Applied Phycology

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The microalga Dunaliella and its applications: a

review

Miguel Barbosa, Leonardo Garcia Inácio, Clélia Afonso & Paulo Maranhão

To cite this article: Miguel Barbosa, Leonardo Garcia Inácio, Clélia Afonso & Paulo Maranhão
(2023) The microalga Dunaliella and its applications: a review, Applied Phycology, 4:1, 99-120,
DOI: 10.1080/26388081.2023.2222318

To link to this article: https://doi.org/10.1080/26388081.2023.2222318

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 British
Phycological
APPLIED PHYCOLOGY
2023, VOL. 4, NO. 1, 99–120
Society
Understanding and using algae
https://doi.org/10.1080/26388081.2023.2222318

The microalga Dunaliella and its applications: a review

a a b b
Miguel Barbosa , Leonardo Garcia Inácio , Clélia Afonso and Paulo Maranhão
a
ESTM, School of Tourism and Maritime Technology, Polytechnic of Leiria, Peniche, Portugal; bMare/Arnet, Estm, Polytechnic of Leiria, Edifício
Cetemares, Peniche, Portugal

ABSTRACT ARTICLE HISTORY

Green microalgae in the genus Dunaliella have become increasingly important in biotechnology Received 11 January 2023
and industry. The high adaptability of Dunaliella to high salinity, as well as its fast growth and Accepted 31 May 2023
production of several metabolites have triggered interest. The attention of industry relates to its KEYWORDS
ability to synthesize several high-value compounds, such as β-carotene, lipids, glycerol, vitamins, Applications; carotenoids;
and proteins. In addition, due to its tolerance to high salinity, contamination is reduced, and it can Dunaliella; lipids; microalgae;
grow in open systems. Dunaliella salina can accumulate up to 25% dry weight in lipids and is the synthesis
most efficient natural source of β-carotene. This review highlights the general characteristics of the
genus, associated with its history, morphology, reproduction, occurrence, and taxonomy. The
metabolic pathways for carotenoid and lipid synthesis are described. Relevant information on
the most common strains is provided as well as the most widely used growth systems and
conditions, and the expression systems under development. Applications of Dunaliella in several
areas of the industry are also highlighted. Thus, this review can serve as a basis for future work and
for the development of environmentally friendly, simple, and highly cost-effective production
methods.

occurrence, metabolism, scientific relevance, most used

Introduction
species and strains, growth conditions and growing
Algae have shown great potential in achieving sustain­ systems, genetic manipulation and specific biotechno­
able and efficient solutions. In addition to their impor­ logical applications. The number of publications asso­
tance in ecosystems, involved in the main ciated with the term [Dunaliella] was analysed in the
biogeochemical cycles (Du, Wang, You, & Zhao, PubMed database (National Center for Biotechnology
2013), they have also been widely used in industry. Information, US National Library of Medicine), from
Specifically, microalgae are recognized as potential the year 2000 to 2019. According to Fig 1, in recent
resources for applications in the food industry, for years, scientific interest and consequently the number of
pharmaceutical compounds, as environmental solutions publications related to this genus have been increasing.
and for the production of specific compounds, among
others. These organisms have many advantages, such as
General description
low production costs, high photosynthetic efficiency,
high resistance, and simple genetics (Chisti, 2007; Koh Dunaliella cells were identified for the first time by
& Ghazoul, 2008; León-Bañares, González-Ballester, Michel F. Dunal (Dunal, 1838) in salt pans in the
Galván, & Fernández, 2004). The great diversity of sub­ south of France and described as reddish unicellular
stances, such as pigments, lipids or proteins, and algae. In 1905, Emanoil C. Teodoresco proposed the
mechanisms that are present in microalgae result partly genus (Teodoresco, 1905). A distinguishing feature of
from their wide diversity of origins, with multiple meta­ these microalgae is their high resistance to various
bolic and physiological strategies (Iglesias et al., 2019). environmental factors, and there is therefore a high
Thus, microalgal biotechnology has developed quickly diversity of strains available for the industry. The
in the last three decades (Spolaore, Joannis-Cassan, study of particular strains has increased our under­
Duran, & Isambert, 2006). standing of the physiological adaptation mechanisms
Our aim here is to report on the genus Dunaliella, a to high salinity, low pH and a wide range of tempera­
well-known microalga used in many industrial applica­ tures (Oren, 2014). Many Dunaliella species have been
tions. We describe the general characteristics of isolated from environments with high salinity and a
Dunaliella are described and also address its ecology, wide range of concentrations of other chemical

CONTACT Miguel Barbosa miguel_luismarques_barbosa@hotmail.com; leonardogarciainacio@hotmail.com

© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted
Manuscript in a repository by the author(s) or with their consent.
100 M. BARBOSA ET AL.

80 and can vary between 5 to 25 μm in length and 3 to 13

y = 2.2955x - 4571.3 μm in width (Hosseini Tafreshi & Shariati, 2009). These
60 R² = 0.8193 cells do not have a rigid cell wall, being delimited by a
thin and elastic membrane that allows more effective
40 morphological adaptation, according to the osmotic
pressure variations of the external environment (Ben-
20
Amotz & Avron, 1990; Ben-Amotz, 1993). The cells
0 contain a single, central, cup-shaped chloroplast with a
central pyranoid surrounded by starch grains
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
(Borowitzka & Siva, 2007).
Dunaliella cells multiply by longitudinal division,
Figure 1. Number of publications related to the term [Dunaliella]
and sexual reproduction by isogamy can occur,
between the years 2000–2019, in PubMed database (NCBI), and
respective linear regression. where two cells merge, forming a zygote (Oren,
2005). It has also been shown that these cells can
lead to the formation of palmelloid or aplanospore
components (Yao et al., 2016). Dunaliella species have cells, if the environmental conditions inhibit growth
already been grown in salinities from 0.5% to 35%, so (Borowitzka & Siva, 2007; Polle, Tran, & Ben-Amotz,
they are widely used in the industry, grown in extreme 2009).
environments (Hadi, Shariati, & Afsharzadeh, 2008). Knowing that Dunaliella species are mostly photo­
Dunaliella‘s adaptation mechanisms to different salt autotrophic, the presence of a wide variety of pigments
concentrations have been shown to be essentially in the cells is expected. In addition to chlorophyll a and
based on the ability to alter the intracellular concentra­ b, several carotenoids such as α and β-carotene, neox­
tion of glycerol, a well-known compatible solute anthin, lutein, violaxanthin and zeaxanthin are found
(Ghoshal, Mach, Agarwal, Goyal, & Goyal, 2002). On (Ben-Amotz & Avron, 1989a).
the other hand, data also indicate that this genus has an Regarding the taxonomy, the genus Dunaliella has
exceptional ability to remove sodium ions, in high sali­ been quite controversial (Polle, Tran, & Ben-Amotz,
nity environments, through a redox sodium pump (Katz 2009). Dunaliella species belong to the phylum
& Pick, 2001). Chlorophyta, class Chlorophyceae, order
Dunaliella species have also been shown to be resis­ Chlamydomonadales and family Dunaliellaceae. In the
tant to high concentrations of other compounds, such as last few years, reassessments of the genus have been
glycerol, and to high light intensities, e.g., 2000 µmol carried out, with 29 species being listed, of which 17
photons m−2 s−1. Dunaliella acidophila can grow in are halophiles: D. parva, D. salina, D. pseudosalina, D.
extremely acidic environments (pH 0–1) and ruineniana, D. gracilis, D. bioculata, D. carpatica, D
Dunaliella antarctica resists temperatures below zero granulata, D. baasbeckingii, D. minuta, D. media, D.
(Hosseini Tafreshi & Shariati, 2009; Pick, 1999), demon­ minutissima, D. terricola, D. viridis, D. asymmetrica, D.
strating its great adaptation capacity. peircei and D. turcomanica. The most researched species
This genus occurs in extreme environments around are D. salina, D. tertiolecta, D. primolecta, D. viridis, D.
the world (Oren, 2009). It has been isolated from cob­ bioculata, D. acidophila, D. parva and D. media
webs in the Atacama Desert, which can be used as a (Borowitzka & Siva, 2007). Dunaliella bardawil is con­
model for studying the evolution of aquatic organisms sidered to be a subspecies of Dunaliella salina
to land organisms (Cereceda, Larrain, Osses, Farías, & (Gonzalez, Coleman, Gomez, & Montoya, 2001).
Egaña, 2008), in saline soils in the Great Salt Plains, According to the NCBI database, the genomes of five
Oklahoma (Buchheim, Kirkwood, Buchheim, strains of this genus have already been sequenced: D. salina
Verghese, & Henley, 2010) and in three Antarctic lakes CCAP 19/18 (GenBank: GCA_002284615.1), in 2017,
(Wright & Burton, 1981). D. salina, D. viridis and D. Dunaliella sp. M2 (GenBank: GCA_004335885.1),
parva are found in the Great Salt Lake, USA (Oren, Dunaliella sp. RO (GenBank: GCA_004335775.1),
2014). D. salina is also found in Lake Tyrrell in Dunaliella sp. WIN1 (GenBank: GCA_004335645.1) and
Australia (Oren, 2014), in lakes in Iran (Hadi, Shariati, Dunaliella sp. YS1 (GenBank: GCA_004335685.1), in 2019.
& Afsharzadeh, 2008), and in halite crystals in Death
Valley, USA, approximately 10–34 ky and 100 ky old
Industrially relevant metabolites
(Schubert, Timofeeff, Lowenstein, & Polle, 2010).
Morphologically, Dunaliella cells are biflagellate, can Dunaliella cells synthesize a wide range of products,
be ovoid, spherical, pyriform, fusiform, or ellipsoidal, among which carotenoids and lipids are the most
 APPLIED PHYCOLOGY 101

relevant. Some processes related to these components Most of the β-carotene industrial production
and their metabolism will be described. (approximately 85%) is still carried out by chemical
synthesis (Ye, Jiang, & Wu, 2008). However, synthetic
β-carotene is essentially made up of all-trans isomers,
Carotenoids
whereas in Dunaliella cells, high amounts of 9-cis iso­
Some species, such as D. salina and D. bardawil, have mers can be found, which have a much more relevant
the ability to accumulate high amounts of carotenoids antioxidant effect (Shaish et al., 2006).
(Ye, Jiang, & Wu, 2008). The most common type of Regarding carotenoid metabolism, studies indicate
carotenoid in Dunaliella cells is β-carotene, but α-car­ that it is strongly related to the synthesis of chlorophyll
otene, lutein, zeaxanthin, cryptoxanthin and neoxanthin (Fu et al., 2013; Lamers et al., 2010). The synthesis of
can also be found (Ben-Amotz, Katz, & Avron, 1982). each of these components can be competitive or coop­
Under specific growth conditions, some strains have the erative, knowing that these processes are controlled by
capacity to accumulate more than 15% of β-carotene by related metabolic pathways (Song et al., 2018).
dry weight (Hosseini Tafreshi & Shariati, 2009). The The first step in the metabolic pathway for carote­
high concentrations of β-carotene give the cells a red noid synthesis is the synthesis of GGPP (geranylgeranyl
colouration (Oren, 2005). pyrophosphate), by the catalyst enzyme GGPS (geranyl­
Specifically, β-carotene has been a widely used com­ geranyl pyrophosphate synthase), from the addition of
ponent in the industry; thus, several strategies have been three IPP molecules (isopentenyl pyrophosphate) to a
developed to maximize its production. Diverse Dunaliella DMAPP molecule (Dimethylallyl pyrophosphate), via
strains have been used for the production of β-carotene in sequential reactions (Hirschberg, 2001). GGPP is the
several countries, such as Australia, the USA and China precursor to carotenoids (Xu et al., 2022).
(Borowitzka, 1999). Dunaliella salina is considered to be For GGPP synthesis to be possible, IPP molecules
the most efficient natural source to produce β-carotene have to be produced. Studies indicate the existence of
(Lamers, Janssen, De Vos, Bino, & Wijffels, 2008). For the two relevant metabolic pathways for the synthesis of IPP
induction of β-carotene accumulation, Dunaliella cells in plants and algae: the mevalonate (MVA) pathway and
should be exposed to high light intensities and growth the non-mevalonate pathway (1-deoxy-D-xylulose 5-
conditions that usually reflect low growth rates, such as phosphate (DXP) pathway or 2-C-methyl-D-erythritol
extreme temperatures, high salinities, and limited nitro­ 4-phosphate (MEP) pathway) (Lange, Rujan, Martin, &
gen (Lamers, Janssen, De Vos, Bino, & Wijffels, 2008). β- Croteau, 2000). The mevalonate pathway, described in
carotene is a component of photosynthesis and is accu­ 1950, starts with acetyl-CoA (Bloch, 1992), with the
mulated in lipid globules in the inter-thylakoid spaces of participating enzymes located in the cytosolic compart­
the chloroplasts in Dunaliella, under stress conditions ment. The vast majority of cytoplasmic IPP is synthe­
(Hadi, Shariati, & Afsharzadeh, 2008). sized by this pathway (Newman & Chappell, 1999). On
The functions of carotenoids have been studied in the the other hand, the non-mevalonate pathway, described
previous work. Knowing their conjugated polyene struc­ in 1980, is responsible for the synthesis of most of the
ture, these components play a very important role in the IPP present in plastids. It starts with the conjugation of
photosynthetic reaction, in terms of energy and oxygen D-glyceraldehyde 3-phosphate (GA3P) and pyruvate via
transport (Siefermann-Harms, Joyard, & Douce, 1978), intermediates. The pathway follows this order: 1-deoxy-
also preventing chlorophyll photo-damage (Ben-Amotz D-xylulose 5-phosphate (DXP), 2-C-methyl-D-erythri­
& Shaish, 1992). On the other hand, carotenoids can also tol 4-phosphate (MEP), 4-diphosphocytidyl−2-C-
suppress singlet oxygen and free radicals (Ye, Jiang, & methyl-D-erythritol (DPME), 4-diphosphocytidyl−2-
Wu, 2008). More than 700 types of carotenoids have been C-methyl-D-erythritol 2-phosphate, 2-C-methyl-D-ery­
described in natural resources (Feltl, Pacakova, Stulik, & thritol 2,4-cyclodiphosphate (MEcDP) and 4-hydroxy
Volka, 2006). Most of the carotenoids are C40 isopre­ −2-methylbut−2-enyl pyrophosphate (HMBPP)
noids, that is, composed of eight isoprene units (Naik, (Seemann, Tse Sum Bui, Wolff, Miginiac-Maslow, &
Chanemougasoundharam, Khurana, & Kalloo, 2003). Rohmer, 2006). Finally, IPP, DMAPP and, conse­
Specifically, β-carotene can be found in several structures quently, GGPP are generated. This pathway has been
since the configuration of each double bond can occur described in bacteria (Rohmer, Knani, Simonin, Sutter,
naturally as trans or cis. The most common isomers are & Sahm, 1993), algae (Schwender, Seemann,
all-trans, 9-cis, 13-cis and 15-cis (Patrick, 2000). Lichtenthaler, & Rohmer, 1996) and plants
Dunaliella cellular β-carotene is composed of a mixture (Lichtenthaler, Schwender, & Müller, 1998). Genes,
of all-trans (42%), 9-cis (41%), 15-cis (10%) and other enzymes, and other components participate in the
isomers (6%) (Borowitzka & Borowitzka, 1989). non-mevalonate pathway (Xu et al., 2022).
102 M. BARBOSA ET AL.

From GGPP, a pathway follows where several caro­ Lipids are essential components in cell structure and
tenoids are synthesized. The synthesis of carotenoids storage, and the variation of the lipid content in micro­
from GGPP is quite common in several organisms. algae is usually due to a response to changes in the
Initially, two GGPP molecules are conjugated to obtain medium composition. Fatty acids (FA) allow the separa­
the first colourless carotenoid, phytoene, from the tion of the cellular content from the extracellular med­
enzyme phytoene synthase (PSY). This enzyme is indi­ ium (Matsumoto, Shioya, & Nagashima, 1984). Sterols
cated as an essential element in the synthesis of carote­ are among the compounds of greatest interest, usually
noids and regulation of carbon flow in this process used to determine the nutritional value of microalgal
(Shewmaker, Sheehy, Daley, Colburn, & Ke, 1999). food products, used as biomarkers of the presence of
Studies indicate that isomerization reactions occur in organic matter in sediments (Volkman et al., 1998), and
this step (Ben-Amotz, Lers, & Avron, 1988). As soon as as indicators of cell proliferation, modulating the activ­
phytoene is formed, denaturation reactions occur, ity of membrane-bound enzymes (Francavilla, Trotta, &
obtaining, in this order: phytofluene, ζ-carotene, neuro­ Luque, 2010).
sporene and lycopene. From the formation of lycopene, Large amounts of fatty acids are found in D. salina,
two pathways for the formation of cyclic or acyclic with about 50% of these being mono- or polyunsatu­
carotenoids can be followed (Schmidt-Dannert, 2000). rated (Cakmak, Kaya, & Asan-Ozusaglam, 2014), prin­
β-carotene is a cyclic carotenoid, so the most prevalent cipally palmitic (C16: 0), alpha linolenic (C18: 3), and
pathway in Dunaliella cells is the one that promotes the oleic acid (C18: 1). Alpha-linolenic acid is one of the
formation of cyclic carotenoids. In the cyclization pro­ best-known ω−3 fatty acids, associated with benefits in
cess, cyclohexene rings are added to one or both ends of brain development, and in the prevention of cardiovas­
the lycopene molecule, from the enzyme lycopene cular diseases (Cakmak, Kaya, & Asan-Ozusaglam,
cyclase (LYC), and the formation of δ-, α-, γ- and β- 2014).
carotene takes place. Two types of the LYC enzyme have Some studies indicate that in eukaryotic algae, the
already been described, the lycopene β-cyclase (LYC-Β) synthesis of fatty acids is simpler than in plants
and lycopene ε-cyclase (LYC-Ε). LYC-Β adds a β ring at (Goncalves, Wilkie, Kirst, & Rathinasabapathi, 2016).
one or two ends of the lycopene to form γ-carotene Three carbon pathways have been identified for bio­
(monocyclic) or β-carotene (dicyclic) molecules. LYC- synthesis in chloroplasts: 1- De novo synthesis directly
Ε only has the ability to add an ε ring to one end of the from CO2 assimilation by the Calvin cycle, the forma­
lycopene generating the δ-carotene (monocyclic) tion of acetyl-CoA by pyruvate dehydrogenase and its
(Cunningham et al., 1996). For the production of α- assimilation via fatty acid synthesis cycle (Fig 3)
carotene, an ε ring (by LYC-E) at one end and a β ring (Coleman & Lee, 2004; Ohlrogge & Browse, 1995); 2-
(by LYC-B) at the other must be added to the lycopene. fatty acid acyl transfer from pre-formed polar lipids,
It is possible to conclude that Dunaliella cells have both particularly from chloroplast galactoglycerolipids
cyclases, as these cells contain α-carotene and β-caro­ (Lippold et al., 2012); 3- Synthesis from the degradation
tene. From these two pathways, other components can of pre-formed starch (Fig 3) (Pick & Avidan, 2017). Pick
be generated, such as α-cryptoxanthin, zeinoxanthin and Avidan (2017) suggest that, in Dunaliella, about
and lutein from α-carotene, and β-cryptoxanthin, zeax­ two-thirds of the triacylglycerol come from the third
anthin, antheraxanthin, violaxanthin and neoxanthin route, one-third from the first, and some residual
from β-carotene according to Fig 2. The metabolic pro­ amounts come from the second route.
cesses previously mentioned were described in detail by
Ye, Jiang, & Wu (2008).
Other products
This genus is also used as a glycerol source.
Lipids
Dunaliella, in hypersaline conditions, produces gly­
In addition to carotenoids, Dunaliella is a source of cerol that helps to maintain the integrity of the
lipids, reaching 25% in dry weight (Liu et al., 2013). In membrane and proteins, with some strains accumu­
general, the production of these compounds is also lating more than 50% dry weight (Ben-Amotz &
associated with non-favourable growth conditions, Avron, 1990), and to maintain osmotic balance.
such as temperature, light intensities (Gim et al., 2014) These organisms have the ability to secrete glycerol
salinities (Takagi, Karseno, & Yoshida, 2006), or with into the medium that functions as a carbon dioxide
the presence of specific compounds in the medium such scavenger (Chow et al., 2013). The mechanisms of
as sodium tungstate (Benhima et al., 2018) glycerol production can vary depending on the
 APPLIED PHYCOLOGY 103

Geranylgeranyl pyrophosphate (GGPP)

Phytoene synthase

Phytoene

Phytoene desaturase

Phytofluene

Phytoene desaturase

ζ-carotene

ζ-carotene desaturase

Neurosporene

ζ-carotene desaturase

Lycopene

Lycopene ε-cyclase Lycopene β-cyclase

δ-carotene γ-carotene

Lycopene β-cyclase Lycopene β-cyclase

α-carotene β-carotene

ε-carotene β-carotene β-carotene hydroxylase

hydroxylase hydroxylase

α-cryptoxanthin Zeinoxanthin β-cryptoxanthin

β-carotene ε-carotene β-carotene hydroxylase

hydroxylase hydroxylase

Lutein Zeaxanthin

Zeaxanthin epoxydase

Antheraxanthin

Zeaxanthin epoxydase

Violaxanthin

Neoxanthin synthase

Neoxanthin

Figure 2. Metabolic pathway of carotenoid production in Dunaliella (Hirschberg et al., 1997; Hirschberg, 2001; Sandmann, 2001; Ye,
Jiang, & Wu, 2008).

growth conditions, with higher production rates dry biomass (Becker, 2007; Sui, Muys, Vermeir,
when cells are exposed to limited light (Ng, Low, D’Adamo, & Vlaeminck, 2019). D. acidophila grows
Chow, & Lee, 2014) and high salinities. D. salina at very low pH and has been used for the production
produces higher glycerol levels than other of the enzyme H+-ATPase, which is one of the main
Dunaliella species (Hadi, Shariati, & Afsharzadeh, proteins in its membrane (Matalin et al., 2021; Sekler
2008). & Pick, 1993). Some strains of D. salina have large
Dunaliella is also used for the production of pro­ amounts of adenosylcobalamin and are identified as
teins, which can represent between 50% and 80% of a possible source of vitamin B12, accumulating about
104 M. BARBOSA ET AL.

Starch pathway De novo pathway

Starch
*Acetyl- CoA

Acetyl-CoA carboxylase

*Glucose Malonyl-CoA

Pyruvate
Malonyl-CoA transacylase
dehydrogenase

*Pyruvate Malonyl-ACP

Fatty acid synthase

*Acyl-ACP

Glycerol 3-
Glycerol 3-phosphate
phosphate

*Lysophosphatidic acid

Lysophosphatidic acid
acyltransferase

*Phosphatidic acid

Phosphatidic acid phosphatase

*Diacylglycerol

Diacylglycerol acyltransferase

Triacylglycerol

Figure 3. The starch and De novo pathway of Triacylglycerol synthesis in green algae. (*the metabolic pathways were simplified to a
better understanding) (Coleman & Lee, 2004; Lippold et al., 2012; Ohlrogge & Browse, 1995; Pick & Avidan, 2017).

50% of dry weight (Kumudha & Sarada, 2016). D. Commonly used species and strains in the
tertiolecta is used to synthesize vitamin E, xantho­ industry
phyll and violaxanthin that have antiproliferative
activities (Pasquet et al., 2011). Several species of Dunaliella have been studied and
Under particular growth conditions, some species widely used in recent years. D. salina is one of the
of Dunaliella have antimicrobial activity against sev­ most used microalgae in the industry. Its most signifi­
eral microorganisms (Kocberber Kilic, Erdem, & cant feature is its ability to accumulate large amounts of
Donmez, 2018). Chang et al. (1993) demonstrated β-carotene and lipids (Yilancioglu et al., 2014). In addi­
that the crude extract of D. primolecta shows anti­ tion, aliphatic and isoprenoid hydrocarbons, sterols,
microbial activity against Staphylococcus aureus, phospholipids and glycolipids have been identified in
Enterobacter aerogenes, Bacillus subtilis and Bacillus D. salina. D. salina has been the target of several studies
cereus. The production of these compounds occurred as a physiological model and for growth optimization
under osmotic stress (Kocberber Kilic, Erdem, & (Besson & Guiraud, 2013; Béchet et al., 2018; Khadim et
Donmez, 2018). Lutein and ferulic acid were the al., 2018). Thus, numerous strains of this species have
main compounds responsible for the antimicrobial been developed and registered, partly due to their halo­
response (Chang et al., 1993). tolerance, that prevents the growth of most competitors
 APPLIED PHYCOLOGY 105

Table 1. Strains used in identified studies, of different species of the genus Dunaliella. (Strain availability: “CCAP” - Culture Collection of
Algae and Protozoa; “DF” - the Marine Biological Association; “SAG” - SAG Culture Collection; “LB” - UTEX Culture Collection; “RCC” -
Roscoff Culture Collection; “CCMP”- NCMA Bigelow Laboratory for Ocean Sciences; “ATCC”- American Type Culture Collection).
Species Strain Reference
1 D. salina CCAP 19/18 (Park, Lee, & Jin, 2013)
2 CCAP 19/26 (Besson, Formosa-Dague, & Guiraud, 2019)
3 DF40 (Monte et al., 2020)
4 SAG 184.80 (Sui, Muys, Vermeir, D’Adamo, & Vlaeminck, 2019; Vanitha, Narayan, Murthy, & Ravishankar, 2007)
5 LB 1644 (Atasever-Arslan, Yilancioglu, Bekaroglu, Taskin, & Cetiner, 2015)
6 RCC3579 (Iglesias et al., 2019)
7 V−101 (Kumudha & Sarada, 2016)
8 MS002 (EL Arroussi et al., 2018)
9 (Lake Tuz, Turkey; Yucatan, Mexico) (Cakmak, Kaya, & Asan-Ozusaglam, 2014; García, Freile-Pelegrín, & Robledo, 2007)
10 D. tertiolecta LB 999 (Lin & Lee, 2017; Lin, Shen, & Lee, 2018; Ng, Low, Chow, & Lee, 2014)
11 CCMP1320 (Thakkar, Mitra, & Wei, 2016)
12 ATCC 30,929 (Takagi, Karseno, & Yoshida, 2006)
13 D. bardawil LB 2538 (Park, Lee, & Jin, 2013)
14 (Sambar Lake, India) (Vanitha, Narayan, Murthy, & Ravishankar, 2007)
15 D. acidophila SAG 19.85 (Sekler & Pick, 1993)
16 RT22 (Puente-Sánchez, Olsson, & Aguilera, 2016)
17 D. maritima CCAP 19/1 (Khramov, Matalin, Karpichev, Balnokin, & Popova, 2019)
18 D. viridis (Yucatan, Mexico) (García, Freile-Pelegrín, & Robledo, 2007)
19 Dunaliella sp. RCC5 (Iglesias et al., 2019)

or predators (Ben-Amotz, Shaish, & Avron, 1991), as development of growth systems and processes asso­
indicated in Table 1. ciated with industrial production, including a need to
make microalgal biorefineries profitable (Monte et al.,
(A) bardawil (considered by some authors as a sub­ 2020). In the production of Dunaliella, systems and
species of D. salina; Gonzalez, Coleman, Gomez, processes that promote the extraction of the largest
& Montoya, 2001) has been grown mainly for number of components in the most efficient way possi­
the production of fatty acids. ble must be developed (Chew et al., 2017). After labora­
(B) tertiolecta has also shown great biotechnological tory evaluations define the potential of a given species,
potential. This species has been the subject of the design of the process, the scale up of the indicated
many studies due to its exceptional ability to photobioreactors and the development of techniques for
accumulate lipids, glycerol and starch the extraction and harvesting of the target components
(Slocombe et al., 2015). It has many advantages, are carried out. This scale-up step is vital for the feasi­
such as high growth rates, efficient use of carbon bility of future applications. The main parameters that
dioxide and high halotolerance, among others influence microalgal productivity are mixing rate, cul­
(Chow et al., 2013; Katz, Paz, & Pick, 2019). ture depth, inoculum volume and the growth conditions
(C) acidophila is one of the most representative (Kroumov et al., 2016).
extreme acidophilic organisms (Aguilera &
Amils, 2005). It can grow at a pH below 1.0,
maintaining its intracellular pH at 7.0 (Pick, Growth systems
1999).
(D) maritima shows high production of ATPases Numerous microalgal growth systems have been devel­
(Khramov, Matalin, Karpichev, Balnokin, & oped, such as large open ponds, circular ponds, raceway
Popova, 2019). ponds, cascade ponds, large bags, tanks, heterotrophic
(E) viridis has applications in the aquaculture feed fermenters, and several kinds of closed photobioreactors
industry (García, Freile-Pelegrín, & Robledo, (Borowitzka, 1999; Pulz, 2001). Within these, two main
2007). Table 1 lists some strains that have been types can be distinguished: open-grown systems and
used commercially. closed-growth systems. The main distinction between
these is the control of the conditions to which the
cultures are exposed. In closed systems, the conditions
are controlled more efficiently. Open growth systems
have been the most widely used in recent years (Vigani
Production methods
et al., 2015). Open ponds represent the most economical
The growing biotechnological applications of microal­ and conventional system for microalgae growth (Ben-
gae have increased interest in the research and Amotz & Avron, 1989a). There are two main types of
106 M. BARBOSA ET AL.

open growth systems, very large ponds (extensive mode) contaminants (Ben-Amotz, 1995). The conditions used
of up to 250 ha (Borowitzka & Borowitzka, 1990), and for the laboratory growth of some species, in some
smaller paddle wheel stirred raceway ponds (Ben- studies, are described in Table 2. The average tempera­
Amotz, 1995). For the growth of Dunaliella in open ture used was 24.7°C, and the most used temperature
systems, it is favourable to establish structures in loca­ was 25°C.
tions with a warmer and drier climate, preferably close Dunaliella species can resist pH values between 1, in
to locations with a high concentration of NaCl. It is also the case of D. acidophila, up to 11, but for optimal growth,
important that there are no sources of contamination they require a pH close to 9 (Hosseini Tafreshi & Shariati,
nearby. These systems are advantageous as they are 2009). In intensive production systems, the pH is kept
considerably cheaper and the media can be quite selec­ close to 7.5 as it tends to increase with the CO2 fixation
tive, given the resistance to high salinity of Dunaliella process (Ben-Amotz, 1995). Considering Table 2, the
cells (Borowitzka & Borowitzka, 1988). However, there average pH used was 7.6 and the most used pH value
are some disadvantages associated with this type of was 7.5.
systems, such as the difficulty of controlling the tem­ In the studies indicated in Table 2, the most used
perature of the medium and the quality of the light to medium was Modified Johnson’s medium (Johnson,
which the cells are exposed, so the productivity tends to Johnson, MacElroy, Speer, & Bruff, 1968), followed by
be lower (Ben-Amotz & Avron, 1989a). F/2 medium (Guillard, 1975) and Walne’s medium
Photobioreactors are very advantageous systems for (Walne, 1970). Carbon dioxide and bicarbonate are
controlling culture parameters and are used for the used as carbon sources for the growth of Dunaliella
growth of microalgae, cyanobacteria, and plants. There (usually photoautotrophic). Ben-Amotz & Avron
are three basic designs in the development of closed- (1989a) indicated the possibility of using sodium bicar­
growth systems: flat plate bioreactors, tubular photo­ bonate as a carbon source. As a source of nitrogen,
bioreactors and ultrathin immobilized configurations nitrate is considered the most efficient. Other sources
(Borowitzka, 1999; Pulz, 2001; Tredici & Zitelli, 1997). of nitrogen, such as ammonia salts or urea, are not
Tubular photobioreactors have been shown to have appropriate and can lead to cell death (Borowitzka,
higher biomass productivities than flat plate bioreactors, 1990). To increase biomass and protein synthesis, a
but the latter have distinct advantages due to low oxygen favourable availability of nitrogen is necessary
accumulation, greater surface area in relation to volume, (Uriarte, Farías, Hawkins, & Bayne, 1993). The source
the possibility of applying turbulent flow, lower cost, of phosphorus that leads to better results is monopotas­
easy maintenance, and bio-encrustation control sium phosphate or monosodium phosphate. For opti­
(Bergmann, Ripplinger, Beyer, & Trösch, 2013). In gen­ mal growth, Dunaliella cells also need sulphate and
eral, closed systems allow precise control of growth several ions, such as Kþ ; Ca2þ ; Mg2þ ; Cl ; Naþ , che­
conditions, enhancing the ability to precisely control lated iron and trace elements (Hosseini Tafreshi &
light quality and the photoperiod (Lamers, Janssen, De Shariati, 2009). For culture conservation, cultures are
Vos, Bino, & Wijffels, 2008). The main disadvantage of usually kept on agar media plates or liquid media, sub­
these systems is the high cost, in relation to open culturing every 1–2 months. Studies also indicate the
systems. possibility of preservation at very low temperatures for
up to 12 months (Taylor & Fletcher, 1998). Colusse,
Mendes, Duarte, Carvalho, & Noseda (2020) directly
Growth conditions
compare the effect on growth, biochemistry, and costs
Species of the genus Dunaliella have great resilience to of the most used media in D. salina cultivation. el
external factors. Studies indicate that some species can Agawany, Kaamoush, El-Zeiny, & Ahmed (2021)
survive at temperatures from 0°C to 45°C, with opti­ demonstrated the effects of heavy metals on D. tertio­
mum growth occurring between 25°C and 35°C (Ben- lecta production.
Amotz, 1995; Jin & Polle, 2009). Borowitzka & Dunaliella cells usually grow under autotrophic con­
Borowitzka (1987) demonstrated that very low night- ditions and light is their most common source of energy.
time temperatures (such as in Hutt Lagoon, Australia) Usually, the cell growth increases with photoperiod (Sui,
can slowdown growth, thus decreasing the process yield. Muys, Vermeir, D’Adamo, & Vlaeminck, 2019). Cell
On the other hand, temperatures close to 40°C promote growth and carotenoid synthesis clearly depend on the
carotenoid synthesis, but also decrease the growth rate quantity and intensity of the light. Ben-Amotz & Avron
(Borowitzka & Borowitzka, 1989). Temperatures above (1989b) demonstrated that the synthesis of carotenoids
40°C can lead to the release of glycerol into the environ­ does not depend on the wavelength of the absorbed
ment, which may be a carbon source for microbial radiation but depends fundamentally on its irradiance.
Table 2. Growth conditions applied to different species of the genus Dunaliella, in the analysed studies.
Light
Nº Species T (ºC) pH Medium Period (h) Irradiance (µmol photons m–2 s –1) NaCl (g l–1) Reference
1 D. salina 20 7.5 Modified Johnson’s medium 12; 24 55 116.90 (Sui, Muys, Vermeir, D’Adamo, & Vlaeminck, 2019)
2 25 7.0 Modified Johnson’s medium 16 90–120 9.90–233.80 (Hadi, Shariati, & Afsharzadeh, 2008)
3 28 7.5 Modified Johnson’s medium 16 80 29.25 (Khadim et al., 2018)
4 29 7.5 Modified Johnson’s medium 12 80 100.00–350.00 (García, Freile-Pelegrín, & Robledo, 2007)
5 20 8.0 F/2 medium 16 - 35.00 (Chae, Kim, & An, 2019)
6 20 8.0 F/2 medium 12 50 35.00 (Gao et al., 2014)
7 24 7.8 F/2 medium (Natural light) 35.00 (Song et al., 2018)
8 20 8.5 Walne’s medium 12 150 35.10–122.70 (Francavilla, Trotta, & Luque, 2010)
9 25 8.5 Walne’s medium 24 150 233.80 (EL Arroussi et al., 2018)
10 30 8.5 AS−100 medium (Natural light) 87.70 (Vanitha, Narayan, Murthy, & Ravishankar, 2007)
11 25 7.5 Modified AS−100 medium 16 45 - (Kumudha & Sarada, 2016)
12 20 8.2 Roscoff seawater 14 150 33.00 (Iglesias et al., 2019)
13 20–21 7.4 Complete Nutritive Conway 12; 16 40 100.00 (Besson, Formosa-Dague, & Guiraud, 2019)
14 25 7.5 Artificial seawater 24 40–400 87.70 (Park, Lee, & Jin, 2013)
15 26 7.5 (Custom) - - 87.70 (Monte et al., 2018)
16 25–35 7.0–8.0 - - - 90.00–120.00 (Monte et al., 2020)
17 D. tertiolecta 25 7.5 ATCC−1174 medium 14 50 29.20 (Lin, Shen, & Lee, 2018)
18 25 7.5 ATCC−1174 medium 14 50 29.20 (Lin & Lee, 2017)
19 25 7.5 ATCC−1174 medium 24 100 29.20–116.90 (Ng, Low, Chow, & Lee, 2014)
20 24 8.0 F/2 medium 24 120–150 35.00 (Benhima et al., 2018)
21 20 8.5 Walne’s medium 12 150 35.10–122.70 (Francavilla, Trotta, & Luque, 2010)
22 19 8.1 Artificial seawater (SOW) 12 120 35.00 (Thakkar, Mitra, & Wei, 2016)
23 24 7.5 BG−11(−N) Medium 24 120–150 29.25 (Pick & Avidan, 2017)
24 28–30 8.0 Modified NORO medium 24 65–150 29.20–58.40 (Takagi, Karseno, & Yoshida, 2006)
25 D. bardawil 25 7.5 Artificial seawater 24 40–400 87.70 (Park, Lee, & Jin, 2013)
26 30 8.5 AS−100 medium (Natural light) 87.70 (Vanitha, Narayan, Murthy, & Ravishankar, 2007)
27 D. viridis 25 7.0 Modified Johnson’s medium 16 90–120 9.90–233.80 (Hadi, Shariati, & Afsharzadeh, 2008)
28 29 7.5 Modified Johnson’s medium 12 80 10.00–35.00 (García, Freile-Pelegrín, & Robledo, 2007)
29 D. acidophila 25 0.5 (Custom) - - 26.90 (Sekler & Pick, 1993)
30 D. parva 25 7.5 Modified Johnson’s medium 16 50 87.70 (Ismaiel, El-Ayouty, Said, & Fathey, 2018)
31 Dunaliella sp. 20 7.5 Modified Johnson’s medium 24 - 25.00 (Çelekli & Dönmez, 2006)
32 30 7.5 Modified Johnson’s medium 24 154 10.00–25.00 (Kocberber Kilic, Erdem, & Donmez, 2018)
33 24 8.5 Walne’s medium 12 30 29.20–292.21 (Jayappriyan, Rajkumar, & Rengasamy, 2011)
34 29 7.8 F/2 medium 24 80 40.00–80.00 (Dahmen-Ben Moussa et al., 2018)
APPLIED PHYCOLOGY
107
108 M. BARBOSA ET AL.

In the growth tests referenced in Table 2, the average of cations (Pirwitz, Rihko-Struckmann, & Sundmacher,
the photoperiod is 18 h, and the most common photo­ 2015), or cationic polymers like chitosan or modified
period was 24 h. The average photon irradiance is 103 starch (Vandamme, Foubert, Fraeye, Meesschaert, &
µmol photons m−2 s−1 and the most used irradiance was Muylaert, 2012). Carotenoids can be extracted from
80 µmol photons m−2 s−1. Recent studies indicate the algal biomass or dry powder, using these methods:
possibility of growing D. salina under heterotrophic and extraction in conventional organic solvents (Ruane,
mixotrophic conditions for the production of lipids. In 1974), extraction of the carotene from the algae in an
this case, the most used carbon sources are glucose, edible oil (Nonomura, 1987), separation of β-carotene
acetate, and glycerol (Capa-Robles, García-Mendoza, isomers via CO2 supercritical fluid extraction (Gamlieli-
& Paniagua-Michel, 2021; Chavoshi & Shariati, 2019; Bonshtein, Korin, & Cohen, 2002), or selective extrac­
Gonabadi, Samadlouie, & Shafafi Zenoozian, 2022). tion by biocompatible organic solvents in two-phase
Dunaliella cells grow in environments with high con­ bioreactors (Hejazi et al., 2002). These conventional
centrations of NaCl. This characteristic is extremely processes are not very efficient, with high costs and
advantageous since most of the contaminating organ­ energy consumption. In the case of cell harvesting
isms, harmful for Dunaliella, do not resist high salinities methods, cells are often damaged, as is the case with
(Ben-Amotz & Avron, 1989a; Butinar, Sonjak, Zalar, centrifugation and filtration (Pragya, Pandey, & Sahoo,
Plemenitaš, & Gunde-Cimerman, 2005). The optimal 2013). Monte et al. (2018) described a membrane-har­
growth of Dunaliella is obtained between concentra­ vesting process for D. salina that allows cell pre-con­
tions of 58.5 g l−1 (1 M) and 116.9 g l−1 (2 M) of NaCl centration, significantly increasing the efficiency of cell
(Hadi, Shariati, & Afsharzadeh, 2008). In the studies harvesting compared to other harvesting processes.
indicated in Table 2, the average NaCl concentration Besson, Formosa-Dague, & Guiraud (2019) also
was 74.6 g l−1, and the most used concentration was described a process of cell harvesting of D. salina by
87.7 g l−1 (1.5 M). D. salina increases its synthesis of flocculation/flotation that can be applied on an indus­
carotenoids and glycerol, when grown in high salinities trial scale. This method allows flocculation without the
(Hadi, Shariati, & Afsharzadeh, 2008). These cells need for chemical flocculants, thus reducing cell damage
respond to the osmotic stress by regulating the flow of and contamination.
carbon between the synthesis of starch in chloroplasts
and the production of glycerol in the cytoplasm (Bental,
Multi-product biorefineries
Pick, Avron, & Degani, 1990). Cycil et al. (2021) also
demonstrated that D. salina grows exposed to low atmo­ The concept of “Blue Biotechnology” has recently
spheric pressures. gained relevance, and consequently, the development
of biorefineries that allow the extraction of several pro­
ducts in a single industrial production has also attracted
Harvesting and component extraction
interest (Nishshanka, Anthonio, Nimarshana,
The development of a sustainable and economically Ariyadasa, & Chang, 2022). Marine microalgae have
viable extraction and harvesting process for microalgal been widely included in these processes and methods
components is a major challenge. The cost represents for multi-product extraction from Dunaliella species
between 20% and 40% of the total production cost have already been described. Francavilla, Kamaterou,
(Mata, Martins, & Caetano, 2010). As such, a universal Intini, Monteleone, & Zabaniotou (2015) verified the
technique cannot be defined; each process depends on production of a total lipid fraction rich in β-carotene,
the species used, its characteristics and the component phytosterols and fatty acids in D. tertiolecta, and eval­
to be extracted (Uduman, Qi, Danquah, Forde, & uated the possibility of extracting, from the residues of
Hoadley, 2010). In the case of Dunaliella cells, there the first process, 45% of bio-oil and 29% biochar by
are three main characteristics that influence the harvest­ pyrolysis. Harvey & Ben-Amotz (2020) demonstrated
ing and extraction process: the absence of a rigid cell the production of 9-cis β-carotene in D. salina, and
wall, the high salinity of the medium and the low cell verified that, after the clean extraction of this com­
densities (Monte et al., 2020). pound, the residual biomass still included 50% of the
Harvesting of microalgae can occur by centrifuga­ lipids present in the cells. In addition, this residual
tion, filtration, flocculation, and flotation (Garg, Li, biomass could also be used to produce animal feed.
Wang, & Schenk, 2012). Component extraction is Monte et al. (2020) proposed a method for extracting
usually done via flocculation by pH modulation several components of D. salina. A possible biorefinery
(Besson & Guiraud, 2013; Vandamme, Foubert, & has been developed that allows efficient fractional
Muylaert, 2013), electrolysis (Wan et al., 2015), metallic extraction of carotenoids, lipids, glycerol and proteins.
 APPLIED PHYCOLOGY 109

This sustainable design allowed the collection of 85% of possible to perform the harvest by removing the product of
carotenoids, 94% of polar lipids and 86% of glycerol. interest and taking advantage of residuals as secondary
products (Hejazi, Holwerda, & Wijffels, 2004). As a haploid
microorganism, the introduced genes are always expressed,
Recombinant gene expression whether dominant or recessive (Jones & Sparks, 2009).
Although reproduction is mainly due to vegetative cell
Dunaliella has been shown to be an advantageous can­
division, sexual reproduction can occur (Borowitzka &
didate for the synthesis of high-value products. The
Siva, 2007). When sexual reproduction occurs between
delay in the use of most eukaryotic algae in these sys­
individuals with different inserted genes, the offspring can
tems is mainly due to the lack of knowledge of specific
receive several genes (Primrose, 1991); however, it is not
promoters for the efficient expression of the introduced
desirable in the production of multi-chain antibodies.
genes. Until recently, there has not been much research
Furthermore, post-translational processing occurs natu­
on the genetic transformation and metabolism of
rally in eukaryotic organisms, which is an advantage.
Dunaliella. As a consequence of advances in genetic
Also, recombinant proteins can be easily removed with
engineering and with the need for higher quality and
cell lysis procedures (Feng, Li, Xu, & Qi, 2014). The great
yield of recombinant proteins, some species such as D.
advantage in comparison with other species is that there are
salina, D. tertiolecta and D. parva have aroused great
no escape mechanisms for genes, making transgenic indi­
interest (Feng, Li, Xu, & Qi, 2014; Ismaiel, El-Ayouty,
viduals easy to control and with a low probability of escape
Said, & Fathey, 2018; Lin, Shen, & Lee, 2018).
of transformed cells into the environment (Barzegari et al.,
Thus, this genus has been the target of mutagenesis and
2010).
genetic manipulation to increase the amounts of carote­
This genus has been the target of several transforma­
noids and other high-value products. Recombination tech­
tion methods, including electroporation (Sun et al.,
nologies allowed heterotrophy by autotrophic algae, thus
2005), particle bombardment (Tan, Qin, Zhang, Jiang,
allowing an increase in biomass per litre and the economic
& Zhao, 2005), glass beads (Feng, Xue, Liu, & Lu, 2009),
value of the algae (Jin & Melis, 2003). Jin & Polle (2009)
and lithium acetate/polyethylene glycol (PEG) mediated
created strains of D. salina with a mutation that allowed the
method (Chai, Chen, Xu, & Xu, 2013).
manipulation of the content of carotenoids, especially zeax­
Many Dunaliella genes have been isolated, character­
anthin, producing 20 times more zeaxanthin than the wild
ized, and transformed (He et al., 2020). Some of these genes
type (Jin & Melis, 2003). Recently, a transgenic strain of D.
express DNA photolyases with the function of repairing
tertiolecta was characterized, with an increase in medium
DNA damage caused by UV radiation (Yi et al., 2006), a
chain length fatty acids (MCFAs) levels (Lin, Shen, & Lee,
nitrate transporter gene named DsNRT2Æ1 (He et al.,
2018). This compound is used in the production of insect
2004), plastid glycerol 3-phosphate dehydrogenases (He
pheromones (Tillman, Seybold, Jurenka, & Blomquist,
et al., 2007), x3 fatty acid desaturases (Lyukevich,
1999), detergents (Knaut & Richtler, 1985), antibiotics
Mouradyan, & Los, 2003), the 5-enolpyruvylshikimate−3-
(Laakel et al., 1994) and surfactants (Ohlrogge, 1994). In
phosphate synthase (Yi et al., 2007), and the sodium-
D. parva transformation with an external PSY gene (phy­
dependent phosphate transporter, DvSPT1 (Li et al.,
toene synthase gene) resulted in carotenoids with greater
2006). In D. maritima, the DmHA2 ATPase gene was
antioxidant capacity (Ismaiel, El-Ayouty, Said, & Fathey,
isolated as responsible for the production of Na+-ATPase
2018).
(Khramov, Matalin, Karpichev, Balnokin, & Popova,
The most used systems in recombination are Escherichia
2019).
coli, yeast, mammalian cells, or transgenic animals.
The genes responsible for carotenogenesis of a carote­
However, all have disadvantages that affect yield and pro­
nogenic strain were identified, extracted, and used to
duction cost. Expression systems with E. coli and yeasts are
genetically manipulate the species (Zhu, Jiang, & Chen,
low-cost, however, these have a reduced ability to make
2008). However, the genes involved in this pathway and
post-translational modifications (Gonçalves et al., 2013; Liu
its regulation are not fully understood (Jin & Polle, 2009).
et al., 2013). Systems of transgenic animals and mammalian
cells are complex and costly and show low yield (Aricescu
& Owens, 2013; Guan et al., 2013). Thus, Dunaliella is a
Industrial applications
competitive alternative to produce recombinant com­
pounds (Barzegari et al., 2010). As it is a microalga that Due to its advantageous characteristics mentioned pre­
does not have a rigid cell wall (Borowitzka & Siva, 2007), viously, Dunaliella is one of the most used species in the
the transformation process is easier, requiring less time to biotechnological industry, and its applications are
scale-up production (Feng, Xue, Liu, & Lu, 2009). Also, it is observed in diverse sectors, such as carotenoid
110 M. BARBOSA ET AL.

production, food and pharmaceutical industry, bioac­ important alternatives for ruminant and crustacean
tive compounds, bioremediation, bioindicators, biofuel feed due to their high mineral and vitamin content,
and antifouling. stimulating optimum growth (Madeira et al., 2017)
and increasing the weight and survival capacity of
shrimps (Supamattaya, Kiriratnikom, Boonyaratpalin,
Carotenoids
& Borowitzka, 2005). Furthermore, due to antimicrobial
Currently, carotenoids are the main product activity against Escherichia coli, Staphylococcus aureus,
obtained from Dunaliella (Duan et al., 2023). In Candida albicans, and Aspergillus niger, Dunaliella
the industry, these components are applied as dyes, extracts can prevent contamination during the produc­
and with the growing demand for natural, healthy, tion of food products (Herrero, Jaime, Martín-Álvarez,
and colourful products, carotenoids are highly Cifuentes, & Ibáñez, 2006).
valued as natural additives for food and cosmetics There is also great potential for the use of compo­
(Ye, Jiang, & Wu, 2008). These compounds are also nents extracted from Dunaliella species in the develop­
applied in animal feeding to improve the colour ment and production of food and feed supplements
manipulation of ornamental fish (Pulz & Gross, (Camacho, Macedo, & Malcata, 2019). Considering the
2004) and eggs (Moulton & Burford, 1990). β-carotene and xanthophylls extracted from these spe­
According to a Fortune Business Insights report the cies, relevant antioxidant and anticancer capacities have
world market for carotenoids reached US $1.4 billion already been demonstrated (Jayappriyan, Rajkumar,
in 2019 and should reach US $1,8 billion by 2027. Venkatakrishnan, Nagaraj, & Rengasamy, 2013; Singh,
β-carotene can be obtained naturally or synthetically, Baranwal, & Reddy, 2016). The use of Dunaliella for
however, the natural form has greater antioxidant and protein production in the food industry is still not very
anti-tumour capabilities (Hu, Lin, Lu, Chou, & Yang, common; however, the great quantity, quality, and
2008). Microalgae are one of the natural sources with nutritional value indicate it as a sustainable source of
the highest production, as well as some vegetables, protein, with an amino acid content similar to soy.
fruits, and fungi (Dufossé et al., 2005). Dunaliella cells Currently, despite its great potential as a protein source
accumulate hundreds of times more β-carotene than a for human consumption, it has mainly been used in feed
carrot cell (Klausner, 1986) in addition to being of supplements (e.g., feeding ornamental fish, shrimp and
higher quality. birds) (Sui & Vlaeminck, 2020). The direct use of
The therapeutic effects of β-carotene are mainly Dunaliella biomass in food products is also potentially
due to its function as a protector of cells against advantageous, having positive effects on the treatment
harmful free radicals and to stimulate the immune of cardiovascular diseases and cancer, with anti-inflam­
system (Ben-Amotz, 1996). In addition, β-carotene is matory properties (Silva et al., 2021).
known to be oxidized by liver enzymes that produce It was also verified that the exopolysaccharides of D.
vitamin A, improving vision and epithelial tissues salina are potential alternatives for the protection and
(Hosseini Tafreshi & Shariati, 2009). This compo­ improvement of agricultural crops, acting as attenuators
nent is also effective in controlling cholesterol levels of biotic and abiotic stress. Positive results have been
(Törnwall et al., 2004), in the prevention and treat­ obtained in tomato plants under osmotic stress
ment of diseases, such as cataracts (Gupta et al., (Arroussi et al., 2018).
2003), cardiovascular problems (Shaish et al., 2006),
and certain cancers (Mayne et al., 1994; Michaud et
Pharmaceutical industry and bioactive compounds
al., 2000; van Poppel, 1993). A recent study proved
the effectiveness of using Dunaliella as a nutraceuti­ Dunaliella produces a large number of bioactive
cal in rats with diet-induced obesity and hepatic compounds, enzymes, and vitamins that are of high
lipid accumulation, without affecting food intake value in applications in the pharmaceutical industry.
(Xu et al., 2022; Yamashita et al., 2020). D. salina produces a unique enzyme, dihydroxy acet­
one reductase, which has already been commercia­
lized (Ben-Amotz & Avron, 1990). The extract has
Food and feed industry
antioxidant, antimicrobial, and antiaging activity
Due to the high production of protein, fatty acids, β- (Cakmak, Kaya, & Asan-Ozusaglam, 2014; Havas et
carotene, and the lack of cell walls, Dunaliella is an al., 2022). D. tertiolecta extracts demonstrated mus­
excellent feed for aquaculture and cattle, and a food cle relaxation, antiserotonin, antihypertensive,
source for humans (Hosseini Tafreshi & Shariati, analgesic, bronchodilator, polysynaptic block, and
2009). Dunaliella products have been identified as antioedema activities (Borowitzka, 1995; Villar,
 APPLIED PHYCOLOGY 111

Laguna, Calleja, & Cadavid, 1992). Moreover, D. (2006) studied the effects and responses of seaweed
primolecta contains several substances with antibiotic when grown in a medium with aluminium and effluent
activity (Chang et al., 1993). Pentapharm (Basel, from a pharmaceutical industrial production. D. primo­
Switzerland) developed a bioactive compound from lecta was the most advantageous alga as a biomarker for
D. salina that stimulates cell proliferation (Stolz & four representative herbicides (Santín-Montanyá,
Obermayer, 2005). Sandín-España, García Baudín, & Coll-Morales, 2007).
In two species of Dunaliella (D. tertiolecta and D. D. salina has also been used as a model to evaluate the
salina) phytosterols (about 1% of dry weight) were toxicity of a typical mutagenic phenol (Chen, Jiang, &
identified and isolated (Francavilla, Trotta, & Luque, Lin, 2007), and the effects of high concentrations of
2010). These compounds are of great value for the microplastics (Chae, Kim, & An, 2019).
pharmaceutical industry as they are precursors of
some bioactive molecules and act in the prevention of
Biofuel production
coronary heart disease through the reduction of choles­
terol (Fernandes & Cabral, 2007). In addition, these Microalgae are identified as potential sources of biomass
compounds have anti-inflammatory activity and may for biofuel production due to their high lipid content
have anti-cancer and anti-oxidative properties and rapid growth (Chisti, 2007). However, these organ­
(Francavilla, Trotta, & Luque, 2010). isms are still not widely applied due to being economic­
The protective effects of D. salina against fibrosar­ ally nonviable at large scale (Benhima et al., 2018).
coma cells are already known (Raja, Hemaiswarya, & However, the interest in applying microalgae in the
Rengasamy, 2007); this species has selective cytotoxic production of biodiesel is increasing (Huntley &
potential in malignant neuroblastoma cells (Atasever- Redalje, 2007). Thus, some species of Dunaliella, such
Arslan, Yilancioglu, Bekaroglu, Taskin, & Cetiner, as D. tertiolecta, D. primolecta and D. salina, are strong
2015). However, in vivo studies are still needed to candidates for these applications, as they can accumu­
expand knowledge of the cytotoxic and anti-tumour late about 40% of their dry weight in the form of lipids
mechanisms. and show high growth rates and high CO2 absorption
rates (Hosseini Tafreshi & Shariati, 2009; Sathya et al.,
2023).
Bioremediation
The treatment of wastewater involves several steps, one
Antifouling
of which may involve the use of microorganisms to
metabolize organic matter. Microalgae can contribute Biofouling is a worldwide phenomenon that causes
to this process by removing inorganic nitrogen, phos­ enormous damage to submerged structures every year
phorus and CO2, products generated by these microor­ (Acevedo et al., 2013). To mitigate this, paints with toxic
ganisms (Talbot & de la Noüe, 1993). Some species of and biocidal components are commonly used
Dunaliella can remove 98% of the metals in a closed (Rittschof, Lai, Kok, & Teo, 2003). However, these com­
system after one week of exposure (Dahmen-Ben pounds have high toxicity for the surrounding environ­
Moussa et al., 2018). D. tertiolecta has heavy metal- ments and are very persistent in time (Thomas &
binding peptides (phytochelatins) and can be used in Brooks, 2010). Thus, natural and environmentally
bioremediation to remove heavy metals from the envir­ friendly compounds with antifouling properties have
onment (Tsuji et al., 2002). Thus, it is a candidate for the been developed. In this context, D. salina emerges as a
third treatment phase of saline wastewater due to its potential producer of these compounds (Gao et al.,
capacity to accumulate NH4+ and PO43- and heavy 2014). Gao et al. (2014) verified that the extract of D.
metals, such as copper and arsenic (Takimura, Fuse, salina, composed of unsaturated and saturated 16- and
Murakami, Kamimura, & Yamaoka, 1996; Yamaoka, 18-carbon fatty acids, has antifouling properties against
Takimura, Fuse, & Murakami, 1999). diatom species and barnacle larvae.

Bioindicators Conclusion
Dunaliella tertiolecta is a good bioindicator for assessing Dunaliella is a genus resistant to several extreme envir­
the ecotoxicity of anthropogenic components in the onments, highlighting hypersaline, low pH and low
environment. This species, as well as other algae, is temperature conditions, often reflected in the produc­
highly sensitive to effluents from urban areas and indus­ tion of compounds, such as carotenoids, lipids, and
tries (Lewis, Weber, & Stanley, 1998). Sacan & Balcioglu glycerol. In addition to these, Dunaliella species
112 M. BARBOSA ET AL.

synthesize compounds with antimicrobial and antipro­ Leonardo Garcia Inácio http://orcid.org/0000-0001-9208-
liferative characteristics. Among the species of this 6136
genus, Dunaliella salina is the most studied and applied Clélia Afonso http://orcid.org/0000-0002-4167-5237
Paulo Maranhão http://orcid.org/0000-0001-5718-0880
in the industry, followed by D. tertiolecta, D. parva, and
D. viridis. These species are usually easy to grow, with
open growth systems being the most used method in Author contributions
industrial production. When grown in photobioreac­
M. Barbosa: research question, literature search and writing;
tors, different conditions are used depending on the
L. G. Inácio: research question, literature search and writing;
purpose of the production. C. Afonso: editing and final draft; P. Maranhão: editing and
With the advent of genetic engineering, these species final draft.
were considered as a biological platform to produce
several recombinant products, presenting advantages
when compared to other organisms, such as yeast or References
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