50 Marine Macro- and Microalgae: An Overview
C18 oleic acid) which make up the oily droplets within which the β-carotene is sequestered (Rabbani et
al. 1998; Mendoza et al. 1999; Lamers et al. 2010). The carotenoid biosynthetic pathway of Dunaliella is
the same as that in other plants (Britton 1988).
The exact function of these high amounts of carotenoids in the cell is not known, but two
complementary hypotheses have been proposed. In nature, D. salina is usually found in shallow salt lakes
where the algae cells are exposed to very high irradiances. D. salina cells with a high carotenoid content
can tolerate much higher irradiances that those with a lower carotenoid content (Gômez Pinchetti et al.
1992). The β-carotene absorbs light in the blue region and cis-β-carotene is an effective quencher of singlet
oxygen and is transformed to trans-β-carotene (Jimenez and Pick 1993) and thus, the β-carotene may also
protect the chloroplast from damage by free oxygen radicals (Shaish et al. 1993). The second hypothesis
proposes that the lipids and associated β-carotene are a carbon sink (Borowitzka and Borowitzka 1988a).
The formation of high amounts of carotenoids occurs when one or more metabolic intermediate pathways
are inhibited by lack of substrate; however, photosynthesis still continues, albeit at a reduced rate. It is
essential for the survival of the cell that photosynthesis continues in order to supply sufficient energy
for essential metabolic processes for survival at the high salinity at which this alga grows, such as Na
+
-
efflux and glycerol synthesis. One by-product of this photosynthesis is 3-phosphoglyceric acid which
is further metabolised and the products of this metabolism must be either stored or excreted. If this
photosynthetically generated product is stored, then it is essential that it does not inhibit cell function.
Triacylglycerols and β-carotene are suitable ‘neutral’ compounds that could serve this function. In fact,
many algae accumulate triacylglycerols (lipids) when their growth is inhibited (Roessler 1988; Ördög et
al. 2013) or, alternatively, they excrete large quantities of organic C once growth limitation sets in (Arad
et al. 1992; Myklestad 1995).
The high salinity brines that D. salina grows also mean that CO 2 solubility is reduced compared
to seawater and the inorganic C equilibrium is shifted towards HCO 3
–
(Lazar et al. 1983). The high
temperatures where D. salina is grown commercially also reduce the solubility of CO 2 further. Although
D. salina has an active CCM mechanism (Booth and Beardall 1991) and an unusual α-type external
carbonic anhydrase that is active over a salinity range of 0–4 M NaCl (Fisher et al. 1996; Premkumar et
al. 2003), large-scale cultures are still severely carbon limited. Some of this limitation can be overcome
by the addition of bicarbonate.
The fact that β-carotene is a secondary metabolite means that conditions to achieve maximum
growth rate (i.e., lower salinities, high nutrients) and the conditions to achieve a high cell β-carotene
content (i.e., high salinities, low nutrients) are incompatible. In practical terms, this means two possible
culture strategies for the production of β-carotene by the culture of D. salina. One strategy is to initially
grow the algae at an optimal salinity for biomass generation (i.e., a salinity of less than 20% w/v NaCl +
sufficient nutrients) and then transferring the cells to a higher salinity under nutrient limiting conditions.
Such a 2-stage process however has the disadvantage that it requires a larger pond area and takes longer,
thus increasing production costs. The alternative strategy is to grow the algae at high irradiances and
at a salinity where β-carotene productivity is highest. Figure 2 shows the effects of salinity on growth
and β-carotene content and the optimum salinity for β-carotene productivity. However, in commercial
production, the algae are usually grown at a higher salinity to ensure reliable production and minimise
the risk of protozoan invasions which can result from a reduction in salinity due to dilution by rainfall
(Borowitzka and Borowitzka 1989). The irradiance received by the algae cells is managed by managing
the areal density of the algae (Grobbelaar 1995).
Haematococcus pluvialis and astaxanthin
The freshwater green alga Haematococcus pluvialis is the best natural source of the oxygenated carotenoid,
astaxanthin, which is accumulated in lipid droplets in the cytoplasm in the aplanospore stage of the alga
(Lang 1968; Wayama et al. 2013). Aplanospore formation and astaxanthin accumulation are induced
by nutrient limitation and other growth-inhibiting induction factors (often called ‘stress’ factors in the
literature) such as high temperature, high light, or increased salinity (Borowitzka et al. 1991; Boussiba
and Vonshak 1991; Vidhyavathi et al. 2008). Haematococcus produces only the 3S,3’S stereoisomer of
C18 oleic acid) which make up the oily droplets within which the β-carotene is sequestered (Rabbani et
al. 1998; Mendoza et al. 1999; Lamers et al. 2010). The carotenoid biosynthetic pathway of Dunaliella is
the same as that in other plants (Britton 1988).
The exact function of these high amounts of carotenoids in the cell is not known, but two
complementary hypotheses have been proposed. In nature, D. salina is usually found in shallow salt lakes
where the algae cells are exposed to very high irradiances. D. salina cells with a high carotenoid content
can tolerate much higher irradiances that those with a lower carotenoid content (Gômez Pinchetti et al.
1992). The β-carotene absorbs light in the blue region and cis-β-carotene is an effective quencher of singlet
oxygen and is transformed to trans-β-carotene (Jimenez and Pick 1993) and thus, the β-carotene may also
protect the chloroplast from damage by free oxygen radicals (Shaish et al. 1993). The second hypothesis
proposes that the lipids and associated β-carotene are a carbon sink (Borowitzka and Borowitzka 1988a).
The formation of high amounts of carotenoids occurs when one or more metabolic intermediate pathways
are inhibited by lack of substrate; however, photosynthesis still continues, albeit at a reduced rate. It is
essential for the survival of the cell that photosynthesis continues in order to supply sufficient energy
for essential metabolic processes for survival at the high salinity at which this alga grows, such as Na
+
-
efflux and glycerol synthesis. One by-product of this photosynthesis is 3-phosphoglyceric acid which
is further metabolised and the products of this metabolism must be either stored or excreted. If this
photosynthetically generated product is stored, then it is essential that it does not inhibit cell function.
Triacylglycerols and β-carotene are suitable ‘neutral’ compounds that could serve this function. In fact,
many algae accumulate triacylglycerols (lipids) when their growth is inhibited (Roessler 1988; Ördög et
al. 2013) or, alternatively, they excrete large quantities of organic C once growth limitation sets in (Arad
et al. 1992; Myklestad 1995).
The high salinity brines that D. salina grows also mean that CO 2 solubility is reduced compared
to seawater and the inorganic C equilibrium is shifted towards HCO 3
–
(Lazar et al. 1983). The high
temperatures where D. salina is grown commercially also reduce the solubility of CO 2 further. Although
D. salina has an active CCM mechanism (Booth and Beardall 1991) and an unusual α-type external
carbonic anhydrase that is active over a salinity range of 0–4 M NaCl (Fisher et al. 1996; Premkumar et
al. 2003), large-scale cultures are still severely carbon limited. Some of this limitation can be overcome
by the addition of bicarbonate.
The fact that β-carotene is a secondary metabolite means that conditions to achieve maximum
growth rate (i.e., lower salinities, high nutrients) and the conditions to achieve a high cell β-carotene
content (i.e., high salinities, low nutrients) are incompatible. In practical terms, this means two possible
culture strategies for the production of β-carotene by the culture of D. salina. One strategy is to initially
grow the algae at an optimal salinity for biomass generation (i.e., a salinity of less than 20% w/v NaCl +
sufficient nutrients) and then transferring the cells to a higher salinity under nutrient limiting conditions.
Such a 2-stage process however has the disadvantage that it requires a larger pond area and takes longer,
thus increasing production costs. The alternative strategy is to grow the algae at high irradiances and
at a salinity where β-carotene productivity is highest. Figure 2 shows the effects of salinity on growth
and β-carotene content and the optimum salinity for β-carotene productivity. However, in commercial
production, the algae are usually grown at a higher salinity to ensure reliable production and minimise
the risk of protozoan invasions which can result from a reduction in salinity due to dilution by rainfall
(Borowitzka and Borowitzka 1989). The irradiance received by the algae cells is managed by managing
the areal density of the algae (Grobbelaar 1995).
Haematococcus pluvialis and astaxanthin
The freshwater green alga Haematococcus pluvialis is the best natural source of the oxygenated carotenoid,
astaxanthin, which is accumulated in lipid droplets in the cytoplasm in the aplanospore stage of the alga
(Lang 1968; Wayama et al. 2013). Aplanospore formation and astaxanthin accumulation are induced
by nutrient limitation and other growth-inhibiting induction factors (often called ‘stress’ factors in the
literature) such as high temperature, high light, or increased salinity (Borowitzka et al. 1991; Boussiba
and Vonshak 1991; Vidhyavathi et al. 2008). Haematococcus produces only the 3S,3’S stereoisomer of