Selection of microalgae species suitable for lipid production and nutritious biomass :Master's thesis - Major : Sciences and management of the environment

THE JOINT ACADEMIC PROGRAM OF EXECUTIVE MASTER IN

SCIENCES AND MANAGEMENT OF THE ENVIRONMENT

BETWEEN INDUSTRIAL UNIVERSITY OF HOCHIMINH CITY

AND LIÈGE UNIVERSITY

DO NGUYEN MINH CHAU

SELECTION OF MICROALGAE SPECIES

SUITABLE FOR LIPID PRODUCTION AND

NUTRITIOUS BIOMASS

Major: EXECUTIVE MASTER IN SCIENCES AND MANAGEMENT OF

THE ENVIRONMENT

MASTER’S THESIS

HOCHIMINH CITY, YEAR 2018

The project was completed at The Industrial University of Hochiminh City

Supervisors:

Gauthier Eppe, Prof Trinh Ngoc Nam, PhD

The thesis was taken at The Industrial University of Hochiminh City date . . . . .

month . . . . year . . . . .

Committee members (name):

1. .........................................................................- Committee Chair

2. .........................................................................- Commissioner

3. .........................................................................- Secretary

(Write full name and signature)

COMMITTEE CHAIR DEAN OF INSTITUTE OF ENVIRONMENTAL

SCIENCE, ENGINEERING AND MANAGEMENT

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ACKNOWLEDGEMENTS

First of all, I would like to deeply express my appreciation to people who supported

and helped me immensely in this study: Prof. Le Hung Anh, PhD. Trinh Ngoc Nam

and Prof. Gauthier Eppe - my thesis’s supervisors. They support me by their

invaluable assistance during the time of doing this study, thanks for their reviews,

constructive advices, patience and encouragement. This study could be finished

successfully with their instructions and motivation.

Also, I am so grateful for the support and an active and friendly environment that

Renewable project gave me. Additionally, all the member of Renewable project and

Institute of Biotechnology and Food Technology help me with encouragement as

well.

Furthermore, with all my deep gratitude for Prof. Jean-Luc Vasel, I highly

appreciate his valuable documents as well as all the help he gave me in Belgium.

Also, thanks to my dear colleague, Ms. Trang Kim Ly who has been with me since

the beginning of our work, Mr. Nguyen Tri Phuong who encourage and support me

mentally and academically and PhD. Thien who support me a lot in this field of

study, I could have conducted this thesis effectively.

Finally, there is no doubt that I would like to give my deepest thanks to my family

for their unconditional love, support and faith in me.

Ho Chi Minh City, August 2018

Author

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ABSTRACT

Microalgae are considered as a third generation of biofuel supply and nutrition

biomass. In Vietnam, microalgae have high biodiversity and high application

potential but the current use is limited. This study performs exploit indigenous

microalgae species with potential for biofuel production. Nine strains were

identified as Chlorella vulgaris (Li9, Li14, Li21, TH3, TH6, TH14), Chlorella

sorokiniana TH21, Halamphora coffeaeformis TH13, Chlorella sp TH38, TH35

strain is belong to Bacillariophyceae. Two strains gave the highest total FAME

results are Chlorella sorokiniana TH21 and Chlorella vulgaris Li9 respectively

11.51% and 3.26%. In addition, CN and CFPP values indicate that lipid quality is

suitable for biofuel production. The corresponding CN values are 69.84 and 190.48

compared to standard EN 14214 with minimum value is 51 and CFPP respectively -

16.2C and -15.8C compared to RD 61/2006 with a maximum value is -10C. The

mineral analysis values of microalgae showed low concentrations, but provided that

heavy metals such as Cu, Cr, Cd and Pd were below acceptable level.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................1

ABSTRACT................................................................................................................2

LIST OF TABLES ......................................................................................................5

LIST OF FIGURES.....................................................................................................6

LIST OF ABBREVIATIONS.....................................................................................8

INTRODUCTION.....................................................................................................10

1. The reason for choosing the topic ......................................................................10

2. Objectives of the study.......................................................................................11

2.1 General Objective .........................................................................................11

2.2 Specific Objectives .......................................................................................11

3. Subject and scope of the study...........................................................................11

3.1 Research subject............................................................................................11

3.2 Research scope..............................................................................................12

4. The meaning of the topic ...................................................................................12

4.1 Significance of research................................................................................12

4.2 Practical implication of research...................................................................12

5. The methodology ...............................................................................................12

5.1 Collecting information .................................................................................12

5.2 Method of sampling analysis........................................................................12

5.3 Comparative method.....................................................................................13

5.4 The main techniques used.............................................................................13

CHAPTER 1 LITERATURE REVIEW ...................................................................14

1.1 Microalgae .......................................................................................................14

1.1.1 Types of cell organization..........................................................................14

1.1.2 Cellular organization..................................................................................15

1.1.3 Ultrastructure and cell division..................................................................16

1.1.4 Microalgal systematics...............................................................................18

1.2 Lipid metabolism in microalgae ......................................................................24

1.3 Application of microalgae on biofuel ..............................................................27

1.4 Application of Microalgae on Cattle feeding...................................................28

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1.5 Condition of aquaculture industrial – shrimp farming: 2010-2014 .................29

1.6 Characteristics of industrial shrimp waste water .............................................31

CHAPTER 2 MATERIAL AND METHODS..........................................................33

2.1 Material ............................................................................................................33

2.1.1 Samples......................................................................................................33

2.1.2 Instruments -Chemical -Laboratory materials...........................................35

2.2 Experiment .......................................................................................................37

2.3 Methods............................................................................................................37

2.3.1 Medium culture and Isolation method .......................................................37

2.3.2 Identification ..............................................................................................38

2.3.3 Analysis of fatty acid composition ............................................................42

2.3.4 Analysis of mineral composition ...............................................................43

CHAPTER 3 RESULTS AND DISCUSSIONS.......................................................44

3.1 Isolation and Preliminary identification by Morphology ................................44

3.2 Identification by DNA sequence......................................................................53

3.3 Lipid content ....................................................................................................81

3.4 Mineral content ................................................................................................83

3.5 Discussions.......................................................................................................84

CHAPTER 4 CONCLUSION...................................................................................89

REFERENCES..........................................................................................................91

APPENDIX 1 ............................................................................................................94

APPENDIX 2 ..........................................................................................................102

APPENDIX 3 ..........................................................................................................109

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LIST OF TABLES

Table 1.1 Physical and chemical characteristics of shrimp pond in Can Gio

District, Ho Chi Minh.[2]..........................................................................32

Table 3.1 Strains of Microalgae [1] ..................................................................44

Table 3.2 Sequence results [1]..........................................................................57

Table 3.3 Define species [1] .............................................................................79

Table AP.1 The composition of Guillard F/2 medium .....................................95

Table AP.2 Number and volume of samples from Ninh Thuan .......................95

Table AP.3 Mineral compound.........................................................................96

Table AP.4 Fatty acid compound......................................................................98

Table AP.5 Compare the sequences of strain TH30 and TH36........................99

Table AP.6 Sequence used for phylogeny tree ...............................................100

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LIST OF FIGURES

Figure 1.1 Electron micrograph of a diving cell of Synechococcus sp.[5].......15

Figure 1.2 Electron micrograph of a cell of Chlorella vulgaris.[5]..................16

Figure 1.3 Electron micrograph of a cell of Microcystis sp.[5]........................20

Figure 1.4 Basic overview of the pathway of carbon capture and lipid

biosynthesis [7] .........................................................................................25

Figure 1.5 The fatty acid and TAG biosynthesis [8] ........................................27

Figure 2.1 Location of Ninh Thuận in Vietnam ...............................................33

Figure 2.2 Sampling diagram. [40]...................................................................34

Figure 2.3 Experiment procedure. [1]...............................................................37

Figure 2.4 Diagram of DNA sequence analysis of microalgae. [1]..................40

Figure 3.1 Electrophoresis results [1] ...............................................................55

Figure 3.2 Phylogenetic tree were created using PAUP software after 1000

rounds of bootstrap. [1].............................................................................71

Figure 3.3 Phylogenetic tree were created using PAUP software after 1000

rounds of bootstrap. [1].............................................................................72

Figure 3.4 Compare of morphology [1]............................................................76

Figure 3.5 Morphology comparison of TH35...................................................78

Figure 3.6 Compare of morphology of strain TH30 and TH36. [1] .................78

Figure 3.7 Comparative chart of mineral composition in microalgae [1] ........83

Figure AP.1 Sequencing and comparison results on Blast of Li9 ..................102

Figure AP.2 Sequencing and comparison results on Blast of Li14 ................102

Figure AP.3 Sequencing and comparison results on Blast of Li21 ................103

Figure AP.4 Sequencing and comparison results on Blast of TH3 ................103

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Figure AP.5 Sequencing and comparison results on Blast of TH6 ................104

Figure AP.6 Sequencing and comparison results on Blast of TH13 ..............104

Figure AP.7 Sequencing and comparison results on Blast of TH14 ..............105

Figure AP.8 Sequencing and comparison results on Blast of TH17 ..............105

Figure AP.9 Sequencing and comparison results on Blast of TH21 ..............106

Figure AP.10 Sequencing and comparison results on Blast of TH24 ............106

Figure AP.11 Sequencing and comparison results on Blast of TH30 ............107

Figure AP.12 Sequencing and comparison results on Blast of TH35 ............107

Figure AP.13 Sequencing and comparison results on Blast of TH36 ............108

Figure AP.14 Sequencing and comparison results on Blast of TH38 ............108

Figure AP.15 Lobocystis planctonica [41] .....................................................109

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LIST OF ABBREVIATIONS

ACC Acetyl-coa carboxylase

ACP Acyl-carrier protein

BLAST Basic local alignment search tool

CFPP Cold flow properties

CN Cetane number

DGAT Diacylglycerol acyltransferase

EDTA Ethylenediaminetetraacetic acid

ER Endoplasmic reticulum

FAME Fatty acid methyl ester

G3P Glycerol-3-phosphate

GCMS Chromatography–mass spectrometry

ICP-OES Inductively coupled plasma optical emission spectrometry

KAS Ketoacyl-acp synthetizes

LCSF Long chain saturated factor

LPA Lysophosphatidate

MGDG Monogalactosyl diglycerid

NCBI National center for biotechnology information search database

PAUP Phylogenetic analysis using parsimony and other methods

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PCR Polymerase chain reaction

PG Phosphatidyl glycerol

PI Phosphatidyl inositol

SQDG Sulphoquinovosyl diglycerid

TAE Tris-acetate

TAG Triacylglyceride