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STATIC CULTURE OF Spirulina platensis IN MODIFIED SAGO
EFFLUENT MEDIUM
NURUL HANA MINARTI BINTI OMAR
This project is submitted in partial fulfillment of
the requirement for the Degree of Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology
UNIVERSITY MALAYSIA SARAWAK
2010
DECLARATION
I hereby declare that no portion of the work referred in this project has been
submitted in support of an application for another degree qualification of this or any
other university or institution of higher learning.
___________________________________
(Nurul Hana Minarti Binti Omar)
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
University Malaysia Sarawak
i
ACKNOWLEDGEMENT
Above all, I would like to express my greatest sincere gratitude and
appreciation to my supervisor, Professor Dr. Kopli bin Bujang for his dedicated
supervision, comments, advice and patience throughout this project. My special
thanks is dedicated to Assoc. Prof. Dr. Cirilo Nalsco Hipolito as my examiner and
giving valuable guidelines for writing this report.
Besides that, I would like to express my appreciation to postgraduate students
of the Biochemistry laboratory, Faculty of Resource Science and Technology, Miss
Merlina Manggi, Miss Rubena Kamal and Mr Ugam Janggu.
Last but not least, I would also like to thank my family for their endless
corroboration and giving me the greatest support while the project is on-going and
not forgetting also my friends and course mates.
ii
ABSTRACT
Effluent from Sago Mill is a polluting wastewater from the starch industries
in Sarawak. The pollution occurs due to the large quantity produced daily rather the
chemical components of the effluent. It is reported that for one kilogram (dry weight)
of starch produced, 20 L of wastewater is generated in the process. This wastewater
needs to be treated before to be discharged into nearby rivers. Therefore, in this
study we proposed to use this wastewater as a media for the cultivation of the alga
Spirulina platensis with the aim to make more attractive its production from an
economical point of view by decreasing the production cost. Spirulina platensis has
commercial importance due to overall nutritional qualities, especially high protein
(70% dry basis) and vitamin contents, particularly B12. To use the sago effluent as
the media for the growing of Spirulina, it needs to be modified. To produce modified
sago effluent (MSE), the sago effluent was pre-treated aerobically by the addition of
the commercial microbial consortium Bakwira MP300 with the aim to degrade the
remained organic materials. Upon cleaned-up the MSE was amended with sodium
bicarbonate (NaHCO3) at different concentrations (0, 4 and 8 g/L). MSE without
amendment of NaHCO3, was used as negative control. The Zarrouks medium being
the best medium for the growth of Spirulina as reported in the literature was used as
positive control to compare the effectiveness of the MSE as a culture medium.
Spirulina platensis was cultivated using MSE amended with and without NaHCO3.
The cultures were analyzed for pH, protein (Bradford method), reducing sugar (DNS
method), starch (Iodine method), total suspended solids (TSS) (APHA method), cell
biomass by dry cell weight (DCW), and OD. The cultures were carried out during 20
iii
days and samples were withdrawn every two days for analysis. The experiment trial
was done by duplicate. The highest pH 10.52 observed was in the MSE amended
with 8 g/L NaHCO3 in the first trial and 9.34 for the second. The highest DCW
obtained after 20 days of cultivation observed was the Zarrouk’s medium with 1095
mg/L and in MSE with 0g/L NaHCO3 with 1445 mg/L in the first and second trial of
experiments respectively. Study on effect of NaHCO3 on the glucose content of
Spirulina, MSE without addition of NaHCO3 (0 g/L NaHCO3) achieved the highest
glucose content with 0.35 g/L in first trial of experiment after 20th
day of cultivation.
It was increased by 133.33% from day 0th
of cultivation with glucose content of 0.15
g/L. While, in second trial the MSE containing 4 g/L NaHCO3 was achieved the
highest glucose content on the 20th
day of cultivation with 0.13 g/L. An increased
was shown by 44.44% from the day 0th
of cultivation with glucose content of 0.09
g/L. Study on the effect of starch shows that the highest starch content for the first
trial of experiment on the 20th
day observed was the MSE containing 8 g/L of
sodium bicarbonate, NaHCO3 with 0.21 g/L of starch content. An increased was
shown by 2000% compare to day 0th
of cultivation with starch content of 0.01 g/L.
While, for the second trial of experiment the highest starch content was achieved in
MSE without the sodium bicarbonate amendment added, 0 g/L NaHCO3 with starch
content of 0.16 g/L on day 20th
of cultivation. An increased was shown by 128.57%
compare to day 0 of cultivation with starch content of 0.07 g/L. Study on effect of
NaHCO3 of Spirulina in Zarrouk’s medium was achieved the highest protein content
at 3.06% and 3.85% in first and second trials of experiments respectively. As
compared to medium of MSE with different concentration of NaHCO3 amendment,
the highest protein content observed was in MSE added with 8 g/L NaHCO3 with
iv
2.31% of protein content in the first trial of experiment, while for the second trial the
highest protein content was achieved by MSE without addition of NaHCO3 (0 g/L
NaHCO3) with 2.92% of protein content.
Key words: Spirulina platensis, modified sago effluent, Zarrouk’s medium, static
culture, single cell protein, dry cell weight, protein content.
v
ABSTRAK
Efluen dari Sagu Mill adalah pencemaran dari sisa-sisa industri kanji di
Sarawak. Pencemaran terjadi disebabkan oleh jumlah besar yang dihasilkan setiap
hari tetapi bukan komponen kimia dari sisa. Hal ini dilaporkan bahawa untuk satu
kilogram (berat kering) daripada tepung yang dihasilkan, 20 L air sisa yang
dihasilkan dalam proses. Air sisa ini perlu dirawat sebelum menjadi dibuang ke
sungai terdekat. Oleh kerana itu, dalam kajian ini menawarkan untuk menggunakan
air sisa ini sebagai bagi penanaman alga Spirulina platensis dengan tujuan untuk
membuat media yang lebih sesuai produksinya dari sudut pandang ekonomi dengan
cara menurunkan kos pengeluaran. Spirulina platensis mempunyai kepentingan
komersial kerana keseluruhan kandungan gizi yang tinggi, terutama protein tinggi
(isi 70% berat kering asa) dan vitamin, khususnya B12. Untuk menggunakan sisa
sagu sebagai media untuk pertumbuhan Spirulina, sisa sagu perlu diubah. Untuk
menghasilkan sagu efluen yang diubah (MSE), sagu efluen dirawat secara pra-
aerobik dengan penambahan konsortium mikroba komersial MP300 Bakwira
dengan tujuan untuk mendegradasi bahan organik tetap. Setelah dibersihkan, MSE
ditambah dengan natrium bikarbonat (NaHCO3) pada kepekatan yang berbeza (0, 4
dan 8 g / L). MSE tanpa perubahan NaHCO3, digunakan sebagai kawalan
negatif. Media Zarrouks menjadi media terbaik bagi pertumbuhan Spirulina yang
dilaporkan dalam laporan sebelum ini digunakan sebagai kawalan positif untuk
membandingkan keberkesanan MSE sebagai kultur media. Spirulina platensis yang
ditanamkan dengan menggunakan MSE yang ditambah dengan dan tanpa NaHCO3.
Kultur dianalisis pH, protein (Kaedah Bradford), kandungan gula (kaedah DNS),
vi
kanji (kaedah Iodine), jumlah pepejal termendap (TSS) (kaedah Apha), sel biojisim
dengan berat sel kering (DCW), dan OD. Pengkulturan yang dilakukan selama 20
hari dan sampel dianalisis setiap dua hari. Sidang percubaan dilakukan dengan 2
kali percubaan. pH tertinggi 10.52 didapati pada MSE yanag ditambah dengan 8
g/L NaHCO3 pada eksperimen percubaan pertama dan 9.34 untuk yang kedua.
Berat kering (DCW) tertinggi yang diperolehi selepas 20 hari pengkulturan adalah
didapati dalam medium Zarrouk dengan 1095 mg/L dan dalam MSE dengan 0 g/L
NaHCO3 dengan 1445 mg/L masing-masing pada eksperimen percubaan pertama
dan kedua. Kajian terhadap kesan NaHCO3 pada kandungan glukosa Spirulina,
MSE tanpa NaHCO3 (0 g/L NaHCO3) mencapai kadar glukosa tertinggi dengan
0.34 g/L dalam eksprimen percubaan pertama selepas 20 hari pengkulturan.
Peningkatan menunjuk 133.33% dari hari ke-0 pengkulturan dengan kandungan
glukosa 0.15 g/L sehingga ke hari 20 pengkulturan. Manakala, dalam eksperiman
percubaan kedua MSE mengandungi 4 g/L NaHCO3 mencatat kandungan glukosa
yang tertinggi pada hari ke-20 pengkulturan dengan 0.13 g/L. Peningkatan tersebut
meunjukkan 44.44% dari hari ke-0 pengkulturan dengan kandungan glukosa 0.09
g/L. Kajian terhadap kandungan kanji dalam Spirulina menunjukkan kadar kanji
yang tertinggi bagi eksperimen percubaan pertama pada hari ke-20 adalah pada
MSE yang mengandungi 8 g/L natrium bikarbonat, NaHCO3 dengan 0.21 g/L kadar
kanji. Ini menunjukkan peningkatan sebanyak 2000% dari hari ke-0 pengkulturan
dengan kadar kanji 0.01 g/L. Manakala, untuk eksperiman percubaan kedua, kadar
kanji yang tertinggi yang didapati pada hari ke-20 adalah pada MSE tanpa
penambahan natrium bikarbonat iaitu 0 g/L NaHCO3 dengan kadar kanji dari 0.16
g/L. Peningkatan ditunjukkan oleh 128.57% pada hari ke-0 dari pengkulturan
vii
dengan kadar kanji dari 0.07 g/L. Kajian terhadap kesan NaHCO3 pada Spirulina
didapati kandungan protein yang tertinggi dicatat oleh Zarrouk medium dengan
3.06% dan 3.85% dalam eksperimen percubaan pertama dan kedua masing-masing.
Perbandingan pada medium MSE yang ditambah dengan kepekatan NaHCO3,
kandungan protein yang tertinggi didapati dalam MSE yang ditambah dengan 8 g/L
NaHCO3 dengan peratus kandungan proteinnya adalah 2.31% dalam eksperimen
percubaan pertama, manakala, dalam eksperimen percubaan kedua pula kandungan
protein yang tertinggi dicatat oleh MSE tanpa penambahan NaHCO3 (0 g/L
NaHCO3) dengan peratus kandungan protein 2.92%.
Kata kunci: Spirulina platensis, sagu efluen yang diubah, media Zarrouk, kultur
statik, protein sel tunggal, berat sel kering, kandungan protein
viii
TABLE OF CONTENTS
TITLE PAGE
ACKNOWLEDGEMENT i
ABSTRACT ii
TABLE OF CONTENTS viii
LIST OF ABBREVIATIONS xi
LIST OF FIGURES xiv
LIST OF TABLES xvi
CHAPTER 1: INTRODUCTION
1.1 Generel Overview 1
1.2 Objectives
3
CHAPTER 2: LITERATURE REVIEW
2.1 Sago 4
2.1.1 Sago Starch 4
2.1.2 Sago Effluent 4
2.2 Spirulina 6
2.2.1 Morphology and Characteristic 6
2.2.2 Nutritional value of Spirulina 8
2.2.2.1 Protein 8
2.2.2.2 Carbohydrate 8
2.2.2.3 Lipids 9
2.2.2.4 Vitamins 10
2.2.3 Application of Spirulina 10
2.2.3.1 Health Benefit Effects 10
2.2.3.1.1 Effects against Hyperlipidemia 10
2.2.3.1.2 Effects on Intestinal Flora 11
2.2.3.1.3 Effects against Vitamin A Deficiency 11
2.2.3.1.4 Potent Cancer-Fighting Phytonutrients 12
2.2.3.1.5 Boosting Immune System Function 12
2.2.3.2 Other Applications of Spirulina 14
2.2.3.2.1 Application of Spirulina in Biofixation
of Carbon Dioxide (CO2).
14
2.2.3.2.2 Application of Spirulina to Improve
Accumulation of Copper, Mercury and
Lead from Wastewater.
15
2.2.3.2.3 Application of Spirulina in Dairy
Industries
16
2.2.3.2.4 The use of Spirulina as Colouring
Compounds
16
2.2.4 Business Plan for Spirulina 17
2.3 Nutrient and Growth Factors of Spirulina platensis 18
2.3.1 Carbon Source 18
2.3.2 Nitrogen Source 19
2.3.3 Chemical Composition of Spirulina 19
2.4 Cultivation of Spirulina 21
ix
2.4.1 pH of Medium 21
2.4.2 Effect of Sodium Bicarbonate (NaHCO3) 22
2.4.3 Influenced of Temperature 23
2.4.4 Effect of Light 23
2.4.5 Mixing rate of culture 24
2.5 Production of Spirulina
24
CHAPTER 3 MATERIALS AND METHODS
3.1 Materials 27
3.1.1 Microorganisms 27
3.1.1.1 Spirulina 27
3.1.1.2 Bakwira MP300 27
3.1.2 Inoculum 28
3.1.3 Modified Sago Effluent Medium 28
3.1.4 Zarrouk’s Medium 29
3.1.5 Concentration of Sodium Bicarbonate (NaCHO3) 31
3.1.6 Static Culture 31
3.1.7 Light Source 32
3.2 METHODS 33
3.2.1 Physical Characteristics 33
3.2.1.1 Total Suspended Solid (TSS) 33
3.2.1.2 Dry Cell Weight (between of biomass) 34
3.2.1.3 pH 35
3.2.2 Chemical Characteristics 35
3.2.2.1 Reducing sugar analysis-DNS Test 35
3.2.2.2 Starch content-Iodine test 36
3.2.2.3 Protein Content-Bradford method 36
3.2.3 Analytical procedure 37
3.2.3.1 Determination of the growth rate of the algae 37
3.2.4 Preparation of Sago Effluent as Growth Medium 38
3.2.4.1 Filtered Sago Effluent 38
3.2.4.2 Treatment of Modified Sago Effluent 39
3.2.5 Cultivation of Spirulina in Modified Sago Effluent 40
3.2.6 List of Experiment 41
3.2.7 Sampling
41
CHAPTER 4 RESULTS
4.1 Characteristics of Sago Effluent 42
4.2 Optical Density of Spirulina platensis 44
4.3 Effects on pH of Spirulina platensis cultured in MSE amended with
different concentration of sodium bicarbonate (NaHCO3) and in
Zarrouk’s medium.
45
4.4 Effects on growth (DCW) of Spirulina platensis in MSE amended
with different concentration of sodium bicarbonate (NaHCO3) and
in Zarrouk’s medium.
48
4.4.1 Comparison on colour of Spirulina cultures on day 0th
of
cultivation until day 20th
of cultivation in first and second
trials of experiments.
53
x
4.5 Glucose content of Spirulina platensis in MSE with different
concentration of Sodium Bicarbonate, NaHCO3.
55
4.6 Starch content of Spirulina platensis in Zarrouk’s medium and in
MSE with different concentration of sodium bicarbonate, NaHCO3.
57
4.7 Comparison of protein content of Spirulina platensis in different
medium.
60
CHAPTER 5 DISCUSSIONS 64
CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS 79
REFERENCES 81
APPENDICES A 91
APPENDICES B 93
APPENDICES C 97
xi
LIST OF ABBREVIATION
BOD Biological Oxygen Demand
BSA Bovine Serum Albumin
C Carbon
CaCl2.2H2O Calcium chloride-di-water
CuSO4.5H2O Copper sulphate-penta-water
COD Chemical Oxygen Demand
Co(NO3)2.6H2O Cobalt nitrate-hexa-water
DCW Dry cell weight
DOS Department Of Statistics
E.coli Escherichia coli
Fe-EDTA Iron-Ethylene Diaminetetraacetic Acid
FeSO4.7H2O Iron sulphate-hepta-water
g Gram
kg Kilogram
GLA γ-linolenic acid
g/L Gram per liter
H3BO3 Boric acid
HCl Hydrochloric acid
HDL High Density Lipoprotein
JCM Japanese Collection of Microorganism
K2HPO4 Di-potassium hydrogen orthophosphate
anhydrous
K2SO4 Potassium sulphate
xii
L Liter
LDL Low Density Lipoprotein
MSE Modified sago effluent
Mg/L Milligram per liter
MgSO4.7H2O Magnesium sulphate-hepta-water
mL Milliliter
mm Millimeter
nm nanometer
MnCl2.4H2O Manganese chloride-tetra-water
N Nitrogen
NaCl Sodium chloride
Na2EDTA.2H2O Sodium Ethylene Diaminetetraacetic Acid-di-
water
NaHCO3 Sodium hydrogen carbonate
NaMoO4.2H2O Sodium molybdate dihydrate
NaNO3 Sodium nitrate
NaOH Sodium hydroxide
OD Optical density
PO4 Phosphate
ppb Part per billion
ppm Part per million
R2 Correlation coefficient
Rpm Revolution per min
tons Tonnes
xiii
Wt. Weight
ZnSO4.7H2O Zinc sulphate-hepta-water
β Beta
˚C Degree Celsius
µ Micron
µm Micro meter
γ gamma
xiv
LIST OF FIGURES
TITTLE PAGE
Figure 2.1 Structural analysis of Spirulina platensis under Olympus light
microscope at 20X.
7
Figure 2.2 Structural analysis of Spirulina platensis under Olympus light
microscope at 40X.
7
Figure 2.3 Scanning electron micrograph of Spirulina platensis.
7
Figure 2.4 Scanning electron micrograph of Nonaxenic of Spirulina platensis
7
Figure 2.5 Spirulina tablets (left), Spirulina powders (right).
25
Figure 3.1 Inoculum of Spirulina platensis in 500ml Erlenmeyer flask using
Zarrouk’s medium after 14 days.
28
Figure 3.2 Cultures of Spirulina platensis in different concentration of sodium
bicarbonate.
32
Figure 3.3 Filtrate of dried sample on Whatman membrane filter paper after
dried at 60oC for 24 hours.
34
Figure 3.4 Measured pH of the MSE.
35
Figure 3.5 Biochrom UV spectrophotometer Libra S12.
38
Figure 3.6 Filtration of the Sago Effluent.
39
Figure 3.7 Aeration of the treated MSE for 3 days.
40
Figure 4.1.1 The difference between Filtered Sago Effluent (FSE) and Modified
Sago Effluent (MSE).
44
Figure 4.1 Effects on pH of Spirulina platensis cultured in MSE amended with
different concentration of sodium bicarbonate (NaHCO3) and in
Zarrouk’s medium for 20 days of cultivation duration for the first
trial of experiment.
45
xv
Figure 4.2 Effects on pH of Spirulina platensis cultured in MSE amended with
different concentration of sodium bicarbonate (NaHCO3) and in
Zarrouk’s medium for 20 days of cultivation duration for the second
trial of experiment.
46
Figure 4.3 Effects on growth (DCW) of Spirulina platensis in MSE amended
with different concentration of sodium bicarbonate (NaHCO3) and
in Zarrouk’s medium for 20 days of cultivation duration for first
trial of experiment.
48
Figure 4.4 Effects on growth (DCW) of Spirulina platensis in MSE amended
with different concentration of sodium bicarbonate (NaHCO3) and
in Zarrouk’s medium for 20 days of cultivation duration for second
trial of experiment.
49
Figure 4.4.1 0th
day (09/02/10) of Cultivation for first trial of experiment.
53
Figure 4.4.2 20th
day (29/02/10) of Cultivation for first trial of experiment.
53
Figure 4.4.3 0th
day (18/03/10) of Cultivation for second trial of experiment.
54
Figure 4.4.4 20th
day (07/04/10) of Cultivation for second trial of experiment.
54
Figure 4.5 Glucose content of Spirulina platensis in MSE with amendment of
0g/L NaHCO3, 4g/L NaHCO3 and 8g/L NaHCO3 for 20 days of
cultivation duration in the first trial experiment.
55
Figure 4.6 Glucose content of Spirulina platensis in MSE with amendment of
0g/L NaHCO3, 4g/L NaHCO3 and 8g/L NaHCO3 for 20 days of
cultivation duration in the second trial experiment.
55
Figure 4.7 Starch content of Spirulina platensis in Zarrouk’s medium and in
MSE with amendment of 0g/L NaHCO3, 4g/L NaHCO3 and 8g/L
NaHCO3 for 20 days of cultivation duration for the first trial
experiment.
57
Figure 4.8 Starch content of Spirulina platensis in Zarrouk’s medium and in
MSE with amendment of 0g/L NaHCO3, 4g/L NaHCO3 and 8g/L
NaHCO3 for 20 days of cultivation duration for the second trial
experiment.
58
xvi
LIST OF TABLES
TITTLE PAGE
Table 3.1
Zarrouk’s medium (modified)
29
Table 3.2 Stock concentration of nutrient solution
29
Table 3.3 Stock concentration of bicarbonate solution
30
Table 3.4 Stock concentration of microelement stock
30
Table 3.5 Stock concentration of Fe-EDTA
30
Table 3.6 40 X stock
31
Table 4.1 Characteristics of Sago effluent before and after treatment
using BakWira MP300 for first trial of experiment.
42
Table 4.2 Characteristics of Sago effluent before and after treatment
using BakWira MP300 for second trial of experiment.
42
Table 4.3 Optical density of Spirulina platensis.
44
Table 4.4 Comparison of protein content of Spirulina platensis in
different medium for first trial of experiment.
60
Table 4.5 Comparison of protein content of Spirulina platensis in
different medium for second trial of experiment.
60
1
CHAPTER 1
INTRODUCTION
1.1 General Overview
“Spirulina is a single-celled, unbranched, helicoidal, and filamentous blue-
green alga” (Belay et al, 1993). According to Kozlenko and Ronald (1998),
Spirulina has a soft cell wall complex which composed of sugars and protein; hence,
it is more easily to digest compare to others algae. Spirulina can be naturally found
in alkaline environment for survival. Proceeding studied performed by Rafiqul et al
(2005) proved that the optimal growth for Spirulina platensis is range at pH9-pH
10.5. Since the metabolism is affected by the high pH because pH is important in
solubility of carbon dioxide and minerals in medium. Thus, other algae are unable to
grow under high pH as Spirulina platensis does. (Berker, 1993 cited in Rafiqul et al,
2005).
According to James et al (1998), Spirulina algae grow ten times faster than
standard plants, and have 70% protein by dry weight. Therefore, Spirulina has
gained considerable popularity in the health food industry and increasingly as a
protein and vitamin supplement to aquaculture diets. The micro algae, Spirulina
seemed to be a good protein source and are comparable with the milk proteins
(Shelef and Soeder, 1980); further research revealed that it is a rich source of
vitamins, essential amino acids, minerals and β-carotene.
2
Besides its nutritional value, Spirulina is also important in medical field. It
find its immense use as anti cancer formulations, diabetes control wound treatment
and to promote skin metabolism (Surekha Rani and Uma Bala, 2006). This is widely
exploited in the manufacturing of beauty products such as anti-wrinkling, anti-
pimple creams, facemasks and high protein shampoos. Moreover, it has been
commercially cultivated for its bluish green pigment, called phycocyanin, which can
be used as a natural colorant for food, cosmetics etc (Liang et al, 2004). Though
several thousand algal forms are available in nature, only a few are amenable to
technology. This is based on their ability to grow in synthetic media, case of
separation and stability to drying and importance of their chemical constituents and
finally the cost effectiveness of the whole system (Venkatraman et al, 1983).
With a deep concern over the probable global food shortage in the years to
come, underutilized plant resources are now being extensively tapped by scientists
throughout the world. In this regard, sago palm is gaining much importance as a crop
par excellence and a starch crop of the 21st century, due to its being an extremely
sustainable plant with an ability to thrive in most soil conditions. The importance
and development of industrial biotechnology processing has led to the utilisation of
microbial enzymes in various applications. One of the important enzymes is
amylase, which hydrolyses starch to glucose. In Malaysia, the use of sago starch has
been increasing, and it is presently being used for the production of glucose (Bujang
and Yusop, 2006). Sago starch represents an alternative cheap carbon source for
fermentation processes that is attractive out of both economic and geographical
considerations (Bujang et al, 2006).
3
Sago palms (Metroxylon sagu) mostly grown in the state of Sarawak in
Malaysia. The sago industry not only contributed as an important source of starch
but also source of economical value in Malaysia. However, the generation of sago
wastewater from the starch extraction in factory brings problems to the environment
as rivers that nearby are contaminated by sago wastewater (Bujang and Yusop,
2006). Waterways are definitely an important natural habitat for aquatic species and
they also provide us an essential source of water for living requirements. Bujang et
al (1996) reported that more than 1,425 tons of sago effluent is produced per week
by a medium sized factory in Sarawak.
1.2 Objectives
From the overview above, the objectives of this study are:
• To maximize the growth of the Spirulina platensis.
• To recognize all the parameters affecting the growth of Spirulina platensis.
• To produce high amount of Spirulina economically.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Sago
2.1.1 Sago Starch
Sago palms occupy over three quarters of the peat land of Sarawak and it is
able to grow well and vigorously in swampy areas (Bujang and Yusop, 2006). “Sago
starch is mainly produced in the sated of Sarawak in Malaysia whereby it will be
extracted from Sago logs through several processing steps such as debarking,
pulping, starch extraction, dewatering, drying and packing” (Bujang and Yusop,
2005). Sago starch has a high commercial value and according to Bujang and Yusop
(2005), the export of sago starch is 61,000 tonnes annually and valued at US $9.15
million for the year 2005.This is because, each of the mother palm can produce many
suckers making replanting not necessary after the mother trunk is cut. Moreover,
Sago palm does not need much care and pesticide.
2.1.2 Sago Effluent
There are two form of the waste produced from the extraction of sago starch
where it can be in solid form or wastewater. As sago effluent is released into nearby
rivers and waterways without any treatment it consequently can cause pollution to
our environment from the extraction process of the sago effluent. However, pollution
5
that occurs is because of the large of production of sago effluents daily but not
because of the chemical components of sago effluent (Bujang and Yusop, 2006).
“For every kilogram (dry weight) of starch produced, it has estimated that 20L of
wastewater is generated in the process” (Bujang et al, 1996). Moreover, according to
DOS (2002), Sarawak has over 30 large sago factories, thus enhancing the
possibility of water pollution from the sago industries.
Previous researches (Bujang et al, 2004) showed that sago effluent can be
aerobically treated with enzyme and microbial amendment such as Bakwira where
the mixed liquor is aerated before being released into the waterways with a reduction
in Chemical Oxygen Demand (COD) by 96% after 32 days.
A study was done by Phang et al (2000), which Spirulina cultivation in
digested sago starch factory wastewater. The objective of the study is to test the high
rate algal pond as a method for the combined treatment of the sago starch factory
wastewater and production of Spirulina for use as animal feed. This is regarding of
high rate alga pond offers a good system for the treatment of wastewater and
production of useful alga biomass (Laliberte et al, 1997). Wastewater arising from
the production of a sago starch has a high carbon to nitrogen ratio which improves
anaerobic fermentation in an up flow packed bed digester, the high carbon to
nitrogen ratio supported growth of Spirulina. Phang et al (2000) reported that sago
effluent contain high ratio in carbon to nitrogen by which this ratio can be increased
by carried fermentation in anaerobic digester. “This digested effluent with an average
C: N: P ration of 24: 0.14: 1 supported growth of Spirulina platensis with an average
6
specific growth rate (µ) of 0.51 per day compared with the average µ of 0.54 per
day in the organic Kosaric medium in a high rate algal pond” (Phang et al, 2000).
2.2 Spirulina
2.2.1 Morphology and Characteristic
“Spirulina is a single-celled, unbranched, helicoidal, and filamentous blue-
green alga” (Belay et al, 1993). It is a cynobacterium that contains chlorophyll a,
carotenoids and some unusual accessory blue pigments and red pigments. Blue
pigments are phycobilins and phycocyanin while red pigment is phycoerythrin. Their
main photosynthetic pigment is phycocyanin, which is blue in colour. Spirulina are
photosynthetic and therefore autotrophic. So, light and temperature are the main
factors that influent the biomass production of Spirulina Platensis (Costa et al,
2004). Thus, Spirulina is a photosynthesizing cyanophyte (blue-green algae) that
grows vigorously in strong sunshine under high temperatures and highly alkaline
conditions. The name of ‘Spirulina’ is actually derived from Latin word which
means spiral in shape. This can be proven by referring to Figure 2.1, Figure 2.2,
Figure 2.3 and Figure 2.4 which is the structural analysis of Spirulina platensis that
had been done by using light microscope. It grows in water, can be harvested and
processed easily and has very high macro- and micro-nutrient contents. Spirulina
reproduce by binary fission (Sanchez et al, 2002).