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