formulation of aquafeed from soya solid waste

FORMULATION OF AQUAFEED FROM SOYA SOLID WASTE (SOYA HAMPAS)

Agus Fery Budi Hartono Bin Mamat Nurhan

(34730)

Bachelor of Science with Honours QK (Resource Biotechnology) 881 2015 Al82 2015

Pusat Khidmat MakJumat Aklldt UlTVEpsm MALAYSIA SARAn

Formulation of Aquafeed from Soya Solid Waste (Soya Hampas)

Agus Fery Budi Hartono bin Mamat Nurhan (34730)

A Thesis Submitted in Partial Fulfillment of the Requirement of the Degree of Bachelor of

Science with Honours (Resource Biotechnology)

Supervisor Dr Lee Kui Soon

Resource Biotechnology

Department of Molecular Biology

r

Faculty of Resource Science ~nd Technology

Universiti Malaysia Sarawak

2015

l

ACKNOWLEDGEMENT

In the first place I would like to praise ALLAH SWT for blessing me with agood health

and perseverance along my final year project I would also like to give my appreciation

towards my parents Mr Mamat Nurhan and Mrs Fatimah for their continuous love and

moral support when conducting this project

Besides thats I would also like to send my appreciation to my supervisor Dr Lee

Kui Soon and my co-supervisor Dr Awang Ahmad Sallehin A Husaini for their generosity

and willingness to guide and leading me to the success of this final year project In spite of

the knowledge they had taught me I am very grateful for their financial supports in

preparing all the essential materials and apparatus for my project

Next I would like to give thousands thanks to all postgraduate students who

assisted me in my final year project They are Mr Shamil Mr Mohammad Khalid Mr

Jackie Wong and Ms Pang Shek Li They provide me with valuable knowledge when

conducting a research in the laboratory along with their kindness in assisting me to achieve

success in this project

Lastly I would like to give thanks to my laboratory mates for their continuous

supports I regain my strength to finish my final year project due to their moral and

technical supports They are Mohammad Irfan Sheikh Imam Simon Peter Mc Marshall

Weddy Ngu Jonathan Chia Fairuz Awatif Dorathy Hanis Hasan and Nur Liyana I had

wonderful moment even though the laboratory works were so hard to be finished Thanks

again to every individual who had contributed for the success of my final year project

J I

DECLARATION

I AgusFery Budi Hartono Bin Mamat Nurhan 34730 Faculty of Resource Science and

Technology hereby declare that the work entitled Formulation of Aquafeed from Soya

Solid Waste (Soya Hampas) is my original work I have not copied from any other

students work or from any other sources except where due reference or acknowledgement

is made explicitly in the text nor has any part been written for me by another person

15 b 20 i5 I

Date submitted Agus Fery Budi Hartono Bin Mamat Nurhan

(34730)

II

Pmat KhirJmat Maklumat Ak d UNTvrll a rr middotSln tALAYSr- SARAW~

TABLE OF CONTENTS

Acknowledgement I

Declaration II

Table of Contents III

List of Abbreviations V

List of Tables VI

List of Figures VII

Abstract

10 INTRODUCTION 2

11 Introduction 2

12 Problem Statement 3

20 LITERATURE REVIEW 4

21 Soybean 4

22 Soya Solid Waste (SSW) 5

23 Red Tilapia (Oreochromis sp) 5

24 Tilapia Aquaculture in Malaysia 6

25 Trichodermareesei 7

26 Trichodermaharzianum 7

27 Aspergillusniger 8

28 Solid State Fermentation (SSF) 8

30 MATERIALS AND METHODS 10

31 Materials 10


312Trichodermareesei Trichodermaharzianumand Aspergillusniger 10

313 Malt Extract Agar (MEA) 10

314 Mineral Salt Media (MSM) 11

315 Hemacytometer 12

316 Model Organism (Tilapia) 12

317 Kjeldahl Apparatus 12

318 SoxhletExtractor 12

32 Methodology 13

111

321 Sampling 13

322 Drying and Grinding 13

323 Spore Culturing 13

324 Preparation of Spore Suspension Culture 14

325 Solid State Fennentation (SSF) 15

326 Chemical Analyses of Samples 15

3261 The Proximate Analysis of Crude Protein 16

3262 The Proximate Analysis of Crude Lipid 17

3263 The Proximate Analysis of Crude Fiber 18

3264 The Proximate Analysis of Ash Matter 19

3265 The Proximate Analysis of Dry Matter and 20

Moisture Content

3266 Nitrogen Free Extract 21

327 Feed Fonnulation 21

328 Feed Production Procedure 21

329 Feeding Procedure 23

3210 Data Collection and FacilityMaintenance 24

3211 Data analysis 26

40 RESULTS 27

41 The Proximate Analyses of Samples 27

42 Growth Perfonnance ofTilapia 30

43 The Diet Fed FCR and FCE of Aquaculture Feed 32

44 The Mortality Rate (MR) and Survival Rate (SR) of Tilapia 36

50 DISCUSSION 37

60 CONCLUSION 42

70 REFERENCES 43

46

APPENDIXB

APPENDIX A

47

I

i

List of Abbreviations

SSW Soya Solid Waste

SSF Solid State Fennentation

MEA Malt Extract Agar

MSM Mineral Salt Media

CP Crude Protein

CF Crude Fiber

CL Crude Lipid

DM Dry Matter

MC Moisture Content

AM Ash Matter

NFE Nitrogen Free Extract

VM Vi tamins Mix

MM Minerals Mix

LBW Living Body Weight

l - BL Body Length

FCR Feed Conversion Ratio

FCE Feed Conversion Efficiency

ANOVA Analysis of Variance

V

List of Tables

Tables Description Page

Table 31 The formula of Mineral Salt Media (MSM) 11

Table 32 The concentration of spore suspension 14

Table 33 The ingredients ratio of four different types of aquaculture 21

feeds

Table 41 The nutritional contents of SSW 28

Table 42 The nutritional contents of aquaculture feeds 29

Table 43 Weekly mean ofLBW measurement (g) 30

Table 44 Weekly mean ofBL measurement (cm) 31

Table 45 Mean weekly consumption of feeds and the total diet fed (g) 33

Table 46 Results of total diet fed weight gain FCR and FCE 34

Table 47 Results ofMR and SR () 36

VI

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description

The BL measurement oftilapias(cm)

The LBW measurement of tilapias (g)

The nutritionals content of SSW

The nutritional contents of aquaculture feeds

Weekly mean ofLBW measurements (g)

Weekly mean ofBL measurements (cm)

Weekly mean of feeds consumption (g)

The diet fed and weight gain of tilapia (g)

The FCR of aquaculture feed

The FCE of aquaculture feed ()

The MR and SR of tilapias ()

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Agus Fery Budi Hartono bin Mamat Nurhan

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Soya solid waste is one of the by-products from the processed soybeans in food manufacturing industries The increase in production rate of freshwater aquaculture sector leads to high demand for aquafeed with complete diets However the cost for raw ingredients in commercially manufactured aquafeed especially fishmeal is expensive Therefore soya solid wastes were utilized as an alternative feed ingredient for source of plant proteins which can reduce the usage of fishmeal in aquafeed productions In this study different types of fungi such as Trichoderma reesei TrichodemlO harzianllm and Aspergillus niger were used to ferment the soya solid wastes by the process of solid state fermentation to improve its nutritional values Soya solid wastes treated with T reesei showed better improvement in the nutritional contents with increased in crude protein and crude lipid level for 575 and 13 respectively and decreased in crude fiber level for 036 compared to the other fungi Thus 10 20 30 and control feeds which contained 10 20 30 of soya solid waste treated with T reessei and absent of soya solid waste respectively were formulated and also tested on tilapias for two weeks The results showed that there were no significant differences (P gt 005) between the growth performances of the tilapias with all types of diet feeds This project was successful since the protein level of all diet feeds were maintained at 30 level and the growth performance of all the tilapias were almost the same

Keywords Soya solid waste solid state fermentation TrichodemlO reesei Trichoderma harzianllm Aspergillus niger

ABSTRAK

Soya hampas menlpakan salah satu produk yang dihasilkan daripada kacang soya yang telah diproses di industri pembuatan makanan Peningkatan dalam produk akuakllltur air tawar menjadi faktor pemintaan yang tinggi terhadap makanan ikan yang mempunyai diet yang lengkap Namun kos bahan mentah yang digunakan secara komersial dalam pembuatan makanan ikan tentam a sekali bahan mentah yang berasaskan isi ikan adalah sangat tinggi Oleh itu soya hampas digunakan sebagai bahan alternatif dalam pembuatan makanan ikan yang bersumberkan daripada protein tumbuhan bagi mengurangkan pengunaan is ikan dalam penghasilan makanan ikan Di dalam kajian ini beberapa jenis kulat yang berbeza telah digunakan iaitu T reesei T harzianum dan A niger bagi tujuan penapaian soya hampas dalam proses fermentasi keadaan pepejal untuk meningkatkan kadar nlltrisi Soya hampas yang ditapai dengan T reesei menunjukkan hasil kadar nutrisi dengan peningkatan dalam protein mentah dan lipid mentah masing-masing dengan nitai 575 dan 13 dan pen unman serat mentah sebanyak 036 berbanding dengan kulat yang lain Lantaran itu 10 20 30 dan makanan ikan kontrol yang mengandlmgi 10 20 30 soya hampas yang ditapai menggunakan T reesei dan tanpa penggunaan soya hampas masing-masing diformulasi dan diuji ke atas ikan tiapia selama dua minggu Hasi kajian menunjukkan bahawa tiada perbezaan yang ketara (Pgt 005) dalam pertumbuhan ikan dengan semuajenis diet makanan yang diuji ke alas ikan tilapia Secara amnya projek ini berjaya dengan hasil yang menunlIkkan kadar protein bagi diet pemakonan berjaya dikekalkan sebanyak 30 dan tumbesaran ke atas ikan lilapia adalah sama tanpa perbezaan ketara

Kllla kund Soya hampas fementasi keadaan pepejal Trichoderma reesei TrichodemlO harzianum

Asoeriillus niier

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10 INTRODUCTION

11 Introduction

Soybean is one of the most important legume plants which have a huge potential in

providing an inexpensive source of protein (Egberongbe et ai 2010) Soybean plants are

economically good in supplying nutrients needed for the individuals in developing country

Due to its quality soybean plants are widely cultured around the world to meet the demand

for the protein sources The soy products also extensively used in animal feed industry in

most of the countries (Foley et ai 2013)

There is a high reliance on complete balanced diet for aquafeed-based aquaculture

productions to sustain the development of the aquatic crops The increase in the cost of the

raw ingredients for aquafeed productions forced most of the farmer around the world to

find alternative strategies to maintain the aquafeeds supply to their crops Fishmeal is

I

among the most expensive ingredient in aqua feed but contributes the main protein source

in the diet Hence the farmer and feed producers tend to looks for alternatives sources of

feeds such as trash fish grain by-products and animal-by-products to be incorporated in

diet of the fish

The previous studies provide the knowledge that the soybeans contain high potential

nutritional content which allows it to be formulated and incorporated into the animal feeds

According to Liu (2000) the soybean products are usually converted to soybean meal

whole soybean flour and soy proteins concentrate and the waste products from the Ii

processed soybean can also be utilized to form aqua feeds

The application of biotechnology focusing on solid state fermentations by microbes

provides an alternatives way to treat the solid wastes from soybean The treated SSW can

be incorporated into aqua feeds as a plant protein sources thus reducing the usage of

2

fishmeal This research focused on improvement of the nutritional contents of SSW and

incorporation of the different levels of treated SSW to the aquafeeds for better growth

performance of the tilapias The objectives of this research are

1 To study the effects of different microbes on solid state fermentation of soya solid

wastes (SSW)

2 To improve the nutritional compositions of soya solid wastes by solid state

fermentation (SSF)

3 To reduce the usage of animal protein by utilizing plant protein as a protein source

in aquafeeds

12 Problem statement

This research was conducted to find an alternative source of protein for aquafeeds

production due to the expensive price of fish meal in the market Thus the soya solid

wastes produced by the food industries in Malaysia were utilized to increase its usability

for the aquaculture sectors However the nutritional contents of SSW were not clearly

stated and need some improvement based on the Crude Protein (CP) Crude Lipid (CL) and

Crude Fiber (CF) level Hence the SSW was treatep with different types of fungi by using

SSF process to enhance the nutritional contents The incorporations of SSW in aqua feed

also provide an alternative ways in supplying the source protein As a result the utilization

of fishmeal as a main protein source can be reduced and the cost for aquafeed productions

can also be decrease

20 LITERATURE REVIEW

21 Soybean

Soybean (Glycine max) is a legume plants that belongs to family Legllminosae (Hossain et

al 2009) Soybeans were believed to originate from China and were being brought first to

United States from China in 1800s In the year 1929 soybeans were brought to United

States for research which results the increase in soybean production (Cromwell 2012)

According to the statistics from USDA in 2015 world soybean production reached

128026 Million Metric Tons from year 2014 to 2015 and United States was the number

one world soybean producer with the annual production estimated at 37712 Million Metric

Tons

Soybean was called the Protein hope of future due to its high protein content which

about 42 - 45 It is one of the most important oil seed crop around the world because

its also contain about 18 - 20 edible oil and 42-46 carbohydrates (Hossain et ai

2009) According to Cromwell in 2012 amino acid profile in soybean also balanced as it

rich in isoleucine valine tryptophan threonine and lysine Besides that soybean plants

have the ability to fix atmospheric nitrogen symbiotically thus contributing approximately

80 - 90 of nitrogen demands (Hossain et ai 2009)

Soybean can be processed to produce soybean meal soy protein concentrate and soy

protein isolates which have been studied intensively due to its ability to supply high

nutrient values The high nutritive values of soybean make them acceptable substitutes for

meat for the vegetarians Lastly the soy products and the waste produced by processed

soybean had also been studied and utilized extensively in the animal feed industry for their

protein source

4

Pusat Khidmat MakJumat Akadem ~ Nrv i1 ~middotAtAYSA SARA

22 Soya Solid Waste (SSW)

Soya solid waste (SSW) is a waste generated from the processed soybean in food

manufacturing industries SSW was normally utilized as an alternative animal feeds as it

still contains some of the protein contents The high fiber content in SSW limits the usage

in non-ruminant feed as the non-ruminant animals such as fish have lack the ability to

digest fiber

The nutritional value of soya solid waste can be improved by fermentation techniques as

shown by the previous studies (Foley et ai 20l3) Soya solid waste shows the great

potential as a substrate for SSF in order to improve the digestibility of the fiber (Yang et ai

2012) SSF of soya solid waste with different fungi promotes the production of enzyme

which results the decreased in crude fiber This shows the potential of SSF and fungi in

improving the digestibility of soya solid waste as non-ruminant feed

23 Red Tilapia (Oreochromis sp)

Oreochromis sp is a tropical species which lives in shallow water Tilapia prefers the

tempemtures that range from 31degC to 36 degC and they are omnivorous fish which feeds on

phytoplankton aquatic plants small vertebrate benthic fauna and bacterial films (F AO

2014)

According to Popma and Masser in 1999 the red tilapia was more popular compared to the

other tilapia species due to appearance that similar to marine red snapper which makes the

market value higher Red tilapias were originally genetic mutants that produced by cross

between mutant reddish-orange female Mozambique tilapia and normal male Nile tilapia

This genetic mutant was called Taiwanese Red Tilapia Tilapia can grow quickly and adapt

5

to the surrounding environment which makes this species a good model organism for this

project Due to their ability to adapt to different habitats tilapias have been widely

introduced into many areas purposely and also accidentally

Furthennore tilapias are easily spawned tolerate poor water quality and grow rapidly in

wann water The characteristics of tilapias along with low input costs attract more fish

fanners to culture them widely in tropical and subtropical countries

24 Tilapia Aquaculture in Malaysia

Tilapias have high protein contents large size and undergo rapid growth which makes

them the major focus in aquaculture industries There are several species of titapia which

are cultured commercially but the Nile tilapia is the most cultured species in worldwide

The production of tilapia in Malaysia reached 52 000 tones in years 2013 The prices

ranged between USD 200 - 375 and USD 470-625 per kg for live tilapia in retail sectors

and supennarket respectively (FAO Globefish 2014) Malaysia is also one of the top

suppliers for live Tilapia and even the fish fillet to the other countries The firm flesh and

mild flavor makes the market for tilapias to expand rapidly in the US (Popma and Masser

1999) Thus thC1 production of tilapias will contribute to the economic growth in

aquaCUlture sectors in Malaysia

6

25 Trichoderma reesei

During the Second World War T reesei was discovered and isolated from Solomon Island

(Peterson and Nevalainen 2012) The cellulolytic ability of T reesei was also discovered

during the Second World War by the deterioration of cotton fabric of the US Army

T ree ei is a mesophilic filamentous fungus which well known for its role in producing the

enzyme cellulase The degradative action of T reesei makes it important in providing

alternative fuel sources as it able to produce enzymes that have potential in hydrolyzing

cellusose-rich biomass to glucose which later on will be converted to ethanol by

fermentation process (Bernhard et ai 2011) The secretions of wide range of hydrolytic

enzymes by T reesei increase their usability in food paper and animal industries

(Kreuszewska et ai 2000)

Previous study also conducted by Lio and Wang in (2012) for the effects of T reesei in

solid state fermentation of soybean Co-culturing of T reesei and Aspergillus oryzae

showed an increase in cellulase and xylanase activity which results in improvement of

protein content and decrease in fiber content of the soybean by-products The findings

suggest the functions of T reesei in feed quality improvement Based on this knowledge

T reesei was used to study its effect in solid state fermentation of SSW

26 Trichoderma harzianum

T harzianl4m is an asexual fungal species from family Moniiaceae It is one of a

saprophytic fungus which occurs in soil especially in the rhizosphere and on an organic

material like decaying wood (European Commission Health amp Consumers Directorateshy

General 2008)

T harzianum have the biological control ability in supporting the growth of plant in the

sustainable soil fertility and also controlling the soil-borne disease (Olabiy et ai 2013) It

is also widely used in agriculture horticulture nursery and protected crops T harzianum

produces a lot of metabolites and one of them have the ability to suppress the pathogen that

will cause harm for the crops or seeds Thus this fungus was beneficial to control the

growth of oil seeds such as soybean and the production of secondary metabolites were

expected to improve the nutritional content of ssw

27 Aspergillus niger

Aspergillus is a genus of mold which reproduces asexually The asexual spores of its

structures are essential taxonomic character which makes them among the successful

groups of mold in natural ecosystems (Bennett 20 I 0) Aspergillus sp also has the abilities

to produce extracellular enzymes organic acids and secondary metabolites

According to Machida and Gomi (2010) A niger has been reported to produce functional

foods such as koji and miso This study will also evaluate the effect of A niger in

improving the nutrient contents and enhancing the digestibility of crude protein in the fish

feed

28 Solid State Fermentation (SSF)

Digestibility of the soybean products can be improved through the fermentation process

which increases the solubility of the soybean proteins (Foley et ai 2013) Fermentation

also results the degradation of macromolecules which leads to the increase of the amount

water soluble components

Solid state fermentation is the process of fermentation which involves grows of

microorganisms on a solid material in the absence of free water (Bhargav et aI 2008) The

reason why the SSF is suitable process for the growth of fungi is mainly because of its low

moisture content which permits the penetration of fungi mycelium through the solid

substrates (Lio and Wang 2012) Fungi are well adapted to SSF due to the ability of their

hyphae can grow on particle surfaces to penetrate into the inter-particles space which result

in colonization of the fungi at the solid substrate According to Chancharoonponga et al

(2012) the fungal mycelium able to penetrates into the solid substrates as four layer

mycelium of penetration Areal hyphae is the first layer aerobic wet hyphae is second

followed by anaerobic wet hyphae and penetrative hyphae Certain enzymes and

metabolites are able to be produced by the microorganisms due to the low humidity in a

SSF

SSF have several advantages over submerged fermentation (SmF) SSF produce high yield

of products compared to SmF and downstream processing are much simpler Aeration

system in SSF also simple thus reduces the energy requirements to run this process In

SSF the growth performances of microbes were more efficient as the SSF mimic the

natural environments for the microbes Hence SSF process was used in this project to

improve the nutritional contents of SSW

9

30 MATERIALS AND METHODS

31 Materials


SSW was collected from 7th Mile Taufu Mill Kuching on 24 October 2014

312 Trichoderma reesei Trichoderma harzianum and Aspergillus niger

T resssei T harzianum and A niger were prepared and used separately to enhance the

nutritional composition of the SSW during the SSF process The stock cultures were

obtained from Molecular Genetic Lab and Microbiology Lab and were sub-cultured in

Animal Biotechnology Lab

313 Malt Extract Agar (MEA)

The MEA was prepared prior to SSF to act as a growth medium for culturing T reesei T

harzianum and A niger

10

314 Mineral Salt Media (MSM)

MSM was prepared by addition of magnesium sulfate (MgS047H20) monopotassium

phosphate (KH2P04) calcium chloride (CaCb) ferum (II) sulfate (FeS04) ammonium

sulfate ((N~)2S04) and sucrose This formula was used based on the previous research by

Omemu et al (2005) The MSM was autoc1aved before used The amount of MSM is

depends on the amount of the samples used in SSF process

Table 31 The formula of Mineral Salt Media (MSM)

Chemicals I Concentration ()

Magnesium sulfate 01

Monopotassium phosphate -

01

Calcium chloride

Ferum (II) sulfate I

I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer

The hemacytometer with the aid of light microscope equipped with screen monitor was

used to measure the concentration of the spore suspension

316 Model Organism (Tilapia)

The Tilapia was selected as the model organism in this research due to the relatively low

price in the market and observable size change during the growth Tilapias were purchased

7that Mile Kuching with their average size and lengths are 260 g and 430 cm

respectively Fish breeding tanks in the Animal Biotech Laboratory are well equipped with

pumps aeration device filter system and water recirculating system for the breeding

purposes of the Tilapias

317 Kjeldabl Apparatus

Kjedahl apparatus were used for the proximate analysis of crude lipid It consists of

digestion block (Gerhardt Gennany) distillation machine (Gerhardt Vapodest 20

Germany) and titration unit for digestion distillation and titration of samples respectively

318 Soxblet Extractor

The Soxhlet extractor was prepared for the proximate analysis of crude lipid content in the

samples The Soxhlet apparatus consist of cellulose thimble heater boiling flask Liebig

condenser and extraction chamber

12

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32 Methodology

321 Sampling

The SSW were obtained from t h Mile Taufu Min Kuching during weekend SSW was

collected after the soybeans had been processed and were placed inside the plastic bags to

be transported to Animal Biotechnology Laboratory UNIMAS The samples were stored

at -20degC to prevent the degradation of samples and for future use

322 Drying and Grinding

Drying process was achieved by exposing the samples under the sunlight and placing some

of the samples inside an oven at 80degC - 90degC Drying is important to remove the moisture

presence in the samples and to make it easier for further storage The dried samples can be

used to detennine the Dry Matter (OM) andMoisture Content (MC) in the sample analysis

323 Spore Culturing

The microbes were cultured on Malt Extract Agar (MEA) plates The plates were

incubated at room temperature for about 7 days until the sporulation has been completed

The stock cultures of the fungi were placed inside refrigerator at -20degC to prevent further

growth Different fungi had been cultured to make comparisons of their ability in

enhancing the nutritional contents of SSW in SSF

13

324 Preparation of Spore Suspension Culture

Spore suspension was done after 7 days of incubating the subculture of the fungi Spore

suspension was prepared as fresh as possible to provide optimum conditions for the fungi

Adequate amount of I Tween 20 solution was poured on the surface of the culture until it

covered all surface of the agar plates Agar plates were shakes gently to ensure the

suspension of the spores with the Tween 20 solution After the spores of the fungi and

Tween 20 solution had been mixed up they were transferred separately into 50 ml Falcon

tube by using pipette

The spores were counted by usmg hemacytometer with an aid of light mIcroscope

equipped with screen monitor to ease the counting process Quantity of spores were

counted and recorded to obtain the concentration of spore suspension in spores per

milliliter (sporesml) by using fonnula shown below

Table 32 The concentration of spore suspension

Aspergillus niger Trichoderma harzianum Trichoderma reesei

508 x 106 195 x 106 309 x 106

Concentration (sporesml) =Mean002 x 1000

14

32S Solid State Fermentation (SSF)

SSF was carried out after the ground SSW had been autoclaved SSW was used as a

substrate whiles the A niger T harzianum and T reesei were used separately as

microorganism for the SSF inside the plastic container 100 g of autoclaved SSW were

weighed and filled into the three flasks The flasks also filled with 20 rnl of inoculum 20

ml of MSM and 30 ml of sterile distilled water each to achieve 70 moisture content The

flasks were then plugged with cotton wools and covered by aluminum foil The

temperature inside the fermentation room was set at room temperature (2SoC - 27degC) for

the incubation purposes

After 7 days of incubation the harvesting of substrates was done by transferring SSW

along with the microbes to the 1000 ml Erlenmeyer flask The flasks also were added with

about 1000 rnl (ratio of I 1 0) distilled water each and rotated at 120 rpm for I hour in room

temperature The mixtures inside the flasks were then filtered by using Muslin clothes to

separates the fungi with the SSW The filtrates (SSW) were dried in an oven soon after the

filtration has been done Lastly the dried SSW was ground for further use such as content

analysis and feed fonnulations

326 Chemical Analyses of Samples

Samples that includes the untreated SSW treated SSW 10 feed 20 feed 30 feed and

control feed were analyzed in this research based on AOAC methods and proximate

analysis by F AO with some modifications in order to identify the nutritional contents

Crude Protein (CP) Crude Lipid (CL) Crude Fiber (CF) Moisture Content (MC) Dry

Matter (DM) Ash Matter (AM) and Nitrogen Free Extract (NFE) were the parameters that

had been analyzed

1~

Pusat Khidmat MakJumat Aklldt UlTVEpsm MALAYSIA SARAn


Agus Fery Budi Hartono bin Mamat Nurhan (34730)

A Thesis Submitted in Partial Fulfillment of the Requirement of the Degree of Bachelor of

Science with Honours (Resource Biotechnology)

Supervisor Dr Lee Kui Soon

Resource Biotechnology

Department of Molecular Biology

r

Faculty of Resource Science ~nd Technology

Universiti Malaysia Sarawak

2015

l

ACKNOWLEDGEMENT





















J I

DECLARATION






15 b 20 i5 I


(34730)

II


TABLE OF CONTENTS

Acknowledgement I

Declaration II



List of Tables VI

List of Figures VII

Abstract

10 INTRODUCTION 2

11 Introduction 2



21 Soybean 4









31 Materials 10









32 Methodology 13

111

321 Sampling 13











Moisture Content







40 RESULTS 27





50 DISCUSSION 37

60 CONCLUSION 42

70 REFERENCES 43

46

APPENDIXB

APPENDIX A

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CP Crude Protein

CF Crude Fiber

CL Crude Lipid

DM Dry Matter

MC Moisture Content

AM Ash Matter


VM Vi tamins Mix

MM Minerals Mix


l - BL Body Length




V

List of Tables





feeds








VI

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Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












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ABSTRACT



ABSTRAK



Asoeriillus niier

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10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















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------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

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ACKNOWLEDGEMENT





















J I

DECLARATION






15 b 20 i5 I


(34730)

II


TABLE OF CONTENTS

Acknowledgement I

Declaration II



List of Tables VI

List of Figures VII

Abstract

10 INTRODUCTION 2

11 Introduction 2



21 Soybean 4









31 Materials 10









32 Methodology 13

111

321 Sampling 13











Moisture Content







40 RESULTS 27





50 DISCUSSION 37

60 CONCLUSION 42

70 REFERENCES 43

46

APPENDIXB

APPENDIX A

47

I

i






CP Crude Protein

CF Crude Fiber

CL Crude Lipid

DM Dry Matter

MC Moisture Content

AM Ash Matter


VM Vi tamins Mix

MM Minerals Mix


l - BL Body Length




V

List of Tables





feeds








VI

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












Page

24

25

28 I

29 I

I 30

31

33

34

35

35

36

I

I

I ~

I

~

VII l

4





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

DECLARATION






15 b 20 i5 I


(34730)

II


TABLE OF CONTENTS

Acknowledgement I

Declaration II



List of Tables VI

List of Figures VII

Abstract

10 INTRODUCTION 2

11 Introduction 2



21 Soybean 4









31 Materials 10









32 Methodology 13

111

321 Sampling 13











Moisture Content







40 RESULTS 27





50 DISCUSSION 37

60 CONCLUSION 42

70 REFERENCES 43

46

APPENDIXB

APPENDIX A

47

I

i






CP Crude Protein

CF Crude Fiber

CL Crude Lipid

DM Dry Matter

MC Moisture Content

AM Ash Matter


VM Vi tamins Mix

MM Minerals Mix


l - BL Body Length




V

List of Tables





feeds








VI

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












Page

24

25

28 I

29 I

I 30

31

33

34

35

35

36

I

I

I ~

I

~

VII l

4





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~


TABLE OF CONTENTS

Acknowledgement I

Declaration II



List of Tables VI

List of Figures VII

Abstract

10 INTRODUCTION 2

11 Introduction 2



21 Soybean 4









31 Materials 10









32 Methodology 13

111

321 Sampling 13











Moisture Content







40 RESULTS 27





50 DISCUSSION 37

60 CONCLUSION 42

70 REFERENCES 43

46

APPENDIXB

APPENDIX A

47

I

i






CP Crude Protein

CF Crude Fiber

CL Crude Lipid

DM Dry Matter

MC Moisture Content

AM Ash Matter


VM Vi tamins Mix

MM Minerals Mix


l - BL Body Length




V

List of Tables





feeds








VI

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












Page

24

25

28 I

29 I

I 30

31

33

34

35

35

36

I

I

I ~

I

~

VII l

4





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

321 Sampling 13











Moisture Content







40 RESULTS 27





50 DISCUSSION 37

60 CONCLUSION 42

70 REFERENCES 43

46

APPENDIXB

APPENDIX A

47

I

i






CP Crude Protein

CF Crude Fiber

CL Crude Lipid

DM Dry Matter

MC Moisture Content

AM Ash Matter


VM Vi tamins Mix

MM Minerals Mix


l - BL Body Length




V

List of Tables





feeds








VI

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












Page

24

25

28 I

29 I

I 30

31

33

34

35

35

36

I

I

I ~

I

~

VII l

4





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

i






CP Crude Protein

CF Crude Fiber

CL Crude Lipid

DM Dry Matter

MC Moisture Content

AM Ash Matter


VM Vi tamins Mix

MM Minerals Mix


l - BL Body Length




V

List of Tables





feeds








VI

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












Page

24

25

28 I

29 I

I 30

31

33

34

35

35

36

I

I

I ~

I

~

VII l

4





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

List of Tables





feeds








VI

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












Page

24

25

28 I

29 I

I 30

31

33

34

35

35

36

I

I

I ~

I

~

VII l

4





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

I

I

Figures

Figure 31

Figure 32

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

List of Figures

Description












Page

24

25

28 I

29 I

I 30

31

33

34

35

35

36

I

I

I ~

I

~

VII l

4





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~





ABSTRACT



ABSTRAK



Asoeriillus niier

1 i

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

-I

10 INTRODUCTION

11 Introduction











I




diet of the fish









2





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~





wastes (SSW)


fermentation (SSF)


in aquafeeds













21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~


21 Soybean








Tons













protein source

4







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~







digest fiber











2014)






5


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~


















6






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~






















General 2008)
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~
















feed

















SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~












SSF







9


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~


31 Materials











10











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~











01

Calcium chloride


I

01

01

Ammonium sulfate 01

Sucrose 10

11

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

315 Hemacytometer


















12

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~

------------------------------------------------------------------------------

32 Methodology

321 Sampling










323 Spore Culturing






13















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~















508 x 106 195 x 106 309 x 106


14























had been analyzed

1~























had been analyzed

1~

formulation of aquafeed from soya solid waste

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