, 0.06 g of

MgS04.7H20, 6.0 g of agar and 0.2 g of each substrate (soluble starch, CMC and citrus

pectin). Distilled water was added to make final volume of 300 mL. Then the MSM agar was

autoclaved for IS minutes at 121°C. The growth of fungi was assayed for the degradation of

the respective carbon sources based on the halo reagent on the agar after treatment of iodine

solution. The test was done in triplicates to reduce the statistical error.

3.4.2 Quantitative Screening Test

The production of enzyme was conducted in 250 mL Erlenmeyer flasks containing

100 mL of Potato Dextrose Broth inoculated with two discs of agar grown with the selective

fungi and sago 'hampas' as the substrate. The submerge culture was run for nine days at 40°C

in a water bath shaker. The samples of the culture were then centrifuged at 8000 rpm for 15

minutes then the supernatant ( crude-enzyme) were used for enzyme assay to test for the

presence of amylolytic, pectinolytic and cellulolytic activity individually. The reducing sugar

analysis for the enzyme assay were done by adding 1 mL of DNS reagent and 1 mL of crude

enzyme in a test tubes done in triplicate. As for the detennination of amylolytic activity, 1

mL of the crude enzyme was added with I mL of 1 % of soluble starch in citrate phosphate

buffer (PH 6.5) (Ekunsaumi, n.d.). The detennination of pectinolytic activity were done by

adding 1 mL of culture supernatant to 1 mL of sodium acetate buffer (PH 5) containing I % of

citrus pectin while as for the detennination of cellulolytic activity, same protocol as

amylolytic enzyme assay with some modification; 1 mL of crude enzyme were added with

0.5 mL of 1 % of carboxymethylcellullose in citrate phosphate buffer (PH 6.5) (Ekunsaumi,

n.d.). The enzymes were assayed for 15 minutes in 90°C water bath. Then, 1 mL of 40% .

(w/v) potassium tartrate (Roschelle salt) was added to the test tubes and the reaction mixtures

7

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were left to cool at room temperature. The absorbance reading of the mixtures was measured

by using spectrophotometer at wavelength of 540 nm (Ekunsaumi, n.d.). The reading were

recorded and compared with glucose standard curve.

3.5 Morphological characterization

Morphological characterization was done by applying one drop of Lactophenol

Cotton Blue solution to mycelia from the selected fungi on a slide. A coverslip was placed on

top of the slide carefully by lowering it gently to prevent air bubbles from trapping under the

coverslip. The slide was then observed under the compound microscope.

3.6 Molecular characterization

3.6.1 Genomic DNA extraction

DNA extraction of the selected fungal isolates was employed by using the extraction

protocol by Cubero et al. (1998). Firstly; the fungi were grown in Potato Dextrose Broth and

incubated for a week in shaking condition. Then, the culture broth was centrifuged and the

pellet was collected. The pellet was then grinded with liquid nitrogen using sterile pestle and

mortar. The extraction buffer was pre-warmed before added to the grinded mycelia in the

Eppendorftube. The extraction buffer (1% w/v CTAB; 1M NaC1; 100 mM Tris; 20 mM

EDTA, 1% w/v polyvinyl polypyrolidone, PVPP) were added to the grinded mycelia in a

tube and mixed by inverting the tube then heated in the water bath at 70°C for 30 minutes.

Next, one volume of chloroform: isoamyl alcohol (24: 1 v/v) was added to the tubes

and mixed by inverting the tubes before centrifuged for 5 minutes at 10000 g at room

temperature. The supernatant was collected and transferred into new tubes and the pellet was

discarded. Next, two volumes of precipitation buffer (1 % w/v CTAB; 50 nM Tris-HO; 10

8

mM EDTA; 40 mM NaCl) were added to the supernatant and mixed well for 2 minutes

before centrifuged for 15 minutes at 13000 g at room temperature and the pellet was

collected. Then, one volume of chlorofonn: isoamyl alcohol was added into the pellet and

then resuspended in 350 III of 1.2 M of NaCI. The mixture was mixed vigorously before

centrifuged for 5 minutes at 10000 g at room temperature. The supernatant was transferred

into new tubes and 0.6 volumes if isopropanol was added. The tubes were mixed thoroughly

and placed in -20°C freezer for 15 minutes.

The final pellet was then collected by centrifugation for 20 minutes at 13000 g at 4°C.

The final wash of the pellet was done by addition of 1 ml of 70% ethanol and the pellet was

recollected by centrifugation for 3 minutes at 13000 g at 4°C. Finally, the pellet was drained

and dried at 50°C and resuspended in TE buffer for gel running.

3.6.2 Agarose Gel Electrophoresis

Approximately 0.5 gram of Agarose powder was dissolved in IX TAE buffer and the

mixture was heated in the microwave until it dissolves completely. Approximately 1 III of

Ethidium Bromide was added into the mixture. Then, a comb was placed in the tray before

the mixture were poured into the tray and left to solidified. The Agarose gel was poured

gently and carefully to prevent air bubbles. As the gel solidified, the comb was removed and

then 4 III of DNA samples and I III of loading dye were loaded into one of the well and 4 III

of marker plus I III of loading dye were loaded into the first well. The electrophoresis process

was carried out at 100 volts for 30 minutes. The DNA band was then visualized under UV

transilluminator and the gel image was captured for result writing.

9

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3.6.3 PCR amplification and purification of PCR product

The genomic DNA extracted template was used to be amplified using the Fennentas

reagents kit. The primer used for the reaction are nS! (forward) and ITS4 (reverse). The

peR product was then analysed using the AGE to check for peR product quality and proceed

with purification of the peR product using the purification kit. The purified peR product

together with the forward and reverse primer involves in the reaction of peR was then sent

for sequencing for identification.

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RESULTS AND DISCUSSION

4.1 Screening of extra cellu'lar enzymes

Two fungal isolates were collected from the Panchor Dayak Hotspring. The fungal

isolated were named as FO 1 and F02. The screening test shows that both the fungi have the

ability to degrade starch, pectin and cellulose. The screening test was done both qualitatively

and quantitatively.

4.1.1 Qualitative Screening

Both of the fungal isolates are able to degrade starch, pectin and cellulose since clear

zone are formed around the fungal colonies 48 hours after the fungus was placed on the

supplemented agar. Halo region are found on the both fungi cultured agar plates medium

supplemented with soluble starch (amylase), citrus pectin (pectinase) and CMC

(carboxymethylcellulase) separately after the application of iodine solution. The result is

summarized as presented in Table 1. The maximum clearing zones approximately 48.67 mm

were estimated for fungi F02 towards pectin substrates. Figure 1 shows the zone of clearance

on pectin supplemented agar plates for F02 isolates after 48 hours of incubation.

11

Table I This table shows the qualitative assessment of fungi strain FO) and F02 for the CMC, starch and pectin

decomposition via the measurement of clear zone around the colony.

Fungal strain Substrate Maximum clearing zone

(mm)

FOI CMC 20± 1

Starch 18 ± 1

Pectin 28± 2

F02 CMC 24 ± 1

Starch 22.33 ± 1.53

Pectin 48.67 ± 1.15

Figure I Zone of clearance on pectin supplemented agar plates for F02 isolates after 48 hours of incubation. The clear zone formation around the colony indicates the secretion of extracellular enzyme.

12

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4.1.2 Quantitative Screening

The quantitative screening was done using the DNS (Dinitrosalicylic acid) method.

The assessment was done based on the enzyme activity of both fungi. Enzyme activity was

done by culturing both fungal in sago ' hampas' minimal media and the enzyme supematanat

was harvested and crude enzyme was used to perform the amylase, pectinase and cellulase

enzyme assays. The result shows that fungus strain FO I has high enzyme activity of pectinase

while strain F02 has high enzyme activity of both cellulase and pectinase. The result can be

summarised in Table 2. Therefore, strain F02 was chosen as the best isolate for the

production of amylase, pectin and cellulase enzyme.

Table 2 This table shows the enzyme activity of both strain FO I and also F02 based on Dinitrosalicylic acid

method.

Enzyme activity (U/mL)

Fungi strain Cellulase Amylase Pectinase

FOI 0.0256 0.0315 0.1660

F02 0.1943 0.0663 0.2058

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4.2 Morphological characterization

Both of the isolated fungal strains were observed under the compound microscope by

using the staining with Lactophenol Cotton Blue solution. The fungi were subcultured onto

PDA and after 2 days of growth, the mycelia of the fungi were collected for observation

under the compound microscope. Figure 2 shows the image of FO 1 strain culture on PDA and

its morphological characteristics under compound microscope. While Figure 3 shows the

image of F02 strain culture on PDA and its morphological characteristics under compound

microscope.

Figure 2 (a) Culture of Fungus strain FO I on PDA (b) Morphological characteristics of strain FO I.

14

The cultured FO I strain fungus on PDA shows the colonies with dusty brown-green

surface pigmentation. It is observed that the conidial head morphology show uniseriate row

of phialides on the upper two third of the vesicle. By observing the morphological

characteristic of strain FO 1, it is expected that FO I strain belong to the genus Aspergillus sp.

As for F02 strain fungus on PDA shows creamy whitish and non-sporulating colonies. The

colonies are also flat. The microscopic morphological also shows that the strain has irregular

branching hyphae. This non-spore formation fungus is therefore a bit difficult to identify to

which genus it belong to.

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4.3 Molecular characterization

4.3.1 Genomic DNA extraction

Due to some limitation during the extraction, DNA of the best isolate for this research

which is F02 strain was not extracted and only FO I was extracted instead for molecular

characterization identification. Genomic DNA of FO I was extracted employing the Cubero et

al. (1998) DNA extraction protocol. The extraction of fungi strain involved the grinding of

the mycelia of the fungus to break the cell walls of the fungus. In this research, the fungi

strain was cultured in Potato Dextrose Broth (PDB) for a week in a shaking condition. The

cultured broth was then centrifuged to obtain the pellet to be grinded. Grinding was done in

sterile condition using liquid nitrogen with pestle and mortar. The first extraction method

following the grinding method is the CTAB precipitation by which the grinded mycelia were

mixed in Eppendorf tube with extraction buffer which has been pre-warmed and incubated in

water bath at 70°C for half an hour. The buffer was pre-warmed to prevent CT AB

precipitation that may reduce the effectiveness of the buffer. Then, chloroform: isoamyl

alcohol (24: I) was added followed by inversion and centrifugation for supernatant collection.

Chloroform: isoamyl alcohol was used to disrupt the cell. Next, precipitation buffer was

added to the supernatant and proceed with centrifugation for pellet collection.

The pellet was then resuspended in 1.2 M NaCI and chloroform: isoamyl alcohol

(24: 1). The mixture was centrifuged and the supernatant was collected and isopropanol was

added. The mixture was placed in -20°C freezer before centrifugation. The final wash of the

final pellet involved the washing with 70% Ethanol and final centrifugation. · The pellet was

then dried and resuspended in TE buffer and stored in -20°C freezer. The extracted genomic

DNA was analysed using Electrophoresis to check for DNA quality. The Electrophoresis

works by separating the DNA base on its size. The result are shown in Figure 3 by which

16

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