Université Abdelmalek Essaadi ‫جامعة عبد المالك السعدي‬

Faculté Des Sciences ‫كلية العلوم‬

Tétouan ‫تطوان‬

Filière: Science de la Vie

Mémoire de Fin d’Etudes (rédigée en anglais):

General Study of Microalgae

Haematococcus pluviaris as example

Présentée par:

Oulad Belayachi Laila

Date de la soutenance:

10 juillet 2017

Jury :

Pr.Abrini Jamal Faculté des sciences de Président

Tétouan
Pr.Skali Senhaji Nadia Faculté des sciences de Examinatrice
Tétouan
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2
General Study of Microalgae
Haematococcus pluviaris as example

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 ‘’Dedication’’
I dedicate this report to my
beloved family, friends and
everyone who stood by my side.

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 ‘’Acknowledgement’’
I want to thank my supervisor
PR.Abrini Jamal for his support
and his guidance through this
project. I also thank Pr.Skali
Senhaji Nadia for her presence
today.

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 ‘’Table of Contents’’

Abstract/Résumé…………….……………………………………………………09

Introduction…………………….…………………………………………………..10

1. MICROALGAE IN GENERAL ..................................................................................................... 11

1.1. Definition ................................................................................................................................................ 11
1.2. Microalgal biodiversity ............................................................................................................................ 11
1.2.1. Main groups .................................................................................................................................. 11
1.2.2. Some well-known species ............................................................................................................. 12
1.3. Morphologic study .................................................................................................................................. 14
1.4. Microalgal metabolism ............................................................................................................................ 16
1.4.1. What is metabolism ...................................................................................................................... 16
1.4.2 Photosynthesis ............................................................................................................................... 17
1.4.2.1. Absorption of Light....................................................................................................... 18
1.4.2.2. Electron Transport ....................................................................................................... 18
1.4.2.3. Generation of ATP ........................................................................................................ 19
1.4.2.4. Carbon Fixation ............................................................................................................ 19
1.4.3. Starch biosynthesis ....................................................................................................................... 20
1.4.4. Fatty acid biosynthesis .................................................................................................................. 21
1.4.5. Biogenesis of lipid droplets (LDs): Chlamydomonas reinhardtii for example ............................... 22
1.5. Cultivation methods ................................................................................................................................ 23
1.5.1. Open cultivation systems: Open ponds or raceways .................................................................... 24
1.5.2. Closed cultivation systems: Photobioreactors .............................................................................. 25
1.6. Microalgal industrial uses ........................................................................................................................ 27
1.6.1. Chemical composition of microalgae ............................................................................................ 27
1.6.2. Human nutrition ............................................................................................................................ 27
1.6.3. Microalgae in cosmetics ................................................................................................................ 28
1.6.4. Production of biodiesel ................................................................................................................. 29

2. HAEMATOCOCCUS PLUVIARIS ................................................................................................ 31

2.1. General characteristics ............................................................................................................................ 31
2.2. Astaxanthin ............................................................................................................................................. 32
2.2.1. Astaxanthin chemical properties and benefits ............................................................................. 32
2.2.2. Astaxanthin production................................................................................................................. 33

General conclusion……………………………………………………………………..34
References…………………………………………………………………….……………35

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 ’’List of Figures’’

-Figure1: Green microalgae under microscope (Anonym, 2013)………………………………......…11

-Figure2: Chlamydomonas under microscope (Melis and Happe, 2001)…………………………..13

-Figure3: Chlorella endosymbiomts inside Paramecium bursaria (Eckardt, 2010)………....13

-Figure4: Illustration detailing organelles present in a typical marine

unicellular microalga (Lodish et al., 2000)…………………………….…………………....…14

-Figure5: The structure of a Chloroplast (Lodish et al., 2000)……………………..……………..……15

-Figure6: Three-dimensional model of the Golgi

complex (Lodish et al., 2000)………………………………………………….………………….….…16

-Figure7: The Chloroplast’s contents (Lodish et al., 2000)………………………………………………..17

-Figure8: Light-dependent reactions of photosynthesis at

the thylakoid membrane (Somepics , 2015)…………………………………………………....…19

-Figure9: Calvin Cycle (Jones, 2010)………………………………………………….…………………………….20

-Figure10: Starch biosynthesis process (Cakir et al., 2015)…….…………………….…………………..21

-Figure11: Overview of metabolic pathways in microalgal

lipid biosynthesis (Shih-Hsin et al, 2014)………………………………………….…….……..23

-Figure12: The possible models of LD biogenesis (Goold et al., 2014)……….………………….…23

-Figure13: Microalgae cultivation in shake-flasks (Wolkers et al., 2011)……….….…………….24

-Figure14: Raceway pond in Netherlands (Wolkers et al., 2011)……………………….…..….…..….25

-Figure15: Horizontal tube reactors filled with microalgae

(Wolkers et al., 2011)……………………….………………………….………………..……..……25

-Figure16: Tree-dimensional tube reactors (Wolkers et al., 2011)…………………..…..…............26

-Figure17: Flat Plate Reactors (Wolkers et al., 2011)…………………..………………..…………….......26

-Figure18: Dunaliella salina and Arthrospira platensis (Wahby, 2016)……..………………..…...28

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-Figure19: Some skin care products that are made of

microalgal powder (Wahby, 2016)…………………………….……….……………………..….29

-Figure20: Production of biodiesel by transesterification (Sheehan et al., 1998)……….……..29

-Figure21: (a) Zoospore; (b) Coccoid green cell; (c) Coccoid green cell with

red-colored lipid droplets (Chekanov et al., 2014)….………………………….…….……31

-Figure22: Astaxanthin structure (Kidd, 2011)…………………………………………….……….…………31

-Figure23: Haematococcus pluvialis cultivation (Wolkers et al., 2011)……………………………33

-Figure24: Astaxanthin powder (Wolkers et al., 2011)…………………………….…..……………..…33

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 Abstract

Microalgae are phytoplanktons that carry out photosynthesis; these microorganisms represent
a huge biodiversity in the world. Due to their eukaryotic organization, they consist of
organelles; include nuclei, mitochondria and chloroplasts, etc. These organelles contribute in
the metabolism by producing either protein, oils or sugar via many biological processes such
as photosynthesis and fatty acids biosynthesis. In order to use this rich biomass in
human/animal nutrition, cosmetics and biodiesel, many cultivation methods have been used,
from open cultivation systems like the open ponds to closed ones like photobioreactors. The
same thing with the special microalgae Haematococcus pluviaris which is capable of
producing a highly antioxidant molecule called astaxanthin. Its production required a strict
method starting with cultivation and ending with harvesting and extraction.

Key-words: Phytoplanktons, biodiesel, astaxanthin.

Résumé

Les microalgues sont des phytoplanctons qui pratiquent la photosynthèse; ces

microorganismes représentent une grande biodiversité dans le monde. Grâce à leur
organisation eucaryotique, ils sont constitués d’organites ; inclus le noyau, les mitochondries
et les chloroplastes, etc. Ces organites contribuent dans le métabolisme par la production soit
de protéines, de lipides, ou de sucre via plusieurs processus biologiques comme la
photosynthèse et la biosynthèse des acides gras. Afin d’utiliser cette riche biomasse en la
nutrition humaine/animale, les cosmétiques et le biodiesel, plusieurs méthodes de culture ont
été utilisées, de systèmes ouverts comme les bassins ouverts à des systèmes fermés comme les
photobioréacteurs. La même chose pour la spéciale microalgue Haematococcus pluviaris qui
est capable de produire une molécule très antioxydant appelé astaxanthin. Sa production
demande une stricte méthode qui commence avec la culture et se termine avec la récolte et
l’extraction.

Mots-clés: Phytoplanctons, biodiesel, astaxanthin.

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 Introduction

Microorganisms in general represent a large biodiversity in the world, from prokaryotic ones
such as bacteria, to eukaryotic ones such as microalgae.

Due to their simple morphology and physiology, bacteria are well studied microorganisms,
unlike microalgae which have complicated organelles and require even more research
(Sheehan et al., 1998).

However, Microalgae are one of the most ancient organisms living on earth and one of the
tiniest plants. They have survived some of earth’s harshest conditions for several billion years.
These unicellular species are capable of performing photosynthesis (converting light energy to
chemical energy), in order to produce not only more than 75% of the atmospheric oxygen, but
also carbohydrates, protein and lipids from 30 to 100 times faster than land plants (Kamyab,
2012 ; Spolaore et al., 2006). That is why they need to be highlighted and studied despite the
high cost of their production (Sheehan et al., 1998).

In order to understand the principal characteristics of microalgae, their biochemical

proprieties and their importance, this study will discuss:

-The microalgal biodiversity by highlighting the main classes and the differences
between them based on pigmentation, life cycle and cellular structure.

-The microalgal morphology by detailing the principal organelles present in a

microalga.

-The microalgal metabolism from photosynthesis process to starch, fatty acids and
lipids biosynthesis.

-The cultivation methods and systems such as open ponds (open system) and
photobioreactors (closed system).

-The use of microalgae in a variety of industries and fields like nutrition, cosmetics
and even in biodiesel production by transesterification.

Moreover, this study will take as an example Haematococcus pluviaris. A special green
microalga that turns red during harsh conditions and can produce a huge proportion of highly
antioxidant molecule which is astaxanthin (a xanthophyll carotenoid); this molecule has an
exceptional range of benefits such as maintaining the immune response etc. (Chekanov et
al.,2014 ; Kidd, 2011).

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1. Microalgae in general
1.1. Definition
Microalgae (figure1) also called phytoplankton by biologists are eukaryotic cells that consist
of organelles, include nuclei, plastids and chlorophyll. Using this chlorophyll, microalgae are
capable of performing photosynthesis in which they use Carbone dioxide (CO2) to grow
photoautotrophically and liberate the atmospheric Oxygen (O2). On a global scale microalgae
produce more than 75% of the Oxygen required for animals and humans.

These unicellular species exist individually or in groups. Normally they are not visible by the
naked eye, but if the water is eutrophic, massive algal blooms occur, changing the water in a
green, brown, blue, or orange liquid mass (Kamyab, 2012 ; Wolkers et al., 2011).

Figure1: Green microalgae under microscope (Anonym, 2013).

1.2. Microalgal biodiversity

1.2.1. Main groups
Microalgae are the most primitive form of plants; they represent a huge biodiversity in the
world. Yet, these microorganisms are not well understood as others. If we compered the
progress seen with other organism we will realize that the microalgae’s study is slower and
difficult because of the limited size of scientific community involved in it (Sheehan et al.,
1998).

However, biologists have categorized microalgae in a variety of classes, mainly distinguished

by their pigmentation, life cycle and basic cellular structure.

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The four most important classes (at least in terms of abundance) are:

The diatoms (Bacillariophyceae):

There are approximately 100,000 species of diatoms that dominate the phytoplankton of the
oceans, but are also found in fresh and brackish water. They contain polymerized silica (Si) in
their cell walls. Due to the presence of high levels of fucoxanthin (a photosynthetic accessory
pigment) diatoms turn to be golden-brown. At lower levels many other xanthophylls are
present, as well as β-carotene, chlorophyll a and chlorophyll c. these microorganisms store
carbon in the form of natural oils or as a polymer of carbohydrates known as chyrsolaminarin
(Sheehan et al., 1998).

Diatoms are characterized of being diploid (having two copies of each chromosome) during
vegetative growth; most algae are haploid (with one copy of each chromosome) except for
brief periods when the cells are reproducing sexually (Sheehan et al., 1998).

The green algae (Chlorophyceae):

Green algae are the evolutionary progenitors of modern plants. They often referred to as
chlorophytes. Those microorganisms are quite abundant especially in freshwater, they can
occur as single cells or as colonies. The main storage compound for green algae is starch or
oils that can be produced under certain conditions (Sheehan et al., 1998).

The blue-green algae (Cyanophyceae):

Much closer to bacteria in structure and organization, they contain no nucleus, no chloroplasts,
and have a different gene structure and an ability of fixing nitrogen from the atmosphere.

There are approximately 2,000 known species found in different habitats (Sheehan et al.,
1998).

The golden algae (Chrysophyceae):

This group of algae is similar to the diatoms in pigmentation and biochemical composition but
they have more complex pigment systems, and can appear yellow, brown or orange in color.
About 1,000 species are known to exist, primarily in freshwater systems. The golden algae
produce natural oils and carbohydrates as storage compounds (Sheehan et al., 1998).

1.2.2. Some well-known species

Chlamydomonas reinhardtii:

The best studied microalga at the physiological, as well as genetic and genomic level is
Chlamydomonas reinhardtii (figure2). It was the first alga to be genetically transformed. This

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microalga can be grown either photoautotrophically, mixotrophically or heterotrophically (Li-
Beisson and Peltier, 2013).

Figure2: Chlamydomonas under microscope (Melis and Happe, 2001).

Chlorella:

Chlorella (figure3) is a genus of single-cell green microalgae; it belongs to the same phylum
Chlorophyta as Chlamydomonas. The cell has a spherical shape, and can be distinguished
from Chlamydomonas because it does not have flagella. Chlorella consists of over 80 species;
it can be isolated from either freshwater or marine environment. It is also an emerging model
for the photosynthetic carbon fixation’s study in the green eukaryotic microalgae (Li-Beisson
and Peltier, 2013).

Figure3 : Chlorella endosymbiomts inside Paramecium bursaria (Eckardt, 2010)

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 Ostreococcus tauri:

The marine pico-eukaryote belongs to Chlorophyta, and is the smallest photosynthetic

unicellular eukaryote, it has an extremely small genome in which it offers great advantages for
functional genetic studies because it represents the minimal genes required to carry out
essential biological functions (Li-Beisson and Peltier, 2013).

Phaeodactylum tricornutum:

Phaeodactylum tricornutum has been developed as a model for diatom research. It is the only
species in the genus Phaeodactylum. Unlike most other diatoms P.tricornutum can grow in
the absence of silicon, and can accumulate significant amount of lipids (Li-Beisson and Peltier,
2013).

Nannochloropsis:

Nannochloropsis consists of six species, of which five are marine and one freshwater. It is
commonly used in aquaculture applications. Upon stress, it can also accumulate significant
amount of neutral lipids thus attracting interests on its development for biofuel production
(Li-Beisson and Peltier, 2013).

1.3. Morphologic study

Figure4: Illustration detailing organelles present in a typical marine unicellular

microalga (Lodish et al., 2000).

The term "morphology" describes the shape or the form of an organism and its parts.
Unicellular algae consist of a single cell (figure4). The single cell contains:

Cell wall: A specialized, rigid extracellular matrix that lies next to the plasma membrane,
protecting a cell and maintaining its shape (Lodish et al., 2000).

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Cytoplasm: Where all the cellular organelles are suspended and are bound together by a lipid
bilayer membrane. The cell membrane surrounds the cytoplasm and it also surrounds the
nucleus and the cellular organelles. The cytoskeleton present in the cytoplasm gives the cell
its shape. The cytoplasm constitutes of dissolved nutrients and it aids to dissolve waste
products. It helps movement of the cellular materials around the cell through a process called
cytoplasmic streaming (Lodish et al., 2000).

Lysosome: Small organelle having an internal pH of 4–5 and containing hydrolytic enzymes
(Lodish et al., 2000).

Ribosome: A large complex comprising several different rRNA molecules and more than 50
proteins, organized into a large subunit and small subunit. The ribosome is the site of the
protein synthesis during the translation phase (Lodish et al., 2000).

Mitochondrion: The main site of ATP production during aerobic metabolism, this organelle is
among the largest in the eukaryotic cell. It contains an independent DNA, and carries
out oxidative phosphorylation, it occupy up to 25% of the volume of the cytoplasm.
Mitochondria have two very different membranes, an outer one and an inner one, separated by
the intermembrane space. The outer membrane composed of about half lipid and half protein
and it is similar to the outer membrane of gram-negative bacteria. The inner membrane, which
is much less permeable, is about 20% lipid and 80% protein, a higher proportion of protein
more than other cellular membranes (Lodish et al., 2000).

Chloroplast: Chloroplasts are the largest and the most characteristic organelles in the cells of
plants and green algae. They are the site of photosynthesis. Like the mitochondrion,
the chloroplast is surrounded by an outer and an inner membrane (figure5). Chloroplasts also
contain an extensive internal system called thylakoids, which are flattened to form disks;
these often are grouped in stacks called grana and embedded in a matrix (the stroma). The
thylakoid membranes contain green pigments (chlorophylls) and other pigments and enzymes
that absorb light and generate ATP during photosynthesis. Part of this ATP is used by
enzymes located in the stroma to convert CO2 into three-carbon intermediates; these are then
exported to the cytosol and converted to sugars (Lodish et al., 2000).

Figure5: The structure of a Chloroplast (Lodish et al., 2000)

Vacuole: A fluid-filled pocket in cell’s cytoplasm that serves varying functions depending on
the cell’s requirements (Lodish et al., 2000).

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Flagellum: A whip-like structure that allows a cell to move (Lodish et al., 2000).

Cytosol: Unstructured aqueous phase of the cytoplasm excluding organelles, membranes, and
insoluble cytoskeletal components (Lodish et al., 2000).

Endoplasmic reticulum: Network of interconnected membranous structures within

the cytoplasm of eukaryotic cells. The rough endoplasmic reticulum (ER), which is associated
with ribosomes, functions in the synthesis and processing of secretory and membrane proteins;
the smooth ER, which lacks ribosomes, functions in lipid synthesis (Lodish et al., 2000).

Nucleus: Large membrane-bounded organelle in eukaryotic cells that

contains DNA organized into chromosomes; synthesis and processing
of RNA and ribosome assembly occur in the nucleus (Lodish et al., 2000).

Golgi complex: Stacks of membranous structures in eukaryotic cells that function in

processing and sorting of proteins and lipids destined for other cellular compartments or for
secretion; also called Golgi apparatus (figure6) (Lodish et al., 2000).

Figure6: Three-dimensional model of the Golgi complex (Lodish et al., 2000).

1.4. Microalgal metabolism

1.4.1. What is metabolism
In general, metabolism is a term that is used to describe all biochemical reactions involved in
maintaining the living state of the cells and the organism. Metabolism can be divided into two
categories:

-Catabolism: The breakdown of molecules to obtain energy.

-Anabolism: The synthesis of all compounds needed by the cells.

Since microalgae are phytoplankton, they are capable of carrying out anabolism by
performing photosynthesis in which they convert inorganic molecules into organic molecules
(Mandal, 2013).

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1.4.2 Photosynthesis
Photosynthesis is the process by which light energy is captured, converted and stored in
simple sugar molecule. This process occurs in chloroplasts. The principal end products are
two carbohydrates: Hexoses (with six-carbon), it is synthesized in the cytosol from three-
carbon precursors generated in the chloroplast, and starch; a large, insoluble glucose polymer,
it is synthesized and stored in the chloroplast (Lodish et al., 2000). The overall reaction of
oxygen-generating photosynthesis is:

Figure7: The Chloroplast’s contents (Lodish et al., 2000).

Chloroplasts are bounded by two membranes (figure7), which do not contain chlorophyll and
do not participate directly in photosynthesis. Of these two membranes, the outer one (very
similar to the outer mitochondrial membrane) contains proteins in which they form very large
aqueous channels. This membrane allows to metabolites of small molecular weight to cross it
easily. The inner membrane, conversely, is the permeability barrier of the chloroplast that
regulates the movement of metabolites into and out of the organelle; his regulation is due to
the existence of transporters. But unlike mitochondria, chloroplasts contain more than two
membranes; a third one called the thylakoid that is the site of photosynthesis (Lodish et al.,
2000).

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In each chloroplast, the thylakoid membrane is believed to constitute a single, interconnected
sheet that forms numerous small flattened vesicles, the thylakoids, which commonly are
arranged in stacks termed grana. The spaces within all the thylakoids constitute a single
continuous compartment, the thylakoid lumen. The thylakoid membrane contains a number of
integral membrane proteins to which are bound several important prosthetic groups and light-
absorbing pigments, most notably chlorophyll (Lodish et al., 2000).

Carbohydrate synthesis occurs in the stroma, the soluble phase between the thylakoid
membrane and the inner membrane.

The photosynthetic process in plants can be divided into four stages, each stage occurs in a
different area of the chloroplast. All four stages of photosynthesis are tightly coupled and
controlled so as to produce the amount of carbohydrate required by the algae. And all
reactions in stages 1 – 3 are catalyzed by proteins in the thylakoid membrane (Lodish et al.,
2000).

1.4.2.1. Absorption of Light

The first stage in photosynthesis is the absorption of light by the chlorophylls of the two
photosystems PSI and PSII (large complexes of proteins and pigments that are optimized to
harvest light). These photosystems remove electrons from water (through the reaction down
below) in order to use them in the next stage (Lodish et al., 2000).

1.4.2.2. Electron Transport

The second stage is about transporting the removed electrons from the water through a chain
of transport molecules in the thylakoid membrane until they reach the ultimate electron
acceptor, usually NADP+, reducing it to NADPH. The transport of electrons is coupled to the
movement of protons from the stroma to the thylakoid lumen, forming a pH gradient across
the thylakoid membrane, in much the same way that a proton-motive force established across
the mitochondrial inner membrane during electron transport (Lodish et al., 2000).

The overall reaction of stages 1 and 2 can be summarized as:

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1.4.2.3. Generation of ATP
In the third stage, protons move down their concentration gradient from the thylakoid lumen
to the stroma through a complex called ATP synthase which couples proton movement to the
synthesis of ATP from ADP and Pi (figure8). This use of the proton-motive force to
synthesize ATP is identical with the analogous process occurring during oxidative
phosphorylation in the mitochondrion (Lodish et al., 2000)

Figure8: Light-dependent reactions of photosynthesis at the thylakoid membrane

(Somepics , 2015).

 Together, these three stages form the light-dependent reactions by which there are a
need to light energy to synthetize the precursors (ATP and NADPH) of the next phase
which it the dark phase or the carbon fixation stage.

1.4.2.4. Carbon Fixation

The ATP and NADPH generated by the second and third stages of photosynthesis provide the
energy and the electrons to drive the synthesis of 3-phosphoglyceric acid from CO2 and H2O
in a cycle called Calvin cycle (figure9). This cycle can be divided into three main stages:

Carbon fixation: A CO2 molecule combines with a ribulose-1,5-biphosphate (RuBP) to

form two molecules with three-carbon atoms which are 3-Phosphoglyceric acid (3-
PGA), this reaction is catalyzed by an enzyme called RuBP carboxylase/oxygenase or
RuBisCo (Lodish et al., 2000).

Reduction: In this stage, NADPH reduces a tree-carbon intermediate to make a

glyceraldehyde-3-phosphate (G3P) (Lodish et al., 2000).

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 Regeneration: RuBP acceptor needs to be recycled. This process required ATP and
some G3P molecules while other ones are been used to form glucose which is the base
of starch,and acetyl-coenzyme A (acetyl-CoA) which is the precursor of fatty acids
biosynthesis (Lodish et al., 2000).

Figure9: Calvin cycle (Jones, 2010)

1.4.3. Starch biosynthesis

Starch is a polymer of glucose residues and it is being synthesized and stored in chloroplasts.
This process starts with Calvin cycle; the formation of glyceraldehyde-3-phosphate (G3P)
leads to synthesize hexose monophosphates; but these hexose monophosphates cannot be
converted directly to starch, that’s why an enzyme called adenylyl glucose-1-phosphate
transferase (AGPase) will catalyzes the first committed step of starch synthesis, it uses
glucose-1-phosphate (G1P) and ATP as substrates that produce ADP-glucose, the activated
form of glucose which is then utilized by starch synthases to form starch (figure10) (Cakir et
al., 2015).

This figure sums up the whole process:

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 Figure10: Starch biosynthesis process (Cakir et al., 2015)

1.4.4. Fatty acid biosynthesis

After the formation of acetyl-CoA -indirectly- from glyceraldehyde-3-phosphate (G3P),
comes the fatty acid synthesis phase in which the acetyl-CoA plays the role of a precursor:

The Formation of Malonyl Coenzyme A Is the Committed Step in Fatty Acid

Synthesis:

Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA. This
irreversible reaction is the committed step in fatty acid synthesis.

The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase (ACC), which contains
a biotin prosthetic group. This enzyme has a major role in fatty acid regulation (Berg et al.,
2002).

Intermediates in Fatty Acid Synthesis Are Attached to an Acyl Carrier Protein

(ACP):

The intermediates in fatty acid synthesis are linked to an acyl carrier protein (ACP); a single
polypeptide chain of 77 residues can be regarded as a giant prosthetic group, a macro CoA
(Berg et al., 2002).

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 The Elongation Cycle in Fatty Acid Synthesis:

The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP and
malonyl ACP. Acetyl transacylase and malonyl transacylase catalyze these reactions:

Malonyl transacylase is highly specific, whereas acetyl transacylase can transfer acyl groups
other than the acetyl unit, though at a much slower rate. Fatty acids with an odd number of
carbon atoms are synthesized starting with propionyl ACP (3 atoms of carbon), which is
formed from propionyl CoA by acetyl transacylase (Berg et al., 2002).

Acetyl ACP and malonyl ACP react to produce acetoacetyl ACP. This acetoacetyl ACP is
reduced to d-3-hydroxybutyryl ACP, and then d-3-hydroxybutyryl ACP is dehydrated to form
crotonyl ACP, which is a trans-Δ2-enoyl ACP. The final step in the cycle reduces crotonyl
ACP to butyryl ACP by NADPH (Berg et al., 2002).

These last three reactions (a reduction, dehydration, and a second reduction) convert
acetoacetyl ACP into butyryl ACP, which completes the first elongation cycle (Berg et al.,
2002).

The elongation cycles continue until C16-acyl ACP is formed. This intermediate is a good
substrate for a thioesterase that hydrolyzes C16-acyl ACP to produce palmitate (16 atoms of
carbon) and ACP. This palmitate will be the precursor in fatty acids synthesis reactions that
will lead to form lipids (Berg et al., 2002).

1.4.5. Biogenesis of lipid droplets (LDs): Chlamydomonas

reinhardtii for example
Lipids (triacylglycerides) are the glycerol and fatty acids esters. In microalgae, it is generally
accepted that these lipid droplets are largely present in the cytosol (figure11). But recently,
two studies suggested the involvement of chloroplasts in the formation of lipid droplets (LDs)
in Chlamydomonas reinhardtii. LDs were observed in both plastid and cytosol in a starch-less
mutant of Chlamydomonas reinhardtii. Based on these studies, a possible model of LDs
biogenesis involving both ER and chloroplasts envelops (figure12) (Goold et al., 2014).

In higher plants, two parallel pathways leading to the sequential acylation of a glycerol-3-
phosphate (G3P) until the formation of diacylglycerols DAGs occur in plastid and also in ER.
The final step of TAG synthesis is the acylation of a DAG molecule to form TAG (Goold et
al., 2014).

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Figure11: Overview of metabolic pathways in microalgal lipid biosynthesis (Shih-
Hsin et al, 2014)

Figure12: The possible models of LD biogenesis (Goold and al., 2014)

TAGs contain twice more energy than starch and protein per weight unit; thus, TAG storage
is the most efficient and compact way to pack cellular energies. In microalgae, when nutrient
is not available (stress conditions), the number and size of lipid droplets increase and the cells
store extra carbon as TAGs, thus prepared for long-term usage (Goold et al., 2014).

1.5. Cultivation methods

Microalgae are one of the most ancient organisms living on earth and one of the tiniest plants
which alone produce about 60 percent of the earth’s oxygen required for animals and humans.
They have survived some of earth’s harshest conditions for several billion years. They are
incredibly robust, and in ideal cultivation conditions, microalgae produce protein and energy
biomass from 30 to 100 times faster than land plants (Kamyab, 2012).

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These microorganisms are relatively easy to cultivate, this is why they have been grown for
decades especially in Asia and North America, in order to use their biomass in different
industries. However, the cultivation concerns a small number of species such as Spirulina and
Chlorella (Wolkers et al., 2011).

The simplest cultivation method in laboratories is based on an illuminated flask placed on a

shaker (figure13), but this method is not as efficient as the cultivation on a large scale by
using other ways to culture microalgae and to harvest their biomass (Wolkers et al., 2011).

This microalgae biomass production is based on the simple scheme:

CO2 + H2O + nutrients + light energy → biomass + O2

Figure13: Microalgae cultivation in shake-flasks (Wolkers et al., 2011)

There are two systems for microalgae’s cultivation on a large scale:

-Open cultivation systems: Outdoor ponds with natural lightening.

-Closed cultivation systems: photobioreactors with either natural lightening or artificial

lightening (Wahby, 2016)

1.5.1. Open cultivation systems: Open ponds or raceways

Open ponds or raceways (figure14) are the most common cultivation systems worldwide.
These shallow ponds use paddle wheels in the annular channels to get mixed. Despite the
advantage of this basic design (low cost) it is very difficult to manage compared to a closed
system, because of the possibility of water evaporation, the contamination and the infection.
Moreover, up to 10% of the energy from solar light can be converted into chemical energy via
photosynthesis, while the remaining part is lost as a heat. This photosynthetic efficiency is
much lower and caused by the limited penetration of sunlight into the bottom of the pond, so
as a result; only the cells near the surface receive a satisfied amount of light to practice
photosynthesis. To solve this problem, researches have created genetic modification to reduce

24
the absorption of light at the surface. This will result to more light penetration into the pond,
thus providing more algae cells with sunlight for photosynthesis (Wolkers et al., 2011).

Figure14: Raceway pond in Netherlands (Wolkers et al., 2011).

1.5.2. Closed cultivation systems: Photobioreactors

Horizontal tube reactors

Horizontal tube reactor (figure15) is a closed culture system, composed of a single layer of
horizontal tubes. They can be illuminated either by natural lightening or by artificial
lightening. It is easier to control the process of photosynthesis and the productivity per square
meter in such a tube reactor than in an open pond by controlling light intensity, nutrients and
gas amount. However, this system has many disadvantages such as the high cost of circulating
the liquid suspension, the low gas exchange and the accumulation of O2. As a result; the
productivity is lower (Wolkers et al., 2011).

Figure15: Horizontal tube reactors filled with microalgae (Wolkers et al., 2011)

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 Three-dimensional tube reactors

Tree-dimensional tube reactors (figure16) system is composed of multiple layers of tubes that
are placed vertically on top of each other and together they form a construction of vertical
panels of tubes. This type of reactor has the same advantages and disadvantages as the
horizontal tube reactor, but its productivity is higher than this one because the tubes are
placed in each other’s shade which makes the light intensity lower (Wolkers et al., 2011).

Figure16: Tree-dimensional tube reactors (Wolkers et al., 2011)

Flat plate reactors

Flat plate reactors (figure17) are closed reactors constructed from series of flat, parallel plates.
These systems are in theory the most productive because there is no accumulation of the toxic
O2 and also the light intensity at the surface is not too high. Nevertheless, the disadvantage of
this closed system is relatively the high amount of energy required for mixing the nutrients in
order to feed each cell (Wolkers et al., 2011).

Figure17: Flat Plate Reactors (Wolkers et al., 2011)

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1.6. Microalgal industrial uses
Microalgae have been used for more than 2000 years to produce a lot of valuable molecules.
However, microalgal biotechnology has known its breakthrough just in the middle of the last
century. Nowadays, there are several commercial applications of microalgae such as
enhancing the nutritional value of food and animal feed, producing biodiesel and
incorporating into cosmetics, etc. Moreover, they are cultivated as a source of highly valuable
molecules. For example, polyunsaturated fatty acid oils (Spolaore et al., 2006).

1.6.1. Chemical composition of microalgae

Microalgae biomass contains three main components:

Carbohydrates: Can be found in the form of starch, glucose, sugars and other
polysaccharides. Their overall digestibility is high, which is why there is no limitation
to using dried whole microalgae in foods or feeds (Spolaore et al., 2006).

Protein: Is one of the main reasons to consider microalgae as an unconventional source

of protein. Moreover, as the cells are capable of synthesizing all amino acids, they can
provide the essential ones to humans and animals (Spolaore et al., 2006).

Natural Oils: Varies between 1% and 70% but can reach 90% of dry weight under
certain conditions. Microalgal lipids are composed of glycerol, sugars or bases
esterified to saturated or unsaturated fatty acids (12 to 22 carbon atoms). Among all
the fatty acids in microalgae, some fatty acids of the ω3 and ω6 families are of
particular interest (Spolaore et al., 2006).

Microalgae also represent a valuable source of nearly all essential vitamins (A, B1, B2,
B6, B12, C, E, nicotinate, biotin, folic acid and pantothenic acid. Microalgae are also rich in
pigments like chlorophyll (0.5% to 1% of dry weight), carotenoids (0.1% to 0.2% of dry).
These molecules have a wide range of commercial applications (Spolaore et al., 2006).

1.6.2. Human nutrition

For human nutrition, microalgae can be used as tablets, capsules and liquids. They can also be
incorporated into pastas, snack foods, candy bars or gums. They can act as a nutritional
supplement or represent a source of natural food colorants thanks to their diverse chemical
properties. The commercial applications are dominated by four strains: Arthrospira, D. salina
(figure18), Chlorella, and Aphanizomenon flosaquae (Spolaore et al., 2006).

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 Figure18: Dunaliella salina and Arthrospira platensis (Wahby, 2016)

Arthrospira has high protein content and an excellent nutritive value. In addition, this
microalga has various possible health-promoting effects such as the alleviation of
hyperlipidemia, suppression of hypertension, protection against renal failure, growth
promotion of intestinal Lactobacillus, and suppression of elevated serum glucose level
(Spolaore et al., 2006).

D. salina is exploited for its β-carotene content that can reach 14% of dry weight and
also for its powder which is an ingredient of dietary supplements and functional foods
(Spolaore et al., 2006).

For Chlorella, the most important substance is β -1, 3-glucan, which is an active
immunostimulator, a free-radical scavenger and a reducer of blood lipids. Moreover,
various other health-promoting effects have been clarified such as efficacy on gastric
ulcers, wounds, and constipation, etc. also, Chlorella can be used as a food additive
(Spolaore et al., 2006).

The last major commercial strain application is Aphanizomenon flosaquae. According

to many research studies, it promotes good overall health when used alone or in
combination with natural food products (Spolaore et al., 2006).

In addition to its use in human nutrition, microalgae can be incorporated into the feed for a
wide variety of animals ranging from fish (aquaculture) to pets and farm animals. In fact, 30%
of the current world algal production is sold for animal feed applications (Spolaore et al.,
2006).

1.6.3. Microalgae in cosmetics

Owing to their global chemical composition, microalgae are generally used in the field of
cosmetics. Their extracts can be mainly found in face and skin care products (anti-aging
cream, sun protection and hair care products, refreshing care products, emollient and as an
anti-irritant in peelers) (figure19). Some microalgal species like Arthrospira and Chlorella are
already established in the skin care market; a protein from the first repairs the signs of early
skin aging, and an extract from the second stimulates collagen synthesis in skin to supports its
tissues and to reduce its wrinkles (Spolaore et al., 2006).

28
 Figure19: Some skin care products that are made of microalgal powder (Wahby,
2016)

1.6.4. Production of biodiesel

Due to increasing interests in the production of renewable energies from microalgae,
microalgal oil metabolism is subjected to intensive research. All eukaryotic microalgae
examined so far can produce oils as energy storage. This important storage can lead to form
biodiesel (Goold et al., 2014).

Biodiesel is diesel fuel that constitutes of long chain alkyl esters; it is usually produced with
lipids (TAGs) and alcohol in a chemical reaction known as transesterification (figure20).
Moreover, its properties are very close to those of petroleum diesel fuel (Kamyab, 2012). It
can be used in unmodified diesel engines, and has advantages over conventional diesel fuel in
that it is renewable and biodegradable (Sheehan et al., 1998).

Figure20: Production of biodiesel by transesterification (Sheehan et al., 1998)

29
There are so many reasons as to why biodiesel is a desirable energy resource:

-It is renewable, sustainably supplied and replaces the declining petroleum resources
that are expected to be exhausted in less than 50 years at the present rate of
consumption.

-It is environmental friendly because it has no net increased release of carbon dioxide
and very low sulfur content (Kamyab, 2012).

Unfortunately, microalgae process has significant limitations and restrictions that will be
minimized intentionally. Those are:

-The requirement to choose highly productive lipid-rich microalgal strains.

-The maintenance of selected species in outdoor culture.

-The high energy inputs required for water pumping, CO2 transfer, mixing the culture
suspension and harvesting/dewatering the microalgal biomass (Kamyab, 2012).

However, biodiesel is a very promising fuel product but it not the only one that can be extract
from microalgae. Biogases such as ethanol and methanol also take a place in today’s
biotechnology and can be used in industry (Sheehan et al., 1998).

30
2. Haematococcus pluviaris
2.1. General characteristics
Haematococcus pluvialis is a unicellular green alga (Chlorophyceae) that has several
characters and unique life, which is divided in two stages:

-The first stage refers to a flagellated motile green cell that is continuously divides and
synthesizes chlorophyll. During this stage full nutrient medium and moderate light
intensity, temperature and pH are required (Panis and Carreon, 2016).

-The second refers to a red non-motile cell, in which its division stops and chlorophyll
levels do not fluctuate. This stage takes place when the conditions become unfavorable,
for example under nutrient depletion, continuous light or high temperature. The red
color is due to accumulation of the red pigment astaxanthin. Because astaxanthin
possesses high antioxidant, its accumulation is considered a survival strategy against
stressful conditions (Ohnuki et al., 2013 ; Panis and Carreon, 2016).

Between these two stages, tree morphological types of cells appear:

Zoospore (figure21: a): It has two flagella and a discoid eyespot near the cytoplasmic
membrane at the cell anterior. It features a cup-shaped chloroplast occupying almost
the entire cell volume (Chekanov et al., 2014).

Non-motile coccoid green cell (figure21: b): Also called palmelloid cell. It possesses a
thick extracellular matrix (0.64–0.8 μm) with no flagella (Chekanov et al., 2014).

Non-motile coccoid green cell with red-colored lipid droplets (figure21: c): Under
adverse environmental conditions, the coccoid cells increased in size (up to 80 μm)
and accumulated red-colored spherical inclusions in the cytoplasm, which tended to
cluster around the nucleus. The red pigment gradually occupied the entire cytoplasm
volume resulting in the formation of resting red cells (Chekanov et al., 2014).

Figure21: (a) Zoospore; (b) Coccoid green cell; (c) Coccoid green cell with red-
colored lipid droplets (Chekanov et al., 2014)

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2.2. Astaxanthin
2.2.1. Astaxanthin chemical properties and benefits
Astaxanthin (C40H52O4) is a pigment that belongs to the xanthophyll subclass of carotenoids.
It has oxygen in its molecular structure, which sets it apart from beta-carotene and other
molecules of the carotene subclass. The astaxanthin molecule has an extended shape, with a
polar structure at both ends of the molecule and a nonpolar zone in the middle (figure22)
(Kidd, 2011).

Figure22: Astaxanthin structure (Kidd, 2011)

Since humans do not make astaxanthin; they depend in their nutrition on seafood such as
crabs, crayfish, lobsters, salmon, shrimp, and trout to cover their needs (usually 40 mg/day or
less). This pigment demonstrates an exceptional range of benefits:

Antioxidant effect: Astaxanthin produces clinically significant antioxidant benefits in

human subjects, especially vulnerable to oxidative stress (excess of free radical
activity over antioxidant capacity), such as smokers, the obese, and the overweight
(Kidd, 2011).

A unique cell membrane antioxidant: Astaxanthin provides cell membranes with

potent protection against free radical or other oxidative attacks. It neutralizes free
radical or other oxidant activity in the nonpolar (hydrophobic) zones of phospholipid
aggregates, as well as along their polar (hydrophilic) boundary zones (Kidd, 2011).

Anti-inflammatory benefits: Astaxanthin can significantly lower C-reactive protein

(CRP), a biomarker of systemic inflammation (Kidd, 2011).

Effect on vision and eye fatigue: Astaxanthin (taken at 6 mg/day) can improve visual
sharpness, even in healthy subjects. Also it might relieve eye fatigue in persons using
computer monitors (Kidd, 2011).

Astaxanthin can also affect positively on immune response, blood circulation, memory and
brain and on mitochondrial function. Its clinical success extends beyond protection against
oxidative stress and inflammation, to demonstrable promise for slowing age-related functional
decline (Kidd, 2011).

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2.2.2. Astaxanthin production
There are several microalgae strains that are reported as potential feedstock to produce
astaxanthin. However, its accumulation inside Haematococcus pluvialis cells exceeds any
other known microalgae species (up to 4% of dry biomass) and thus it is the most preferred
one for large-scale natural astaxanthin production (Panis and Carreon, 2016).

In order to produce the astaxnthin, three principal steps are required:

Cultivation: A hybrid system for photoautotrophic cultivation comprised by a

photobioreactor (PBR) fence and a raceway pond complex was assumed for the ‘green’ and
the ‘red stage’ respectively (figure23). For the green stage, a horizontal tubular PBR fence
was used, since controllable environmental and nutritional conditions that can be achieved in
the PBR, facilitate optimal growth. As for the red stage, the raceway pond complex was
selected principally in order to create unfavorable environment to stimulate the accumulation
of astaxanthin (Panis and Carreon, 2016).

Figure23: Haematococcus pluvialis cultivation (Wolkers et al., 2011)

Harvesting: Microalgae harvesting currently involves mechanical (gravity sedimentation,

flotation, filtration and centrifugation), chemical (coagulation/flocculation), biological (bio-
flocculation) and to a lesser extent, electrical methods (electrophoresis) (Panis and Carreon,
2016).

Extraction: This phase is about extracting the algal powder (figure24) and it can be divided
into three main processes: Cell disruption (by using bead milling), dehydration (by using
spray drying that has been labeled as the most appropriate method to dry high-value
microlagal products) and recovery of the desired metabolite (astaxanthin) (by using
supercritical CO2 extraction at a temperature of 60 °C and a pressure of 30 MPa, while using
ethanol as a co-solvent) (Panis and Carreon, 2016).

Figure24: Astaxanthin powder (Wolkers et al., 2011)

33
 General conclusion

In general, microalgae have shown a lot of benefits on so many levels. These microorganisms
have a promising future in the biotechnical industry, from producing carbohydrates, proteins
and natural oils, to producing biodiesel via transesterification. This last process can help
saving the environment by abandoning the petroleum fuels and depending even more on algal
biomass as natural resource. Unfortunately, the high cost of producing this biodiesel prevents
it from becoming economically competitive, which is why it is necessary for future research
to find another sheep method to produce it.

As for the red pigment astaxanthin, the Chlorophyte Haematococcus pluvialis accumulates
large quantities of it as a stock; this pigment has demonstrates several positive effects of
human health, so as a result, it is acceptable to say that this antioxidant molecule is also a very
promising product in many ways.

In order to exploit microalgae in the best way possible, researches must look for better
methods either to cultivate them or to extract their biomass without wasting any of its
amounts.

34
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