GROWTH OF oligosporus IN SAGO EFFLUENT AT DIFFERENT AERATION

Nadia Shuhada Bt Zanarizam @ Zahuzir

Bachelor of Science with Honours (Resource Biotechnology) 2015 ---=----ý"'ý ftsst Kbidmet Msklnmat Akademik [1NII,TRCiii rIA 1,AvcT A V1 R AWAK

Growth of Rhizopus oligosporus in Sago Effluent at Different Aeration

Nadia Shuhada bt Zanarizam @ Zahuzir

(37417)

A report submitted in partial fulfillment of the Final Year Project (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 ACKNOWLEDGEMENT

Alhamdulillah, all praises to Allah S.W. T., The Most Greatest and The Most

Merciful for His blessing and strength He gave to me in completing this thesis. Special

appreciation goes to my supervisor, Dr. Micky Vincent and my co-supervisor Dr. Devagi

Kanakaraju for their supervision and for being extremely supportive towards me. Their

valuable help, suggestions, and constructive comments throughout the experimental and

thesis work have significantly contributed to the success of this research.

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

Faculty of Resource Science and Technology, Universiti Malaysia Sarawak for their help

and support towards my research project.

My deepest gratitude goes to my parent, Mr Zanarizam @ Zahuzir bin Mansor, Mrs

Wan Saudah bt Wan Abdullah and also to my siblings, Mr Muhammad Khairul Amirin and Ms Nadia Syakirah for their endless love, prayers and encouragement. Sincere thanks to all my friends, course mates, and laboratory mates especially Ms Fazidah bt Junaidi, Ms

Shahirah Safin bt Amran and Ms Nur Aglily Riana bt Nasar for their kindness and moral support they given to me along the journey. When I felt down, their support will always give me strength to face all the problems and complication happened.

Lastly, it is impossible to list all the individuals that have been with me throughout this research. Thus, f dedicate my outmost gratitude to all of them. Thank you very much.

ii DECLARATION

I hereby declare that this Final Year Project Report entitled "Growth of Rhizopus oligosporus in Sago Effluent at Different Aeration" is based on my original work except

for quotations and citations, which have been duly acknowledged. I also declare that it has

not been submitted in support of an application for another degree or qualification to this or any other university or institute of higher learning.

(Nadia Shuhada ht Z.anarizam (c,r Zahuzir)

JiJNE 201 5

III eusat Khidmat MMaUucnatflkademik UMYERSM MALAYSIASARAWAK

TABLE OF CONTENTS

TITLE PAGE

ACKNOWLEDGEMENT 11

DECLARATION III TABLE OF CONTENTS IV

LIST OF ABBREVIATIONS vi LIST OF TABLES V11

LISTS OF FIGURES viii LIST OF APPENDICES X ABSTRACT I

CHAPTER 1: INTRODUCTION 1. 1 Introduction 2 1.2 Problem Statement 4

CHAPTER 2: LITERATURE REVIEW

2.1 Rhi_opus oligosporus 5 2.2 Sago Effluent 8 2.3 Submerged 10 2.4 Aeration 13 2.5 High Fungal Biomass (HPFB) 16

CHAPTER 3: METHODS 3.1. Pretreatment of substrate 19 3.2. Microorganism 19 3.3. Submerged Fermentation Process 20

3.3.1 Effect of Amount of Aeration on Submerged 21 Fermentation 3.4 Dry Cell Weight Analysis 21 3.5 Phenol-Sulphuric Analysis 22 3.6 Nitrate Analysis 23 3.7 Nitrite Analysis 24

IV CHAPTER 4: RESULT AND DISCUSSION 4.1 Propagation Stage 25

4.2 Accumulation of R. oligosporus 30 4.3 Phenol-Sulphuric Analysis 40 4.4 Nitrate Analysis 43 4.5 Nitrite Analysis 47

CHAPTER 5: CONCLUSION AND RECOMMENDATION 51

REFERENCES 53 APPENDICES:

Appendix I : Propagation stage of R. oligosporus 57 Appendix II : Accumulation stage of R. oligosporus 58 Appendix III Phenol-sulphuric analysis result 61 Appendix IV Nitrate analysis result 63 Appendix V Nitrite analysis result 65

V LIST OF ABBREVIATIONS

BOD Biological oxygen demand

COD Chemical oxygen demand

HPFB High protein fungal biomass

Kg Kilogram

LB Luria broth mL Milliliter

OD Optical density

R. oligosporus Rhi opus oligosporus rpm Revolution per minute

SmF Submerged fermentation

SSF Solid state fermentation t Tons

TSS Total suspended solid vvm Volume per volume per minute

YMB Yeast malt broth

°C Degree celsius

µm Micrometer

vi LIST OF TABLES

Page

Table I Feed ingredient composition, dry matter basis. 18

Table 2 Wet biomass and dry biomass concentration during 27 propagation stage of R. oligosporus.

Table 3 Wet biomass of R. oligosporus during accumulation stage for 35 0.00, 0.50, 0.75 and 1.00 vvm.

Table 4 Dry biomass of R. oligosporus during accumulation stage for 37 0.00, 0.50, 0.75 and 1.00 vvm.

Table 5 Reading of phenol-sulphuric analysis. 41

Table 6 Concentration of nitrate at different day for 0.00, 0.50, 0.75 45 and 1.00 vvm.

Table 7 Concentration of nitrite at different day for 0.00, 0.50, 0.75 49 and 1.00 vvm.

VII LIST OF FIGURES

Page

Figure I Sago palm (Metroxylon sagu) in Mukah, Sarawak. 2

Figure 2 Morphology of R. oligosporus under microscope. 5

Figure 3 Production of in industrial scale. 6

Figure 4 Sample of commercial animal feed pellet that available in 7 market.

Figure 5 A process of harvesting a sago palm for sago production. 8

Figure 6 Water pollution caused by discharged of waste water. 9

Figure 7 Mycelial biomass has been collected after submerged 10 fermentation.

Figure 8 Industrial fermenter used in the food and beverage industry. 11

Figure 9 Aeration supplied to the fermentor during fermentation of 13 beer.

Figure 10 Fungal biomass is processed produce dried fungal biomass 16 product.

Figure 11 Submerged fermentation set-up. 20

Figure 12 Submerged fermentation of R. oligosporus at day 0. 21

Figure 13 Phenol-sulphuric test to identify the total carbohydrate in 22 sample.

Figure 14 API Liquid nitrate test kit used to identify the concentration 23 on nitrate.

Figure 15 API Liquid nitrite test kit used to identify the concentration 24 on nitrite.

Figure 16 Propagation stage of R. oligosporiis from day Ito day 6. 26

Figure 17 Graph of wet biomass of R. oligosporus against day during 28 the propagation stage.

Figure 18 Graph of dry biomass of R. oligosporus against day during 29 the propagation stage.

Figure 19 The accumulation stage ot'R. oligosporlus for 0.00 vvm. 31

viii --

Figure 20 The accumulation stage of R. oligosporus for 0.50 vvm. 32

Figure 21 The accumulation stage of R. oligosporus for 0.75 vvm. 33

Figure 22 The accumulation stage of R. oligosporus for 1.00 vvm. 34

Figure 23 Dry biomass of R. oligosporus after submerged fermentation. 36

Figure 24 Graph of wet biomass of R. oligosporus at different day 38 during accumulation stage.

Figure 25 Graph of dry biomass of R. oligosporus at different day 38 during accumulation stage.

Figure 26 The graph of concentration against day for phenol-sulphuric 42 analysis.

Figure 27 The changes of colour for nitrate analysis at different day. 44

Figure 28 The graph of concentration against day for nitrate analysis. 46

Figure 29 The changes of colour for nitrite analysis at different day. 48

Figure 30 The graph of concentration against day for nitrite analysis. 50

ix LIST OF APPENDICES

Page

Appendix A Wet biomass for propagation stage of R. oligosporus from 57 day 0 to day 6.

Appendix B Dry biomass for propagation stage of R. oligosporus from 57 day 0 to day 6.

Appendix C Wet biomass for accumulation stage of R. oligosporus for 58 0.00 vvm from day 0 to day 6.

Appendix D Wet biomass for accumulation stage of R. oligosporus for 58 0.50 vvm from day 0 to day 6.

Appendix E Wet biomass for accumulation stage of R. oligosporus for 58 0.75 vvm from day 0 to day 6.

Appendix F Wet biomass for accumulation stage of R. oligosporus for 59 1.00 vvm from day 0 to day 6.

Appendix G Dry biomass for accumulation stage of R. oligosporus for 59 0.00 vvm from day 0 to day 6.

Appendix 11 Dry biomass for accumulation stage of R. oligosporus for 59 0.50 vvm from day 0 to day 6.

Appendix I Dry biomass for accumulation stage of R. oligosporus for 60 0.75 vvm from day 0 to day 6.

Appendix J Dry biomass for accumulation stage of R. oligosporus for 60 1.00 vvm from day 0 to day 6.

Appendix K Data of phenol-sulphuric analysis for 0.00 vvm from day 0 to 61 day 6.

Appendix L Data of phenol-sulphuric analysis for 0.50 vvm from day 0 to 61 day 6.

Appendix M Data of phenol-sulphuric analysis for 0.75 vvm from day 0 to 61 day 6.

Appendix N Data of phenol-sulphuric analysis for 1.00 vvm from day 0 to 62 day 6.

Appendix 0 Data of nitrate analysis for 0.00 vvm from day 0 to day 6. 63

Appendix P Data of nitrate analysis for 0.50 vvm from day 0 to day 6. 63

x Appendix Q Data of nitrate analysis for 0.75 vvm from day 0 to day 6. 64

Appendix R Data of nitrate analysis for 1.00 vvm from day 0 to day 6. 64

Appendix S Data of nitrite analysis for 0.00 vvm from day 0 to day 6. 65

Appendix T Data of nitrite analysis for 0.50 vvm from day 0 to day 6. 65

Appendix U Data of nitrite analysis for 0.75 vvm from day 0 to day 6. 65

Appendix V Data of nitrite analysis for 1.00 vvm from day 0 to day 6. 66

xi Growth of Rhizopus oligosporus in Sago Effluent at Different Aeration

Nadia Shuhada bt Zanarizam @ Zahuzir

Resource Biotechnology Faculty of Resource Science and Technology University Malaysia Sarawak

ABSTRACT

In recent years. there are increasing levels of pollution caused by industrial wastewater. Sago effluent discharged into the river contains hazardous compound, toxic chemical and organic materials. In order to overcome this problem, organic materials from sago effluent must be removed by using submerged fermentation. R. oligosporus was grown in sago effluent to treat waste water and convert organic matters into useful product. In this study, different amount of aerations were tested for the growth of R. oligosporus in sago effluent which were 0.00, 0.50. 0.75 and 1.00 vvm. Aeration of 1.00 vvm was identified to produce the highest biomass production which was 77.0% or 104.333 g/L for wet biomass and 66.2% or 3.790 g/L for drv biomass and subsequently 0.75, 0.50 and 0.00 vvm. This shown that the microorganisms need sufficient aeration for their growth. There were three analysis conducted in this study which were phenol-sulphuric, nitrate and nitrite analysis. The phenol-sulphuric analysis revealed the total carbohydrates in the sample decreased along this study from 19.919 g/l, to 0.334 g/l. for 1.00 vvm which was 98.3%. In addition, this study showed the concentration of nitrate was decreased from 0.267 g/I_ to 0.260 g/L and nitrite was decreased from 0.056 g/l, to 0.031 g/I. against the day of fermentation. It proved that the growth of R. oligosporus in sago effluent had the potential to treat the waste water. At the same time, this study can produce beneficial product which was high protein fungal biomass (HPFB) which can used as animal feedstock. It can he concluded that this study could be the solution to the pollution problem as it can minimize the damage on environment.

Keywords : Rlri:opus oligosporus. sago effluent, submerged fermentation (SMF), water treatment, high protein lüngal biomass (HPFR).

ABSTRAK

Sejctk kehelakangan ini. tulrap pencenraran nreningkat disebabkan oleh air sisa perindustrian. Efluen sagu yang dilepaskan ke dalam sungai mengandungi sebatian berbahaya, bahan kiniia toksik dan bahan organik rung tinggi. l/ntuk nrengatasi masa/ah ini, bahan-bahan organik daripada efluen saga harus dihapuskan dengan metýggunakan /ernaentasi substrat cecair. Pertumbuhan R. oligosporus di dalam efluen sagu menrhantu turtttk mercnrat air sisa dan menukar bahan organik kepada produk yang berguna. Dalam kajian ini. jtunlah pengudaraan yang berbe-a telah dikaji untuk pertunrbuhan R. oligosporus dalam efluen sagu iaitu 0.00, 0.50. 0.75 dan 1.00 rvm. Pengudaraan 1.00 vvnt telah dikenalpasti untuk ntenghasilkan pengeluaran biomass tertinggi sebanpak 77.0% iaitu 104.333 g/L bagi biornas hasah dan 66.2% iaitu 3.790 g l, turtttk hionrus kering dan seterusnya diikuti 0.75, 0.50 dan 0.00 vrm. lni nnenunjukkan bahml'a mikroorganisma memerlrrkan pengudaraan yang niencukupi untuk pertrmrbuhan niereka. Junrlah perrgudcrraan Yang tidak nrencukupi boleh mernbantutkan pertumbulran mikroarganisnta. Terdapat tiga ana/isis yang dija/unkcrn dalant kajian ini iaitu analisis fenol-stt fa"ik, nitrat dan nitrit. Analisis fenol-srtlf: trik ntenwnjukkan balrmi'a jumlah karbohidrat dalani sanrpel ntenururr sebanyak 98.3% sepanjang kajian ini duripaclu 19.919 kepuda 0.334 1.00 Di im. kajian ini menunjukkan kepekatan gl gýL untuk vvnt . sc'npirrg nitrat urerurrun daripada 0.267 gL kepada 0.260 gýL dan nitrit telah ment'"un daripada 0.056 g/L kepada 0. 031 g 1, seining dengan hari, fermentasi. lni ntentbuktikan bahawa pertumbuhan R. oligosporus dalani Pada kajian ini dapat e/lrren .saga nnenrpur. rai potensi untuk menalvat air sisa. 'rasa yang sanra, ntenghusilkan produk benmarrfatat iaint biomas kulat berprotin tinggi (HPFB) yang boleh digunakan sebagai hahun nrentulr haiu'an. Duput drsrmprtlkarr bahmra kajian ini dapat menjadi pen>.velesaian kepada masalah perrcemuran kerana ia boleh inengurangkan kerosakan ke atas alani sekitar.

Kam kunci : Rhi: opus oligosporus, efluen saga, fer, nentasi substrat cecair (SMF), rawatan air, biomas kulat beºprotin tinggi (HPFB).

1 CHAPTER I

INTRODUCTION

1.1 Introduction

Malaysia is one of the world supplier of sago starch which is commonly produced in large quantities especially in Sarawak (Karim et al., 2008). Sago starch is extracted from the

Metroxj, lon sagt{ or commonly known as sago palm as presented by Figure 1. Sago palm is a tropical crops that grow in peat swamps which can tolerate wet growing state. It produces high amount of starch in its trunk which is 4 times more productive rather than paddy (Karim et al., 2008). According to Awang-Adeni (2010), sago is the highest starch producer between all starch crops in the world. Starch is widely used in the pharmaceutical, plastics, paper, adhesive beverage and textile industries (Omojola et al.,

2012). Sago starch, particularly in Sarawak, can also be used to produce many types of food such as crackers and bread (Mohamed et al., 2008).

Figure 1 Sago palm (Afetroxylon sogt, ) in Mukah, Sarawak. (Retrieved from http: //www. craunresearch. com. mv/I l'I'MI. /Information/Info%20(intro). html)

2 In recent years, there are increasing levels of pollution caused by industrial wastewater.

This valuable resource can be recovered by reusing them. According to Kanu et al. (2011),

rapid growth of certain industries especially sago industry causes water pollution which is

due to the effluent discharged. Sago industry requires several washing cycles during

processing the sago which subsequently produce 30, 000 to 35, 000 L of effluent for each

single tons of sago produce (Anbukumar et al., 2014). At the same time, food processing

plant produces large quantities of waste materials since starch is one of the polysaccharide

that store the energy in plant (Jin et al., 1999).

The sago effluent discharged into the river contains hazardous compound and toxic

chemical (Dubey, 2013). According to Anbukumar et al. (2014), sago effluent also

contains high organic materials with 1, 000 to 5, 000 mg/L of COD during off-season and

5, 000 to 7, 000 mg/L during the season. The effluent is believed to affect some characteristics of the environment such as BOD, COD and temperature (Awg-Adeni et al.,

2010). This will cause great damages to the environment.

In this study, Rhi=opus oligosporus was grown in sago effluent and underwent submerged fermentation to treat and convert organic matters to beneficial product.

The specific objectives of this study are

1. To observe the growth of the R. oligosporus at different amount of aeration in sago

effluent which are 0.00, 0.50, 0.75 and 1.00 vvm.

2. To determine the best amount of aeration for enhancing the growth of R.

oligosporus.

3. To observe the ability of R. oligosporus in treating the sago effluent by using the

submerged fermentation.

3 1.2 Problem Statement

The high amount of the sago effluent dumped into the water bodies by the sago industries

causes pollution and damage to the environment. In order to overcome this problem,

organic materials from sago effluent must be removed by using submerged fermentation.

R. oligosporrrs grown in sago effluent to treat and convert organic matters into useful

product. The treatment of R. oligosporus on sago effluent could be the solution to the

pollution problem as it can minimize the damage on environment and produce products

which can be used as protein sources and feedstock (Vikineswary et al., 1997).

4 Nuut Khidmzt Maklumat Akadeniik UNIVERSITIMALAYSIA SAKAWAK

CHAPTER 2

LITERATURE REVIEW

2.1 Rhizopus oligosporus

Rhi_opus oligosporus is a from the genus Rhi_opus, family and order

of (Jennessen et al., 2008). The morphology of R. oligosporus can be seen in

Figure 2 below. This fungus is widely used in the production of tempeh as the starter

culture. According to Babu et al. (2009), R. oligosporus is the dominant fungus used compare to other fungus such as Mucor spp. and R. ory_ae during tempeh production. It grows well at high temperature between 34 to 45 °C and exhibit proteolytic and lipolytic activities which enhance the desirable properties in the tempeh.

Figure 2 Morphology of R. oligosporus under microscope. (Retrieved from http: //fineartamerica. com/ featured/fruiting-bodies-of-rhizopus-oligosporus-power-and-syredhtml )

5 R. oligosporus can produce many types of such as proteases, carbohydrases and

lipases which hydrolyze the constituents of the (Jennessen et al., 2008). This will

lead to the development of desirable flavor, aroma and taste of the tempeh as shown in

Figure 3. This fungus also produces some metabolites that helps to inhibit gram-positive

bacteria and other , including harmful microorganisms such as Staphylococcus

aureus therefore reducing the risk of intestinal infections among the consumers. R.

oligosporus is actually produced in many morphological forms but during the culture

process, the hyphae is not completely active (Miszkiewicz et al., 2004).

Figure 3 Production oftempeh in industrial scale. (Retrieved from http: //en. wikipedia. org/wiki/Tempeh)

According to Jennessen et al. (2008), R. oligospa-us is related to the production of unwanted metabolites, pathogenesis and food fermentation. R. oligosporus can also be used to treat waste water such as sago effluent. Fungi is used to treat the wastewater it does compared to bacteria as it is low cost owing to the use of raw materials and not

6 required specific nutrients. At the same time, R. oligosporus can produce biomass and

benefical . The biomass has been proven to be harmless and are suitable as

feedstock for animals (Vikineswary et al., 1997). For instance, as stated by Getha et al.

(1998), the biomass of R. oligosporus is used widely in aquaculture such as used for the

prawn feed as the right supplement as in Figure 4.

Figure 4 Sample of commercial animal feed pellet that available in market. (Retrieved from http: //www. hydronix. com/applications/moisture-inranimal_feed. php)

R. oligosporus is an ideal food for the animal feed as it has high protein content which contains all the essential amino acids that required by the human and animals nutrition

(Moore & Chiu, 2001). Fungal biomass is easily digested and low fat as well as provide dietary fibre. The important characteristics of fungal biomass is it is free of cholesterol.

The price of the animal feed depends on the protein content in the biomass. The higher protein content, the higher quality of the biomass as well as higher price in the market.

7 2.2 Sago Effluent

Sago industry is important particularly in India. Generally, sago is derived from tapioca tubers, which is converted into comercial sago by using indigenous technology with the use of large amounts of water (Anbukumar et al., 2014). Sago effluent is generated by the production of the sago starch through debarking and processing of sago trunk as shown in

Figure 5 (Karim et al.. 2008). In this process, it is estimated that 5, 000 to 6, 000 L of sago effluent is produced per day. Sago effluent contains high amount of organic components either in suspended state or dissolved state (Anbukumar et al., 2014). When sago effluent is stored for a few days, it will resulted in off colouring due to oxidation, and obnoxious odours (Bhaskar & Prasada, 2014). Sago effluent has the potential to alter the ecosystem when it is exposed to the soil.

from http: //en. Figure 5 A process of harvesting a sago palm for sago production. (Retrieved wikipedia. org/wiki/Sago)

8 In Malaysia, Sarawak is the major exporter of sago which exports up to 40, 000 t sago a

year to countries such as Taiwan, Japan and Singapore (Zainab et al., 2013). Sago palm

yields high amount of starch and will grow well in the swamp and peat areas. The amount

of starch is estimated between 150 to 300 kg by one palm. According to Awg-Adeni et al.

(2010), single sago starch will produce 7 t of sago pith waste daily. This sago residue

produced which consists of sago hampas and effluent usually discharged into the nearby

rivers. This activity will cause pollution to the river as the sago effluent contains high

amount of organic materials, as well as high content of COD and BOD as presented in

Figure 6 below.

Figure 6 Water pollution caused by discharged of waste water. (Retrieved from http: //w,A, w. bioenergyconsult. com/tag/malaysia/)

In order to overcome this problem, the sago effluent is used as the medium for the R. oligospoi"us to grow in order to treat it by removing all the organic materials. Previous study by Awg-Adeni et al. (2010), sago residue has been converted into valuable product such as enzyme, animal feed and fermented sugar. The conversion does not only minimize the pollution but also provide economic benefits for the country. 2.3 Submerged Fermentation (SmF)

Fermentation is the conversion of complex substrate into smaller compound with the

presence of microorganisms such as fungi (Subramaniyam & Vimala, 2012). There are two

techniques of fermentation that is suitable for this study which are submerged fermentation

(SmF) and solid state fermentation (SSF). Submerged fermentation is the cultivation of

microorganisms such as R. oligosporus with the presence of free water under the controlled conditions (Mienda, 201 1) as shown in Figure 7. This is done to produce desired product such as fuels, food, pharmaceutical products and industrial enzymes. Different types of microorganisms used like fungi, bacteria and yeast will produce different types of enzyme.

For instance, hydrolytic enzymes are formed by fungal cultures as the fungi use enzyme for their own growth.

Figure 7 M1vicelialhiomass has been collected aller submerged fermentation. (Retrieved from http: //v,1vw. intechopen. com/books/advances-in-applied-biotechnology/biotechnology led-cultivation-of-mushrooms) -of-agricultural-wastes-recycling-through-control

10 Submerged fermentation can produce industrial enzyme in large amount. The industry grows selected microorganisms whether fungi or bacteria in closed vessels which containing high concentration of oxygen and rich broth of nutrient (Renge et al., 2012).

Microorganisms will break down the nutrient and release the enzyme into the solution. The production of microbial enzyme increases rapidly as the presence of the development of large-scale fermentation technologies. According to Renge et al. (2012), industry that related to submerged fermentation commonly takes place in large fermenter about 1, 000 cubic meters such as in Figure 8. Most of the industrial enzymes are secreted by the selected microorganism in order to the nitrogen and carbon sources.

Figure 8 Industrial fermenter used in the food and beverage industry. (Retrieved from http: //www. corevdelta. com/food-beverage-gallery)

11 Submerged fermentation commonly involves continuous and fed-batch fermentation. For

the continuous process, sterilized nutrient is added into the fermenter at the same rate as

the fermentation broth is removed from the process (Gutierrez-Correa & Villena, 2015).

Continuous process will produce steady product all the time. During the growth of the

biomass, sterilized nutrient is added into the fermenter in the fed-batch fermentation.

The selection of substrate is important for the cultivation of an organisms for submerged

fermentation as the organism react differently for each substrate used. Each substrate

provide different types of nutrient for the organism. According to Subramaniyam and

Vimala (2012), some of the widely used substrates are sago effluent, liquid media,

vegetable and fruit juice, molasses and soluble sugar. There are a few parameters that are

controlled under the submerged fermentation such as amount of aeration, pH, substrate,

temperature, moisture and nutritional factors (Manpreet et al., 2005).

Submerged fermentation has been proven to have many advantages compared to other types of fermentation. One of the advantage of this type of fermentation is the purification process of the product is easier and simpler (Subramaniyam & Vimala, 2012). In addition, the substrate used for this process usually provide sufficient nutrient for the growth of the microorganisms. Moreover, the condition of the environment for submerged fermentation is provided similarly to the original habitat for fungi. Hence, fungi can be grow easily on the medium provided.

12