Characterization of vietnamese microalgae strains for aquaculture wastewater treatment and biomass valorization

They were subjected to characterization in terms of growth, protein content, pigment and fatty acid content and profiles.. Those two strains were further characterized in terms of growt

UNIVERSITY OF LIEGE GENETICS AND PHYSIOLOGY OF MICROALGAE LABORATORY

Characterization of Vietnamese microalgae strains for aquaculture wastewater treatment and biomass

valorization

LUU THAO NGUYEN

A thesis submitted in fulfillment of the requirements for the degree of

Doctor in Sciences Academic year: 2019 - 2020

Promoter: Prof Claire Remacle

Co – promoter: Prof Gauthier Eppe

Acknowledgement

My gratitude firstly goes to Académie de Recherche et d’Enseignement Supérieur CCD, Brussels, Belgium) for its financial support during my thesis under the project

(ARES-“RENEWABLE” The research was performed in the laboratories of Genetics and Physiology

of Microalgae (InBios/Phytosystems Research Unit), and Laboratory of Mass Spectrometry (MolSys Research Unit) in Liege University, and Industrial University of Ho Chi Minh City

I would like to express my deepest gratitude to Prof Claire Remacle and Prof Gauthier Eppe for their dedicated supervision This work could not be completed without their valuable guidance as well as scientific orientation I also want to thank Prof Le Hung Anh for his great support during my study in the laboratory of Institute of Environmental Science, Engineering and Management,Industrial University of Ho Chi Minh City Prof Spiros Agathos, partner of the RENEWABLE project, is also acknowledged for his careful reading of the draft of the manuscript

I would like to thank all of my colleagues in Liege University and Industrial University of Ho Chi Minh City for their helps, kindness, encouragement and especially their sharing experiences

Last but not least, I am indebted to my family members, especially my mom and my wife, who help me take care of my two loved children during my study in Liege, Belgium

Summary

The study aims to isolate microalgal strains from shrimp – farm wastewater and select some

of them suitable for production of biomass, biodiesel and other valuable products We have

isolated 4 isolates, namely NL3, NL4, NL6 and NL12 The four isolates were preliminarily

identified using molecular techniques based on partial sequence of 18S rRNA gene and

classified into four different genera belonging to two phyla, Desmodesmus, Chlorella,

Nannochloris (Chlorophyta) and Nannochloropsis (Eustigmatophyta) They were subjected to

characterization in terms of growth, protein content, pigment and fatty acid content and

profiles As a result, isolates NL3 and NL6 were chosen for the project The two strains were further identified based on the whole sequence of 18S rDNA – ITS1-5.8S-ITS2 Isolate NL3 was identified as Desmodesmus sp NL3, which is proposed to be a new species with two group

I introns (S516 and S1046) while isolate nl6 was 100% identical to Nannochloropsis salina (D12, accession number JX185299.1) from Shandong in China, so named Nannochloropsis

salina NL6 Those two strains were further characterized in terms of growth, protein, pigment,

fatty acid profiles and salinity tolerance (10‰, 20‰, 30‰ and 35‰) The two strains are

tolerant to the different salinities studied Nannochloropsis salina NL6 was characterized by a

high percentage of fatty acids (40% DW at the end of exponential phase) and a fatty acid profile

suitable for biodiesel production Desmodesmus sp NL3 was characterized by high protein

(40% DW), biomass (1.54 g/L), and lutein (7 mg/g DW) contents at the end of the exponential

phase Notably, Nannochloropsis salina NL6 (N salina NL6) was able to produce

eicosapentaenoic acid (EPA) and astaxanthin Their performance on the production of these compounds did not change much across the four salinities The two strains, therefore, fit the project goals

Keywords: Microalga; Fatty acid; Vietnam; Nannochloropsis; Desmodesmus; Chlorella;

Nannochloris

Table of contents:

1 Introduction 12

1.2.2.1 Chlorophyta (Green algae) 16

1.2.2.2 Rhodophyta (Red algae) 16

1.2.2.3 Bacillariophyta (diatoms) 17

1.2.2.4 Eustigmatophyta 18

1.3 Algal cultivation system 20 1.3.1 Closed photobioreactors (PBRs) 20 1.3.1.1 Tubular photobioreactors 20

1.3.1.2 Flat plate PBRs 21

1.3.2 High rate algal pond (HRAP) 23 1.3.3 Comparison of PBRs and HRAPs 26 1.4 High value components of microalgae 27 1.4.1 Proteins 27 1.4.2 Lipids 28 1.4.3 Carotenoids 31 1.5 Application of microalgae 34 1.5.1 Animal feedstock 34 1.5.2 Microalgae in aquaculture 35 1.5.3 Microalgae used for production of Human food 36 1.5.5 Biofuel production 40 1.6 Bottlenecks in large - scale production of microalgae 42 2 Aim 46 3 Materials and methods 47 3.1 Materials 48 3.2 Methods 48 3.2.1 Isolation and purification of microalgae 48 3.2.2 DNA isolation and phylogenetic analyses 48 3.2.2.1 DNA isolation 48

3.2.2.2 Polymerase chain reaction (PCR) assay 49

3.2.2.3 DNA agarose electrophoresis 51

3.2.2.4 Phylogenetic analyses 51

3.2.3 Microalgal growth and biomass analysis 52

3.2.3.1 Microalgal growth 52

3.2.3.2 Biomass analysis 52

3.2.3.2.1 Dry weight 52 3.2.3.2.2 Fatty Acid Methyl Ester Analysis (FAME) analysis and estimation of biodiesel fuel properties 53 3.2.3.2.3 Protein analysis 56 3.2.3.2.4 Pigment analysis 57 4 Results and discussion 58 4.1 Isolation, identification and preliminary characterization of four microalgal isolates 59 4.1.1 Isolation and identification 59 4.1.2 Microalgal growth 68 4.1.3 Biomass analysis 70 4.1.3.1 Pigment profiles and protein content 71

4.1.3.2 Fatty acid profiles 72

4.2 Identification and characterization of strain NL3 74 4.2.1 Sequencing of the rDNA-ITS Region 74 4.2.2 Phylogenetic analysis of the NL3 sequence 77 4.2.3 Characterization 78 4.2.3.1 Microalgal growth in TAP medium 78

4.2.3.2 Microalgal growth in TAP medium at different salinities 78

4.2.3.3 Effect of growth phase and salinity condition on fatty acid profile 79

4.2.3.4 Effect of salinity condition on pigment profile 81

4.2.3.5 Effect of salinity condition and growth phase on biodiesel quality 82

4.3 Identification and characterization of strain NL6 83 4.3.1 Sequencing of the rDNA –ITS Region 83 4.3.2 Characterization 83 4.3.2.1 Microalgal growth 83

4.3.2.2 Effect of growth phase, salinity and light intensity condition on fatty acid profile 86 4.3.2.3 Effect of growth phase, salinity and light intensity condition on biodiesel quality 89 4.3.2.4 Effect of growth phase on pigment profile and protein content 91

5 General discussion and conclusion 93

6 Publication 98

7 Scientific activities 111

7.1 Participation at The 9th International Conference on Algal Biomass, Biofuels and Bioproducts,

17 – 19 June, 2019, Boulder, CO, USA (Poster), https://orbi.uliege.be/handle/2268/237197 112 7.2 Participation at AlgaEurope Conference, 3-5 December 2019, Paris, France (Poster),

References 114

List of Tables

Table 1.1 Summary of some characteristics of Chlorophyta, Rhodophyta, Bacillariophyta and

Eustigmatophyta 19

Table 1.2 Comparison of PBRs and HRAPs (Mata et al., 2010) 26

Table 1.3 Protein content in some microalgal species 28

Table 1.4 Oil productivitis from microalgae and other oil plants (Chisti, 2007) 29

Table 1.5 Lipid content in some microalgal species 31

Table 3.1 Primers used in this study 51

Table 3.2 FAME external standards preparation using (F.A.M.E Mix C8 – C24, SUPELCO, USA) 54 Table 3.3 Bovine serum albumin (BSA) standard preparation 56

Table 4.1 Isolates based on partial sequence of 18S rRNA gene amplified using primers NS1 and NS4 60 Table 4.2 Parameters of the four isolates 70

Table 4.3 Pigment profiles and protein contents in exponential phase for the four strains at light intensity of 50 µmol m2 s-1 72

Table 4.4 Fatty acid profiles in exponential phase of the four strains at light intensity of 50 µmol m2 s-1 74

Table 4.5 Biomass and protein contents of Desmodesmus sp NL3 in different salinities at light intensity of 200 µmol m-2s-1 79

Table 4.6 Fatty acid profiles of Desmodesmus sp NL3 in different growth phases at 200 µmol m-2s-1 80

Table 4.7 Fatty acid profiles of Desmodesmus sp NL3 in different salinities at light intensity of 200 µmol m-2s-1 81

Table 4.8 Pigment profiles of Desmodesmus sp NL3 in different salinities at light intensity of 200 µmol m-2s-1 82

Table 4.9 Biodiesel properties of Desmodesmus sp NL3 in different salinities and different growth phases at light intensity of 200 µmol m-2s-1 83

Table 4.10 Fatty acid profiles of N salina NL6 in different growth phases and light intensities 86 Table 4.11 Fatty acid profiles of N salina NL6 in different salinities at light intensity of 200 µmol m-2s-1 88

Table 4.12 Biodiesel properties of N salina NL6 in different growth phases, salinities and light intensities 90

Table 4.13 Pigment profiles and protein contents of of N salina NL6 in different growth

phases 92

List of Figures

Figure 1.1 Cellular structure of a typical green microalga, such as Chlamydomonas

reinhardtii (Shukla & Karthik, 2015) 15

Figure 1.2 A model of a horizontal tubular photobioreactor (Chisti, 2007) 21

Figure 1.3 The flat panel PhotoBioReactor (PBR) system (Lindblad et al., 2019) 23

Figure 1.4 A model of open raceway pond (Chisti, 2007) 24

Figure 1.5 Algal raceway pond of RENEWABLE project in the central province of Ninh Thuan, Vietnam The system is equipped with airlift device responsible for mixing and aerating 26 Figure 1.6 Chemical structures of some carotenoid compounds found in microalgae (Gong & Bassi, 2016) 33

Figure 1.7 The microalgae production process coupled with wastewater treatment (Zerrouki & Henni, 2019) 38

Figure 1.8 Conversion of oil into biodiesel via Transesterification (Chisti, 2007) 41

Figure 3.1 Summary of primers used and their annealing positions along 18S rDNA – ITS sequence 50 Figure 3.2 The TIC chromatogram of FAMEs (A) and mass spectra of ion fragments of peak at the retention time of 24.6 min, corresponding to the C18:1 (10-Octadecanoic acid, methyl ester) (B) The retention time, area of peak and its ion fragments are the data used to identify and quantify the content of FAME based on the FAME external standards stated in table 3.2 and database 55

Figure 3.3 The chromatogram of pigments: pigment components are separated on the column and presented as peaks Peaks are detected using UV detector and identified based on comparison with the retention time of pigment standards 57

Figure 4.1 Strains NL3, NL4, NL6 and NL12 on solid medium and under light microscope at magnification of 60X 62

Figure 4.2 Amplicons of strains NL3, NL4, NL6 and NL12 using primers NS1 and ITS4 and the ladder (Thermo Scientific) on agarose gel 1% 63

Figure 4.3 18S ribosomal RNA gene, complete sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence (3492 nucleotides) of Desmodesmus sp NL3 64

Figure 4.4 18S ribosomal RNA gene, complete sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence (2411 nucleotides) of Chlorella sp NL4 65

Figure 4.5 18S ribosomal RNA gene, complete sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence (2600 nucleotides) of N salina NL6 66

Figure 4.6 18S ribosomal RNA gene, complete sequence and partial sequence of internal transcribed spacer 1 (1784 nucleotides) of Nannochloris sp NL12 67

Figure 4.7 Phylogeny of isolate NL3, NL4, NL6 and NL12 along with their closely related

sequences obtained from NCBI database The rooted phylogenetic tree was constructed based

on 18S rDNA sequence Bootstrap values are the number mentioned on the branch nodes of the tree (1000 replicates) Thalassiosira antarctica strain T1 (EF140621.1) is defined as the outgroup to root the tree 68

Figure 4.8 Growth curves of the four strains in F/2 medium (24‰ NaCl) (Chlorella sp NL4,

Nannochloropsis sp NL6, Nannochloris sp NL12) and TAP medium (Desmodesmus sp NL3) under constant illumination with light intensity of 50 µmol m2 s-1 The arrow indicates the time to harvest sample for analysis 69

Figure 4.9 Predicted secondary structure of the group I intron S516 of Desmodesmus sp

NL3, located in the 18S rDNA Paired elements are indicated as P1 to P9, the intron sequence

is in upper case letters, the exon sequence in lower case letters 76

Figure 4.10 Phylogenetic position of isolate NL3 within Scenedesmaceae The phylogenetic

tree was built on a selected set of microalgal rDNA (18S–ITS1-5.8S-ITS2) sequences

available in GenBank, using maximum likelihood (ML), neighbor-joining (NJ) and maximum parsimony (MP) Bootstrap values after 1000 replicates are indicated at nodes for

ML/NJ/MP Filled and open circles indicate 100 or >95% BV support with all methods, respectively; hyphen, node not supported 77

Figure 4.11 Growth curve of Desmodesmus sp NL3 in TAP at light intensity of 200 µmol m

-2s-1 The arrow indicates the time to harvest sample for analysis 78

Figure 4.12 A Color change of N salina NL6 from green in exponential to yellow in

stationary phase, B Agar plate of nl6 after 10 days of growth C Agar plate of nl6 after several weeks of growth 84

Figure 4.13 Growth curve of N salina NL6 in F/2 (24‰ NaCl) at light intensity of 50 (A)

and 200 (B) µmol m-2s-1 The arrow indicates the time to harvest sample for analysis 85

Figure 4.14 Growth curve of N salina NL6 in F/2 at different salinities under 200 µmol m-2s

-1 The arrow indicates the time to harvest sample for analysis (n=2) 88

List of abbreviations

ADU Average degree of unsaturation

BLAST Basic Local Alignment Search Tool

BOD Biochemical oxygen demand

BSA Bovine serum albumin

EAA Essential amino acid

EPA Eicosapentaenoic acid

FAME Fatty Acid Methyl Ester

FAO Food and Agriculture Organization of the United Nations

HHV Higher heating value

HRAP High rate algal pond

IV Iodine value

kV Kinematic viscosity

LHCs Light-harvesting complexes

MUFAs Monounsaturated fatty acids

NCBI National Center for Biotechnology Information

PBRs Closed photobioreactors

PUFAs Polyunsaturated fatty acids

SFA Saturated fatty acids

SG Specific gravity

TAGs Triglycerides

TFA Total fatty acid

TSS Total suspended solids

UNU United Nations University

WHO World Health Organization

1 Introduction

1.1 Context

The economy of Vietnam has continuously improved and developed over the past three decades since 1986 when the government applied economic and political reforms Through this process, Vietnam has recently become a lower middle-income country, the poverty rate decreased by 3% and the GDP (Gross domestic product) growth rate increased from 6.8% in 2017 to 7.1%

in 2018 (The World Bank, 2019) However, agriculture development is still backward and causes pollution Among agricultural activities, aquaculture is one of the main sectors contributing to the economic development Indeed, Vietnam, with a 3,200 - km coastline spreading from the south to the north, is attractive for aquaculture and fishery activities which directly bring jobs to over 400,000 people in capture fisheries and two million people in support industries and related services However, the wastewaters from those activities are not treated properly and are released to the water environments, including the rivers and the sea Vietnam

is the number one exporter of value – added shrimps to Japan, the United States of America and the EU-28 and is the leader producer of black tiger shrimps in the world with around 300,000 metric tons per year (Cong, 2017) There are 685,000 hectares in the whole country used for shrimp cultivation most of which are in the Mekong Delta (604,000 hectares) (Phuong

et al 2015) Production of one ton of black tiger shrimps in Can Gio, Ho Chi Minh City was estimated to release 30 kg of Ntotal, 3.7 kg of Ptotal, 4.8 kg N-NH3, 259 kg BOD (Biochemical oxygen demand), 769 kg COD (Chemical oxygen demand), and 1,170 kg TSS (Total suspended solids) (Anh et al., 2010) A study has reported the pollution loads on shrimp farm sediment in Ca Mau province was 8.4 ± 3.3 kg N/ton and 5.9 ± 2.5 kg P/ton (Manh & Nga, 2011) Apart from being rich in nutrients, wastewaters from shrimp farms also contain viruses, bacteria, chemicals, and drug residues exposing the aquatic environment to a significant threat (Cong, 2017)

According to the Ministry of Agriculture and Rural Development, a plan to develop brackish shrimp industry out to 2030 was made in September 2018 of which the goal is to improve the productivity, production volume and the commercial value of shrimp products In particular, the shrimp production is planned to be doubled, reaching 13 million tons and by 2030 the export value of brackish water shrimp will increase by three times to reach US $12 billion (PECH-Committee, 2018)

To partially contribute to the sustainable agricultural development of Vietnam, the Renewable project (Removal of nutrients in wastewater treatment via microalgae and biofuel/biomass production for environmental sustainability in Vietnam) aims at the protection of coastal surface water and groundwater by using microalgae for the treatment of wastewater produced

by shrimp farming activities to reduce its pollution After this treatment, the biomass of microalgae could be utilized for the production of animal feedstock, biodiesel, and some high value products The study in this thesis is the first part of the Renewable project, in which some microalgae species have been isolated and screened for characteristics suitable for the second part of the project related to the study on wastewater treatment

1.2 Microalgae and classification

1.2.1 General information on microalgae

Microalgae are unicellular photosynthetic organisms able to survive and grow in various environmental conditions (Mata et al., 2010; Brennan & Owende, 2010; Tan et al., 2018), including fresh water, salt water, ice or hot acidic springs, for example (Rajkumar & Sobri Takriff, 2016) The majority of them is photosynthetic and autotrophic Through photosynthesis, microalgae use the sunlight energy and carbon dioxide to convert them into sugars like glucose which is then stored into polymers such as starch, which can be used for metabolism and growth of all life forms on the Earth directly or indirectly (Masojidek et al., 2004) Notably, marine microalgae are responsible for 50% of the total photosynthesis

activities that occur on our planet (Camacho Rubio et al., 2003) Few of them are heterotrophic (Enzing et al., 2014) and use organic matter as energy source (Van Den Hoek et al., 1995; De Martino et al., 2011) Basically, a eukaryotic microalgal cell is made up of compartments, including nucleus, chloroplasts, endoplasmic reticulum, mitochondria, vacuoles, Golgi apparatus, cytoplasm and cell wall (Lamond & Earnshaw, 1998; Tomaselli, 2004; Jensen & Leister, 2014) Owing to their simple reproductive and cell growth system, some microalgae species exhibit a high growth rate Microalgae are ubiquitous in the world and there have been more than 30,000 species identified (Mata et al., 2010)

Figure 1.1 Cellular structure of a typical green microalga, such as Chlamydomonas

reinhardtii (Shukla & Karthik, 2015)

1.2.2 Presentation of algae with commercial interest

We present here four major groups of microalgae

1.2.2.1 Chlorophyta (Green algae)

Green algae are a large group of organisms varying greatly in morphology, with unicellular or multicellular forms (Tomaselli, 2004) Species in this group may exist as coccoid, unicellular

or colonial flagellates, multicellular or multinucleate filaments Cellulose is sometimes a constituent of their cell walls (Tomaselli, 2004) There are more than 7,500 species of green algae living in a variety of habitats, including freshwater, marine, or even terrestrial environments This makes them the most diverse among different algae groups (Arora & Sahoo, 2015) They are subdivided into four classes, including Micromonadophyceae, Charophyceae, Ulvophyceae and Chlorophyceae (Tomaselli, 2004) Pigments include

chlorophylls a , b and carotenoids associated to membrane proteins complexes called

light-harvesting complexes (LHCs) in the thylakoids of the chloroplasts, which are surrounded by two membranes (Heimann & Huerlimann, 2015) Eyespots to detect light and pyrenoids where starch is accumulated are present in their chloroplasts The origin of green algae was plausibly caused by an endosymbiotic event between 1 and 1.5 billion years ago (Hedges et al., 2004; Yoon et al., 2004) In this process, a heterotrophic eukaryotic host cell captured a photosynthetic prokaryote (cyanobacterium) which later turned into a photosynthetic plastid (Archibald, 2009; Keeling, 2010) Nowadays, there are certain members in this phylum used

for commercial purposes, such as Dunaliella for biomass and β-carotene production Amotz, 2007), Chlorella, Dunaliella and Haematococcus in cosmetics (Borowitzka, 2013) and

(Ben-Haematococcus for astaxanthin used as a colouring agent in fish muscles (Ambati et al., 2014)

1.2.2.2 Rhodophyta (Red algae)

Fourteen genera are filamentous (<1 mm), such as Rhodaphanes, Bangiopsis, Chroodactylon, and Stylonema They account for a great number of seaweeds inhabiting mostly in temperate and tropical regions (Tomaselli, 2004) The three other genera, including Rhodosorus,

Rhodospora and Rufusia are unicellular (Vis & Sheath, 1993; West et al., 2014) They represent

6,500–10,000 species (Woelkerling, 1990), mostly marine (Sheath, 1984) In addition to

chlorophyll a, they possess phycobilins (red and blue) (Woelkerling, 1990) associated in

phycobilisomes in the thylakoid membranes of the chloroplasts and accumulate floridean starch (β-1,4-linked glucan) as storage product in the cytosol (Tomaselli, 2004).A microfibrillar layer

of cellulose or xylan and amorphous polysaccharidic mucilages (agar or carrageenans) are the constituents of their cell wall (Tomaselli, 2004) Agar and carrageenan are the products that make red algae commercially utilized (Tomaselli, 2004) Among members of Rhodophyta,

species in the genera Porphyridium and Rhodella have been drawing increased interest in their

valuable compounds, including extracellular polysaccharides, phycobilins and long chain polyunsaturated fatty acids (PUFAs) (Guihéneuf & Stengel, 2015; Pignolet et al., 2013)

1.2.2.3 Bacillariophyta (Diatoms)

Diatoms are a large group containing organisms which are golden brown and unicellular

Golden brown is the result of their pigment composition, including chlorophylls a, c 1 and c 2 ,

fucoxanthin and β-carotene They can be phototrophic, auxotrophic for vitamin B12 or colourless heterotrophic (Tomaselli, 2004; Vancaester et al., 2020) Nowadays, about 17,000 species (12,000 living and 5,000 extinct) from 500 genera of diatoms (350 living and 150 extinct) have been discovered (Hoek & Mann, 2009; Williams & Kociolek, 2011) Diatoms are subdivided into two main subgroups, including the pennate diatoms with bilateral symmetry and the central diatoms with radial symmetry A large number of the central diatoms are found

in marine environments, where their roles in the food chains are important (Tomaselli, 2004) Both freshwater and marine environments can be habitats of diatoms Most of them are unicellular, while few are colonial (Heimann & Huerlimann, 2015) Many epiphyte diatoms are found living on the surface of aquatic organisms including plants, molluscs, turtles, fishes, seaweeds etc (Kumar et al., 2015) Chrysolaminarin (β-1,3-linked glucan) is accumulated as

storage product in cytosolic vacuoles (Chiovitti et al., 2004) Their siliceous cell walls, called the frustules, enclose cytoplasm Frustules are variable in structure and ornamentation They are the important features to be used for diatom classification (Tomaselli, 2004) Recently, diatoms have been used in some fields, such as waste degradation (Marchetti & Cassar, 2009), toxicity testing (Atazadeh et al., 2009), biomineralization (Yamazaki et al., 2010), and nutritional applications (Bozarth et al., 2009) The highest nutritional values of diatoms come from PUFAs, such as eicosapentaenoic acid (EPA), arachidonic acid, and docosahexaenoic acid (Bozarth et al., 2009)

1.2.2.4 Eustigmatophyta

Members in this phylum can be characterized by their pigment profiles They are unicellular

organisms which only have chlorophylls a and completely lack chlorophylls b or c (Eliáš et al.,

2017) Their habitats are diverse, including marine, freshwater, and terrestrial environments (Heimann & Huerlimann, 2015) Unlike higher plants and green algae, the light harvesting

complexes proteins bind only chlorophyll a and carotenoids (violaxanthin, antheraxanthin,

zeaxanthin and vaucheriaxanthin (Simionato et al., 2011) Eicosapentaenoic acid (EPA) has been found in cytoplasm and photosynthetic lamella lipids of some species, especially

Monodus subterraneus (Cohen, 1999) Another important organism in this group is Nannochloropsis Owing to its high content of EPA, Nannochloropsis has been considered as

a source of PUFAs (Zittelli et al., 2007) Nannnochloropsis has been also used for large – scale

production of biodiesel (Moazami et al., 2012) There have been five species identified in the

genus Nannochloropsis, of which N limnetica is a freshwater species (Suda et al., 2002)

Table 1.1Summary of some characteristics of Chlorophyta, Rhodophyta, Bacillariophyta and Eustigmatophyta

or multinucleate filaments

Freshwater, marine and terrestrial environments

Chlorophylls

a, b and

carotenoids

- Biomass and carotene production from

β-Dunaliella

- Cosmetics from

Chlorella, Dunaliella and Haematococcus

- Astaxanthin from

Haematococcus

Rhodophyta Filamentous Mostly

marine

Phycobilins pigments (red and blue)

- Extracellular polysaccharides, phycobilins and

Chlorophylls

a, c 1 and c 2, fucoxanthin and β-carotene

- Waste degradation

- Toxicity testing

- Biomineralization

- Production of cadmium metal Eustigmatophyta Unicellular Marine,

freshwater, and terrestrial environments

Chlorophyll a

and carotenoids

- PUFAs from

Nannochloropsis

- Large – scale production of biodiesel using biomass of

Nannochloropsis

1.3 Algal cultivation system

Two main categories of cultivation system can be distinguished, namely, open and closed systems Open reactors include open ponds and raceway ponds also known as high rate algal ponds Closed systems are usually referred to as photobioreactors

1.3.1 Closed photobioreactors (PBRs)

1.3.1.1 Tubular photobioreactors

Tubular PBRs are the most common system among closed cultivation systems (Ting et al.,

2017) There are three types of tubular PBRs, including vertical (Pirt et al., 1983), horizontal

(Molina et al., 2001), or near-horizontal systems (Tredici & Zittelli, 1998) A tubular PBR is a growth system composed of an array called solar collector Solar collectors are used to capture sunlight They are made up of straight transparent tubes (Chisti, 2007) Those tubes are made

of either plastic or glass (Brennan & Owende, 2010) The use of transparent materials aims to favor the optimal light penetration into the culture This ensures high biomass productivity achieved through photosynthesis with biomass concentration of 1 kg/m3 (Faried et al., 2017) These tubes are called solar collector tubes The diameter of the solar collector tube is limited

to less than 0.1m because it is impossible for light to penetrate too deeply into the culture medium This enables the light to sufficiently penetrate into the culture medium, ensuring the high biomass productivity (Chisti, 2007) Air pumps or airlift pumps are responsible for aeration and mixing (Kiran et al., 2014) They continuously circulate the microalgal culture within the system and help achieve the mass transfer of gases (CO2 and oxygen) (Tan et al., 2018) The microalgal culture is continuously circulated between the reservoir and the solar collector (Chisti, 2007) The orientation of solar collector is adjusted to capture maximum amount of sunlight (Molina Grima et al., 1999; Mirón et al., 1999) Solar collector tubes are arranged parallel with each other and sometimes arranged into a fence – like form to maximize the number of tubes in a given area North–South orientation is typically applied in the design

of the culture system (Chisti, 2007) The color of the grounds under the solar collector is recommended to be white They can be painted or covered with white sheets of plastic (Tredici, 1999) Photosynthesis is known to produce oxygen and the production rate of oxygen under conditions of high irradiance can be 10 g·m−3·min−1 Photosynthesis can be inhibited by the high amount of dissolved oxygen in the culture medium In addition, microalgal cells may face bleaching because of photooxidation which may happen when there are high concentrations of dissolved oxygen and high sunlight intensity at the same time The recommended level of oxygen is less than 400% of air saturation Therefore, the microalgal culture in the tubular photobioreactor design is always pushed back to the degassing column for the removal of excessive oxygen The water temperature can be controlled by pumping the cooling water through the heat exchanger in the degassing column (Chisti, 2007)

Figure 1.2A model of a horizontal tubular PBR (Chisti, 2007)

1.3.1.2 Flat plate PBRs

Flat plate PBR is another type of PBR that can be positioned horizontally or vertically on the ground (Lee, 2001) Flat plate PBRs can be divided into two types, including indoor type illuminated with artificial light or outdoor illuminated with sunlight (Ting et al., 2017) and

minimum light path (Singh & Sharma, 2012) Flat plate PBR is the growth system which has cuboidal shape (Singh & Sharma, 2012) Microalgae grow inside the system by passing through the flat panels (Faried et al., 2017) The materials of the panels are transparent, including glass, plexiglass, polycarbonate and plastic bags (Ting et al., 2017) Transparent flat plates with thickness of a few millimeters favor the sufficient radiance penetration to the culture medium, promoting the microalgae growth For example, outdoor semi-continuous cultivation in flat-plate PBR could reach a biomass productivity of 0.25–3.64 g·L−1·d−1 if a light path of 1.2 to 12.3 cm is used (Lee, 2001) The advantages of flat panel PBRs are high surface area to volume ratio for illumination, flexible design for scale-up process and low amount of oxygen accumulation (Ting et al., 2017) (Debowski et al., 2012) Many researchers are interested in flat-plate PBRs because of their high surface-area-to-volume ratio character and high cell densities (Brennan & Owende, 2010) Motor or bubbling air device are set up for agitation (Faried et al., 2017) However, there are still some main limitations in the design of this system, including less control in cultivation temperature, some degree of wall growth and many compartments required for scale-up (Kiran et al., 2014) Indeed, scaling up PBRs remains a challenge because it is difficult to keep the different variables (temperature, mixing, light, mass transfer) in the desired range Nowadays, most reactors are designed and scaled up thanks to semi-empirical methods (Huang et al., 2017) Therefore, some researches are conducted in order to improve current PBRs to achieve larger mass concentration, growth rate and lower energy use and capital cost (Li et al., 2015; Lee et al., 2014) By optimizing the operational conditions and the design of PBRs, it will be possible to use them at a commercial scale in the future A recent study has identified the principal features that a PBR should have for being commercially efficient; these characteristics include a highly illuminated surface/volume ratio

in order to increase the solar irradiance, easy control of the temperature, a good mixing and effective mass transfer and, finally, low operating and capital costs (Huang et al., 2017)

Given the fact that PBRs have a higher productivity and are less likely to produce contaminated culture, this type of cultivation system can be combined with high rate algal pond (HRAP) having lower operational and capital costs The first step is the microalgae cultivation within the PBR to produce a sufficient amount of sufficiently pure microalgae that will be injected in the HRAP during the second step The PBR is thus used for seeding while the HRAP is used for microalgae growing and production

Figure 1.3 The flat panel PBR system (Lindblad et al., 2019)

1.3.2 High rate algal pond (HRAP)

Raceway ponds or high rate algal ponds is one type of open, shallow (from 0.2 to 0.6 m) cultivation system which is made of a closed loop recirculation channel (Chisti, 2007) They were first investigated in the 50’s The material used for construction of the system is inexpensive (Faried et al., 2017) Concrete can be used to construct the pond which may be lined with white plastic (Tan et al., 2018) The pond may be designed to contain one or a group

of channels (Ting et al., 2017) Baffles in the flow channel are used to direct the flow of culture

at the bends (Chisti, 2007) When the circulation of broth through the loop is completed,

collection of microalgae can be done at the harvesting port which is located at the backmost of the paddlewheel (Tan et al., 2018) The paddlewheel is set to run continuously to prevent sedimentation (Chisti, 2007) The largest open raceway pond in the world resides on an area of

444, 000 m2 at Calipatria, CA (USA) It is used for cultivation of the cyanobacterium Spirulina

(Faried et al., 2017)

Figure 1.4 A model of open raceway pond (Chisti, 2007)

Some features of HRAPs may be optimized in order to obtain a productivity as high as possible Among these, depth has been identified as being one of the most important characteristics in HRAP as it is the main factor determining the amount of light received by the microalgae and the frequency at which biomass is exposed to the light Areal productivity of culture systems can be increased between 134 and 200% by doubling the depth from 200 to 400 mm (Sutherland et al., 2014) Improvement of the mixing has also been studied in the late 90’s (Mihalyfalvy et al., 1998) due to the finding that microalgae situated in the deeper layers were not contributing to the treatment process while the microalgae situated in the superficial layer were suffering from photosaturation Inducing a vertical mixing resulted in a maximal exposure

of the microalgae to the light and, consequently, in optimizing the photosynthetic activity of

the biomass These cycles of exposure and non-exposure to the light are referred as light-dark cycles Furthermore, higher photosynthetic activity obviously results in higher carbon dioxide utilization by the microalgae

Other research has been conducted on the design of HRAPs and focused on the reduction of energy losses due to water resistance It appeared that the main energy losses take place in the curve so that a re-design of the curves may be a solution to reduce the energy requirements for water circulation (Liffman et al., 2013) Some other re-design criteria have been identified such

as reduction of water depth as previously mentioned, increase in culture concentration in order

to ease downstream processing, substitution of the traditional paddlewheel mixing by a more energy-efficient pumping system or the reduction of the flow velocity while maintaining a suitable mixing of the microalgae

Although open raceway ponds are a cost - effective system, it is at risk of contamination with unwanted algae and other microorganisms feeding upon algal production (Balat, 2011; Chisti, 2007) but also due to the fact that they are highly exposed to their environment Moreover, they have a limited exposure to light, a complex carbon management and, hence, a lower long-term productivity as compared to closed PBRs (Chisti, 2007) Open raceway ponds also have problems with significant water loss through evaporation Due to this, the fixation of carbon dioxide by microalgae in raceway ponds is less efficient than in PBRs (Chisti, 2007) Poor mixing combined with dark zones in the system may result in low biomass concentration in raceway ponds (Chisti, 2007) Because of these problems, the pond is currently designed and constructed with greenhouse to prevent debris, pollution, water loss and rainfall The cascading system is better than one - channel raceway pond because the residence time of culture in cascading system is longer (Dragone et al., 2010; Rawat et al., 2011)

In 2018, in the framework of the RENEWABLE project, a HRAP pilot was installed at the Marine Breeding Center in the province of Ninh Thuan in Vietnam It has been decided to

improve the shape of the curves in order to reduce head losses as can be seen on the Figure 1.5 here below The system is equipped with an airlift system specifically designed by Prof J-L Vasel The air flowrates were tested, optimized and a model was developed in order to simulate the hydrodynamics, the gas transfers and the biological phenomena occurring within the HRAP All this research is part of a second PhD thesis undertaken in parallel to the work presented in this manuscript

Figure 1.5 Algal raceway pond of RENEWABLE project in the central province of Ninh

Thuan, Vietnam The system is equipped with an airlift device responsible for mixing and

aerating

1.3.3 Comparison of PBRs and HRAPs

The main features of both PBRs and high rate algal ponds are summarized in the Table 1.2 below

Table 1.2Comparison of PBRs and HRAPs (Mata et al., 2010)

Operation regime Batch & Semi-continuous Batch & Semi-Continuous

1.4 High value components of microalgae

1.4.1 Proteins

Contributing to 40 – 70% of microalgal biomass, algal proteins are now considered as one of

the major products interesting for human or animal nutrition (Chew et al., 2017) They have

been recommended by WHO (World Health Organization), FAO (Food and Agriculture Organization of the United Nations) and UNU (United Nations University) to be used because

of their well - balanced essential amino acid (EAA) content which suits human consumption requirement (Chronakis & Madsen, 2011) Indeed, microalgae are able to synthesize all amino acids and their protein quality is demonstrated to be higher than that from conventional plants (Becker, 2007) A lack of one or more than one EAAs is typical of plant – based proteins, including histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Young & Pellett, 1994) Furthermore, plants have been known to beharder digested due to their high content of insoluble polysaccharides (Bleakley & Hayes, 2017)

Several studies have confirmed the elevated accumulation of protein in some microalgae

species For example, protein content may account for 50 – 60% of dry weight in Chlorella (Kovač et al., 2013) Protein productivity in microalgae is also much higher than in

conventional plants In particular, the protein productivity of 4–15 tons/ha/year has been recorded in microalgae while 1.1 tons/ha/year, 1–2 tons/ha/year and 0.6–1.2 tons/ha/year are the protein productivities in wheat, pulse legumes and soybean, respectively (Van Krimpen et al., 2013) Besides, some previous studies have shown some medical benefits from microalgal proteins, including antihypertensive (Murray & FitzGerald, 2007), immunomodulating (Morris et al., 2008), anti-cancer (Sheih et al., 2009), hepatoprotective (Kang et al., 2012) and anticoagulant activities (Athukorala et al., 2007)

Table 1.3Protein content in some microalgal species

Phylum

Chlorophyta

Bacillariophyta Navicula sp 33 (Villarruel-López et al., 2017)

Cyanobacteria

1.4.2 Lipids

Microalgae as lipid producers have drawn much attention from many scientists (Han et al., 2015) because their lipid productivity is much higher than that in oil plants (Table 1.4) (Chisti, 2007) However, the theoretical lipid productivity of 136,900 L/ha in microalgae calculated

by Chisti (2007) is much beyond optimistic Under outdoor cultivation condition, the highest biomass productivity of microalgae recorded is around 100 tons/ha/year (Rodolfi et al., 2009) Given a microalgal species with a high lipid concentration of 50% (DW), the highest oil productivity can only be around 50,000 L/ha/year

In classification, lipids are subdivided into polar and neutral lipids Phospholipids and glycolipids belong to polar lipids while acylglycerides (tri, di- and monoglycerides) and free fatty acids are neutral lipids In microalgae, neutral lipids are used as energy source and polar lipids are the constituents of cell membranes (Halim et al., 2012) In terms of fatty acid compositions, microalgal oil mainly contains certain given unsaturated fatty acids, such as

palmitoleic acid (C16:1)1, oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) and the saturated fatty acid palmitic acid (C16:0) Stearic acid (C18:0) is present as small amount Notably, some microalgae have the ability to produce high amount of polyunsaturated

fatty acids (PUFAs) For instance, Aurantiochytrium sp., Schizochytrium limacinum, and

Porphyridium cruentum have been found to produce C22:6 [42% total fatty acid (TFA)], C22:5

+ C22:6 (39.4% TFA) and C20:5 (25% TFA), respectively (Sajjadi et al., 2018)

Table 1.4Oil productivities from microalgae and other oil plants (Chisti, 2007; Rodolfi et al.,

(Porphyridium, Dunaliella, Isochrysis, Nannochloropsis, Tetraselmis, Phaeodactylum,

Chlorella and Schizochytrium) and the cultivation conditions (Bellou et al., 2014) For

instance, lipid content in Chlorella vulgaris and Botryococcus braunii may be in the range of

12% to 26% and 14% to 75%, respectively (D’Alessandro & Antoniosi Filho, 2016) There are several stress methods routinely used for the enhancement of lipid production, including nutrient depletion, light intensities, temperature, salinity, and pH (Bartley et al., 2014; Chu et

1 CXX:n where XX represents the number of carbon atoms and n the degree of unsaturation along the aliphatic chain

al., 2015; Suyono et al., 2015) For instance, under nitrogen limited condition, the lipid content

of Nannochloropsis sp F&M-M24 phototrophically cultured was double (Rodolfi et al., 2009) while that of Chlorella vulgaris NIES reached 89% of DW under heterotrophically cultivated

condition (Shen et al., 2015) Sulfur depletion is also used to enhance the production of lipids

in microalgae, as in case of Chlamydomonas reinhardtii CC-124 and Chlamydomonas

reinhardtii CC-125 (Cakmak et al., 2012) In another study, salinity stress was applied to

increase the lipid accumulation with the highest lipid productivities found in Chlorella sp

CG12 (52.8 mg L-1 day-1) and Desmodesmus sp GS12 (55.2 mg L-1 day-1) (Srivastava et al., 2017) Those two strains were cultivated in the salinity of 25 mM CaCl2 In Chlorella sp L1,

high light intensity of 400 µmol photon m-2 s-1 favored the accumulation of neutral lipid while low light intensity of 60 µmol photon m-2 s-1 increased the accumulation of triglycerides (TAG) (He et al., 2015) In some cases, a combination of stresses can be used to enhance the lipid accumulation In a recent study by Fan & Zheng (2017), the lipid accumulation of

Chlamydomonas reinhardtii was enhanced under heterotrophic culture with a combination of

salt and light stress (Fan & Zheng, 2017).In general, nitrogen starvation is proved to be the most effective condition for the lipid accumulation (Belotti et al., 2013) In addition, genetic modification of microalgae is another approach which could control accurately the mechanisms engaged in the lipid production enhancement under normal condition (Xue et al., 2015; Lim & Schenk, 2017)

Table 1.5Lipid content in some microalgal species

Lipid content

Chlorophyta

1.4.3 Carotenoids

Most of carotenoids are pigments belonging to lipophilic compounds, whose colors may be yellow, orange or red and are the most diverse and ubiquitous pigments in nature (Sasso et al., 2012), (Varela et al., 2015) A large amount of them is present in some phytoplanktons, such

as cyanobacteria, and microalgae (Orosa et al., 2000; Huang et al., 2016) There have been over

700 carotenoids found and classified into two major groups, including xanthophylls containing oxygen and non – oxygen hydrocarbons called carotenes (Armstrong & Hearst, 1996; Britton

et al., 2004) (Lohr, 2012), of which less than 30 carotenoids are mainly involved in photosynthesis (Varela et al., 2015) They bind to the light harvesting complexes (LHCs) and are mostly found in the thylakoid membranes (Nisar et al., 2015) In microalgae, β-carotene, lycopene, astaxanthin, zeaxanthin, violaxanthin, and lutein are major carotenoid compounds,

of which β-carotene, lutein, and astaxanthin have been most investigated (Del Campo et al., 2007)

By function, carotenoids are classified into primary and secondary carotenoids Primary carotenoids are defined as structural and functional components of the photosystems, such as lutein, neoxanthin and violaxanthin Secondary carotenoids such as astaxanthin, ketolutein,

canthaxanthin, adonixanthin, echinenone and β-carotene are involved in cell protective mechanisms They are produced in large amounts in response to specific environmental stimuli Unlike primary carotenoids, the secondary ones are not bound to the photosystems, but suspended in oily droplets They constitute the layer to protect the cells from the stress conditions and are the cause of pink or red colors in stressed algae (Begum et al., 2016; Wang

et al., 2014; Jin et al., 2003; Guedes et al., 2011; Mulders et al., 2014)

Several carotenoids are considered as most value – added products in microalgae The first valuable pigment from microalgae is astaxanthin (a keto – carotenoids) Astaxanthin is a powerful antioxidant, whose activity is 10 times and 500 times stronger than that of β-carotene and α -tocopherol, respectively (Dufossé, 2007) In humans, astaxanthin was proven to be effective on cancers, inflammatory diseases, metabolic syndrome, diabetes, diabetic nephropathy, neurodegenerative diseases and eye diseases (Yuan et al., 2011) Some

microalgae, such as Haematococcus pluvialis, Chlorella zofingiensis and Chlorococcum sp are considered as astaxanthin producers due to their high content of astaxanthin (Del Campo et

al., 2004; Zhao et al., 2009; Yuan et al., 2011; Cysewski & Lorenz, 2007) Of those,

Haematococcus pluvialis is the best producer with high astaxanthin content of 5% DW

(Wayama et al., 2013) The routine uses of astaxanthin in nutraceutical, cosmetics, food and feed industries have been reported (Guerin et al., 2003) Lutein is another important pigment from microalgae, which is commonly found in species belonging to Chlorophyceae, such as

Chlorella sp., Scenedesmus sp and Muriellopsis sp (Del Campo et al., 2007; Wu et al., 2007)

Of those, Chlorella sp was found to produce high amount of lutein when heterotrophically

grown in the laboratory (31.4 mg L-1 ) (Wei et al., 2008) This has helped Chlorella sp to

become another potential producer of lutein for commercial purposes (Wu et al., 2007) In epidemiological studies, lutein has been identified as an effective agent, which can prevent a

large number of human diseases (Sathasivam et al., 2019) In some studies, high irradiance under high salinity and low nitrogen are the conditions to be used for carotenoids production (Hosseini Tafreshi & Shariati, 2009; Francavilla et al., 2010) For example, the carotenoid concentration of 40 mg L-1 was obtained when D tertiolecta was grown under the condition of

high light intensity of 300 µmol m-2 s-1 and nitrogen starvation (Kim et al., 2013)

Desmodesmus sp F51 under the condition of excessively high light intensity of 600 μmol m-2

s-1, sufficient nitrogen, and high temperature of 350 C could produce 3.6 mg lutein L-1 day-1

(Xie et al., 2013) In another study by Hejazi et al (2004), D salina reached the β-carotene

productivity of 2.5 mg m-2 day-1 when cultured under high light intensity (Hejazi et al., 2004)

With great potential in the global market, the global sales of astaxanthin (synthetic and natural astaxanthin) in 2014 was $447 million with high market value ranging from $2500–7000/kg (Koller et al., 2014; Pérez-López et al., 2014; Industry Experts, 2015) The global market of lutein has noted a significant growth A recent survey showed that the global market value of carotenoids was estimated to reach 1.53 billion USD by 2021 (Sathasivam & Ki, 2018)

Figure 1.6 Chemical structures of some carotenoid compounds found in microalgae (Gong &

Bassi, 2016)

1.5 Application of microalgae

1.5.1 Animal feedstock

For animals, feed quality is an important factor affecting their health (Pulz & Gross, 2004) The use of microalgae as animal feeds has been studied (Beacham et al., 2014; Bhardwaj et al., 2014) and nutritional and toxicological assessments have proven that algal biomass could be used as feed supplements (Becker, 2004) Microalgae as animal feeds affect positively on animal physiology (Certik & Shimizu, 1999) Aside from carbohydrates and proteins (Keffer

& Kleinheinz, 2002), they also supply animals with natural vitamins, minerals, and fatty acids

By that, their immune reaction and reproduction are improved (Varfolomeev & Wasserman, 2011) The use of microalgae as supplement to animal feed brings out some benefits For

example, fatty acid profiles of milk in dairy cattle fed with Chlorella vulgaris are modified

Their saturated fatty acid content is reduced while the amount of docosapentaenoic acid (DHA) increases (Kouřimská et al., 2014) The use of feed supplemented with microalgae for lambs and horses results in the increase in fatty acid content of their meat (Cooper et al., 2004; Hess

et al., 2012) and Arthrospira platensis as additive to feed promotes weight gain in pigs and poultry (Simkus et al., 2013) There has been frequent use of Spirulina as feed supplements

because its nutrient profile and digestibility are extremely good It is easily digested because

of its low content of carbohydrates (Yaakob et al., 2014) Spirulina possesses a special nutrient

profile consisting of B-complex vitamins, minerals, proteins, γ-linolenic acid and super oxidants (such as β-carotene, vitamin E, trace elements) and several unknown bioactive

anti-compounds (Kulshreshtha et al., 2008) A recent study has shown that Spirulina can

significantly affect weight, growth and body conformation in sheep (Holman, 2012) A study

on the effect of Porphyridium sp as feed supplement on metabolism of chicken has shown a

decrease of 10% in egg yolk cholesterol and an increase in carotenoid content (Harun et al.,

2010; Kiron et al., 2012) Microalgae used for animal feed production account for 30% of the current algal production (Becker, 2004)

1.5.2 Microalgae in aquaculture

The utilization of microalgae in aquaculture can bring out some benefits, such as color development in farmed salmonids (Muller-Feuga, 2000); fish feed (Brown et al., 1997); induction of important biological activities of red aquatic species (Muller-Feuga, 2000) and improvement of immune system in fish (Pulz & Gross, 2004) As feed for aquatic animals,

there have been several species most widely used, including Chlorella, Tetraselmis, Isochrysis,

Pavlova, Phaeodactylum, Chaetoceros, Nannochloropsis, Skeletonema, and Thalassiosira

(Yamaguchi, 1996; Apt & Behrens, 1999; Yamaguchi, 1996; Muller-Feuga, 2000; Borowitzka,

1997) For instance, Chaetoceros, Thalassiosira, Tetraselmis, Isochrysis, and Nannochloropsis

have been used for larval cultivation, in which larval organisms can be fed directly with

microalgae or they are fed with Artemia, rotifers, and Daphnia grown using microalgae (Sirakov et al., 2015) In fish production, Nannochloropsis sp., Pavlova sp and Isochrysis sp

have been the source of nutrition for commercial marine fishes The fishes have a better growth and balanced nutrition when fed with the combined diet of some microalgae species rather than

a single one (Spolaore et al., 2006) Aside from providing nutrients, microalgae as feed also help enhance the pigments in several aquatic animals Pigmentation in domesticated shrimps, red sea breams, salmons, trouts, sea urchins, lobsters and ornamental fishes is favored when they are grown using feed containing additives, including astaxanthin, canthaxanthin and β-carotene (Skjånes et al., 2013) Furthermore, pigments from microalgae also favor the growth

of aquatic animals as in the case of astaxanthin, which is commonly found in Nannochloropsis

sp and Haematococcus pluvialis It shows the useful contribution to the growth and survival

of shrimps, salmons and trouts during their early feeding period (Bazyar Lakeh et al., 2010; Lorenz & Cysewski, 2000; Niu et al., 2009) Ease of cultivation, non – toxic species, rich -

nutritional value, suitable cell size and shape and digestible cell wall are important criteria for microalgae to be used in aquaculture (Patil et al., 2007) The market price of microalgae biomass used for animal and fish feed is of 5–20€/kg (Wijffels, 2008)

1.5.3 Microalgae used for production of human food

Over thousands of years, humans have been using microalgae as one of their food sources (Milledge, 2011) An archaeological study in Chile has found that humans began consuming algae 14,000 years ago (Dillehay et al., 2008) Indeed, microalgae have been consumed

worldwide for a long time In Africa, Kanembu people used Spirulina naturally grown in Lake

Chad as food hundreds or thousands of years ago (Liang et al., 2004) In Mexico, the Spanish

noted the use of cyanobacteria to prepare sun – dried cake among people native to Lake Texcoco (Echlin, 1966) In China, Nostoc flagelliforme was used for consumption by the

Chinese 2000 years ago (Echlin, 1966)

Nowadays, algae are widely used and recommended by WHO to be consumed worldwide owing to their high nutritional value and health benefits (Borowitzka, 2013; Rathinam Raja et al., 2018) and are considered safe for human consumption by the U.S Food and Drug Administration (U S Food and Drug Administration, 2018) The functional value of microalgae in food generally comes from their high contents of proteins, polyunsaturated fatty acids, polysaccharides, pigments, vitamins, minerals, phenolic compounds, volatile compounds, and sterols (Andrade, 2018) For example, DHA is responsible for the correction

of brain and eye development in infants as well as necessary for cardiovascular health in adults (Spolaore et al., 2006; Cottin et al., 2011) EPA is found to regulate the biological functions and take a role in preventing and treating some human diseases, including heart and inflammatory diseases (Wen & Chen, 2003) The microalgal pigments have been characterized

by anti-inflammatory, antihypertensive, anticancer, antioxidant, antidepressing, and antiaging features (Khazi et al., 2018; Chronakis et al., 2000; Soares et al., 2016) Among algal pigments,

astaxanthin and lutein are the two major ones Astaxanthin from Haematococcus has been used

as dietary supplement for human and no adverse side-effect from using it has been documented

so far (Capelli & Cysewski, 2012; Yang et al., 2013) while lutein has a variety of applications

in human, such as colorant in food production (Jin et al., 2003)

Recently, Aphanizomenon flos-aquae, Chlorella sp., Dunaliella salina (D salina), Dunaliella

tertiolecta (D tertiolecta) and Spirulina platensis (S platensis) (Soletto et al., 2005;

Rangel-Yagui et al., 2004) have been species widely used for human consumption Of those,

Spirulina (Arthrospira) and Chlorella are the two dominant species in the microalgal market

(Sathasivam et al., 2019) They are now commercialized and available in the market as a health food, whose forms can be tablets, capsules and liquids (Pulz & Gross, 2004) They may also

be included in the recipes for common foods, including mayonnaises, gelled desserts, biscuits, pasta, breakfast cereals, beverages, candy bars or gums, snack foods etc (Suganya et al., 2016; Gouveia et al., 2009; Spolaore et al., 2006) Their market price is in the range of 30 to 300 €

kg−1 depending on the strains (Martín-Girela et al., 2020) Every year, around 5,000 tons of dry matter is commercially produced (Raja et al., 2008) Currently, several countries are getting deeply involved in microalgal manufacturing, such as Australia, China, South Korea, Malaysia, Singapore, Taiwan, USA, Netherlands, Spain, Portugal, France, Denmark, Japan and some African countries (Raja et al., 2008)

1.5.4 Microalgae used in wastewater treatment

Because microalgae are able to absorb nutrients and turn them into biomass, their use in wastewater treatment has been regarded as the approach which is favorable (Chinnasamy et al., 2010), environmentally friendly and cost effective (Liu et al., 2013) In comparison with other conventional treatment processes, this method is a more cost-effective approach where solar energy, nutrients in wastewater and oxygenation from the photosynthesis are all suited as well

as free for the growth of microalgae during the wastewater treatment (Oswald, 2003) Therefore, algae – based wastewater treatment and their biomass are now drawing global attention (Renuka et al., 2015) To be chosen as suitable for use in wastewater treatment, microalgae must be screened for some features, including high growth rate, high removal rate

of nutrients, being highly adaptable to a variety of wastewater types and local climate, and have high biomass productivity Li et al., 2019) Notably, not only do microalgae remove nutrients from wastewater, but also CO2 because microalgae grow faster if the amount of CO2 is higher

(Chiu et al., 2009) than the level of 400 ppm generally present in the air (Fazal et al., 2018) N

oculata NCTU-3, for example, has the optimum growth in the culture supplemented with 2%

CO2 (Chiu et al., 2009) Microalgae, therefore, can exploit the amount of CO2 released from industrial activities or soluble carbonates at commercial scale (Fazal et al., 2018)

Figure 1.7 The microalgae production process coupled with wastewater treatment (Zerrouki

& Henni, 2019)

Microalgae can be used to treat domestic wastewater as well as wastewaters from agricultural and industrial activities (Zerrouki & Henni, 2019) The minor presence of heavy metals in several types of industrial wastewaters can be removed using microalgae Microalgae can assimilate the heavy metals using micronutrient transporters, and ultimately detoxify them in specific cellular compartments (Kaplan, 2007) There have been several types of industrial wastewaters reported to be treated by microalgae, in which they serve as nutrient sources (Li

et al., 2019) For instance, wastewaters sourced from the olive oil mill and paper industries

have been demonstrated to be treated by some microalgae, including Chlorella,

Ankistrodesmus and Scenedesmus (Rawat et al., 2011) Chlamydomonas sp TAI-2 was proven

to be able to remove 100% NH4-N and NO3-N, and 33% PO4-P from wastewater in an industrial park (Wu et al., 2012) The nutrient removal rate of greater than 96% was observed during the treatment process of carpet mill effluents using algal strains native to Dalton, North Central

Georgia, USA (Chinnasamy et al., 2010) Scenedesmus obliquus has been reported to be able

to completely remove all the pollutants in a variety of wastewater sources, such as poultry, swine and cattle breeding, brewery and dairy industries, and urban effluents (Ferreira et al.,

2017) The cultivation of Chlorella sp in brewery wastewater resulted in the complete removal

of nitrogen, phosphorus, and organic carbon and a considerable growth of Chlorella sp recorded (Subramaniyam et al., 2016) In another study, Desmodesmus sp CHX1 has been

shown to be able to remove 78.46% of nitrogen and 91.66% of phosphorus from piggery wastewater (Luo et al., 2019) Concerning the biomass productivity of microalgae in wastewater treatment, Chinnasamy et al (2010) cultivated 15 native algal isolates in 950 – liter raceway ponds using wastewater consisting of 85–90% carpet industry effluent and 10–15% municipal sewage and reported a biomass productivity of 9.2–17.8 tons/ha/year(Chinnasamy

et al., 2010) The microalgal biomass from wastewater treatment processes can be applied in several fields as mentioned in Figure 1.7

1.5.5 Biofuel production

The increasingly growing population, fast industrialization, and the escalating use of fossil fuels are factors responsible for the worldwide energy crisis and global warming, which humankind is experiencing nowadays (Rupprecht, 2009) This along with the depletion of fossil fuels have rapidly increased the worldwide interest in alternative renewable sources (Behera et al., 2015) Furthermore, the need for fossil fuels is estimated to increase by forty - percent from 2010 to 2040 (Bhore, 2014) Feedstocks sourced from photosynthetic organisms, including land plants and aquatic microalgae are materials to produce biofuels, which can meet the global demand for energy as well as the carbon neutrality, and make carbon dioxide (CO2) sequestration from the atmosphere feasible (Stephenson et al., 2011; Ravindran et al., 2017) However, the use of microalgae as a sustainably alternative source of biofuel is considered as

a better option since it will lead to the reduction in the utilization of land and water as well as the immoderate amount of harmful pesticides (Raheem et al., 2018) Researchers, governments, and local and international entrepreneurs are thus resting their interest on microalgae (Medipally et al., 2015) The capacity of microalgae in conversion of solar energy into biomass is higher than terrestrial plants on the basis of land area (Klok et al., 2014) Some oily microalgae species can accumulate TAGs at a level greater than 70% of dry cell weight

(Scott et al., 2010) Nowadays, Chlorella sp., Nannochloropsis sp and Scenedesmus sp are

among some microalgae used for biofuel production owing to their high lipid productivity and high growth rate (Nascimento et al., 2013; Moazami et al., 2012; Oncel, 2013; Milano et al., 2016)

There are currently three generations of biofuels The first generation is crop – based biofuel while the second and the third ones are nonfood lignocellulosic material - based and algae – based biofuels, respectively (Ho et al., 2014) In biodiesel production, TAGs will go through a process called transesterification, in which the reaction between TAG with an alcohol