growth of rhizopus oligosporus of rhizopus... · 2016. 2. 24. · growth of rhizopus oligosporus in...

GROWTH OF Rhizopus oligosporus IN SAGO EFFLUENT AT DIFFERENT PH

Nur AqJily Riana binti Nasar

Bachelor of Science with Honours (Resource Biotechnology)

2015

Growth of Rhizopus oligosporus in Sago Effluent at Different pH

Nur Aqlily Riana binti Nasar

(37883)

A report submitted in partial fulfillment of the Final Year Project 2 (STF3015)

Supervisor: Dr. Micky Vincent

Co-supervisor: Dr. Devagi Kanakaraju

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

University Malaysia Sarawak

2015

I

ACKNOWLEDGEMENT

Assalamualaikum, first and foremost, I would like to bring all the glory to Allah S.W.T. for

His abundant love and grace in the process of completing my final year project. I also

would like to thank my supervisor, Dr Micky Vincent and co-supervisor, Dr Devagi

Kanakaraju for giving me this opportunity to carry out this Final Year Project under his

supervision. He always assisted and gives opinion to all of us during our Final Year

Project. The help and guidance given by him time to time shall carry me long way in the

journey of life on which I am about to embark.

My deepest gratitude goes to my parents, Mr Nasar Ibrahim and Mrs Laila Deris and also

my brothers, Mr Qayyum, Mr Qawiem and Mr Iyad for their love, prayer, encouragement

and support from beginning till the end. The constant encouragement they give to keep me

going and their love to empower me that never fails all the time without which this project

would not be possible. Thank you for supporting me.

Besides that, I would like to express my appreciation to postgraduate student, Ms Fazidah

for her patient guardian and opinion throughout my research period. She never fails to

support us during the Final Year Project. I also would like to express my thanks to

laboratory mates, Ms Nadia Shuhada, Ms Shahirah Safin, Ms Khairunnisa, Ms Fatiyah, Ms

Ummi Syahida, Ms Lailatul Munirah for their support and opinion when I faced problem

in my experiments. Not to forget my friends Ms Suhailah, Ms Aisyah, Ms Amira Shafiza

and Ms Qeela for their emotional support, useful opinion throughout the experiment. I

gratefully acknowledge UNIMAS for the complete and convenient laboratories facilities

for all of the final year students to carry out experiment and completed this project.

Lastly, I dedicate my utmost gratitude to all individuals that have been indirectly

contributed in this research. Your kindness means a lot to me. Thank you very much.

II

DECLARATION

I hereby declare this Final Year Project entitled “Growth of Rhizopus oligosporus in

Sago Effluent at Different pH” is based on my original work except for quotations and

citation, which have been duly acknowledged. I also declare that it has not been previously

submitted for any other degree at UNIMAS or other institutions.

_________________________

(Nur Aqlily Riana binti Nasar)

JUNE 2015

III

TABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION II

TABLE OF CONTENT III

LIST OF ABBREVIATIONS V

LIST OF TABLES VI

LIST OF FIGURES VII

LIST OF APPENDICES IX

ABSTRACT

XI

CHAPTER 1: INTRODUCTION

1.1 Introduction 1

1.2 Problem Statement

2

CHAPTER 2: LITERATURE REVIEW

2.1 Sago Effluent 3

2.2 Submerged Fermentation Process 5

2.3 Rhizopus oligosporus 6

2.4 pH 8

2.5 High Protein Fungal Biomass (HPFB)

9

CHAPTER 3: METHODOLOGY

3.1 Pre-treatment of Substrate 11

3.2 Microorganism 11

3.3 Submerged Fermentation Process 12

3.4 Effect of pH on Submerged Fermentation by R. oligosporus 14

3.5 Dry Cell Weight Analysis 15

3.6 Phenol Sulphuric Test (Total Carbohydrate Analysis) 16

3.7 Nitrate Analysis 17

3.8 Nitrite Analysis

18

CHAPTER 4: RESULT AND DISCUSSION

4.1 Propagation stage of R. oligosporus 19

4.2 Biomass Accumulation of R. oligosporus 24

4.3 Phenol Sulphuric Test (Total Carbohydrate Analysis) 34

4.4 Nitrate Analysis 36

4.5 Nitrite Analysis 40

IV

CHAPTER 5: CONCLUSION AND RECOMMENDATION 44

REFERENCES 45

APPENDICES:

Appendix I: Propagation stage of R. oligosporus 47

Appendix II: Biomass Accumulation of R. oligosporus 48

Appendix III: Result of Phenol Sulphuric Test 51

(Total Carbohydrate Analysis)

Appendix IV: Result of Nitrate Analysis 53

Appendix V: Result of Nitrite Analysis 55

V

LIST OF ABBREVIATIONS

R. oligosporus

BOD

COD

g

h

L

min

s

mL

NSP

pH

rpm

SmF

SSF

TSS

YMB

µm

˚C

%

Rhizopus oligosporus

Biological Oxygen Demand

Chemical Oxygen Demand

Gram

Hour

Liter

Minute

Second

Milliliter

Non-starch Polysaccharide

Power of Hydrogen

Revolution per minute

Submerged Fermentation

Solid State Fermentation

Total Suspended Solid

Yeast Malt Broth

Micrometre

Degree Celsius

Percentage

VI

LIST OF TABLES

Page

Table 1 Ratio on solution mixture of citric acid and trisodium citrate dihydrate on

pH 4, 5 and 6.

14

Table 2 Wet and dry biomass of R. oligosporus at propagation stage from day 0 to

day 6.

21

Table 3 Wet biomass of R. oligosporus in the sago effluent at control replicate,

pH 4, 5 and 6 during the accumulation stage from day 0 to day 6.

29

Table 4 Dry biomass of R. oligosporus in the sago effluent at control replicate,

pH 4, 5 and 6 during the accumulation stage from day 0 to day 6.

31

Table 5 Data of phenol sulphuric test at control replicate, pH 4, 5 and 6 during the

accumulation stage from day 0 to day 6.

34

Table 6 Data of nitrate analysis during the accumulation stage at control replicate,

pH 4, 5 and 6 from day 0 to day 6.

38

Table 7 Data of nitrite analysis during the accumulation stage at control replicate,

pH 4, 5 and 6 from day 0 to day 6.

42

VII

LIST OF FIGURES

Page

Figure 1 Sago palm (Metroxylan sagu) in Sarawak, Malaysia.

4

Figure 2 Morphology of R. oligosporus under microscope.

6

Figure 3 Range of pH value from pH 0 to 14.

8

Figure 4 Biomass of R. oligosporus in Yeast Malt Broth (YMB) solution.

9

Figure 5 Submerged Fermentation Set-up.

12

Figure 6 R. oligosporus in the sago effluent at day 0 during the accumulation

stage.

13

Figure 7 Phenol sulphuric test of sago effluent at control replicate, pH 4, 5 and

6 during the accumulation stage.

16

Figure 8 API nitrate test kit for nitrate analysis.

17

Figure 9 API nitrite test kit for nitrite analysis.

18

Figure 10 Propagation of R. oligosporus. (A) day 1 of propagation; (B) day 2;

(C) day 3; (D) day 4; (E) day 5; (F) day 6.

20

Figure 11 Graph of wet biomass of R. oligosporus at propagation stage from day

0 to day 7.

22

Figure 12 Graph of dry biomass of R. oligosporus at propagation stage from day

0 to day 7.

23

Figure 13 Accumulation stage of R. oligosporus at control replicate. (A) day 1;

(B) day 2; (C) day 3; (D) day 4; (E) day 5; (F) day 6.

25

Figure 14 Accumulation stage of R. oligosporus at pH 4. (A) day 1; (B) day 2;


26



27



28

Figure 17 Wet biomass of R. oligosporus at control replicate, pH 4, 5 and 6

during the accumulation stage from day 0 to day 6.

30

Figure 18 Dry biomass of R. oligosporus at control replicate, pH 4, 5 and 6 H


32

VIII

Figure 19 Dry biomass of R. oligosporus in sago effluent during the

accumulation stage.

33

Figure 20 Graph of phenol sulphuric test at control replicate, pH 4, 5 and 6


35

Figure 21 Changes in colour of nitrate analysis at different pH during the

accumulation from day 0 to day 6. (A) control replicate; (B) pH 4; (C)

pH 5; (D) pH 6.

37

Figure 22 Graph of nitrate analysis during the accumulation stage at control

replicate, pH 4, 5 and 6 from day 0 to day 6.

39

Figure 23 Changes in colour of nitrite analysis at different pH during the

accumulation from day 0 to day 6. (A) control replicate; (B) pH 4; (C)

pH 5; (D) pH 6.

41

Figure 24 Graph of nitrite analysis during the accumulation stage at control

replicate, pH 4, 5 and 6 from day 0 to day 6.

43

IX

LIST OF APPENDICES

Page

Appendix A Wet biomass of R. oligosporus at propagation stage from day 0 to

day 7.

47

Appendix B Dry biomass of R. oligosporus at propagation stage from day 0 to

day 7.

47

Appendix C Wet biomass of R. oligosporus at control replicate during the


48

Appendix D Wet biomass of R. oligosporus at pH 4 during the accumulation

stage from day 0 to day 6.

48

Appendix E Wet biomass of R. oligosporus at pH 5 during the accumulation


48

Appendix F Wet biomass of R. oligosporus at pH 6 during the accumulation


49

Appendix G Dry biomass of R. oligosporus at control replicate during the


49

Appendix H Dry biomass of R. oligosporus at pH 4 during the accumulation


49

Appendix I Dry biomass of R. oligosporus at pH 5 during the accumulation


50

Appendix J Dry biomass of R. oligosporus at pH 6 during the accumulation


50

Appendix K Data of phenol sulphuric test at control replicate during the


51

Appendix L Data of phenol sulphuric test at pH 4 during the accumulation stage

from day 0 to day 6.

51

Appendix M Data of phenol sulphuric test at pH 5 during the accumulation stage


51

Appendix N Data of phenol sulphuric test at pH 6 during the accumulation stage


52

Appendix O Data of nitrate analysis at control replicate during the accumulation


53

X

Appendix P Data of nitrate analysis at pH 4 during the accumulation stage from

day 0 to day 6.

53

Appendix Q Data of nitrate analysis at pH 5 during the accumulation stage from

day 0 to day 6.

53

Appendix R Data of nitrate analysis at pH 6 during the accumulation stage from

day 0 to day 6.

54

Appendix S Data of nitrite analysis at control replicate during the accumulation


55

Appendix T Data of nitrite analysis at pH 4 during the accumulation stage from

day 0 to day 6.

55

Appendix U Data of nitrite analysis at pH 5 during the accumulation stage from

day 0 to day 6.

55

Appendix V Data of nitrite analysis at pH 6 during the accumulation stage from

day 0 to day 6.

56

XI

Growth of Rhizopus oligosporus in Sago Effluent at Different pH

Nur Aqlily Riana binti Nasar

Resource Biotechnology

Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

The discharge of large amounts sago effluent into the river leads to severe water pollution. A study

on the growth of R. oligosporus in the sago effluent at different pH was carried out. R. oligosporus

was grown for the treatment of sago effluent with the production of high protein fungal biomass

(HPFB) via submerged fermentation (SmF). The objectives of this study were to investigate the

optimum pH for the growth of R. oligosporus in sago effluent and the effectiveness of SmF by R.

oligosporus for water treatment. Sago effluent was adjusted to pH 4, 5, 6 and one replicate with no

adjustment of pH. The optimum pH for R. oligosporus was notable at pH 4. The highest wet and

dry biomass production of pH 4 were 98.67 g/L and 3.80 g/L respectively. There were three

analysis that were successfully carried out which were phenol sulphuric test, nitrate and nitrite

analysis. There was 98% reduction of carbohydrate concentration in sago effluent. Besides, the

nitrate concentration was reduced from 0.266 to 0.257 g/L in pH 4. Concentration of nitrite was

decreased from 0.040 to 0.029 g/L in pH 4. In addition to the production of HPFB, the decreasing

of starch, nitrate and nitrite concentration would lead to a potential benefits to the environment.

Keywords: Rhizopus oligosporus, sago effluent, submerged fermentation (SmF), high protein

fungal biomass (HPFB), water treatment

ABSTRAK

Pembuangan efluen sagu yang tinggi ke dalam sungai membawa kepada pencemaran air yang

membimbangkan. Satu kajian mengenai pertumbuhan R. oligosporus di dalam efluen sagu pada

pH yang berbeza telah dijalankan. R. oligosporus ditumbuhkan untuk merawat efluen sagu

bersama penghasilan biomas kulat tinggi protin melalui fermentasi substrat cecair. Objektif kajian

ini adalah untuk mengenalpasti pH yang optimum untuk pertumbuhan R. oligosporus di dalam

efluen sagu dan keberkesanan fermentasi substrat cecair oleh R. oligosporus untuk rawatan air.

Efluen sagu ditukarkan kepada pH 4, 5, 6 dan terdapat satu replika efluen sagu yang mana pH

tidak diubah. pH yang optimum dikesan pada pH 4. Terdapat tiga analisis yang berjaya dijalankan

iaitu ujian fenol sulfurik, nitrat dan nitrit analisis. Terdapat pengurangan sebanyak 98% kepekatan

karbohidrat di dalam efluen sagu. Selain itu, kepekatan nitrat berkurang dari 0.266 kepada 0.257

g/L dalam pH 4. Kepekatan nitrit berkurang dari 0.040 kepada 0.029 g/L dalam pH 4. Berikutan

penghasilan biomas kulat tinggi protin, pengurangan kepekatan kanji, nitrat dan nitrit, akan

membawa kepada keuntungan kepada alam sekitar.

Kata Kunci: Rhizopus oligosporus, efluen sagu, fermentasi substrat cecair, biomas kulat tinggi

protin, rawatan air

1

CHAPTER 1

INTRODUCTION

1.1 Introduction

The main exporter for sago is Sarawak, which is located in east Malaysia. The largest

plantation area for sago is in Mukah, Sarawak (Awg-Adeni et al., 2010). Sago palm is

easily to plant because it can resist stress condition such as strong wind, fire, floods and

drought (Singhal et al., 2008). Sago palm consist of large fibrous root system that function

to remove pollutant, heavy metal, faecal toxin and trap slit loads (Singhal et al., 2008).

According to Singhal et al. (2008), sago starch is widely used compared to other starches

because it has low production cost and high yields. There are 73% of amylopectin and 27%

of amylose in sago starch (Singhal et al., 2008). Sago effluent from factories is usually

disposed into nearby rivers, which can lead to river water pollution (Awg-Adeni et al.,

2010). Based on Awg-Adeni et al. (2010), sago effluent contains high Chemical Oxygen

Demand (COD), Biological Oxygen Demand (BOD) and Total Suspended Solid (TSS).

Submerged fermentation (SmF) can be defined as the process of microorganism’s

cultivation in nutrient broth in the form of liquid (Subramaniyam & Vimala, 2012). Based

on Subramaniyam and Vimala (2012), SmF is different from solid state fermentation (SSF)

because SSF is using nutrient broth that is in solid state. Many Asian countries use the

SmF process because it is the best alternative that gives efficient production of metabolite

such as ganoderic acid and polysaccharide (Fang & Zhong, 2002). In this study, Rhizopus

oligosporus was used to treat sago effluent. R. oligosporus was grown in sago effluent at

different pH of 4, 5, and 6 to find the optimum pH by using SmF. R oligosporus is one of

the fungi that provide high protein fungal biomass (Femi-Ola et al., 2013). Besides,

2

biomass of fungus usually considered as potential source of single cell protein (Ferreira et

al., 2014). In addition to water treatment, biomass of R. oligosporus was converted into

value-added product such as aquaculture feed as supplementary protein source

(Vikineswary et al., 1997). For the water treatment, phenol sulphuric test, nitrate and nitrite

analysis were conducted in order to identify concentration of nitrate, nitrite and total

carbohydrate in the water.

The purpose of this study is to observe the growth of R. oligosporus in sago effluent at

different pH.

The specific objectives of this study are:

1. To observe the growth of R. oligosporus at different pH in sago effluent.

2. To identify the optimum pH for R. oligosporus in sago effluent.

3. To determine the effectiveness of submerged fermentation (SmF) process by R.

oligosporus to treat sago effluent.

1.2 Problem Statement

Sago effluent is abundant since it occurs naturally in large amount. Sago effluent is usually

disposed into the river and causes water pollution since it has high organic material,

causing COD, BOD and TSS of the water resources to be increased. By growing R.

oligosporus in sago effluent, total organic load may be reduced. Besides, SmF can be used

to provide high value product such as enzyme and animal feeds (Awg-Adeni et al., 2010).

SmF process is widely used since it is easy to set up and monitor.

3

CHAPTER 2

LITERATURE REVIEW

2.1 Sago effluent

Sago is extracted from Metroxylan sagu (Figure 1) or also known as sago palm (Awg-

Adeni et al., 2010). According to Wee et al. (2011), Malaysia which is the biggest exporter

of sago, exports 25,000 to 40,000 tons of sago yearly. It is estimated that 7 tons of the sago

effluent is produced daily (Awg-Adeni et al., 2010). Vikineswary et al. (1997) stated that

sago effluent which consists of high organic load is drained into the river and it will cause

increasing of Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD) and

Total Suspended Solid (TSS). Based on Awg-Adeni et al. (2010), other than starch, sago

effluent consists of hemicellulose, lignin and cellulose that are non-starch polysaccharide

(NSP) pollutants. Sago effluent has horrible odour and irritating colour because it contains

high organic material (Ruban, 2013).

Figure 1 Sago palm (Metroxylan sagu) in Sarawak, Malaysia (Retrieved from www.craunresearch.com).

4

According to Vikineswary et al. (1997), sago effluent can be divided into three parts,

which are, wastewater, sago hampas and bark. Wastewater is known as liquid residue

while sago hampas and barks are solid residue (Awg-Adeni et al., 2010). There are some

treatments that can be used to treat wastewater using fungi and bacteria (Vikineswary et

al., 1997). Based on Singhal et al. (2008), Rhodopseudomonas palustris which is an

indigenous strain isolated from the wastewater of rice-noodle factory was grown in sago

effluent in order to produce biomass. Currently, fungus such as Rhizopus oligosporus is

used because it consumes low cost and its biomass can be converted into value-added

product such as aquaculture feed. R. oligosporus is grown using submerged fermentation

(SmF) process to reduce COD, BOD and TSS. Sago effluent will act as substrate for the

fermentation process to grow R. oligosporus. Thus, river water pollution can be reduced.

5

2.2 Submerged Fermentation Process

Submerged fermentation (SmF) can be defined as microorganism cultivation in liquid

substrate (Subramaniyam & Vimala, 2012). SmF usually occurs in the present of water.

Example of the liquid substrate is sago effluent. According to Subramaniyam and Vimala

(2012), utilization of substrate is rapid, therefore nutrient need to replace constantly. This

fermentation technique is the best for microorganism that need a special condition such as

high content of moisture (Subramaniyam & Vimala, 2012). In this process, there are

several factors that can influence fermentation process for examples, pH, aeration, nitrogen

concentration, temperature and this entire factor can give higher impact to the rate of

fermentation and yield of the product (Chisti et al., 1999). Based on Chisti et al. (1999),

substrate such as liquid media, wastewater and soluble sugar are usually used in the SmF

process. If solid substrate is used in SmF, it needs to be in the slurry form by suspending it

in large amount of water.

There are five types of submerged fermentation bioreactor which are bubble column, airlift

fermenter, stirred-tank fermenter, trickle-bed fermenter and fluidized-bed fermenter (Chisti

et al., 1999). These types of bioreactor are the major bioreactors that are used in SmF. SmF

are usually used in the production of yoghurt, wine, soy sauce and beer. The extraction of

the secondary metabolites mainly will be performed by SmF (Subramaniyam & Vimala,

2012). SmF is usually used because it is easy to handle and easy to monitor. According to

Sandhya et al. (2005), there is advantage in SmF which is easy recovery of mycelia, spores

and extracellular enzymes but the extraction of enzyme is less stable and product is diluted

compared to SSF. Based on Subramaniyam and Vimala (2012), the other advantage is, the

purification technique of SmF is easier compared to SSF.

6

2.3 Rhizopus oligosporus

R. oligosporus is a fungus that belongs to the family Mucoraceae. It is a filamentous

fungus (Takaya et al., 1998). Figure 2 shows the morphology of R. oligosporus under

microscope. Most of the other Rhizopus taxa consist of 5% of irregular spores compared to

R. oligosporus that consists of larger irregular spores in the range of 10 to 31% (Jennessen

et al., 2008). This fungus can be used in the production of metabolite, fermentation of food

and pathogenesis (Jennessen et al., 2008). R. oligosporus is used in the production of

tempeh, which is a well-known fermented food made from soybean (Jennessen et al.,

2008). R. oligosporus produce fluffy white mycelia that able to bind the bean together.

According to Jennessen et al. (2008), enzyme that is released by this fungus is important to

human because it is protein-rich product and consumable. Based on Kovaĉ and Raspor

(1997), filamentous fungi are widely used in the production of food since it able to

improve taste of food, nutritional value and protect food from spoilage. Fermentation by R.

oligosporus is classified into two types of process which is industrial fermentation and

traditional fermentation (Kovaĉ & Raspor, 1997).

Figure 2 Morphology of R. oligosporus under microscope (Retrieved from www.mycology.adelaide.edu).

7

R. oligosporus is commonly used because it is low cost and also offer other advantages

such as constraining growth of tumor, anaemia diseases, hypertension, lowering

cholesterol level, and reducing diarrhea infection (Patrick et al., 2007). Instead of involve

in food fermentation, R. oligosporus also helps in producing enzyme and treating

wastewater (Jennessen et al., 2008). Filamentous fungi are used to treat wastewater and it

can convert the wastewater into useful biomass that can be used as animal feed especially

aquaculture life and source for human diet (Kovaĉ & Raspor, 1997). Compared to bacteria,

fungi are more effective in metabolizing complex carbohydrate for example starch (Kovaĉ

& Raspor, 1997).

8

2.4 pH

pH is known as logarithmic measure of the hydrogen ion concentration. pH is stands for

power of Hydrogen. pH is one of the parameter that is widely used in the growth rate of

microorganism. It is usually used as a parameter to determine the optimum pH for the

highest growth rate of the microorganism. The usual pH value is in the range of 0 to 14

(Figure 3). Neutral pH can be shown in the solution of pH 7. Solutions that are above pH 7

are alkaline and less than 7 is considered acidic.

Figure 3 Range of pH value from pH 0 to 14 (Retrieved from www.eoeartht.com)

Buffer is used to resist changes in pH through the addition of small quantity of strong acid

or strong base (Russo & Hanania, 1987). Therefore, buffer is added to maintain the pH.

Buffer that is suitable for pH 4 and 5 is Citrate buffer, while for pH 7 is Tris buffer

(Kulevich et al., 2014). Buffer for pH 6 is Acetate buffer. Example of function of buffer is

buffer allowing living organism to live, for example R. oligosporus.

9

2.5 High Protein Fungal Biomass (HPFB)

High protein fungal biomass (HPFB) can be retrieve from the starch processing wastewater

(Jin et al., 2002). Fungus such as R. oligosporus and Aspergillus oryzae can produce HPFB

from starch processing water even though without nutrient supplementation (Jin et al.,

2002). Figure 4 shows fungal biomass of R. oligosporus in Yeast Malt Broth (YMB)

solution. The fungal biomass that is produced by this fungus contains more than 45%

protein which is suitable for the animal consumption (Jin et al., 2002). pH can influence

the biomass production of the fungus. The biomass product that has been produced from

the submerged fermentation process is easily separated and efficiently harvested (Jin et al.,

2002).

Figure 4 Biomass of R. oligosporus in Yeast Malt Broth (YMB) solution (Retrieved from www2.hawaii.edu).

10

The valuable product such as microbial biomass protein (MBP) is produced from the

biotechnological treatment which can purify the effluent for example sago effluent (Jin et

al., 2002). The effluent that has been purified can be used for agricultural irrigation or

industrial use (Jin et al., 2002).

11

CHAPTER 3

METHODOLOGY

3.1 Pre-treatment of Substrate

Sago effluent was collected from Herdsen Sago Mill, Pusa Sarawak. Firstly, a 710 µm

mesh size filter was used to filter sago effluent to isolate it from sago hampas. Suspended

solid was removed because only supernatant are required. The most suitable sago effluent

medium for strain collection is 50% (v/v) of supernatant. Sago effluent was transferred into

2000 ml Schott bottle (SCHOTT Duran Laboratory glass bottle, SCHOTT, Germany). The

bottle was autoclaved first at 121 oC for 15 min.

3.2 Microorganism

R. oligosporus was obtained from Microbiology Laboratory from Department of Molecular

Biology, UNIMAS. From isolation process in tempeh, the strain of R .oligosporus was

obtained and it was cultured on Sabouraud agar and was incubated for 72 hours at ambient

temperature. Then, from each plate, colonies were picked and subculture on Yeast Malt

Broth (YMB) (Himeidia, India). The agar containing colonies were incubated for 72 hr at

ambient temperature.

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