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EFFECTS OF CASTOR OIL ON Saccharomyces cerevisiae GROWTH AND FERMENTING ACTIVITIES DURING

ANAEROBIC CONDITION

Siti Hajar Binti Mohamad Jufri

QK 613 S13

Bachelor of Science with Honours 8613 lOll (Resource Biotechnology)

2012

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ACKNOWLEDGEMENT

In the name of Allah, the Most Gracious and the Most Merciful. Alhamdulillah, all

praises to Allah for the strengths and His blessing in completing this thesis. First and

foremost, I would like to express my sincere gratitude to my research supervisor Dr. Micky

Vincent for the continuous support, patient guidance and useful critiques throughout the

experimental and thesis works which have contributed to the success of this research. I

would also like to extend my thanks to my co-supervisor, Madam Dayang Salwani Awang

Adeni for her support and knowledge regarding this project.

My sincere thanks go to my fellow labmates and coursemates for their kindness and

moral supports. Assistance provided by all the staffs and technicians especially Mr. Leo

Bungin and Mr. Azis Ajirn for their co-operations are greatly appreciated.

Last but not least, I would like to thank: my beloved parents; Mr. Mohamad Jufri

Bin Md Nooh and Mrs. Roziyaton Binti Ahmad Subari and also to my family members for

their encouragement and prayers. Also to those who indirectly contributed in this research.

Thank you very much.

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ACKNOWLEDGEMENT

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

CHAPTER 1

CHAPTER 2

Pusat Khidmat MakJumat Akademik VNlVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS

I

II

IV

V

VI

1

INTRODUCTION

t.l Introduction 2

1.2 Objectives 3

LITERATURE REVIEWS

2.1 Castor Tree and Castor Oil (Ricinus communis L.) 4

2.2 Saccharomyces cerevisiae 6

2.3 Anaerobic Fermentation 7

2.4 Bioethanol 7

2.5 High Performance Liquid Chromatography (HPLC) 8

MATERIAL AND METHODS

3.1 Materials 10

3.2 Solubility Test 11

3.3 Culture Preparation ofSaccharomyces cerevisiae 11

3.4 Anaerobic Fermentation 13

3.5 Sample Processing 15

3.6 Viable Cell Count 15

3.7 Analytical Method 19

3.7.1 High Performance Liquid Chromatography (HPLC) 19

3.7.2 Data Analysis 19

CHAPTER 3

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CHAPTER 4

CHAPTER 5

CHAPTER 6

REFERENCES

APPENDIX

RESULTS

4.1 Solubility Test 20

4.2 Viable Cell Count 21

4.3 Data Analysis from High Perfonnance Liquid 25

Chromatography (HPLC)

4.3.1 Ethanol Yield 25

4.3.2 Glucose Concentration 29

4.3.3 Concentration of Lactic Acid 33

4.3.4 Concentration of Acetic Acid 37

DISCUSSIONS 41

CONCLUSION 47

48

50

iii

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rpm

g

ml

l·tI

Jlm

mm

gil

kglm3

HPLC

S. cerevisiae

ATCC

PBS

LB

C02

EMP

pH

ATP

GDP

RFS

TMTC

TFTC

TEY

LDH

LIST OF ABBREVIATIONS

Percentage

Degree celcius

Revolutions per minute

Gram

Milliliter

Microliter

Micrometer

Milimiter

Gram per litre

Kilogram per cubic meter

High Performance Liquid Chromatography

Saccharomyces cerevisiae

American Type Culture Collection

Phophate Buffered Saline

Luria Betani

Carbon dioxide

Embden-Meyerhof-Parnas

Potential ofhydrogen

Adenosine Triphosphate

Gross Domestic Product

Renewable Fuels Standard

Too many to count

Too few to count

Theoretical ethanol yield

Lactate dehydrogenase

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13

LIST OF TABLES

Table Page

Table 1 Main ingredients of the fermentation broth

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LIST OF FIGURES

Figure Page

Figure 1 Constitution equation ofcastor oil 5

Figure 2 A Shimadzu High Performance Liquid Chromatography (HPLC) System 9

Figure 3 Culture at 0 hour 12

Figure 4 Culture after 24 hours 12

Figure 5 Colour changes of fermentation broth 14

Figure 6 Serial dilution for viable cell count 16

Figure 7 Yeast colonies too few too count 17

Figure 8 Yeast colonies too many too count 17

Figure 9 Yeast colonies with range between 30-300 colonies 18

Figure 10 Amount ofethanol absorb by castor oil 20

Figure 11 Time course ofS. cerevisiae concentration (5% glucose) 21

Figure 12 Time course ofS. cerevisiae concentration (1 0% glucose) 22

Figure 13 Time course of S. cerevisiae concentration (15% glucose) 23

Figure 14 S. cerevisiae concentration in broths with castor oil vs. no castor oil 24

Figure 15 Time course of ethanol production (5% glucose) 25

Figure 16 Time course ofethanol production (10% glucose) 26

Figure 17 Time course ofethanol production (15% glucose) 27

Figure 18 Ethanol production in broths with castor oil vs. no castor oil 28

Figure 19 Time course of glucose concentration (5% glucose) 29

Figure 20 Time course ofglucose concentration (1 0% glucose) 30

Figure 21 Time course ofglucose concentration (15% glucose) 31

Figure 22 Glucose concentration in broths with castor oil vs. no castor oil 32

Figure 23 Time course oflactic acid concentration (5% glucose) 33

Figure 24 Time course of lactic acid concentration (10% glucose) 34

Figure 25 Time course oflactic acid concentration (15% glucose) 35

Figure 26 Lactic acid production in broths with castor oil vs. no castor oil 36

Figure 27 Time course of acetic acid concentration (5% glucose) 37

Figure 28 Time course of acetic acid concentration (10.% glucose) 38

Figure 29 Time course of acetic acid concentration ( 15% glucose) 39

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Figure 30 Acetic acid concentration in broths with castor oil vs. no castor oil 40

vii

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Effects of castor oil on Saccharomyces cerevisiae growth and fermenting activities during anaerobic condition

Siti Hajar Binti Mohamad Jufri

Resource Biotechnology Programme Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Liquid bioenergy, especially ethanol is aggressively being devefoped due to the depletion of petroleum in the world. However, bioethanol production faces problems like the accumulation of ethanol in the fermentation broth. In this study, the production of ethanol was performed using Saccharomyces cerevisiae (ATCC 24859), while simultaneously using castor oil that functions as a selective separator to absorb and separate the ethanol from the fermentation broth. Castor (Ricinus communis L.) plant, family member of Euphorbiaceae is a versatile plant that can produce this specialized oil. An experiment was designed to identify and determine whether the presence of castor oil has affects toward the growth and fermentation activities ofS. cerevisiae. Therefore, the capability ofcastor oil as selective separator was observed along this study. During the study, several analytical procedures were used such as High Performance Liquid Chromatography (HPLC) and plate counting of S. cerevisiae. The presence of castor oil was determined to have the capability acted as selective separator between ethanol and fermentation broth due to overall results gained from the study shown that ethanol production for broth with castor oil has lower amount compared to the broth without castor oil at three different concentrations of glucose (5%, 10%, 15%). According to the results obtained from this study, the best concentration of glucose that was effective for production of ethanol during anaerobic fermentation with the assist of S. cerevisiae was 5% due to high ethanol production (76.86%) with the quickest glucose utilization, lowest acetic acid (1.74 gil) and lactic acid (4.83 gil). The result from the experiment is expected to confer a huge impact towards a better enhancement of producing ethanol throughout the process of anaerobic fermentation.

Key Words: Castor oil, Saccharomyces cerevisiae ATCC 24859, anaerobic fermentation, glucose, ethanol.

ABSTRAK

Biotenaga cecair terutamanya etanol sedang dijalankan secara disebabkan oleh pengurangan sumber petroleum di dunia. Walaubagaimanapun, penghasilan bioetanol berhadapan dengan masalah iaitu pengumpulan etanol di dalam kaldu penapaian. Dalam kajian ini, penghasilan etanol dilakukan dengan menggunakan Saccharomyces cerevisiae (ATCC 24859), dan secara serentak menggunakan minyak kastor yang beifungsi sebagai pemisah selektif untuk menyerap dan memisahkan etanol daripada kaldu penapaian. Tumbuhan kastor (Ricinus communis L'), tergolong dalam keluarga Euphorbiaceae merupakan tumbuhan serbaguna yang berpotensi menghasilkan minyak khusus ini. Satu kajian telah direka untuk mengenalpasti dan menentukan sama ada kehadiran minyak kastor memberi kesan terhadap pertumbuhan dan aktiviti­aktiviti penapaian oleh ~. cerevisiae. Oleh sebab itu, sepanjang kajian ini, keupayaan minyak castor sebagai pemisah selektif telah diperhatikan. Semasa kajian dijalankan, beberapa prosedur analisis telah digunakan seperti Kromatograji Cecair Berprestasi Tinggi (HPLC) dan pengiraan viabel sel. Kehadiran minyak kastor telah dikenalpasti mempunyai kebolehan sebagai pemisah selektif antara etanol dan kaldu penapaian berdasarkan keputusan keseluruhan membuktikan bahawa jumlah penghasilan etanol bagi kaldu yang mengandungi minyak kastor lebih rendah berbanding kaldu yang tidak mengandungi minyak kasto pada tiga kepekatan glukosa yang berbeza (5%, 10%, 15%). Berdasarkan keputusan yang diperoleh daripada kajian ini, kepekatan glukosa yang berkesan untuk penghasilan etanol semasa penapaian secara anaerobik dengan bantuan ~. cerevisiae adalah 5% disebabkan penghasi/an etanol yang tinggi (76.85%) dengan kadar penggunaan glukosa yang cepat, serta kepekatan terendah bagi asid asetik (1.74 gil) dan asid laktik (4.83 gil). Hasi/ daripada kajian ini dijangka memberi kesan yang besar terhadap penghasi/an etanol melalui proses penapaian anaerobik supaya menjadi lebih baik.

Kata Kunci: Minyak kastor, Saccharomyces cerevisiae ATCC 24859, penapaian anaerobik, glukosa, elano/.

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CHAPTER 1

INTRODUCTION

1.1 Introduction

The major source of fuel, especially for transportation purposes, is petroleum based. Yet,

petrol oil is restricted and exhaustion of the supply is occurring faster than predicted.

Therefore, currently this concern has built up a new enthusiasm in the yield of biomass and

bioenergy (Bai et at., 2007). Bai and co-workers (2007) also stated that utilization of

petroleum products excessively has threatened human society's sustainability. As a result,

production of ethanol as an alternative fuel that is environmental friendly is one of the

greatest solution to maintain the over consumption of petroleum resources. In 2010, the

demand for ethanol has increased tremendously due to improvement in biofuel economics

and the execution of Renewable Fuels Standard (RFS 2) (Urbanchuk, 2011). During the

same year, 12 billion gallons of conventional bioethanol was produced in USA. Ethanol

now has become a part of manufacturing sector that gives extensive value to agricultural

supplies produced in United States as well as a significant contribution to American

economy in tenns of employment, income and Gross Domestic Product (GDP) supported

by the industry (Urbanchuk, 2011).

Currently, anaerobic fermentation is the frequently and most preferred process used around

the world for the production of ethanol. Anaerobic fermentation contributes to about 80%

of ethanol production worldwide while the remaining is from the synthesis of ethylene, a

product fonned from petroleum. However, there are few drawbacks with the fermentation

process, especially with the accumulation ofethanol in the fermentation broth. Therefore, a

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solution is required to overcome the problem, hence increase the amount of ethanol

production during the fennentation process. Thus, in this study, castor oil was chosen to be

evaluated in by acting as a selective ethanol separator in the broth. Anaerobic fennentation

was perfonned using Saccharomyces cerevisiae (ATCC 24859) for the conversion of

glucose to ethanol and then, the castor oil was added to aid in the separation of ethanol

from the fennentation broth.

1.2 Objectives

The objectives for this study were:

1) To study the effects of castor oil on S. cerevisiae ATCC 24859 growth during

anaerobic fennentation.

2) To detennine the effects of castor oil on the fennentation activities of S. cerevisiae

(ATCC 24859) during anaerobic fennentation.

3) To determine the best concentration of glucose for effective ethanol production

during anaerobic fennentation by S. cerevisiae (ATCC 24859).

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CHAPTER 2

LITERATURE REVIEWS

2.1 Castor Tree and Castor Oil

Castor tree (Ricinus communis L.) is a type of plant that has been identified as a source of

oil for biodiesel production (Cesar & Batalha, 20 I 0). The castor plant belongs to the family

of Euphorbiaceae, a tropical spurge family which can be found in temperate countries in

the world. Castor plant grows well in humid tropics area to sub-tropical dry zones with

750-1000 rnm optimal precipitation and temperature of 15-38 0c. This plant can grow until

the age of 4 years and reach the height above 10m. Several varieties are known to grow

until 60-120 cm of height in a year (Scholz & Silva, 2008). The castor plant initiates the

yielding of fruits from the sixth month onwards throughout the whole year and then the

quantity of production will decrease significantly after the third germination, which is

suitable time for replanting the castor plant (Cesar & Batalha, 2010).

Based on Scholz et al. (2008), the process for harvesting castor oil is slightly complex, as

one need to be selective when cutting the ripe fruit by hand and subsequently remove the

capsule. More than five separate harvesting techniques are practiced according to various

stages of fruit ripeness. The process of sorting out and cleaning of the castor seed is

followed by pressing it until the oil is extracted. There are some differences in the

percentage of oil produce which depends on the temperature when the seeds are pressed.

The yield of the oil is 30-36% of the castor seed's mass during cold weather. Meanwhile,

during wanner condition, the oil of the seed is about 38-48% of the mass of the seeds. The

oil is very adaptable and suitable for phannaceutical as well-as cosmetic purposes.

4 , ­

Pusat Khidmat MakJumat Akademik· ., UNlVERSm MALAYSIA SARAWAK

Castor oil is fragrance-free, viscous and non-drying with yellow-green to yellow-brown in

colour (Scholz et ai., 2008). Castor oil also has several chemical properties that are unique

in comparison to other oils. It consists ofhigh proportion of unsaturated fatty acid which is

unique from other vegetable oils. It has a triglyceride of various fatty acids, where about

80-90% of the fatty acids consist of ricinoleic acid, 3-6% linoleic acid, 2-4% oleic acid and

1-5% saturated fatty acids. The presence of ricinoleic acid unsaturated fatty acid with the

hydroxyl functional group at carbon atom number 12 (Figure 1) in castor oil confer an

extraordinarily high viscosity of castor oil with the presence of the hydroxyl groups, the oil

is more stable, hence preventing the fonnation of hydroperoxides. More importantly, the

hydroxyl group also gives the oil high affinity towards ethanol (Ogunniyi, 2006).

CH2-0-C (O)-R CH2-0-C (O)-R

CH-O-C(O)-R + CH-O-C(O)-R

CH2-0-C(O)-R CH2-0-C(O)-R'

R == -(CH2)7-CH= CH-CH2-CH(OH)-(CH2)5-CH3

R' = other fatty acid derivatives

Figure 1: Constitution equation of castor oil

The world's largest manufacturer and exporter of castor oil is India, meanwhile China has

become the largest buyer of Indian's castor oil which then followed by US and Japan

(Soumitra, 2010). In Malaysia, the large-scale cultivation of castor oil started three years

ago by the Seremban-based Casa Kinabalu Sdn. Bhd. The same cultivation is expected to

be operational in Kuching as mentioned by the chief executive officer, Sam Chai. Sam also

mentioned that the oil extracted from the castor seed will be sold to a refmery in China

(Wong, 2010).

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2.2 Saccharomyces cerevisiae

Many microorganisms have been used for the production of ethanol, and the most

significant species is Saccharomyces cerevisiae. S. cerevisiae is a unicellular fungus and it

is generally known as yeast, brewer's yeast and lager beer yeast (Dombek et al., 1987).

Kadar et al. (2003) mentioned that S. cerevisiae is an ideal microorganism for the

production of ethanol as it has high rate of glycolysis. Santangelo (2006) reported that S.

cerevisiae is a model system of glucose response in the eukaryotes. This is because yeast

share many signal transduction components with the complex multicellular eukaryotes for

glucose detection. S. cerevisiae then will gain energy from glucose, which they are able to

break down through anaerobic fermentation and aerobic respiration (Madigan et al., 2006).

According to Yan and Tanaka (2006), S. cerevisiae produces ethanol in the fermentation

broth until the concentration reaches about 18% before further fermentation activities stop.

This ethanol tolerance is higher compared to other microorganisms such as Zymomonas

mobilis. Yan et al., (2006) also mentioned that S. cerevisiae has the ability to grow well in

glucose concentration at various temperatures, pH, and high concentration ofglucose.

6

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2.3 Anaerobic Fermentation

Glycolysis is the main metabolic pathway that is vital for the fermentation process

according to Bai et al. (2007). In Embden-Meyerhof-Parnas or EMP pathway, two

molecules of pyuruvate are produced by metabolizing one molecule of glucose. The

pyruvate is then reduced to ethanol by the anaerobic condition with the release of C02 and

also two A TPs. These A TPs are important due to the purpose of the microorganisms for

managing biosynthesis of yeast cells, thus showed that the yield of ethanol is frrmly related

with growth of the yeast cell producing co-product (Bai et al., 2007).

Ethanol fermentation process is the bioconversion of glucose to ethanol which often using

S. cerevisiae (Yan et aI., 2006). The main fennentation product is ethanol, as other than

that is the release ofcarbon dioxide, C02. According to Bai et al. (2007), instead ofethanol

and C02 being produced, other various products may also fonned during fennentation

which are lactic acid and acetic acid.

2.4 Bioethanol

Liquid biofuels are commonly used in the transportation sector. These liquid biofuels

include ethanol, methanol, plant oils and methyl esters (Uriarte, 2010). Ethanol is a type of

alcohol that consists of molecular oxygen that encourages complete combustion. It has

many similarities with gasoline that enable ethanol to be the gasoline's substitute or even

alternative fuels. The similarities between ethanol and gasoline is based on the densities of

the oil which is nearly identical except that the energy content of ethanol is about 30%

lower than gasoline. Bioethanol is highly functional and beneficial in turn to "reduce the

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consumption of fossil fuels and also lessening the emissions of carbon dioxide from the

main greenhouse gas (Erdei et al., 2010). To produce ethanol, fermentation process is the

most preferable method currently, various feedstocks from carbohydrates sources such as

sugar cane, corn and cellulose are used (Aden, 2007; Uriarte, 2010).

2.5 High Performance Liquid Chromatography (HPLC)

Hjgh Performance Liquid Chromatography is a chromatography system that able to detect

and analyze the presence of many biological compound such as cellulose, glucose and

fennentation products such as ethanol, lactic acid and acetic acid (Vincent et al., 2011).

HPLC also assist in the adsorption, partition, exchange of ion, exclusion and affinity

chromatography which allow it faster and better result of resolution (Wilson & Walker,

2005). A microprocessor controls the systems in order to allow dedicated, continuous

chromatographic separations. Figure 2 shows a typical HPLC system (ShimadzulLC-20A,

Tokyo, Japan).

The mechanism of this chromatographic procedure is defined as separation between the

mobile and stationary phase. In order to separate the components of the mixture, the HPLC

utilizes the liquid mobile phase, while the stationary phase can either be liquid or solid

phase. The components that are needed to be analyzed are dissolved in a solvent and flow

through the chromatographic column under high pressure, hence, separated into its

components. The amount of resolution is based on the interactions between the stationary

phase and solute components which can be manipulated with various choices of both

solvents and stationary phases (Standardbase techniques, n.d). According to Lindsay et al.

(1992), HPLC is very reliable compared to other chromatographic techniques. This is due

8

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to a relatively easy separations and analyses when compared to other forms of

chromatographic procedures.

Figure 2: A Shimadzu High Performance Liquid Chromatography (HPLC) System

9

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CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

I) Castor oil

2) 0-(+)- Glucose (SIGMA, USA)

3) Luria broth (SIGMA, USA)

4) Saccharomyces cerevisiae (ATCC 24859)

5) lOX YP medium solution

1. 100 gil Yeast extract (CONDA, Spain)

11. 200 gil Bacteriological peptone (CONDA, Spain)

6) 1 L Phosphate Buffered Saline (PBS)

1. 80 g Sodium chloride (MERCK, Germany)

11. 2 g Potassium chloride (J.T. Baker, USA)

iii. 11.45 g Di-sodium hydrogen orthophosphate (MERCK, Germany)

iv. 2.4 g Potassium hydrogen phosphate (BDH, GDR TM)

7) Plate count agar (MERCK, Germany)

8) Sterile distilled water

10

3.1 Solubility Test

10 ml of castor oil was pipetted each into 11 different 100 ml conical flasks. Different

variables concentration of water and ethanol were used in the experiment and was done in

duplicate as to get the average result for ethanol's solubility. The mixture from the

different ratio of absolute ethanol and water concentration was poured into the conical

flask containing castor oil. The samples were then incubated in the shaker machine for

overnight at 120 rpm and 37°C. After 24 hours, two layers were formed which can be seen

tom the side of the conical flasks, separating ethanol and water. The conical flasks

containing the samples were shaked and 1.5 ml from each samples were pipetted into 1.5

ml Eppendorf tubes. The tubes were centrifuged for 2 minutes at 14000 rpm to allow the

water to be at the upper layer. After that, 200 J..11 of the water was pipetted from each

sample into a new Eppendorftubes and took for HPLC analysis. If the samples were not

directly analyzed, they need to be preserved in freezer at -4°C.

3.3 Culture Preparation of Saccharomyces cerevisiae

The S. cerevisiae culture was prepared by growing the stock in a sterile flask containing

Luria bertani (LB) broth. The culture was then placed inside an incubator shaker at 32°C

and 120 rpm agitation for 24 hours (Vincent et al., 2011). After the overnight incubation

period, the culture was aliquoted into two 50 ml tubes and centrifuged using Kubota 8800

(Kubota Corporation, Japan) at 7000 rpm for 3 minutes to obtain the cell pellet and

harvested into the fermentation broths.

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Figure 3: Culture at 0 hour

Figure 4: Culture after 24 hours

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3.4 Anaerobic Fermentation

Fermentation was done in two different 250 ml bottles for replicate containing 150 ml

batch culture each. Glucose powder, yeast extract and bacteriological peptone were used to

make the fermentation broth. Then, distilled water was added to dissolve all the reagent

materials until the total volume of broth reached 150 ml. The concentration ofglucose used

in this study was diversified which were listed in the Table 1. The sample was then

inoculated aseptically with the S. cerevisiae suspension, and 15 ml of castor oil was

pipetted onto fermentation broth. The bottles containing broth were placed in an incubator

shaker (Innova™4000 incubator shaker, New Brunswick Scientific) at 125 rpm and

incubated at 37°C for 5 days (Vincent et aI., 2011).

Table 1: Main ingredients of the fermentation broth

Materials ml

Glucose 5%,10%,15% 106S. cerevisiae - 108/ml

lOX YP solution Added until 150 ml Total 150

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Figure 5: Colour changes of broth fermentation A) Broth at 6 h B) Broth at 120 h C) Broth samples

14

3.5 Sample Processing

3 ml sample from the fermentation broth was taken aseptically and aliquoted into two 1.5

ml microcentrifuge tubes. The samples were taken at 0 hour, 6 hours, 12 hours, 24 hours,

48 hours, 72 hours, 96 hours, and 120 hours. 1.5 ml of the samples were used for direct

plate counts (Reynolds & Farinha, 2005), and another 1.5 ml of the sample was kept for

High Performance Liquid Chromatography (HPLC) analysis. For analysis purposes, the

sample was centrifuged for 3 minutes at 14 000 rpm in a centrifuge (EBA2I- Hettich

Zentrifugen). The supernatant was then filtered using 0.45 11m syringe filter and kept at -4

°C before HPLC analysis (Dowe and McMillan, 2008).

3.6 Viable Cell Count

Viable cell count was done to quantify the concentration of yeast in the fermentation broth.

The petri plates were labeled according to the number of dilution needed for the plate

count. Six series serial dilution was prepared using the original sample from the broth.

Initial dilution was done by transferring aseptically 500 III of broth sample to test tube

containing 450 III sterile phosphate buffered saline (PBS) solution. The dilution was then

vortexed to distribute the yeast evenly and break up any clumps that may formed in the

solution (Reynolds & Farinha, 2005).

The process was repeated until achieving 1 O~ dilution. The last three dilutions were then

used for plate count. To perform the plate count, the samples were mixed wen and 300 III

were aseptically transferred to petri plates and spread evenly using a glass hockey stick on

the plate count agar. The plate was then sealed using parafilm, inverted and incubated at 37

°C for 24 hours (Reynolds and Farinha, 2005). After 24 hours of incubation period, all of

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the petri plates were collected and the colonies fonned were counted. Plates with more than

300 colonies were deemed as too many to count (TMTC). Meanwhile, plates with less than

30 colonies were discarded as too few to count (TFTC). The number of yeast (CFU) per

milliliter or gram of sample was calculated by dividing number of colonies with dilution

factors mUltiplied by the amount of diluted broth added to the agar.

Figure 6: Serial dilution for viable cell count

16