Flow Culture of Spirulina in Filtered Sago Effluent (FSE)
Aryati Almi Binti Ibrahim
(18068)
This project is submitted
in fulfillment of the requirements for the Degree of Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
2010
DECLARATION
I hereby declare that no portion of this dissertation has been submitted in support of an
application for another degree of qualification of this or any other university or institution of
higher learning.
……………………………………..
(ARYATI ALMI BINTI IBRAHIM)
Resource Biotechnology Programme
Department of Molecular Biology
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ACKNOWLEDGEMENT
Thank you for all His Gracious that I am able to finish this project. I am very thankful to my
supervisor Prof. Dr. Kopli Bujang for his advice, supports, and knowledge given towards
completion for this project. To all the lecturers, thank you for all the knowledge and wisdom
you have given to me for the past three years in UNIMAS.
I would like to express my appreciation to Puan Dayang Salwani, Dr. Cirilo, Miss Rubena
Malfia Kamal and Miss Merlina Manggi for their guide and knowledge during the lab work.
Not forgetting as well to all laboratories assistants of FRST for their co-operation.
Lastly, I would like to mention my appreciation to Nurhamizah Bt Merali, Mohamad Sufiyan
Bin Saiful Ikhwan and other fellow course mate for their co-operation, assistance and
supports. To my family and friends, your moral support is very meaningful to me.
Thank you.
Flow Culture of Spirulina in Filtered Sago Effluent
Aryati Almi Bt. Ibrahim
Resource Biotechnology
Molecular Biology Department
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A flow culture system study was carried out to evaluate the cultivating of Spirulina in filtered sago
effluent under sunlight. Cultivation of Spirulina in FSE amended with 4 g/L sodium bicarbonate
(NaHCO3) was compared with cultivation in FSE without any amendment by the measurement of dry
cell weight (DCW). Both trials were observed for 21 days at pH 9.5. It was shown that culture in FSE
amended with 4 g/L sodium bicarbonate has higher biomass although the biomass produced from
Zarrouk medium (standard medium) was higher.
Keywords: Flow culture, Spirulina, filtered sago effluent, sodium bicarbonate, dry cell weight.
ABSTRAK
Satu kajian secara penggunaan sistem kultur aliran telah dilakukan untuk mengkaji pengkulturan
Spirulina dalam effluen sago di bawah cahaya matahari. Perbandingan Spirulina yang dikultur dalam
FSE dengan 4 g/L sodium bicarbonate (NaHCO3) dan FSE dikaji secara sel berat kering (DCW).
Kedua-dua dikultur selama 21 hari pada pH 9.5 dan menunjukkan kultur dalam FSE 4 g/L sodium
bicarbonate menghasilkan biomass yang lebih tinggi dari FSE. Bagaimanapun, biomass yang terhasil
daripada kedua-dua kultur tidak setinggi kultur dalam medium Zarrouk.
Kata kunci: Kultur aliran, Spirulina, effluen sago yang ditapis, sodium bicarbonate, sel berat kering.
LIST OF ABBREVIATIONS
APHA American Public Health Association
DCW Dry cell weight
FSE Filtered sago effluent
g/L Gram per liter
mg/L Milligram per liter
NaHCO3 Sodium bicarbonate
TSS Total suspended solid
LIST OF TABLES
Table 1 Component of Zarrouk’s medium (modified) 7
Table 2 Stock concentration of nutrient solution 8
Table 3 Stock concentration of bicarbonate solution 8
Table 4 Stock concentration of microelement stock 8
Table 5 Stock concentration of Fe-EDTA 9
Table 6 Stock concentration of 40 X stock 9
Table 7 Characteristics of the sago effluent 15
Table 8 Comparison protein of Spirulina cultured in 24
FSE, FSE supplemented with 4 g/L
NaHCO3 and Zarrouk’s medium
LIST OF FIGURES
Figure 1 Setup of flow culture system. 11
Figure 2 Time course of biomass concentration for Spirulina cultivated 17
in Zarrouk medium for 20 days.
Figure 3 Time course of biomass concentration for Spirulina cultivated 18
in FSE supplemented 4 g/L NaHCO3 for 20 days.
Figure 4 Time course of reducing sugar for Spirulina cultivated in 19
FSE supplemented 4 g/L NaHCO3 for 20 days.
Figure 5 Time course of starch for Spirulina cultivated in FSE 19
supplemented 4 g/L NaHCO3 for 20 days.
Figure 6 Time course of biomass concentration for Spirulina cultivated 20
in FSE without supplemented for 20 days.
Figure 7 Time course of reducing sugar for Spirulina cultivated in FSE 21
without supplemented for 20 days.
Figure 8 Time course of starch for Spirulina cultivated in FSE without 21
supplemented for 20 days.
Figure 9 Time course of Spirulina in FSE, FSE amended with 4g/L 22
NaHCO3 and Zarrouk’s medium for 20 days.
ACKNOWLEDGMENT
i
ABSTRACT
ii
ABSTRAK
ii
LIST OF ABBREVIATIONS
iii
LIST OF TABLES
iv
LIST OF FIGURES
v
1.0 INTRODUCTION
1
1.1 General Overview 1
1.2 Objectives
2
2.0 LITERATURE REVIEW
3
2.1 Sago Palm 3
2.2 Sago Effluent 3
2.3 Sago Industry in Sarawak 4
2.4 Spirulina Algae 4
2.5 Sodium Bicarbonate
5
3.0 MATERIALS AND METHODS
6
3.1 Preparation sago effluent 6
3.1.1 Total suspended solid 6
3.1.2 Filtration sago effluent 6
3.2 Spirulina Culture 7
3.3 Preperation of Zarrouk Medium 7
3.4 Cultivation of Spirulina 10
3.4.1 Zarrouk Medium 10
3.4.2 Filtered Sago Effluent 10
3.4.3 Filtered Sago Effluent Amendment with Sodium Bicarbonate 10
3.5 Growth of Spirulina in Flow Culture 11
3.6 pH 12
3.7 Analysis 12
3.7.1 Reducing Sugar 12
3.7.2 Starch Content 13
3.7.3 Dry Cell Weight Measurement 13
3.7.4 Protein
14
4.0 RESULTS AND DISCUSSIONS
15
4.1 Characteristic of Sago Effluent 15
4.1.1 Total Suspended Solid 15
4.1.2 pH 16
4.2 Cultivation of Spirulina in Zarrouk Medium 17
4.3 Cultivation of Spirulina in Filtered Sago Effluent Amendment
with Sodium Bicarbonate (NahCO3)
18
4.4 Cultivation of Spirulina in Filtered Sago Effluent 20
4.5 Growth of Spirulina in Filtered Sago Effluents and Zarrouk
medium
22
4.5.1 Overall Analysis 22
4.6 Protein Analysis
24
5.0 CONCLUSION
25
REFERENCES
26
APPENDIX A
29
APPENDIX B 31
1.0 INTRODUCTION
1.1 General Overview
Sago palm grows well with the minimum care in swamp and peat areas. It has high starch
yield where one palm may yield between 150 kg to 300 kg of starch. Sarawak exports up to
40,000 tons sago a year and the effluent resulting from sago debarking and processing often
discharged to nearby rivers. A typical sago mill consumes about 1,000 logs per day,
generating a minimum of 400 tons of slurry effluent which contains about 5% solids. For the
past couple of years, the potential sago waste solid have been exploited in the sago effluent to
look at the possible generation of biofuel and other by- product. One of other by-product is
investigated algae Spirulina culture on the filtered sago effluent (Bujang, 2008).
Spirulina have received greater attention as a source of human food and poultry feed. Seen it
has traditionally consumed by tribal people in Central Africa and Mexico. A large number of
formulations are available for the preparation of novel food using Spirulina powder (Hills,
1980). It has a great use in fermented food like “pastalina”. The biliprotiens present in
Spirulina are also used in immuno diagnostics (Venkataraman, 1989). Spirulina has also been
claimed to have healing effect on patients suffering from pancreatitis, cirrhosis and hepatitis
and also acts as a prophylactic against cancer (Beeker and Venkataraman, 1984). The United
Nations World Food Conferences in 1974 declared that Spirulina was “the best food for
tomorrow”.
In this study, Spirulina was grown in filtered sago effluent amended with sodium bicarbonate
(NaHCO3) using flow culture.
1.2 Objective of the Study
1. To study the growth of Spirulina in filtered sago effluent using flow culture.
2. To determine the effects of adding 4 g/L sodium bicarbonate to the growth of Spirulina
in filtered sago effluent (FSE).
3. To determine the biomass of Spirulina by using dry cell weight method.
2.0 LITERATURE REVIEW
2.1 Sago Palm
Sago palm is a metroxylon species and is found from Thailand, Malaysia, Samoa and Fiji. The
natural habitat of metroxylon is tropical lowland forest and freshwater swamps. The palms are
often found growing in the freshwater margin at the back of mangrove swamps, extending
inland as far as slow moving freshwater flows. Mextroxylon species stand between 9 to 33 m
in height. Generally the species tolerate salinity and pro-longed flooding, acidic and wet soils
(McClatchey et al, 2004).
2.2 Sago Effluent
The waste generated from sago mills are bark “hampas” and wastewater. Generally, the
wastewater was acidic, high in organic load and low in nutrient. Starch extraction from sago
pulp produces large amount of wastewater. Depending on the capacity of the mill, almost 400
liters of wastewater and 12 kilograms of dry solids is generating from each log and for every
kilograms of starch produce, approximately 20 liters wastewater is generated (Bujang, 1997).
In Sarawak, large volumes of sago effluent may present a serious pollution problem (Tie and
Lim, 1991). This is because most sago factory do not performed any wastewater treatment
before releasing the effluent to the environment.
2.3 Sago Industries in Sarawak
Sago palm has the greatest potential to be Malaysian producer of starch. Sago starch ranks as
the fifth highest agriculture revenue after pepper, palm oil, cocoa and rubber in Malaysia.
Besides, it is also act as a raw material of the natural resource of starch includes potato, corn,
tapioca and wheat.
The sago industries in Malaysia are concentrated mainly in Sarawak, much lesser in
Peninsular Malaysia due to the demands for land and capital for oil palm plantations. About
25,000 to 40,000 tons of sago products exported per year in the sago industries that give good
impact to the agricultural export commodities in the economy Sarawak. Sago palm production
capacity varies from 2-5 tons of dry starch/ha in the wild regions to 10-25 tons/ha in cultivated
plantations (Singhal et al, 2008).
2.3 Spirulina Algae
Spirulina is symbiotic, multicellular and filamentous blue-green microalgae with symbiotic
that fix nitrogen with air. Spirulina can be rod or disk-shaped. The main photosynthetic
pigment is phycocyanin, which is blue colour. Spirulina are photosynthetic and therefore
autotrophic. The trichomes have length of 50µm -500µm and width of 3µm to 4µm. The
helical shape of the filamentous is characteristic of the genius and it is maintained only in
liquid environment and or culture medium. The presence gas-filled vacuoles in the cell,
together with the helical shape of the filaments, result in floating mats. Spirulina is found in
soil, marshes, freshwater, brackish water, seawater and thermal spring. (Ahsan et al, 2008).
Spirulina has the highest protein contain (60-70 percent) of any natural food, far more than
fish (15 to 20 percent), soybean (35 percent), milk powder (35 percent), fresh egg (12 percent)
and grains (8-14 percent). It also has 20 percent carbohydrate, 5 percent fat, 7 percent minerals
and 6 percent moisture. Besides, Spirulina is thus a low fat, low calorie, cholesterol-free
sources of protein unlike meat (rich in fat) or diary product. The other rich source contain in
this blue-green alga is ß-carotene, thiamine, riboflavin and it is the richest sources of vitamin
B12 (Durand-Chastel, 1980). The general composition of Spirulina does not indicate the
presence of any harmful compound and shows promising properties as a food and feed
ingredient (Beeker and Venkataraman, 1984).
2.4 Sodium Bicarbonate NaHCO3
Sodium bicarbonate is the chemical compound with the formula NaHCO3 and it is soluble in
water. Sodium bicarbonate is a white solid that is crystalline but often appears as a fine
powder. Sodium bicarbonate can be added as a simple solution for raising the pH balance of
water to increasing the total alkalinity .It is component of mineral natron and is found
dissolved in many mineral springs. The natural mineral is form nahcolite. It is also produced
artificial (Wikipedia, 2009).
3.0 MATERIALS AND METHODS
3.1 Preparation of Sago Effluent
3.1.1Total Suspended Solid
The TSS of the effluent was determined using standard method 2540D (APHA, 1995). 20 mL
sample was filtered using filter membrane no. 1 (110nm). After that, the filter paper was dried
at 70ºC. Then, the filter paper was cooled in desiccator to balance the temperature before
weighting. TSS was determined as:
TSS (mg/L) = (A-B) x 1000
Sample volume (mL)
Where, A is weight of sample after drying on filter paper
B is the weight of filter paper
3.1.2 Filtration of Sago Effluent
Sample of sago effluent was obtained from a commercial sago process, Herdson Sago
Sdn.Bhd., located in Pusa Sarawak. The sample was filtered using 710µm mesh size of filter
in order to separate it from sago ‘hampas’.
3.2 Spirulina Culture
The Spirulina strain was obtained from the laboratory and maintained in Zarrouk medium.
3.3 Preparation of Zarrouk Medium
Preparation of the components Zarrouk’s medium is using ingredients based on Tables 1, 2, 3,
4, 5, and 6. Each of the components of Zarrouk’s medium except for bicarbonate solution was
prepared and stored at 4°C. These components were mixed for used to form Zarrouk’s
medium.
Table 1: Component of Zarrouk’s medium (modified)
Component Stock conc., g/l ml stock/liter medium
Nutrient solution Refer to table 3.2 478
Bicarbonate solution Refer to table 3.3 500
CaCl2.2H2O 4.0 10
MgSO4.7H2O 20.0 10
Microelement stock Refer to table 3.4 1
Fe-EDTA stock Refer to table 3.5 1
The various components was mixed aseptically and dispensed into sterile containers.
Table 2: Stock concentration of nutrient solution
Component Amount, ml
40 X Stock (refer to table 3.6) 25
Distilled water 453
The components were autoclaved and allowed to cool to temperature room.
Table 3: Stock concentration of bicarbonate solution
Component Conc., g/500 ml
NaHCO3 16.8
K2HPO4 1.0
The ingredients were added to about 400ml distilled water and then made the final volume to
500ml. The mixed solution was autoclaved.
Table 4: Stock concentration of microelement stock
Compound Conc., g/liter stock
H3BO3 2.86
MnCl2.4H2O 1.81
ZnSO4.7H2O 0.222
CuSO4.5H2O 0.08
NaMoO4.2H2O 0.23
Co(NO3)2.6H2O 0.046
Each of the components was dissolved separately and mixed to make the final volume. The
final stock was sterile-filtered and stored in refrigerator.
Table 5: Stock concentration of Fe-EDTA
Component Conc., g/liter stock
Na2EDTA.2H2O 27.9
FeSO4.7H2O 24.5
The solution was bubbled with air for 24 hours until the solution was clear yellow-brown. The
stock was sterile filtered and stored the refrigerator.
Table 6: 40 X stock
Component Conc., g/liter stock
NaNO3 100
K2SO4 40
NaCl 40
The stock was stored in the refrigerator.
3.4 Cultivation of Spirulina
3.4.1 Zarrouk Medium
10% inoculum of Spirulina was used and cultivated about 1 L in 1000ml shake flaks. Then it
was exposed under sunlight with aeration pump. Zarrouk medium were used as control for this
project.
3.4.2 Filtered Sago Effluent
Sago effluent was filtered using 750µm stainless steel filter. Then, filtered sago effluent was
centrifuge (HITACHI) at 6000 rpm for 10 minutes. This is to reduce interferences that cause
by remaining solid in the sago wastewater. The filtered sago effluent was adjusted to pH 9-10
with 1M NaOH because it is suitable range pH for Spirulina growth. Trial was performed in
flow culture with total volume 14 L under sunlight with aquarium pump.
3.4.3 Filtered Sago Effluent Amendment with Sodium Bicarbonate
The cultivation process was the same as in section 3.4.2 but amended with sodium bicarbonate
at 4 gL-1
.
3.5 Growth of Spirulina in Flow Culture
The flow culture system consists of 2 transparent perspex growth chamber arranged at
different height on adjustable iron frame table, as in Figure 1. The height between first tank
and the second tank adjusted to 15 cm and connected to each other by silicone tube. To create
the flow, an aquarium pump was use and put in the first tank. Both of the tanks were filled
with the same amount of filtered sago effluent prior to pump tank 1 to tank 2. The flow from
tank 2 will return back to tank 1 by gravity, when the tank fills and overflow through the top
tube and flow back to tank 1. The trial was run for 20 days under sunlight condition.
Figure 1: Setup of flow culture. The perspex tanks were arranged at a height of 15 cm in
order for the flow of liquid from the 2 tank to 1 tank to flow by gravity.
3.6 pH
The sample pH was measured using pH meter (ADWA, AD1030).
3.7 Analysis
3.7.1 Reducing Sugar
The analysis of reducing sugar was determined according to Dinitrosalicylic Acid (DNS)
method (Miller, 1959). About 3 ml sample mixed with 3 ml of DNS solutions in a test tube.
Then the mixture was boiled for 15 minutes and let cool down before added 1 ml Rochelle
salts. Absorbance was measured at 575 nm using UV-Visible spectrophotometer (Libra S12)
and the amount of reducing sugar is calculated as follows:
Reducing sugar, g/L = OD 575nm
Standard glucose slope
3.7.2 Starch
The content of starch was performed based on iodine-starch colorimetric method (Nakamura,
1979). The sample was heated to 60-70oC until the residue are dissolved. 1 ml sample
transferred into test tube and added with 100 l iodine solution. Then the mixture was added
with 8.9 ml dH20 until the volume to 10 ml. The measurement was made using UV-Visible
spectrophotometer (Libra S12) and the amount of starch is calculated as follows:
Starch, g/L = OD 590nm
Standard starch slope
3.7.3 Dry Cell Weight Measurement
20 ml sample was filtered on a filter paper. The filter paper was dried at 70oC for 24 hours and
cooled at desiccator.
Dry Cell Weight (mg/L) = (A-B) x 1000
Sample volume (mL)
Where, A is weight of sample after drying on filter paper
B is the weight of filter paper