universiti putra malaysia - psasir.upm.edu.mypsasir.upm.edu.my/id/eprint/39345/1/ib 2012 16r.pdf ·...

UNIVERSITI PUTRA MALAYSIA

NURUL SALMA BINTI ADENAN

IB 2012 16

LIPID PRODUCTION IN MARINE MICROALGAE UNDER DIFFERENT SALINITY, TEMPERATURE AND NUTRIENT LEVELS

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LIPID PRODUCTION IN MARINE MICROALGAE UNDER DIFFERENT

SALINITY, TEMPERATURE AND NUTRIENT LEVELS

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirements for the Degree of Master of Science

JULY 2012

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Master of Science

LIPID PRODUCTION IN MARINE MICROALGAE UNDER DIFFERENT

SALINITY, TEMPERATURE AND NUTRIENT LEVELS

By


July 2012

Chairman: Professor Fatimah Md. Yusoff, PhD

Institute: Bioscience

The marine microalgae Chlorella sp. (UPMC-A0013) and Chaetoceros calcitrans

(UPMC-A0010) contain relatively high lipid levels (15.0 to 20.0% of dry weight)

that can contribute as important components for the formulation of feed in

aquaculture industry. However, lipid levels in microalgae vary according to the

culture conditions. This study was carried out to determine the various environmental

factors that control the growth and lipid contents of marine Chlorella sp. and C.

calcitrans by subjecting the algae to different levels of stress in terms of salinity and

temperature changes and nutrient depletion in their early stationary phase.

The effects of salinity stress on lipid production of Chlorella sp. and C. calcitrans

were performed by culturing both microalgae species in Conway medium of various

salinity levels (15, 20, 25, 30, 35 and 40‰). The cultures were centrifuged and

resuspended into culture medium of lower salinity levels of -5‰ (S1i) and -10‰

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(S2i) from the initial salinity levels in their early stationary phase. The highest

growth rate (P<0.05) of Chlorella sp. and C. calcitrans were observed at 25‰

(μ=0.37 day-1

) and 30‰ (μ=0.28 day-1

), respectively. Salinity changes from S30 to

S130 and S230 increased P<0.05) the production of total lipid in Chlorella sp. to

10.0% and 19.0% of d.w., respectively. Total lipid of C. calcitrans increased

significantly (P<0.05) to 13.2% of d.w. when stressed to S230.

Prior to the temperature stress, marine Chlorella sp. and C. calcitrans were cultivated

at 20, 25, 30 and 35oC (Ti). Then, the microalgae were shifted to higher temperature

of +5oC (TSi) from the initial temperature levels in their early stationary phase.

Before stress, Chlorella sp. showed the highest growth rate (µ= 0.35 day-1

, P<0.05)

at 25oC (T25). Total lipid of Chlorella sp. significantly increase (P<0.05) from 31.0%

of d.w. (T25) to 41.0% of d.w (TS25). Diatom, C. calcitrans showed the highest

growth rate at T30 (µ= 0.26 day-1

) and lipid content increased significantly (P<0.05)

from 31.0% to 39.0% after stress (TS30).

The effects of nutrient stress on lipid production was determined by culturing the

algae into Nitrogen and Phosphorus deprived Conway medium (25.0% reduction =

N25 and P25; 50.0% reduction = N50 and P50; 75.0% reduction = N75 and P75) in their

early stationary phase. Lipid concentrations significantly increased (P<0.05) by 9.0%

in both cultures subjected to 75% nitrogen and phosphorus reduction (N75 and P75).

This study illustrated that various growth conditions such as salinity and temperature

changes, and reduction in nutrient concentration enhanced lipid content of Chlorella

sp. and C. calcitrans.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Sarjana Sains

PENGHASILAN LIPID OLEH MIKROALGA MARIN DI BAWAH TAHAP

KEMASINAN, SUHU DAN NUTRIEN BERBEZA

Oleh


Julai 2012

Pengerusi : Professor Fatimah Md. Yusoff, PhD

Institut : Biosains

marin mikroalga, Chlorella sp. (UPMC-AA0013) dan C. calcitrans (UPMC-

AA0010) mengandungi kadar lemak yang tinggi (15-20% berat kering) yang boleh

diaplikasikan sebagai komponen penting dalam pembuatan dan penghasilan makanan

bagi industri akuakultur. Namun begitu, kadar lipid yang dihasilkan bergantung

kepada kaedah pengkulturan. Dengan itu, penentuan pelbagai faktor pengkulturan

yang boleh mengawal kadar pertumbuhan dan penghasilan lipid adalah penting.

Kadar pertumbuhan dan penghasilan lipid oleh Chlorella sp. dan C. calcitrans

dilakukan dengan mengaplikasikan beberapa tahap tekanan pada pembolehubah

pertumbuhan, yang merangkumi perubahan tahap kemasinan dan suhu, serta

pengurangan nutrisi di awal fasa pertumbuhan mendatar.

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Kajian kesan perubahan tahap kemasinan terhadap pengeluaran lipid bagi Chlorella

sp. dan C. calcitrans dengan mengkultur kedua-dua mikroalga di dalam media

Conway yang mengandungi tahap kemasinan asal (Si) yang berbeza iaitu 15, 20, 25,

30, 35 dan 40‰. Kultur diemparkan dan dilarutkan ke dalam media yang

mempunyai tahap kemasinan yang rendah, -5‰ (S1i) dan -10‰ (S2i) dari kepekatan

asal. Chlorella sp. dan C. calcitrans menunjukkan kadar pertumbuhan yang tinggi

pada 25‰ (μ = 0.37 hari-1

) dan 30‰ (μ=0.28 hari-1

). Chlorella sp. menunjukkan

peningkatan pengeluaran lipid kepada 10.0% and 19.0% berat kering setelah berlaku

perubahan tahap kemasinan dari S30 to S130 dan S230. Jumlah pengeluaran lipid bagi

C. calcitrans meningkat kepada 13.2% berat kering setelah tahap kemasinan diubah

ke S230.

Chlorella sp. dan C. calcitrans dikultur pada suhu 20, 25, 30 and 35oC. Kemudian,

peningkatan suhu sebanyak +5oC (TSi) dari suhu asal (Ti) dilakukan pada peringkat

awal fasa pertumbuhan mendatar. Kadar pertumbuhan Chlorella sp. adalah µ= 0.35

day-1

(P<0.05) pada suhu 25oC sebelum suhu dinaikkan. Kadar lipid meningkat dari

31.0% berat kering pada TS25 ke 41.0% berat kering selepas suhu dinaikkan ke 30oC

(TS25). Chaetoceros calcitrans menunjukkan kadar pertumbuhan yang tinggi pada

suhu T30 (µ= 0.26 hari-1

). Selepas suhu dinaikkan, kadar lipid meningkat dari 31.0%

ke 39.0% berat kering pada TS30.

Dalam menentukan kesan pengurangan nutrisi terhadap kadar penghasilan lemak,

Chlorella sp. marin dan C. calcitrans dikultur ke dalam media Conway dan

dilarutkan ke dalam kultur media yang mengandungi beberapa siri kepekatan

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nitrogen dan fosforus yang dikurangkan (pengurangan 25.0% = N25 dan P25;

pengurangan 50.0% = N50 dan P50; pengurangan 75.0% = N75 dan P75) pada awal fasa

pertumbuhan mendatar. Peratusan lipid bagi kedua-dua spesis didapati meningkat

sebanyak 9.0% berat kering apabila kepekatan nitrogen dan fosforus dikurangkan

sehingga 75.0%. Kajian ini menunjukkan perubahan tahap kemasinan dan suhu serta

pengurangan kadar nutrisi dapat meningkatkan penghasilan peratusan lipid yang

tinggi dalam Chlorella sp. marin dan C. calcitrans.

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ACKNOWLEDGEMENTS

Assalamualaikum,

First of all thank you Allah, for your love and guidance I have successfully

completed my laboratory work and finally, this thesis.

My sincere gratitude goes to my supervisors, Prof. Dr Fatimah Md. Yusoff and Prof.

Dato’ Dr Mohamed Shariff Mohamed Din for your guidance and encouragement

throughout the research. Special thanks also extended to all my colleagues for their

assistance, encouragement and friendship, I love you guys. To lecturers, researchers

and science officers who were involved directly or indirectly throughout research,

thank you for you help and advice.

Last but not least, I would like to acknowledge with thank and love to my family and

husband for all their love, support and the tremendous efforts they have put to

materialize this thesis.

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I certify that an Examination Committee has met on 26th

July 2012 to conduct the

final examination of Nurul Salma Binti Adenan on her Master in Science thesis

entitled “Lipid Production In Marine Microalgae Under Different Salinity,

Temperature And Nutrient Levels” in accordance with Universiti Pertanian Malaysia

(Higher Degree) Act 1980. The committee recommends that the student be awarded

the Master of Science.

Members of the Examination committee were as follows:

Aziz Arshad, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Mohamad Pauzi Zakaria, PhD

Professor

Faculty of Environmental Science


(Internal examiner)

Che Roos Saad, PhD

Associate Professor

Faculty Agriculture


(Internal examiner)

Wan Maznah Wan Omar, PhD

Associate Professor

School of Biological Sciences

Universiti Sains Malaysia

Malaysia

(External examiner)

SEOW HENG FONG Professor and Deputy Dean

School of Graduate Studies


Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Fatimah binti Md. Yusoff, PhD

Professor

Institute of Bioscience


(Chairman)

Mohamed Shariff bin Mohamed Din, PhD

Professor

Faculty of Veterinary Medicine


(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.


Date: 26 July 2012

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TABLE OF CONTENTS

CHAPTER

1 INTRODUCTION

1.1 Background of study 1

1.2 Problem statement 3

1.3 Objectives 4

2 LITERATURE REVIEW

2.1 Biology and distributions of microalgae 5

2.2 Application of microalgae 7

2.3 History of microalgae in aquaculture industry 8

2.4 Bioactive compounds of microalgae 10

2.5 Fatty acid proportions in microalgae 11

2.6 Microalgae as a source of beneficial nutrients for

aquaculture

13

2.7 Lipid biosynthesis in microalgae 14

2.8 Factors influencing the increment of microalgae lipid

production

15

2.8.1 Salinity 16

2.8.2 Temperature 17

2.8.3 Nitrogen deprivation 18

2.8.4 Phosphorus deprivation 19

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vii

APPROVAL viii

DECLARATION x

TABLE OF CONTENTS xi

LIST OF TABLES xiv

LIST OF FIGURES xvi

LIST OF ABBREVIATIONS xix

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3 GENERAL METHODOLOGY

3.1 Algal cultures 21

3.1.1 Cultural condition of the starter culture 23

3.2 Microalgae identification 23

3.2.1 Light microscope observation 23

3.2.2 Molecular DNA identification equal 24

3.2.2.1 Polymerase chain reaction and sequencing 25

3.3 Experimental Design 26

3.4 Statistical Analyses 27

3.5 Determination of cell concentration 29

3.5.1 Optical density 29

3.5.2 Cell counts 29

3.5.3 Biomass estimation 30

3.5.4 Estimation of growth rate 30

3.6 Biochemical analysis 31

3.6.1 Total Lipid Determination 31

3.6.2 Determination of fatty acids 32

3.6.3 Proximate analysis 33

3.6.3.1 Determination of protein 34

3.6.3.2 Determination of carbohydrate 35

4 EFFECTS OF DIFFERENT SALINITY LEVELS ON

LIPID PRODUCTION OF MARINE MICROALGAE

4.1 Introduction 36

4.2 Materials and methods 37

4.2.1 Experimental design 37

4.2.2 Algal production 39

4.3 Results 39

4.4 Discussion 52

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5 EFFECTS OF DIFFERENT TEMPERATURE LEVELS

ON LIPID PRODUCTION OF MARINE MICROALGAE

5.1 Introduction 57




5.3 Results 59

5.4 Discussion 71

6 EFFECTS OF NITROGEN REDUCTION ON LIPID

PRODUCTION OF MARINE MICROALGAE

6.1 Introduction 76




6.3 Results 79

6.4 Discussion 87

7 EFFECTS OF PHOSPHORUS REDUCTION ON LIPID

PRODUCTION OF MARINE MICROALGAE

7.1 Introduction 93




7.3 Results 96

7.4 Discussion 105

8 SUMMARY, GENERAL CONCLUSION &

RECOMMENDATION

109

REFERENCES 113

APPENDICES 134

BIODATA OF STUDENT 141

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