Selection of microalgae species suitable for lipid production and nutritious biomass masters thesis major sciences and management of the environment

This study performs exploit indigenous microalgae species with potential for biofuel production.. Because of these microalgae cells which content lipid, carbohydrate and protein, for eac

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

The project was completed at The Industrial University of Hochiminh City

Supervisors:

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

(Write full name and signature)

SCIENCE, ENGINEERING AND MANAGEMENT

1

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

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

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

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

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

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.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

LIST OF ABBREVIATIONS

ACP Acyl-carrier protein

KAS Ketoacyl-acp synthetizes

PCR Polymerase chain reaction

INTRODUCTION

1 The reason for choosing the topic

For a recent century, people have started to find a new generation of energy which are a renewable energy that comes from geothermal, hydropower, solar energy, wave power, wind power and so on And one of the sources of renewable energy that has plenty advantages is microalgae such as:

 The rapid growth of microalgae (2-5 days) with the content about 50% of lipid compared to total mass Microalgae has a potential higher than corn and grass 6-12 times [3] Potential of triglycerides production is higher (45-220 times) than terrestrial plants [4];

 The requirement of small areas for breeding and reusing the land without ability for farming Facing with the overpopulation, microalgae farming becomes a good resolution to increase the productivity and decrease the land requirement;

 The high biodiversity The selection the suitable strain depends on the type

of products (biodiesel, bioethanol, biogas or feeding cattle….) Because of these microalgae cells which content lipid, carbohydrate and protein, for each purpose, we choose the strain and covert to biofuel;

 Microalgae can be cultivated on various conditions such as marine, brackish water, fresh water and even wastewater These convert nitrogen and phosphate in wastewater to biomass and perform photosynthesis by carbon dioxide

The lipid recover are not only interesting but microalgae also have a potential for animal feeding due to the high nutritious biomass Therefore, many applications of microalgae were introduced as a safe source more than adding chemical into the cattle feeding

With the recognition of a huge application of microalgae in renewable energy as

strain of microalgae In this study, I perform the isolation and identification of microalgae in wastewater of shrimp farming in the middle of Vietnam, and compare the productivity of lipid recover from biomass

We conducted a selection of microalgae for the purpose of lipid recovery and nutritious biomass including the following:

 Isolate and identify microalgae from Ninh Thuan Province;

 Analysis the lipid content;

 Analysis the nutritious content

2 Objectives of the study

 Compare the productivity of lipid recover from biomass

 Classify the strains to apply on biofuel and cattle feed

3 Subject and scope of the study

3.1 Research subject

The research subject of this thesis are the microalgae present in shrimp pond at Ninh Thuan province

4.2 Practical implication of research

The thesis contributes to the development of biodiversity of microalgae species at Ninh Thuan province, which provides potential strains for the effective recovery of biomass

5 The methodology

5.1 Collecting information

Document information collection, data on:

 Natural conditions of Ninh Thuan province, the situation and characteristics

of aquaculture industrial in Ninh Thuan

 The ecological characteristics of microalgae in general and distribution of microalgae in Ninh Thuan province

 The condition of biomass application in renewable energy and cattle feeding

5.2 Method of sampling analysis

Sample survey for Isolation

Sampling at shrimp pond in Ninh Thuan province:

 The number of samples: 4 samples/time of x 4 = 16 samples

Sampling schedule

Sample survey for lipid and nutritious analysis

Sampling the biomass of microalgae isolated:

 Sampling volume: 5g

 The number of samples: Number of samples isolated x 5g

 The analysis parameters: lipid content, nutritious content

5.3 Comparative method

Base on the results of analysis, DNA sequence will be compare on National Center for Biotechnology Information (NCBI)

5.4 The main techniques used

 Finch TV software is to edit the genetic code

 Seaview software for comparison between selected genetic code

 Mega 5.0 software and PAUP 4.0 software to calculate and construct the phylogenetic tree

CHAPTER 1 LITERATURE REVIEW

In 2001, "List of plant species in Vietnam" has shown about 2000 microalgae species in Vietnam whereas it has been about 40.000 species in the world discovered and predicted only about 11% of total global available species Microalgae are mainly distributed in the waters, but are also found on the surface of all type of soils Although they are generally free-living, a certain number of microalgae live in symbiotic association with a variety of other organisms

1.1.1 Types of cell organization

Microalgae have different types of cell organization: unicellular flagellate and unicellular non- flagellate, colonial flagellate and colonial non-flagellate and filamentous (unbranched and branched) most of microalgae are phototrophic while some are heterotrophs depending on the environment conditions

Most of the unicellular Cyanobacteria are nonmotile but gliding and swimming

motility may occur Swimming motility are found in a Synechococcus sp and

Baeocytes may have a gliding motility In motile forms, the motility is due to the presence of flagella The colonial cells may be organized into coenobic forms with a

fixed number of cells in the colony (e.g Scenedesmus), or into non-coenobic forms with a variable number of cells (e.g Pediastrum) The filamentous type are

organized in nonmotile but zoospores and gametes are expected

no membrane-bounded organelles (Fig 1.1)

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

In longitudinal section Abbreviations: cw - cell wall, t - thylakoids, cs - carboxysomes, n - nucleoplasm with DNA fibrils Scale = 0.5 µm

The eukaryotic microalgae possess a true membrane-bounded nucleus, which contains the major part of the genome distributed on a set of chromosomes, and the nucleolus They have cytoplasm divided into compartments and membrane-bounded organelles (Golgi body, mitochondria, endoplasmic reticulum, vacuoles, centrioles and plastids) devoted to specific functions (Fig 1.2) Many microalgae are uninucleate, those with multinucleate cellular organization (coenocytic) usually have a peripheric cytoplasm containing nuclei and chloroplasts, which are the most important plastids

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

In longitudinal section Abbreviations: cw – cell wall, ch – cup shaped chloroplast, t – thylakoids, st – starch grains (leucoplasts), n – nucleus, nl – nucleolus, m – mitochondria Scale ¼ 1 mm

1.1.3 Ultrastructure and cell division

1.1.3.1 Prokaryotes

Cyanobacteria and Prochlorophytes have a four layered cell wall which is of the Gram-negative type; the structural part consists of a peptidoglycan layer, outside which there is a lipopolysaccharide layer Beneath the cell wall there is the plasma membrane, or plasmalemma It is a thin unit membrane of about 8nm thickness In the cyanobacterial cell, thylakoids are the most evident membrane system; they lie free in the cytoplasm and contain the photosynthetic apparatus The cell inclusions

of cyanobacteria are the glycogen granules, cyanophycin granules, carboxysomes, polyphosphate granules, lipid droplets, gas vacuoles, and ribosomes

Cell division through binary fission, with constriction of all the wall layers that grow inward, or invagination of the plasma membrane and peptidoglycan layer without involvement of the outer membrane Cell division may also occur by multiple fission leading to the formation of baeocytes A very particular type of cell division, similar to budding, occurs in Chamaesiphon Cyanobacteria also reproduce

by fragmentation (hormogonia) Moreover, some filamentous genera produce akinetes Although the cyanobacteria have no evident sexual reproduction, genetic recombination by transformation or conjugation may occur

1.1.3.2 Eukaryotes

The microalgal cell wall is generally composed of a microfibrillar layer of cellulose, which may be surrounded by an amorphous layer The cell wall is secreted by the Golgi apparatus It may be silicified or calcified, and it may be strengthened with plates and scales Some species are naked, lacking the cell wall Outside the outer amorphous layer there may occur a laminated polysaccharide investment The nature of the outer cell wall layers supports polysaccharide production (alginates, agar and carrageenans) from various macro algae as well as from the microalga

Porphyridium

The plasma membrane is a thin unit membrane that bounds the cytoplasm The Chryptophyta do not possess a cell wall but there is an outer cell wall covering the cytoplasm, called periplast In the Euglenophyta the proteinaceous outer covering is called pellicle

The cytoplasm contains the nucleus and different kinds of organelles and compartments formed by invagination of the plasma membrane and endoplasmic reticulum Among the organelles there are: chloroplast, Golgi apparatus, endoplasmic reticulum, ribosomes, mitochondria, vacuoles, contractile vacuoles, plastids, lipid globules, flagella, and microtubules The nucleus is bounded by a double nuclear membrane; it contains the nucleolus and several DNA molecules distributed among the chromosomes, and undergoes division by mitosis

The chloroplast contains a series of flattened vesicles, or thylakoids, containing the chlorophylls, and a surrounding matrix, or stroma Thylakoids also contain phycobiliproteins in phycobilisomes in the Rhodophyta, whereas in the Cryptophyta the phycobiliproteins are dispersed within the thylakoids Thylakoids can be free or grouped in bands Pyrenoids can occur within chloroplasts A double membrane

envelops the chloroplast; in some algal division besides this double membrane one

or two membranes of endoplasmic reticulum are present

Cell division and reproduction

Vegetative reproduction by cell division is widespread in the algae to an increase in cell or colony size Other types of asexual reproduction occur by fragmentation and

by production of spores Although sexual reproduction occurs most of the species, it

is not a universal feature in algae It involves the combination of gametes, often having different morphology and dimension, from two organisms of the same species

1.1.4 Microalgal systematics

1.1.4.1 Classification

The hierarchy of biological classification has various ideas, according to one of those concepts, there are two kingdoms and microalgae belong to plant kingdom According to Copeland, there are four kingdoms, microalgae belongs to Mesoroans kingdom along with fungi and protozoa According to Whittaker, red algae, brown algae and some of green algae belong to Plant kingdom while others belong to Mesoroans kingdom In 1975, Gordon introduced a system with 19 kingdoms with algae are defined as 7 kingdoms such as: Rhodophyta, Euglenophyta, Haptophyta, Gryptophyta, Dinophyta, Eustigmatophyta, Geterocontophyta and green algae belongs to Plant kingdom Gordon F Leedale also diveded them into 12 phylums as: Rhodophycophyta, Cryptophycophyta, Dinophycophyta, Haptpphycophyta, Chrysophycophyta, Xanthophycophyta, Eustigmatophycophyta, Bacillariophycophyta, Phaeophycophyta, Euglenophycophyta, Chlorophycophyta, Charophyta

The current systems of classification of algae are based on the following main criteria: kinds of pigments, chemical nature of storage products and cell wall constituents Additional criteria take into consideration the following cytological

flagella, scheme and path of nuclear and cell division, presence of an envelope of endoplasmic reticulum around the chloroplast, and possible connection between the endoplasmic reticulum and the nuclear membrane

Lee (1989) was one of the first scientists to stress the phylogenetic importance of the additional membranes around the chloroplast envelope He separated the algal divisions into four groups

 The first group includes the prokaryotic algae: Cyanobacteria and Prochlorophyta The other groups are classified with respect to the evolution of the chloroplast, and include the eukaryotic algae, which probably acquired the chloroplast along different evolutionary events

 The second group, which includes Glaucophyta, Rhodophyta and Chlorophyta, has the chloroplast surrounded only by two chloroplast membranes

 The third and fourth group have the chloroplast surrounded respectively by one (Dinophyta and Euglenophyta) or two additional membranes of the endoplasmic reticulum (Cryptophyta, Chrysophyta, Prymnesiophyta, Bacillariophyta, Xanthophyta, Eustigmatophyta, Raphidophyta and Phaeophyta) The phylum Prochlorophyta contains chlorophylls a and b and, according to Castenholz (2001), the described genera (Prochloron, Prochlorothrix and Prochlorococcus) are included in the phylum Cyanobacteria

1.1.4.2 General description of major Divisions and Classes

1.1.4.2.1 Prokaryotes

Cyanobacteria (Cyanophyta and Prochlorophyta)

The Cyanophyta and the Prochlorophyta are prokaryotic algae that contain chlorophyll a The traditional name of blue-green algae for the Cyanophyceae is due

to the presence of phycocyanin and phycoerythrin, which usually mask the

chlorophyll pigmentation The main storage product is glycogen (a-1,4-linked glucan) The cells may occur singly or in filaments, unbranched or branched, with uniseriate or multiseriate arrangment The cells may aggregate to form colonies, which are surrounded by a firm or amorphous mucilage Filaments may have cells differentiated into heterocysts and/or akinetes Some planktonic forms can float owing to the presence of gas vacuoles, and most of the filamentous forms have gliding motility The Cyanophyceae have a cosmopolitan distribution and inhabit marine and freshwater environments, moist soils and rocks, either as freeliving or as symbiotic organisms

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

In cross-section Abbreviations: cw – cell wall, t – thylakoids, n – nucleoplasm, cs – carboxysomes, cy – cyanophycin granule and ph – poly-hydroxyalkanoate granules Scale ¼ 0:5 mm

1.1.4.2.2 Eukaryotes

Rhodophyta

The class Rhodophyceae, or red algae, includes multicellular and filamentous forms, whereas unicellular species are less represented These algae have chlorophyll a and d, phycobiliproteins (phycoerythrin and phycocyanin), and floridean starch (a-1,4-linked glucan) as storage products accumulated in the

distributed mostly in temperate and tropical regions The red microalga Porphyridium is an important source of sulphated polysaccharides, and of polyunsaturated fatty acids, such as arachidonic acid

Chlorophyta

The Chlorophyta, or green algae, embrace a large group of organisms with a great morphological variability, ranging from microscopic to macroscopic forms They comprise four classes: Micromonadophyceae, Charophyceae, Ulvophyceae and Chlorophyceae They have chlorophyll a and b and several carotenoids, that may be synthesized and accumulated outside the chloroplast under conditions of nitrogen deficiency and/or other stress, colouring the alga orange or red The storage product

is starch (a-1,4-linked glucan) Cell walls generally contain cellulose Some species are naked Chloroplasts may have an eyespot and pyrenoids The group includes coccoid, unicellular or colonial flagellates, multicellular or multinucleate filaments The green algae are cosmopolitan They are primarily freshwater, but a great number grow in marine, terrestrial and subaerial habitats Some species occur in symbiotic associations, mostly with lichens

Dinophyta

The class of Dinophyceae, formerly named Pyrrophyceae, includes a diverse assemblage of unicellular biflagellate planktonic algae of marine and freshwater habitats They have chlorophylls a and c2 and carotenoids Starch is accumulated in the cytoplasm outside the chloroplast The cell is composed of two parts, an epicone and a hypocone, and divided by the transverse girdle Perpendicular to the latter there is a longitudinal groove A layer of flat vesicles usually containing the cellulose plates surrounds the cell wall The Dinophyceae can produce large blooms, red or brown tides that colour the water and are highly toxic They are the main contributors to marine bioluminescence The dinoflagellates are a potential source of ω-3 unsaturated fatty acids, eicosapentaenoic and docosaesaenoic acids

Chrysophyta

The Chrysophyta or golden-brown algae include two classes: the Chrysophyceae and the Synurophyceae They mainly occur in freshwaters, especially in oligotrophic waters low in calcium The chloroplasts contain chlorophylls a and c1,

c2, fucoxanthin and β-carotene, which are responsible of the goldenbrown colour The storage product is chrysolaminarin (b-1,3-linked glucan), which is accumulated

in a cytoplasmic vesicle Most of the species are unicellular or colonial Cells usually have two different apical flagella, one smooth, the other hairy, and contain two parietal chloroplasts with an eyespot The cell wall is often lacking, or composed of cellulose Silicified scales, polysaccharidic envelopes, or loricas and various cytoplasmic processes may occur external to the cell wall Characteristic of the chrysophyta is the formation of special resting spores, statospores, enclosed in a silicified wall Some species require vitamins and growth substances Ochromonas malhamensis, which requires vitamin B12 to grow, has been used as an assay organism for this vitamin

Prymnesiophyta

The Prymnesiophyta, also named Haptophyta for the presence of a thin filamentous appendage between two smooth flagella, called haptonema, include only the class Prymnesiophyceae The cells are flagellate and have chlorophyll a, c1 and c2 and fucoxanthin as the major carotenoid The storage product is chrysolaminarin (b-1,3-linked glucan) These microalgae are widespread in marine environment, forming a major part of marine phytoplankton Since many years, Prymnesium parvum is known for producing a potent exotoxin lethal for fish and molluscs Recently, toxic blooms of Chrysochromulina polylepsis have been reported Strains of Isochrysis and Pavlova are investigated as a source of polyunsaturated fatty acids (PUFAs)

Bacillariophyta

The class of Bacillariophyceae, or diatoms, includes a very conspicuous number of golden brown unicellular organisms The diatoms live mostly singly or attached to

one another in chains of cells or in colonial aggregations, in aquatic and terrestrial

habitats Their colour is due to the masking of the green colour of chlorophylls a, c 1 and c 2 by the brown and yellow pigmentation of the fucoxanthin and β-carotene The storage product is chrysolaminarin (β-1,3-linked glucan) Lipids are also present The cytoplasm is enclosed in a siliceous cell wall, the frustule, showing different structures and ornamentation, which are used as key features for diatom classification The frustule consists of two overlapping halves joined by a girdle The upper longer and wider half (epitheca) fits on the lower half (hypotheca) as the cover of a box Cells contain two parietal chloroplasts; the nucleus is laterally or centrally placed and suspended by protoplasmic threads Pyrenoids are sometimes present Some diatoms may have a central raphe, or fissure Diatoms with a raphe possess gliding motility There are two major groups of diatoms: the pennate diatoms with bilateral symmetry and the central diatoms with radial symmetry The latter are mostly widespread in marine environments, where they have a key role in food chains Diatoms include photoautotrophic, auxotrophic and colourless heterotrophic species Deposits of fossil diatoms, known as diatomaceous earth, have many industrial uses (filtration and absorption processes), while commercial uses of living cells are mainly related to the aquaculture, since some diatoms contain significative amounts of PUFAs, especially eicosapentaenoic acid

Xanthophyta

The class of Xanthophyceae, also known as Tribophyceae, comprises freshwater and terrestrial species Only few members are marine The yellowgreen colour is

due to the presence of both chlorophyll a and carotenoids Chlorophyll c may also

be present, whereas fucoxanthin is absent Most species are unicellular or colonial The storage product is chrysolaminarin (β-1,3-linked glucan) Mannitol and glucose are also accumulated in plastids

Eustigmatophyta

The class of Eustigmatophyceae includes unicellular and coccoid organisms producing a small number of zoospores and living in freshwaters and soil The

chloroplast have chlorophyll a; violaxanthin is the major light-harvesting carotenoid

pigment The cell wall is polysaccharidic Unlike the Chrysophyta, the eyespot is not enclosed in the chloroplast Cytoplasm and photosynthetic lamella lipids of many species are promising sources of eicosapentaenoic acid, particularly in Monodus subterraneus

Rhaphidophyta

The class of Rhaphidophyceae, or chloromodas, includes a small group of

unicellular flagellate containing chlorophyll a, c 1 and c 2, and some carotenoids, often fucoxanthin in marine species and heteroxanthin in freshwater species The cells are naked and have two different apical or subapical flagella; there is no eyespot The cytoplasm is subdivided into a peripheral part occupied by a layer of many discoid chloroplasts and vacuoles, and a central part containing the nucleus

and the mitochondria Species of marine genera (e.g Fibrocapsa japonica) may

give rise to massive blooms, red tides, that cause serious damages to fish-farming

1.2 Lipid metabolism in microalgae

Microalgae contains lipid which have ingredients being equivalent to the components of vegetable oil Lipid content on algae is about 20-40% per dry matter while in some typical conditions, it can reach to 85% per dry matter In microalgae, lipid is an ester of glycerol, a long chain fatty acid C14-C22, saturated fatty acid or

unsaturated fatty acid For an example, Spirulina has a high value of unsaturated

fatty acid about 25-60% per total In Eukaryote algae, Triglyceride is an important compound of accumulation and can reach to 80% per total lipid Some featured groups of lipid include: Sulphoquinovosyl diglyceride (SQDG), monogalactosyl diglyceride (MGDG), digalatosyl diglyceride (SQDG), lecithin, phosphatidylglycerol (PG) and phosphatidyl inositol (PI) Besides, some improtant unsaturated long chain fatty acids include acid linoleic (C18:2), linolenic (C18:3), arachidonic (C20:4) and eicosapentaenoic (C20:5)

Microalgae consume sunlight to convert CO2 to bioenergy such as food and other products However, in stress conditions, with poor nitrogen, algae will adapt to accumulate lipid in cells [6] All nitrogen and CO2 consuming from environment is used for enzyme synthetic required for living activity and cell structure

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

[7]

(i) = acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS)

(ii) = fatty acid thioesterases and acyl-CoA synthetases

(iii) = TAG biosynthesis enzymes, including acyl-CoA:diacylglycerol acyltransferase (DGAT)

(iv) = oil body formation

(v) = ADP-glucose pyrophosphorylase and starch synthase

Free fatty acid is synthetized on chloroplast and exported to Endoplasmic reticulum (ER) to convert to triacylglyceride (TAG) and then they bud off into oil bodies in the cytosol (figure 1.4)

TAG biosynthesis pathway on microalgae cell includes 3 step [8]:

1) The biotin-dependent carboxylation of acetyl-CoA to form malonyl-CoA by acetyl-CoA carboxylase (ACC) ACC is a protein from E.coli, containing four subunits which are encoded by genes accA, accB, accC, accD that are located at different positions on the chromosome

2) Once malonyl-CoA is synthesized It is transferred by ACP transacylase to the acyl-carrier protein (ACP) of the fatty acid synthase (FAS) multi enzyme complex FAS catalyzes fatty acid elongation by condensing malonyl-CoA molecules and acetyl-CoA or condensing multi acetyl-CoA to form acetyl-ACP Then, the acetyl-group is transferred to another subunit of the FAS, the ketoacyl-ACP synthetizes (KAS)-enzyme which catalyzes the condensation of malonyl-ACP or the growing acyl chain to form ketobutyryl-ACP or ketoacetyl-ACP, after that, they condensed with another malonyl-CoA This cycle is repeated until the saturated chain of a palmitic (16:0) or a stearic acid (18:0) is formed At last, ACP-thioesterase cleaves the acyl chain and liberates the fatty acid

3) The first step of TAG synthesis is the condensation (acylation) of phosphate (G3P) with an acyl-CoA to form lysophosphatidate (LPA), which is

glycerol-3-catalyzed by acyl-CoA (glycerol-sn-3-phosphate acyl-transferase (GPAT)) The

LPA is then further condensed, catalyzed by acyl-CoA: phosphate acyltransferase (GPAT), with another acyl-CoA to produce phosphatidate (PA) At last, synthesis of TAG is catalyzed by acyl-CoA: diacylglycerol acyltransferase (DGAT) TAGs can then be stored in oil bodies

acylglycerol-sn-3-Figure 1.5 The fatty acid and TAG biosynthesis [8]

1.3 Application of microalgae on biofuel

Microalgae are concerned as a new source for various application fields People select the microalgae from a wild environment for applying to biofuel production, wastewater treatment and so on

In the report of Meilin He in 2011 [9], they are collected the samples from natural water bodies to isolate strains and compare the effect of H2 production:

 Fifty-two strains have their potential to produce H2 under anaerobic conditions

 Twenty-eight strains include 13 strains of green algae in fresh water, 12 marine blue algae and 3 cyanobacteria to evolve H2 production under sulfur

or nitrogen deprivation

The samples from various marine, freshwater and terrestrial environment around China have been collected and studied in 3 years to select the most effective strains for H2 production Two freshwater strains (Chlorella protothecoides and

Parietochloris incise) and eight marine strains (Chlorella capsulata, Chlorella autotrophica, Tetraselmis helgolandica, Tetraselmis tetrathele, Tetraselmis striata, Nannochloropsis sp., Pyramimonas sp and Dunaliella apiculata) were found and

presented as H2 production strains Under test conditions, freshwater strains were proved to be a higher H2 producer compared to marine strains

In other researchs, people attempted to find strains that could grow in domestic wastewater under dark conditions with high lipid content [42] Three strains were

isolated: Scenedesmus sp ZTY2, Scenedesmus sp ZTY3 and Chlorella sp ZTY4

The density increased 203% for Scenedesmus and 60.5% for Chlorella After

11-day cultivation, the lipid content of Scenedesmus sp ZTY2, Scenedesmus sp ZTY3 and Chlorella sp ZTY4 reached 69.1%, 55.3% and 79.2%, respectively The DOC removal efficiencies of the three strains ranged from 52.9% (Scenedesmus sp ZTY3)

to 64.4% (Chlorella sp ZTY4)

Beside of biofuel, microalgae are also a source for cattle feeding

1.4 Application of Microalgae on Cattle feeding

Compared to other microorganisms, Spirulina (Arthrospira platensis)can be

cultivated in high saline water and alkaline conditions which give an advantage to function as a feedstock for livestock feed [10] Recently, studies regarding growth and body conformation responses of genetically divergent Australian sheep to Spirulina supplementation revealed that Spirulina could be increase in weight, growth and body conformation significantly [11] Feeding lipid-encapsulated algae supplements may increase n-3 content in milk fat without adversely affecting milk

fat yield [12] The annual Porphyra sp harvest worldwide has been estimated to be worth of US$2.5 billion Porphyra sp primarily contains vitamin A, vitamin B,

vitamin C, beta-carotene, as well as essential minerals including iodine Besides

that, a study on Laminaria digitata suggested that algae supplemented feed has

increased the pig weight up to 10% on a daily basis [13, 14]

Poultry feed

Institute für Getreideverarbeitung (Bergholz-Rehbrücke, Germany) produces a

natural feed with the algae Chlorella sp and Arthrospira sp called Algrow [15]

Aquaculture feed

Phorphidium valderianum was successfully used as feed for aquaculture (based on

its nutritional performance and non-toxic properties) [13] The diatom Thalassiosira

pseudonana is widely cultivated to feed variety of mollusks, including the Pacific

oyster Crassostrea gigas and rock scallops [14]

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

Following Ministry of agriculture and rural development, institute of economics and planning of aquatic resources - Report on the summary of the planning on the development of aquatic animals in the middle province toward 2020 and orientations to 2030 [16]

The area of aquaculture in the central provinces of Vietnam increased at an average growth rate of 3.06% per year

 The area of saltwater and brackish water in the central provinces continued

to increase at an average rate of 5.08% per year, including the increasing of shrimp farming area, lobster farming, marine fish farming

 The area of freshwater tended to decrease slightly 0.04% per year The production of freshwater in those provinces in this period rose to an average rate of 6% per year Of which, traditional fish production always accounts

for the largest share (in 2014, accounting for 58.2% of total freshwater production in central provinces)

In this period, aquaculture productivity in the Central provinces ranged around 3.3 tones/ha Saltwater and brackish water yields decreased 2.86% per year from 4.4 tons/ha in 2010 to 3.9 tons/ha in 2014; the productivity of sweet-raising during this period increased at an annual average of 6.05% from 1.4 tones/ha in 2010 to 1.7 tones/ha in 2014 The increase of aquaculture area of the region tends to increase not much and gradually stable due to several reasons:

3.1- There are overlapping developments of aquaculture with other economic sectors such as tourism, industry, urban development

 Polluted environment directly affects the aquaculture, disease outbreaks occur more frequently, leading to many regions not investing in production

Salinity and brackish productivity tend to decrease in the period 2010-2014 In

2010, the productivity of brackish-water aquaculture was 4.4 tons/ha, down to 3.9 tons/ha in 2014 This was due to:

 Some shrimp farming areas develop spontaneously leading to environmental pollution and epidemics that affect production efficiency

 Cage culture in cages in the bays in recent years has also encountered many difficulties, some areas are located in the area of tourism activities, transportation should not increase the farming area but According to the new planning, they must be relocated to the gates of the bay, causing difficulties for production As lobster farming area in Vung Ro, cage farming area in Cam Ranh

In the period 2010-2014, the shift from black tiger prawn to TCT is due to the following reasons:

 Due to the long tiger shrimp culture period, 4-5 months/crop on average, disease situation on tiger shrimp is quite complicated, causing serious damage to farmers

 Many shrimp farming areas have been converted to TCT shrimp (tôm thẻ chân trắng-Whiteleg shrimp) because they are easy to feed and can be raised with high density and high productivity or mixed with others

1.6 Characteristics of industrial shrimp waste water

According to research on wastewater samples from Shrimp farming in Can Gio District, Ho Chi Minh City is analyzed to determine the physical and chemical criteria [2] The analysis results are presented in Table 1.1 Average daily, day prawn ponds will replace about 15-20 cm of water level in the pond There will be about 150-200 m3 of effluent discharged from each 1000 m2 pond Analytical results show that the composition of shrimp wastewater in Can Gio area contains all necessary factors for the development of microalgae The study by Hillebrand and Sommer [17] determined that the environment with N and P supply balance, an optimal ratio of C: N: P of 46.1: 7.7: 1, was the best fit for the development of microalgae In the shrimp culture wastewater, the N: P ratio was 6.5: 1, which was lower than the optimal ratio However, total nitrogen and total phosphorus content

of 15.7 mg/L and 2.4 mg/L, respectively, are still suitable for the development of microalgae Other physicochemical characteristics such as pH, conductivity, salinity, alkalinity, COD, BOD, and micronutrient compositions within the

tolerance range of the microalgae used in the study were S.platensis, C.vulgaris and

D.tertiolecta [18]

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

CHAPTER 2 MATERIAL AND METHODS

2.1 Material

2.1.1 Samples

Microalgae samples were collected at Ninh Thuan Province, Central of Vietnam

Figure 2.1 Location of Ninh Thuận in Vietnam

Samples are collected from 5 different points for each of 5 ponds Each point is collected at 0.5 meter from the water surface for about 500 mL The samples are collected every three months and four times per year at January, April, July and October (table AP.1 - Appendix 1)

The sample after collected are transferred to lab for preservation and analysis

Figure 2.2 Sampling diagram [1]

Location: Shrimp pond in Ninh Thuan province – SEED SEASONING CENTER I

NINH THUAN

Number of sample: Take 5 samples for 5 ponds from the Shrimp farm including 4

ponds of cultivation and disposal pond

Combination pattern: Collect 4 points at the side lake and 1 point at the middle of

the lake (show in figure AP.6) at 0.5 meter from the water surface for about 500

mL Samples from 5 points were mix together about 2.5 liter

Treatment and preservation: Samples after collected are stored with ice through

the transfer time back to laboratory, after that, samples are kept in fridge at 2℃ for the next step

2.1.2 Instruments -Chemical -Laboratory materials

Instruments

3 Electrophoresis and Blotting Bio-rad

6

Basic equipment and tools in

biochemistry and biochemical

research

-

Chemical

2 NaH2PO4.H2O Guangzhou Jiahuan Appliance Technology Co.,Ltd

3 Na2SiO3.9H2O Xilong chemical Co.Ltd

4 Na2C10H14O8N2.H2O Xilong chemical Co.Ltd

5 CoCl2.6H2O Xilong chemical Co.Ltd

6 CuSO4.5H2O Xilong chemical Co.Ltd

7 FeCl3.6H2O Xilong chemical Co.Ltd

No Chemical Supplier

8 MnCl2.4H2O Xilong chemical Co.Ltd

9 Na2MoO4.2H2O Xilong chemical Co.Ltd

10 ZnSO4.7H2O Xilong chemical Co.Ltd

11 Thiamin HCl Himedia Laboratories

14

Plant genomic DNA

purification mini kit

#K0791

Thermo scientiific

2.2 Experiment

The samples are cultivated by Guillard F/2 culture medium on the petri dish at temperature 25oC to 27oC with white fluorescent light for 1-3 week The expect collection from 20 to 30 strains are identified and analyzed the lipid and nutrient to select the best strain which high productivity

2.3 Methods

2.3.1 Medium culture and Isolation method

The samples collected at the field are introduced to Guillard F/2 culture medium to grow up the microalgae population for sever analysis The samples were inoculated into 30 cm3 Guillard F/2 culture medium in a 50 cm3 centrifuge tube and incubated

Figure 2.3 Experiment procedure [1]

on orbital shakers machine at 27C with continuous white fluorescent light for 1-2 weeks Several samples was made and observed under microscope to define the suitable one which increases microalgae’s population for isolation step

The microalgae were isolated by streaking on agar plates with Guillard F/2 culture medium At first, putting on 0.1 mL of the sample after treating on over the surface

of the agar medium and spreading evenly The petri dishes are incubated at room temperature with white light After 5 - 10 days, the microalgae colonies will grow

on the surface of agar

Observed the characteristics of microalgae colonies by visualization and under the microscope to select the separated colonies introduces these strains into a new culture medium for pure breeding The process can be repeated many times until the strains are pure without bacterial and fungi either

2.3.2 Identification

2.3.2.1 Identification by traditional method

The traditional method to classify and identify microalgae is based on morphology, nutritional form and ecological characteristics Aside from identify by gene sequence, identify by traditional method still play an important role Due to Stackebrandt et al (2002), typical phenotypes are one of an important criteria to assess new identify group Based on that, microalgae is identified by comparing the typical phenotype and structure which observed on light microscope with information on internet, combined with classification from Van den Hoek [19]

2.3.2.2 Identification by 18S rDNA gene sequencing

The ribosome DNA sequence (rDNA) of microorganism taking 0.3-0.4% of all sequence is a low evolute area and high conservation characteristics According to Stackebrandt (2002), 18S rDNA has a high value on classification and identification new species if no more than 5% nucleotide is unidentified Strains is determined as